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11860102 | DESCRIPTION OF EMBODIMENTS In the following description, although embodiments of the present invention will be explained, it should be reminded that the present invention is not limited by the embodiments of the present invention explained below. <An Overview of One Embodiment of the Present Invention > FIG.1is a flowchart for explaining an overview of one embodiment of the present invention. First, test sample (tobacco raw materials), which contains known quantities of four kinds of TSNAs is prepared (S01); and fluorescence fingerprint of the above known test sample is measured to obtain fluorescence fingerprint information (S02). In this regard, when obtaining the fluorescence fingerprint information, it is desired to measure fluorescence via a filter for reducing intensity of light in a specific wavelength range. Further, it is preferable that the specific wavelength range be set to that equal to or greater than 400 nm. Details thereof will be explained later (refer to [A preferred embodiment of the present invention]). Next, a pre-process is applied to the obtained fluorescence fingerprint information as necessary (S03). In this regard, the above pre-process may be omitted. Details of the pre-process will be explained later. Next, the relationship between the obtained fluorescence fingerprint information and the total amount of the four kinds of TSNAs contained in the test sample is modeled to create an inference model (a calibration curve) (S04). Specifically, in this modelling, an estimation equation, in which the obtained fluorescence fingerprint information is set as an explanatory variable and total amount of the four kinds of TSNAs is set as response variable, is constructed by using various multivariate analysis techniques and data mining techniques; and a calibration curve (a regression equation) for inferring total amount of the four kinds of TSNAs in the test sample from the fluorescence fingerprint information is constructed. In this regard, the algorithm used for constructing an estimation equation may be a machine learning algorithm that is versatile and able to handle a nonlinear phenomenon, such as a Support Vector Machine (SVM), Random Forest (RF), a neural network, or the like. An example of a multivariate analysis technique used for modelling will be explained later. The inference model (the calibration curve) constructed as explained above is verified to confirm its effectiveness (S05). By using the inference model (the calibration curve), the effectiveness of which has been confirmed, and based on fluorescence fingerprint information of an unknown processed raw material (a tobacco raw material), a total amount of the four kinds of TSNAs contained as components in the unknown processed raw material (the tobacco raw material) is inferred (S06) The contained quantities of the four kinds of TSNAs are inferred, based on the inferred total amount of the four kinds of TSNAs and a known abundance ratio of each TSNA (S07). <Overview of Another Embodiment of the Present Invention> FIG.8is a block diagram for explaining an overview of another embodiment of the present invention. A TSNAs quantification apparatus100for quantifying TSNAs in a processed raw material comprises: a preprocessing means110that inputs fluorescence fingerprint information consisting of excitation wavelength, fluorescent wavelength, and fluorescence intensity data of samples (tobacco raw materials), and preprocesses the inputted fluorescence fingerprint information; an inference model creating means120that obtains a calibration curve by receiving an output from the preprocessing means110as an input, setting the preprocessed fluorescence fingerprint information as an explanatory variable, and setting total amount of four kinds of TSNAs contained in the test samples as a response variable; a total amount inferring means130that infers total amount of the four kinds of TSNAs contained as components in the unknown processed raw material (tobacco raw materials), based on the calibration curve obtained by the inference model creating means120and fluorescence fingerprint information of the unknown processed raw material (tobacco raw materials); and a contained quantity inferring means140that infers the contained quantities of the four kinds of TSNAs, based on the inferred total quantity of the four kinds of TSNAs and a known abundance ratio among the four kinds of TSNAs. In this regard, the preprocessing means110may be omitted. First, using a known fluorescence spectrophotometer or the like, fluorescence fingerprint information of a test sample, which contains the known kinds and quantities of components, is obtained. In this regard, when obtaining the fluorescence fingerprint information, it is desired to measure fluorescence via a filter for reducing intensity of light in a specific wavelength range. Further, it is preferable that the specific wavelength range be set to that equal to or greater than 400 nm. Details thereof will be explained later (refer to [A preferred embodiment of the present invention]). Next, the obtained fluorescence fingerprint information is inputted to the preprocessing means110to preprocess the inputted fluorescence fingerprint information. In this regard, the above preprocessing may be omitted. Details of the preprocessing, in the case that it is performed, will be explained later. Next, by the inference model creating means120, the relationship between the preprocessed fluorescence fingerprint information and total amount of the four kinds of TSNAs is modeled to create an inference model (a calibration curve). The above modeling is similar to that in the above-explained embodiment of the present invention. Thereafter, the thus created inference model (the calibration curve) is verified to confirm its effectiveness; and the inference model (the calibration curve), the effectiveness of which is confirmed, is stored in a memory or the like which is not shown in the figure. The total amount inferring means130infers, using the inference model (the calibration curve), the effectiveness of which is confirmed, total amount of the four kinds of TSNAs contained as components in an unknown processed raw material (tobacco raw material) based on fluorescence fingerprint information of the unknown processed raw material (tobacco raw material). In this regard, although it is desirable to preprocess, by the preprocessing means110, fluorescence fingerprint information of the unknown sample (the TSNAs quantification apparatus100inFIG.8adopts a configuration such as that explained above), it may be possible to omit the above preprocessing according to necessity. The contained quantity inferring means140infers the contained quantities of the four kinds of TSNAs, based on the inferred total amount of the four kinds of TSNAs and a known abundance ratio among the respective TSNAs. In this regard, information of the known abundance ratio among the respective TSNAs may be stored in the contained quantity inferring means140or the TSNAs quantification apparatus100in advance, or may be supplied from the outside to the TSNAs quantification apparatus100. <Multivariate Analyses Used in Modeling > Regarding multivariate analysis techniques used in modeling, various types of analysis methods such as PLS (Partial Least Squares) regression analysis, multiple regression analysis, principal component regression analysis, least squares method may be used. The PLS regression analysis is a technique for extracting principal components in such a manner that covariance between principal components and response variables becomes the maximum, and the technique is effective in the case that strong correlation exists among explanatory variables (in the case that multicollinearity exists). The principal component regression analysis is a technique for extracting principal components in such a manner that variance of the principal components is maximized; wherein principal component analysis is performed using explanatory variables only, and multiple regression analysis using the least-squares method is performed between the obtained principal components and the response variables. The multiple regression analysis is a technique wherein the least squares method is applied between explanatory variables and response variables, and it has a characteristic different from that of the principal component regression analysis. Since each of the above analysis techniques is well known and the present invention does not require special processing when performing a modelling process, explanation of details of contents of the processing will be omitted; however, explanation of the PLS will be provided later in relation to the process for creating the calibration curve. <Regarding the Technique for Recognizing a Total of Four Kinds of TSNAs as a Peak of a Fluorescence Fingerprint and Extracting the Peak > As explained above, it becomes possible, for the present invention, to quantify the total amount of the four kinds of TSNAs in consideration of quantitative value of components such as NNK and NAB that is difficult to quantify by itself, by adopting the technique for recognizing the total of the four kinds of TSNAs as a peak of a fluorescence fingerprint and extracting the peak; and quantifying the four kinds of TSNAs based on the total amount and the known abundance ratio among the respective TSNAs. With reference toFIGS.5and6, it will be explained that, when the four peaks of the four kinds of TSNAs are viewed collectively as a single peak, the shapes of the single-peaks of tobacco raw materials become approximately similar to each other despite differences between the heights of the respective single-peaks, in the case that the tobacco raw materials belong to the same tobacco species. FIG.5is a figure representing an example of a fluorescence fingerprint of a sample. As the sample, burley was used; and the total amount of the four kinds of TSNAs contained in the sample was 2.856 μg/g. It should be reminded that a process based on an adjustment condition that will be explained later, has been applied to the sample. FIG.6is a figure representing an example of a fluorescence fingerprint of an authentic sample including TSNAs reagents. The authentic sample was prepared by mixing reagents of the four kinds of TSNAs with an ethanol solution, wherein the ratio among the four reagents was set to be approximately the same as the ratio among the four kinds of TSNAs contained in Burley; and the total amount of the four kinds of TSNAs contained in the authentic sample was 1.28 μg/g. It should be reminded that the measurement conditions for the fluorescence fingerprints are the same for the both cases, and excitation light is 200-600 nm, fluorescence is 200-700 nm, resolution is 2.5 nm, slit width is 2.5 nm and photomultiplier-tube sensitivity is 950 V. As a result of comparison between the fluorescence fingerprint information inFIG.5and the fluorescence fingerprint information inFIG.6, the following findings could be obtained roughly: The fluorescence fingerprint information of the four kinds of TSNAs observed in the authentic sample inFIG.6(refer to the region of the elliptical frame to which symbol “A′” has been assigned inFIG.6(“region A′”)) could be observed in the sample inFIG.5(refer to the region of the elliptical frame to which symbol “A” has been assigned inFIG.5(“region A”)). When the region A′ inFIG.6is compared with the region A inFIG.5, it can be observed that the shapes of the both regions are approximately similar to each other, although there is a difference in the peak height of the fluorescence fingerprint information. InFIG.5, saturation has been occurring in many bands due to the influence of fluorescence of other materials in the sample (since sensitivity is set to the maximum value); however, the region A is identifiable even in the state explained above, so that it is considered that effect of contaminants thereon is low. It is considered that, in actual measurement, it will be effective if differentiation of fluorescence fingerprint information of TSNAs is achieved, by deleting fluorescence fingerprint information of fluorescence in a band in which occurrence of saturation is predicted. Further, regarding preprocessing, it is considered that it will be effective if execution of a process, wherein saturation relating to a second derivative and so on may affect other wavelengths, is avoided. In the following description, respective steps of one embodiment of the present invention will be explained. [Preparing Test Samples (Tobacco Raw Materials)] Regarding respective samples that contain known quantities of the four kinds of TSNAs, they were pulverized to a particle size of 1 mm diameter or less, and, thereafter, are sufficiently mixed, and the resultant mixtures were prepared as test samples. Since the four kinds of TSNAs could be localized in a tobacco raw material, it is preferable that the sample be pulverized to a certain particle size (1 mm diameter or less) and sufficiently mixed before performing measurement, and, thereafter, fluorescence fingerprint be obtained. In this regard, the quantities of the four kinds of TSNAs in respective samples were quantified in advance by using a high performance liquid chromatograph (HPLC-MS/MS). Further, a sample stored in advance for stabilizing the water content was used as the test sample. For making the water content in the test sample constant, it is preferable to store it under a harmony condition (the inside of a room in which temperature is 22 degrees Celsius and humidity is 60 percent) for 24 hours or more. By keeping the water content constant in advance as explained above, shifting of the peak is suppressed. [Obtaining Fluorescence Fingerprint Information] For obtaining fluorescence fingerprint information of the test samples, F-7000 manufactured by Hitachi High-Tech Science Corporation was used as the fluorescence fingerprint measuring device, and a reflection method (Front Face) was used when performing measurement. The measurement conditions were as follows: excitation light of 200-600 nm, fluorescence of 200-700 nm, resolution of 2.5 nm, slit width 2.5 nm, and photomultiplier-tube sensitivity of 950 V. In this regard, when the resolution of 2.5 nm is taken into consideration, an error of at least approximately 5 nm is allowed with respect to the measured wavelength. [Preprocessing Fluorescence Fingerprint Information] While, when obtaining fluorescence fingerprint information of a sample, it is possible to use measured values of a fluorescence fingerprint (a fluorescence spectrum for each excitation wavelength) as they stand, it is required to perform various pre-processes, as necessary. As a preprocessing technique for removing a noise from a measured fluorescence fingerprint and obtaining effective fluorescence fingerprint information, one or a combination of a process for removing non-fluorescent components, a process for removing scattered light, and a process for removing low-sensitivity regions, for example, may be adopted. Further, one or a combination of the following operation processes that may be applied to the obtained fluorescence fingerprint information may be adopted as a pre-process: centering, normalization, standardization, baseline correction, smoothing, auto-scaling, logarithmic conversion (log 10), secondary differentiation, and so on. Further, regarding the processing order when plural operation processes are combined, the following example order may be adopted: logarithmic conversion (Log 10)==>secondary differentiation==>normalization (normalize)==>auto-scaling (autoscale). Also, as the processes for removing wavelengths that do not have contribution to component information, the following example techniques may be adopted; and, since the following respective processing techniques are known, explanation about them will be omitted: (a) Variable important projection (VIP) (b) Interval PLS (iPLS) (c) Genetic algorithms (GA) (d) Jack-knife analysis (e) Forward interval PLS (f) Backward interval PLS (biPLS) (g) Synergy interval PLS (siPLS) (h) LASSO type method The application order of preprocessing may be set appropriately; however, in view of efficiency of processing, it is preferable that the processes such as a process for removing non-fluorescent components, a process for removing scattered light, and a process for removing low-sensitivity regions, and so on be preceded. In the above preprocessing, it is possible to use dedicated software such as Matlab, PLS#toolbox, and so on. It should be reminded that, although only auto-scaling only is used as the preprocessing in the present embodiment, the technique used in the preprocessing is not necessarily limited to the above technique. [Creation and Verification of a Calibration Curve] Specifically, a calibration curve is created using the PLS regression analysis (this may be simply referred to as “PLS”), for example, wherein the obtained fluorescence fingerprint information is an explanatory variable and the total amount of the four kinds of TSNAs is a response variable. An outline of the PLS regression analysis used when creating the calibration curve will be briefly explained below. In the PLS, the explanatory variable X (matrix) and the response variable y (vector) satisfy the following two basic formulas (1) and (2): X=TPT+E(1) y=Tq+f(2) In the above formulas, T denotes a latent variable (matrix), P denotes a loading (matrix), E denotes a residual of the explanatory variable X (matrix), q denotes a coefficient (vector), f denotes a residual of the response variables y (vector), and PTdenotes a transposed matrix of P. In this connection, the PLS does not directly use information of the explanatory variables X for modeling the response variable y, but the PLS converts a part of the information of the explanatory variable X to the latent variable t, and models the response variable y using the latent variable t. In this regard, the number of latent variables may be determined by using, as an index, a predictive explanatory variance value obtained by performing cross-validation, for example. Further, a latent variable may sometimes be referred to as a principal component. Especially, in the case of a single component model, (1) and (2) shown above can be represented by (3) and (4) shown below: X=t1p1T+E(3) y=t1q1+f(4) In the above formulas, t1denotes a latent variables (vector), p1denotes a loading (vector), and q1denotes a coefficient (scalar). Now, if it is supposed that t1is represented by a linear combination of X, (5) shown below holds: t1=Xw1(5) In the above formula, w1denotes a normalized weight vector. The PLS is a method for obtaining t1that maximizes covariance yTt1of y and t1, under the condition that the norm of w1is 1 (|w1|=1); and, for calculation of t1, the so-called method of Lagrange multiplier may be used. Since the calculation technique using method of Lagrange multiplier is well known, details of calculation are omitted; and only results of calculation with respect to w1, p1, and q1are shown as those represented by (6), (7), and (8) below: w1=XTy/|XTy|(6) p1XTt1/t1Tt1(7) q1=yTt1/t1Tt1(8) In this regard, t1in equations (7) and (8) is a vector calculated by substituting w1, that is obtained by using equation (6), in equation (5). A technique similar to the above technique can be used for calculation in a multi-component model, and, since the calculation technique is well known, its details will be omitted. For creating a calibration curve and verifying the created calibration curve, a plurality of samples, which contain the known quantities of the four kinds of TSNAs in the samples are prepared separately as a sample group for calibration to be used for creating the calibration curve, and as a sample group for validation to be used for verifying the calibration curve to confirm its effectiveness. In this regard, in the present example, 40 samples were prepared as samples for calibration, and 19 samples were prepared for samples for validation. Further, although the number of wavelengths is limited by VIP to approximately 1000 wavelength, the present invention is not limited thereto. To the calibration sample group, the above-explained PLS regression analysis (for example, the number of latent factors is 6), for example, is applied, and a calibration curve for inferring the total amount of the four kinds of TSNAs from obtained fluorescence fingerprint information is created. It should be reminded that the total amount of the four kinds of TSNAs in each sample used for creating the calibration curve is obtained by summing the quantities of the four kinds of TSNAs contained in each sample previously quantified by use of a high performance liquid chromatograph (HPLC-MS/MS). Next, for the validation sample group, the total amount of the four kinds of TSNAs is inferred by use of the calibration curve, from the obtained fluorescence fingerprint information, to thereby verify the calibration curve. FIG.7is a graph, wherein the horizontal axis corresponds to measured values (chemical analysis values) obtained by use of a high performance liquid chromatograph (HPLC-MS/MS), the vertical axis corresponds to inferred values of total amount of the four kinds of TSNAs obtained by use of fluorescence fingerprint information; and points corresponding to respective samples belonging to the validation sample group are plotted in the graph. Regarding the calibration sample group, the coefficient of determination R2=0.99 (SEC=0.08 μg/g), and there is high correlation between chemical analysis values and inferred values according to the calibration curve, so that it is confirmed that accuracy of inference is satisfactory. Further, according toFIG.7, regarding the accuracy of inference with respect to the validation sample group, the coefficient of determination R2=0.86 (SEP=0.22 μg/g), so that effectiveness of the calibration curve has been confirmed. [Inference of the Total Amount of the Four Kinds of TSNAs in a Sample (Tobacco Raw Material) That Contain Unknown Quantities of the Four Kinds of TSNAs] By using a calibration curve, effectiveness of which has been confirmed, and based on fluorescence fingerprint information of a sample (tobacco raw material) that contains unknown quantities of the four kinds of TSNAs, the total amount of the four kinds of TSNAs contained in the sample is inferred. It should be reminded that, regarding the sample that contains unknown with respect quantities of the four kinds of TSNAs, although it is possible to omit preprocessing applied to the obtained fluorescence fingerprint information, it is also possible to perform preprocessing having contents identical to those in the preprocessing performed when obtaining the calibration curve. [Inference of Respective Quantities of the Four Kinds of TSNAs Contained in an Unknown Sample] The contained quantities of the four kinds of TSNAs are inferred based on the inferred total amount of the four kinds of TSNAs and a known abundance ratio between respective TSNAs. In this connection, as explained above, abundance ratios among respective TSNAs are approximately constant in the case that the same species of tobacco is used; so that, if it is supposed that the known abundance ratio among respective TSNAs is a:b:c:d, the contained quantities of the four kinds of TSNAs can be calculated by multiplying the total amount of the four kinds of TSNAs by a/(a+b+c+d), b/(a+b+c+d), c/(a+b+c+d), and d/(a+b+c+d), respectively. [One Preferred Embodiment of the Present Invention] The inventors of the present invention have further performed detailed analysis, and found that further improvement in accuracy of inference can be expected by measuring fluorescence via a filter for reducing intensity of light in a specific wavelength range, in a fluorescence fingerprint information obtaining process such as that in the embodiment explained above. In the following description, the above matter will be explained in detail. FIG.9is a figure representing an example of a fluorescence fingerprint of an authentic sample obtained by use of the present embodiment, wherein the authentic sample contains TSNAs reagents similar to that inFIG.6. In this regard, the authentic sample was prepared by mixing reagents of the four kinds of TSNAs with an ethanol solution, wherein the ratio among the reagents was approximately the same as the ratio between those in Burley; and the total of the four kinds of TSNAs contained in the authentic sample was 1.28 n/g. The region in the elliptical frame, to which symbol “A″” is assigned, inFIG.9(“region A″”) is the region corresponding to the region “A′” inFIG.6. Further, the conditions of measurement of the fluorescence fingerprint are the same as those inFIG.6except for measurement of fluorescence via a filter, with excitation light of 200-600 nm, fluorescence of 200-700 nm, resolution of 2.5 nm, slit width of 2.5 nm, and photomultiplier-tube sensitivity of 950 V. The filter used has a function for reducing the intensity of light in a wavelength range of 400 nm or more. In other words, the filter used is that having a function for reducing, according to a predetermined light reducing rate, light in a wavelength range approximately corresponding to visible light in the light (florescence) emitted from the sample. FIG.10is a schematic diagram for explaining an overview of arrangement of a filter which has a function such as that explained above. It should be reminded that the filter is not necessarily one, and may be a combination of a band pass filter, which allows light in a predetermined wavelength range to pass through it, and a light reducing filter. Further, the wavelength range and the light reducing rate may be set appropriately according to the kind, the characteristics of a sample. It should be reminded that, in the preprocessing performed when obtaining fluorescence fingerprint information, and in processes for creating and verifying the calibration curve using the obtained fluorescence fingerprint information, the techniques such as those explained above are used, so that details thereof will be omitted. FIG.11is a graph wherein the horizontal axis corresponds to measured values (chemical analysis values) obtained by use of a high performance liquid chromatograph (HPLC-MS/MS), the vertical axis corresponds to inferred values of total amount of the four kinds of TSNAs according to fluorescence fingerprint information obtained by use of the present embodiment, and points corresponding to respective samples belonging to the validation sample group are plotted. Regarding the graph inFIG.11, it should be reminded that the calibration sample group and the validation sample group, that are used, are the same as those used in the case in which the results relating toFIG.7were obtained, and that the calibration curve is also created by applying the PLS regression analysis (the number of latent factors is 3) in a manner similar to that relating toFIG.7. According toFIG.11, regarding accuracy of inference in the validation sample group, has a coefficient of determination R2=0.92 (SEP=0.19 μg/g), and there is high correlation between chemical analysis values and inferred values according to the calibration curve, so that satisfactory accuracy of inference has been achieved. WhenFIG.11is compared withFIG.7, it is considered that further improvement of accuracy of inference can be expected by using the present embodiment, and the present embodiment may be applied more preferably to inference of the total amount of the four kinds of TSNAs contained in a sample. In this regard, a technique similar to the above-explained technique may be adopted for inference of the contained quantities of the four kinds of TSNAs, based on the inferred total amount of the four kinds of TSNAs and the known abundance ratio among respective TSNAs. It should be reminded that the present invention may also be adopted in various embodiments, which are different from the above-explained embodiments, within the scope of the technical ideas recited in the claims. REFERENCE SIGNS LIST 100: TSNAs quantification apparatus110: Preprocessing means120: Inference model creating means130: Total amount inferring means140: Contained quantity inferring means | 28,520 |
11860103 | DETAILED DESCRIPTION The following description discloses embodiments of a Raman spectrometer that is particularly suited to be carried into the field for use. The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The appended drawings are incorporated herein and constitute a part of the detailed description. The detailed description includes specific details for the purpose of providing a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. In some instances, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology. Like, or substantially similar, components are labeled with identical element numbers for ease of understanding. As used within this disclosure, the term “light” means electromagnetic energy having a wavelength within the range of 10 nanometers (nm) to 1 millimeter (mm). In certain embodiments, this range is preferably 300-1100 nm. In certain embodiments, this range is preferably 700-1100 nm. In certain embodiments, this range is preferably 10-400 nm (ultraviolet). In certain embodiments, this range is preferably 400-700 nm (visible). In certain embodiments, this range is preferably 700 nm to 1 mm (infrared). As used within this disclosure, the terms “frequency” (f) and “wavelength” (λ) should be considered interchangeable in characterizing a beam of light unless explicitly stated otherwise, as they are related by the equation λ=c/f, wherein “c” is the speed of light, and a value of either parameter is uniquely associated with a respective value of the other parameter. Furthermore, the terms “wavelength” and “wave number” should be considered interchangeable in characterizing a spectral response unless explicitly stated otherwise, as they are inversely related to each other and a value of either parameter is uniquely associated with a respective value of the other parameter. FIG.1illustrates diffraction of a coherent monochromatic light beam100at a slit110in a plate112, according to certain aspects of the present disclosure. The white lines in the incident beam100represent the “valleys” of the sinusoidal wave of the light, shown to the right for reference. The light that passes through the slit110is diffracted, also referred to as “scattered,” with the diffracted light102propagating as a spherical wave toward a surface120as seen inFIG.1. FIG.2depicts the intensity distribution200of diffracted light102on surface120, according to certain aspects of the present disclosure. The central peak210is centered under the incident beam100and flanked on both sides by secondary lobes (maxima)220and dark lines (minima)230, with the intensity of each succeeding lobe222decreasing as the lateral distance from the center increases. The pattern250formed on the surface120is governed by equation 1: sin(θ1)=1.22(λ/d) Equation 1: wherein:θ1is the angular position of the first order diffraction minima (the first dark ring)λ is the wavelength of the incident lightd is the width of the slit FIG.3depicts the refractive and reflective scattering of modes of an incident beam of monochromatic light, according to certain aspects of the present disclosure. A beam100of coherent light strikes the grating300at an angle θincrelative to an axis302that is perpendicular to the plane of the grating300. A portion of the incident beam100is transmissively diffracted into various modes at various angles θmtrnrelative to the incident beam100, wherein “M” is the mode number. For example, beam320is the 0th-order mode (primary) transmitted mode, beams322A,322B are the 1st-order transmitted modes, and beams324A,324B are the 2nd-order transmitted modes. The 0th-order mode is at an angle θ0trnwhile the 1st-order modes322A,322B are at ±θ1trnrelative to beam100. The angles θmtrnare dependent upon the frequency of the incident light, the mode order, the geometry, and the index of refraction of the material of the grating300as shown in Equation 2. This equation presumes air on both sides of the grating300. sinθm=sinθinc-mλΛEquation2 wherein:λ is the wavelength of the incident lightΛ A is the spacing of the lines of the gratingm is the order of the refracted rayθ is the angle from perpendicular to the grating, the subscript “inc” indicates the incident ray and the subscript “m” indicates the mthrefracted ray A portion of the incident beam100may be reflectively diffracted into various modes at various angles θmrefrelative to axis302. FIG.4illustrates Rayleigh scattering, according to certain aspects of the present disclosure. Raman spectroscopy relies upon inelastic scattering of photons. An incident beam of monochromatic light introduces energy into the molecules of a sample material and excites the system. The material emits the absorbed energy at frequencies associated with the various energy states of its molecules. The shift in energy gives information about the vibrational modes in the system. FIG.5illustrates an example Raman spectrum, according to certain aspects of the present disclosure. The light emitted by a material is plotted as intensity vs. frequency shift relative to the frequency of the source light, referred to as the “Raman shift,” traditionally measured in a unit called the wavenumber, which is the number of waves per cm (cm−1). The spikes of the plot are associated with vibrational modes of chemical bonds in one of the component materials. These spikes are referred to as “Raman bands” and the frequency and relative intensities of the bands allow us to identify the material by comparison of their spectral “signature” with a library of reference signatures of known materials. Heavy atoms and weak bonds have low Raman shifts. Light atoms and strong bonds have high Raman shifts. The plot ofFIG.5is the Raman spectrum of polystyrene. The high frequency carbon-hydrogen (C—H) bonds have a resonant frequency that creates the Raman band at about 3000 cm−1. The carbon-carbon (C—C) bonds create the small Raman band at around 800 cm−1. The C—H vibrations have a higher frequency than the C—C vibrations because hydrogen is lighter than carbon. The vibrations of a complex molecule partly consist of many simple diatomic vibrations while also showing the vibrational modes of larger groups of atoms, such as the expanding/contracting “breathing mode” of the aromatic carbon rings in polystyrene that appears at 1000 cm−1. FIG.6depicts a schematic representative of a conventional Raman spectrometer600, according to certain aspects of the present disclosure. A sample602to be characterized is placed against a surface of a sample holder610. A partially reflective mirror630deflects a portion of beam of source light634emitted by a light source632to travel toward the sample602as illumination beam636. The scattered light emitted by the sample602passes through the sample holder610and the sample lens620to the mirror630, where a portion604of the scattered light continues through a spatial filter, e.g., a slit,642. The sample lens310focuses the light604on the spatial filter642. The light606that has passed through the spatial filter642is formed into a unidirectional beam608by the collimating lens650. An excitation filter660blocks the transmission of the light from source632. The beam of filtered light608strikes the transmissive diffraction grating670. The diffracted light609coming out of the grating670is focused by the final focus lens680onto surface692of a detector690, which may be a charge-coupled device (CCD) sensor or camera. FIG.7depicts a block diagram700of an exemplary Raman spectrometer, according to certain aspects of the present disclosure. An instrument body702is configured to accept a holder710that has a compartment712configured to accept a sample of a material. In certain embodiments, the holder710includes a specimen plate720, a sample lens array722, a slit array724, and a collimating lens array726. In certain embodiments, the specimen plate forms a portion of the compartment712. In certain embodiments, the holder710comprises a lid714configured to selectably close over the accepted sample and permanently prevent removal of the sample from the holder compartment712. In certain embodiments, closure of the lid714is a non-reversable event wherein the lid714cannot be opened again without damage to the lid714or holder710, i.e., evidence of tampering. This feature makes the holder710into a tamper-resistant sample container that can be archived for later retrieval and re-examination. In certain embodiments, the lid714is hingedly attached to the body of the holder710. The instrument body702is coupled to a transmissive grating730, a focusing lens732, and a detector740. The detector740is communicatively coupled to a processor750and configured to provide information about the Raman spectrum of this sample, i.e., the sample signature, to the processor750. The processor750can store the sample signature and associated data, e.g., a date, a sample ID, a location such as where the sample was collected, a field designator, a user name, etc., in the memory760. The processor750is coupled to a comm module752that is communicatively coupled to a server790that may be at a remote location or implemented as a virtual device on a “cloud” server. In certain embodiments, the server790is implemented as a software service. The body702is also coupled to a user interface754, for example a color graphics display with an overlaid touchscreen. The body702is also coupled to a power supply770that provides power to all of the electronic components of the apparatus and, in certain embodiments, received information from one of more of the components. In certain embodiments, the body702is also coupled to a GPS module756that provides location information to the processor750. Light782from the light source780is guided to the sample, which is shown as contained in compartment712having a lid714in this example. The sample is stimulated by the light782to emit light, a portion of which passes through the sample plate720, the sample lens array722, the slit array724, and the collimating lens array726to the grating730. The light is refracted by the grating726and a portion of the refracted light passes through the final focus lens732to the detector740. Detector740creates data that is provided to the processor750. The processor850is communicatively coupled a memory760via a bidirectional path. In certain embodiments, the memory760contains instructions that, when transferred to the processor750and executed by the processor750, cause the processor750to receive the data from the detector740, compare the received data with a portion of one or more reference files, and determine an attribute of the sample. In certain embodiments, memory760also contains the one or more reference files that are respectively associated with one or more materials and the instructions comprise instructions to transfer a portion of the files to the processor750. In certain embodiments, the one of more reference files are stored on the server790that is communicatively connected to the processor750through the comm module752, for example over a wired and/or wireless network. In certain embodiments, the light source780emitting light at a determined frequency. In certain embodiments, the frequency is in the infrared band. In certain embodiments, the frequency is in the visible band. In certain embodiments, the frequency is in the ultraviolet band. In certain embodiments, the light source780comprises an optical filter (not shown inFIG.7) that passes light only in a selected band having a frequency bandwidth. In certain embodiments, the source780emits light in a band having a bandwidth that is less than or equal to 5 nm. In certain embodiments, the source780emits light in a band having a bandwidth that is less than or equal to 2 nm. In certain embodiments, the light source780comprises a plurality of sources each emitting light at a different frequency. FIG.8depicts a flow chart800of an exemplary method of use, according to certain aspects of the present disclosure. In the first step810, a user places a sample of a material in the holder. This step may include closing a lid over the sample and, in certain embodiments, closing of the lid may be a one-time action, i.e., a non-reversable event. In step820, the user places the holder in the instrument body. In step822, the user performs set-up and data entry operations. In certain embodiments, one of steps820and822includes the instrument retrieving a unique identifier (ID) from the holder that was placed in the body and storing this ID. In certain embodiments, one of steps820and822includes determination of the current physical location of the instrument using a locating system, e.g., an internal global position system (GPB) module and storage of the location. Once the instrument is fully configured for this sample, the user initiates the analysis of the sample in step830. In certain embodiments, step830comprises one of more of collection of multiple spectral signatures using different frequencies of source light and collection of multiple spectral signatures using different optical filters to modify the light emitted by the sample. In certain embodiments, step830comprises stimulation of the sample, for example by exposure of the sample to one or more of a magnetic field, an electrostatic field, and a radio frequency (RF) field. In certain embodiments, step830comprises introduction of a fluid into the sample. After the spectral signatures are collected, the instrument transfers a portion of the data, which includes one or more of the spectral signatures and information entered by the user and determined by the instrument, to a server that may be remote. Software on the server analyzes the data in step840, compares the results of the analysis to a library of signatures in step842, and identifies a match between the sample and the materials of the library in step844. In step846, the software on the server analyzes the spectral signatures and determines an amount of the matched material in the sample. In certain embodiments, the analysis determines an amount present only for a pre-determined material. In certain embodiments, the analysis may calculate a ratio of the amount of one material to the amount of another material. Step850stores the results of the analysis and the data in a memory on the server. In certain embodiments, the memory is located separate from the server. In certain embodiments, the results are sent to the instrument and stored in a memory in the instrument or a removable drive, e.g., a thumb drive, attached to the instrument. The results are sent to the instrument in step860and provided to the user on the user interface. In certain embodiments, step860includes providing the information on one of a personal computer, a laptop, a tablet, a smart phone, or other display. FIG.9Adepicts an exemplary embodiment of an optical system900of the apparatus, according to certain aspects of the present disclosure. In this embodiment, the system900comprises a holder910, an optical filter920, a grating930, a focusing lens940, and a detector950. The optical filter920is configured to block the wavelength of the excitation light. The grating930separates the light emitted by the sample into its various wavelengths. The focusing lens940focuses each separated wavelength onto the detector950in a spatially separated position. FIG.9Bdepicts the passage of light emitted by the sample (not visible as it is located within the holder910) through the optical system900, according to certain aspects of the present disclosure. A single beam960of collimated light being emitted from the holder910is shown for clarity, although there are a plurality of adjacent beams of light coming from the holder910that are collimated and parallel to each other. After passing through the filter920, the filtered light962strikes the grating930and a portion is transmissively refracted into a refracted beam964. Each of the matching-wavelength spectral sub-component beams964of the plurality of adjacent beams of light that are exiting the grating930are still collimated and parallel to each other, i.e., all sub-components at the same wavelength will enter the focusing lens940at the same angle. For example, the green portions from the multiple beams are all collimated and parallel to each other as they enter the focusing lens940. The focusing lens940focuses the spectral sub-component beams964into converging sub-component beams966that have foci on a plurality of spatially separate locations on the detector950. In certain embodiments, the focusing lens940comprises multiple elements for focusing and beam shaping. In certain embodiments, the focusing lens940comprises one or more of a curved mirror and a flat mirror. In certain embodiments, the detector950comprises one or more of a linear 1D array of sensing elements, e.g., pixels, and a 2D array of sensing elements. FIG.10Adepicts an exemplary embodiment of a single-use holder910, according to certain aspects of the present disclosure. The holder910has a frame912on which is printed a unique identifier914, e.g., a matrix code. In certain embodiments, the identifier914comprises a human-readable code. In certain embodiments, the identifier914comprises an electronic device, e.g., a ROM chip or an RFD chip, that stores the identifier. FIG.10Bdepicts an optical assembly1000removed from the cavity916of the holder. The cavity916is adjacent to a compartment (not visible inFIG.10B) of the frame912that is configured to accept a sample of a material such that the sample is pressed against the optical assembly1000. FIG.10Cdepicts an exploded view of an exemplary optical assembly1000, according to certain aspects of the present disclosure. In this example, optical assembly1000comprises one or more of a sample plate1010, a sample lens array1020, a slit array1030, and a collimating lens array1040, each coupled to at least one of the adjacent component and to the frame912. In this embodiment, the sample plate1010is an optically clear planar sheet that is disposed, when the optical assembly1000is mounted in the frame912, proximate to the sample compartment such that the sample is in contact with a surface of the sample plate1010. In certain embodiments, optical assembly1000comprises a sample plate1010, a slit array1030, and a collimating lens array1040. Other embodiments of the sample plate are discussed with respect toFIGS.12A and12B. The sample lens array1020comprises a plurality of focusing elements1022that are mounted in a frame1024with a set-back1026that provides clearance for the height of the focusing elements1022as well as a portion of a separation of the focusing elements1022from the next component. In certain embodiments, the focusing elements1022comprise one or more of spherical, aspherical, and diffractive optical components. In certain embodiments, the plurality of focusing elements1022are configured to collect light from a respective plurality of regions of the surface of the sample and produce a respective plurality of beams of light. The slit array1030comprises one or more slits each having a width. In certain embodiments, a portion of the plurality of focusing elements1022is arranged in a straight row that is parallel to a slit of the slit array1030and the focusing elements of the row are configured to focus the respective beams of light on the slit. In certain embodiments, the plurality of focusing elements and the plurality of slits are arranged in a non-rectilinear pattern, e.g., concentric circles. The collimating lens array1040comprises a plurality of collimating lenses1042mounted in a frame1044with a set-back1046that provides clearance for the height of the collimating lenses1042as well as a portion of a separation of the collimating lenses1042from the next component. In certain embodiments, a portion of the plurality of collimating lenses1042is arranged in a straight row that is parallel to a slit of the slit array1030. Each collimating lens1042is configured to accept the refracted light emanating from one of the slits and modify the light to form a collimated beam of light. All of the modified plurality of beams of light are collimated in a common direction. In certain embodiments, the diameter of the individual focusing elements1022and/or the collimating lenses1042is less than 125 μm. In certain embodiments, the focusing elements1022and/or the collimating lenses1042are holographic lenses. In certain embodiments, the use of holographic lenses in place of conventional lenses provides a 10× improvement in light capture. In certain embodiments, the use of holographic lenses in place of conventional lenses provides a 50× improvement in light capture. In certain embodiments, the use of holographic lenses in place of conventional lenses provides a 100× improvement in light capture. In certain embodiments, the separation of the sample plate1010from the sample lens array1020is less than 5 mm. In certain embodiments, the separation of the sample plate1010from the sample lens array1020is less than 2 mm. In certain embodiments, the separation of the sample lens array1020and the slit array1030is less than 5 mm. In certain embodiments, the separation of the sample lens array1020and the slit array1030is less than 2 mm. In certain embodiments, the separation of the slit array1030and the collimating lens array is less than 5 mm. In certain embodiments, the separation of the slit array1030and the collimating lens array is less than 2 mm. FIG.10Ddepicts another embodiment of a disposable holder, according to certain aspects of the present disclosure. The optical assembly1000ofFIG.10Cis replaced by optical assembly1050that replaces sample lens array1020with a sample lens array1060and the collimating lens array1040with a collimating lens array1070. Each column of individual lenses1022and1042is replaced by a cylindrical lens1062and1072. Each cylindrical lens1062focuses light on one of the slits of slit array1030and the light emerging from each slit is collimated by one of the cylindrical lenses1072. With the cylindrical lens arrays of optical assembly1050, the sample lens array1060captures light reflected from respective parallel areas of the sample and focuses the light onto a respective slit of slit array1030. Compared to a conventional spectrometer, this arrangement captures light from a much larger area of the sample, in certain embodiments 2×, 5×, 10×, 20×, 50×, and 100× the amount of light, and consequently the intensity of the light from the sample is increased by approximately the same amount. An increase in intensity creates a stronger signal from the detector, thereby improving the measurement of small signals, e.g., values of Raman peaks. The collimating lens array1070captures light that emerges from a respective slit of slit array1030and collimates the light in a direction common to all the cylindrical lenses1072. With reference toFIG.9B, the grating930will disperse the light from all collimating lenses1072at common angles, e.g., light of a common frequency is collimated in a single direction. The focusing lens940will focus light of a common frequency to a common location on the sensor950. By gathering light from a large area of the sample, the intensity of the light at the sensor is proportional to a total area of the parallel areas of the sample from which reflected light is captured by the sample lens array1060. This increase in intensity is effectively optical amplification increases the sensitivity of the analyzer in the same way that a larger diameter telescope is able detect dimmer stars by collecting more light from the sky. In certain embodiments, the intensity of the light at the sensor is 10× that of a conventional spectrometer having a single slit. In certain embodiments, the intensity of the light at the sensor is 20× that of a conventional spectrometer having a single slit. In certain embodiments, the intensity of the light at the sensor is 50× that of a conventional spectrometer having a single slit. In certain embodiments, the intensity of the light at the sensor is 100× that of a conventional spectrometer having a single slit. In certain embodiments, the holder910, or910A, is separable from the main spectrometer, which contains one or more of the optical filter920, the grating930, the focusing lens940, and the detector950as well as one or more of a light source, a power supply, a user interface, a processor, a memory, and other components (not shown inFIG.9A) necessary to measure aspects of the light provided by the holder910. In certain embodiments, one or more of the optical filter920, the grating930, the focusing lens940are provided as part of the holder910and therefore not part of the main spectrometer. In certain aspects, the body912comprises an interface configured to detachably mate with a spectrometer. In this manner, a plurality of holders910, or910A, may be used to capture a respective plurality of samples, wherein each holder910is sequentially mated to the spectrometer and the sample of that holder analyzed. FIG.11Adepicts an exemplary embodiment of a sample plate1100, according to certain aspects of the present disclosure. The sample plate1100comprises a channel1102that is configured to accept a liquid sample (not shown inFIG.11A). In certain embodiments, the channel1102passes through the width of the sample plate1100so as to form a passage through which a liquid sample can flow, thus enabling continuous monitoring of a stream to be periodically tested. In certain embodiments, the channel1102is a sealed compartment with entrance and exit ports (not shown inFIG.11A) so as to facilitate introduction of a liquid sample into the channel1102and removal of air. FIG.11Bdepicts an exemplary embodiment of a sample plate1110, according to certain aspects of the present disclosure. In certain embodiments, the sample plate1110comprises an actuator1112at least partially embedded in the sample plate1110. In certain embodiments, the actuator1112is selected from the group of a temperature-control element, a filtering element, and a stimulation element. In certain embodiments, the temperature-control element can perform at least one of heating or cooling the sample. In certain embodiments, the filtering element can selectively allow or block selected frequencies of light. In certain embodiments, the stimulation element generates one of a magnetic field, an electrostatic field, and a dynamically oscillating electric field, e.g., a radiofrequency (RF) field. In certain embodiments, the sample plate1110comprises a coating1114on one or more surfaces. In certain embodiments, the coating1114functions as one or more of an optical filter, an electric shield, an antenna, and an electric conductor that may be patterned. FIG.11Cdepicts an exemplary embodiment of a sample plate1120, according to certain aspects of the present disclosure. In certain embodiments, the sample plate1120comprises a reservoir1124embedded within the body1122of the sample plate1120and configured to contain a fluid1126and a pump1128fluidically coupled between the reservoir1124and a surface of the sample plate1120and configured to selectably expel a portion of the fluid1126from the sample plate. FIG.12A-12Bdepicts exemplary means of providing illumination to the sample, according to certain aspects of the present disclosure. FIG.12Adepicts a schematic of certain aspects of a novel Raman spectrometer1200, according to certain aspects of the present disclosure. In this exemplary embodiment, an incident ray1210of coherent, monochromatic, unidirectional illuminating light strikes a transmissive diffraction grating930at an angle1230. In certain embodiments, angle1230is selected such that one of the mode rays1220is directed along the optical axis1202of the spectrometer1200. In certain embodiments, the angle1230is selected to direct a higher-order mode ray, for example a 1st-order ray1222, along the optical axis1202. In certain embodiments, the angle1230is selected to direct the primary ray along the optical axis1202. One advantage of the novel arrangement of the light source (not shown inFIG.12A) is the elimination of the partially reflective mirror630shown inFIG.6. As the light in a conventional spectrometer must be first reflected and then transmitted by the mirror630, there is a loss of energy, normally about 50%, of the scattered light coming from the sample. Although the selected mode with have only a portion of the energy of the incident beam1210, there is no energy loss in the optical path from the sample to the grating930. A second advantage of the spectrometer1200is the more compact arrangement of components, as the light source is now generally aligned with the long dimension of the device, while a conventional spectrometer600has a laser light source632, which may be large and heavy, positioned on one side. Repositioning the source632in a conventional design requires additional optical elements, for example folding mirrors and rigid supporting structure, that add weight and cost. Light passing through grating920from a first surface to a second surface on the opposite side of the grating920from the first surface is described as passing through the grating920in a first direction, regardless of the angle of the path of the light to a perpendicular reference axis, such as axis1202. Similarly, light passing through grating920from the second surface to the first surface is described as passing through the grating830in a second direction regardless of whether the path of the light traveling in the second direction is parallel to the path of the light traveling in the first direction. The use of “first direction” and “second direction” are meant only to convey the general direction of transmission from one surface to another. FIG.12Bdepicts another exemplary embodiment of means of providing illumination of the sample, according the certain aspects of the present disclosure. In certain embodiments, a beam of illuminating light is provided via a fiber optic cable1280, or functional equivalent, that passes through openings1262in the frame1260of holder1250and then into a receiving port1272of the sample plate1270. This type of side illumination is known in optics and provides light output across the planar surface of the sample plate1272. In certain embodiments, the beam of illuminating light is provided via a fiber optic cable1290, or functional equivalent, that passes through the holder1250from a backside and mates with a diffuser (not visible inFIG.12B) within the frame1260and disperses the light across the planar surface of the sample plate1270. In certain embodiments, the illumination light is modulated, for example by driving the light source with a square wave, thereby producing periods of illumination of the sample, i.e., when the source is on, separated by intervals of dark, i.e., when the source is off. Sensing of the output of the detector is synchronized with the square wave, for example by recording the output only while the source is off and adding the recordings of multiple dark intervals. In certain embodiments, sensing of the output of the detector occurs during portions of both the illuminated periods and the dark periods and the respective sets of measurements are compared during analysis. Certain embodiments of the disclosed Raman spectrometer incorporate a novel arrangement of a light source that introduces the light into the optical path of the apparatus by passing the light through the transmissive diffraction grating in direction opposite the direction of the light passing from the sample to the detector. This novel arrangement beneficially reduces the size and complexity of the optical path by eliminating components that are critical in conventional spectrometers. Certain embodiments of the disclosed Raman spectrometer consolidate critical elements of the optical path into a single-use holder. Miniaturization of the optical elements and the use of arrays of lenses in place of single lenses enables precise alignment without requiring complex alignment techniques during manufacturing. Material Analysis Cannabidiol (CBD) is a phytocannabinoid discovered in 1940. It is one of 113 identified cannabinoids in cannabis plants, along with tetrahydrocannabinol (THC), and accounts for up to 40% of the plant's extract. CBD has been found to have beneficial medical effects, including relief from pain and stiffness. CBD may be supplied as an oil, a powder, or as a liquid suspension. The mechanism of action for its biological effects has not been determined. CBD does not have the psychoactivity of THC and is not listed as a proscribed substance. Hemp is a subspecies of cannabis that contains significant levels of CBD and low levels of THC. The 2018 United States Farm Bill removed hemp and hemp extracts (including CBD) from the Controlled Substances Act. THC, on the other hand, is still listed under Schedule I under the Controlled Substances Act. Federal law classifies a plant as hemp and therefore exempt from the Controlled Substances Act if the THC content, specifically delta-9 tetrahydrocannabinol, is ≤0.3% by weight. This is echoed in the California Code of Regulations that require that hemp crops be tested for THC content prior to harvest and that a hemp crop found to contain more than this amount of THC must be destroyed. FIGS.13A-13Bare the 2D representations of the chemical structures of CBD and THC, respectively. It can be seen that while there is a great deal of structure in common, there are small differences in the composition and bond structure between CBD and THC that produce different Raman responses. FIGS.14A-14Cpresent experimental results provided by the Raman analyzer disclosed herein. Graph1400depicts the spectral response of a sample of CBD powder having ≤0.3% THC when illuminated with a source light having a wavelength of 515 nm. The x-axis is the frequency of the response in nanometers while the y-axis is an intensity. The response curve includes a large generally smooth portion1410that is the photoluminescence of the sample combined with narrow spikes1402,1404that are Raman peaks associated with the chemical structure of CBD. In this experiment, peak1402has an intensity of approximately 60,000 and peak1404has an intensity of approximately 18000. Graph1420depicts the spectral response of a sample of plant material containing approximately 47% by weight of THC when illuminated with a source beam of light having a wavelength of 515 nm. The curve includes the photoluminescence1412, which has a markedly different shape that the photoluminescence1410of the CBD sample, and Raman spikes1422and1424. In this experiment, peak1422has an intensity of approximately 100,000 and peak1424has an intensity of approximately 75000. Graph1440depicts the spectral response of a sample consisting of a mixture of 40% of the powder of graph1400and 60% of the plant material of graph1420when illuminated with a source beam of light having a wavelength of 515 nm. Peaks1442,1444correspond to peaks1402,1404and peak1446corresponds to peak1422. Peak1442has an intensity of approximately 24000, peak1444has an intensity of approximately 7000, and peak1446has an intensity of approximately 64000. The shoulder1448corresponds to the peak1424but does not provide a distinct peak. In certain embodiments, the ratios of the intensities of corresponding peaks provides a measure of the amount of the associated material in the sample. In this case, the values of corresponding peaks and their respective ratios are shown in Table 1. TABLE 1PEAK-AINTENSITYPEAK-BINTENSITYRATIO1442240001402600000.414466400014221050000.6 The ratio of peaks1442/1402represents the relative amount of the CBD powder in the mixture of graph1440and the ratio of peaks1446/1422represents the relative amount of the THC-containing plant material powder in the mixture. The respective ratios of 0.4, 0.6 are complementary and together support an assessment that the mixture is approximately 40% CBD powder and 60% plant material. This data demonstrates the principle of establishing “fingerprints” of two reference materials and then being able to determine the proportions of a mixture of the two materials using this analyzer disclosed herein. In certain embodiments, the determination compares the magnitudes of one or more Raman peaks of a measurement of a sample of the mixture to the fingerprints. In certain embodiments, the determination compares attributes of the overall response curve to the fingerprints. In certain embodiments, the attribute is a curve value at a specific wavelength or wave number. In certain embodiments, the fingerprint is a computational prediction of a wavelength or wave number of a Raman peak based on one or more aspects of the chemical structure of a reference material. It must be noted that the measurement made by the disclosed apparatus is based on the surface area of the sample illuminated by the light source and examined by the sensor of the disclosed apparatus while the limit of THC content is provided as a weight percentage. Conversion of the sensed results to a weight percentage is accomplished by use of reference samples that have been characterized by an accepted standard process, for example liquid chromatography coupled with mass spectrometry. In the example ofFIGS.14A-14C, the THC content of the plant material was determined by a third party using an accepted laboratory process and the THC content of the mixture of graph1440is therefore 60% of 47%=0.28% by weight. The wavelengths of the various attributes of the spectral response of a sample are related to the wavelength of the source light. In addition, the shape and overall intensity of the photoluminescence may be different depending on the wavelength of the source beam. The graphs ofFIG.14A-14C, for example, are produced by a source beam of 515 nm. In certain embodiments, the frequency of the source beam is selected to maximize the visibility of a Raman peak, i.e., to provide the greatest difference between the intensity of a Raman peak and the intensity of the photoluminescence at and around the frequency of that Raman peak. In certain embodiments, the frequency of the source beam is selected to maximize the visibility of a particular Raman peak that is characteristic of a target molecule. In certain embodiments, measurements are made with a plurality of light sources having a respective plurality of wavelengths. In certain embodiments, the plurality of wavelengths are selected to each maximize the visibility of one of a plurality of Raman peaks characteristic of a target molecule. In certain embodiments, the intensity of a Raman peak is determined only when the source light is the wavelengths selected to maximize the visibility of that Raman peak. In certain embodiments, the intensity of a Raman peak of a reference sample is determined at the same wavelengths as selected to maximize the sample. In certain embodiments, the ratios of a plurality of Raman peaks to their corresponding peak of a reference standard made with the same wavelengths of source light are combined to produce a composite value of the amount of a target material present in a sample. In certain embodiments, the intent of the analysis of the spectral response is not to identify a material within a sample and is simply to determine the amount of a pre-determined molecule is present. For example, a sample of a hemp plant is analyzed to determine the amount of THC present in the sample. In another example, a sample of a food product is analyzed to determine the amount of a pre-determined pesticide in the sample. In another example, a sample of a wine is analyzed to determine the amounts of pre-determined molecules in the sample, wherein the pre-determined molecules are associated with the one or more of the taste, smell, and texture of wine. Sample Holder FIG.15Adepicts an exemplary sample holder1500in an open configuration, according to certain aspects of the present disclosure. The holder1500has a body1510with an access opening1512into a sample compartment1514. The cap1520has an interior surface1522and is connected to the body1510by a strap1524. In certain embodiments, the cap1520is separate from the body1510. FIG.15Bdepicts sample holder1500in a closed configuration, wherein the surface1522of cap1520seals the opening1512. In certain embodiments, the materials of one or more of the body1510, the contact surface of opening1512, cap1520, and surface1522are selected to maintain a controlled environment in the compartment1514. In certain embodiments, one or more of the body1510, the contact surface of opening1512, cap1520, and surface1522comprise a coating. In certain embodiments, the contact surface of opening1512and surface1522comprise a sealing material, for example a gasket, to enhance the sealing of the opening1512. Tamper-Evident Sample Holder FIG.16Adepicts an exemplary tamper-evident sample holder1600, according to certain aspects of the present disclosure. In certain embodiments, the holder1600comprises a body1610having a sample compartment1614, a closure compartment1616, and a window1622providing visibility into one or more of the sample compartment1614and the closure compartment1616. There is an access opening1618to the sample compartment1614and a latch opening1620to the closure compartment1616. There is a cap1630with a body1632coupled to a non-return fitment1634. In certain embodiments, the sample compartment1614is separated from the closure compartment1616such that a sample placed in the sample compartment1614does not obscure the visibility of the latch opening1620through the window1622. In certain embodiments, there is no access into the closure compartment1616regardless of whether the cap1630is installed on the body1612. In certain embodiments, window1622is replaced by separate windows (not shown) respectively covering the sample compartment1614and closure compartment1616. In certain embodiments, the window1622is implemented as an opening (not shown) from the exterior into the latching compartment1616that provides visibility of the inside of latching opening1620while preventing access to the interior of the latching compartment as well as preventing a broken-off fitment1634(seeFIG.16C) from coming out of the latching compartment1616. In certain embodiments, a portion of window1622comprises a transparent material and is sealed to the body1612such that a sample in compartment1614is protected from contamination. FIG.16Bdepicts the holder1600with the cap1632disposed in the closure position relative to the body1610, wherein the body1632is configured to seal the access opening1618. The fitment1634has passed through the latching opening1620to a latched position within the closure compartment. The fitment1634is visible through the window1622while in the latched position such that a user can verify that the fitment1634is intact and in the intact location so as to verify that the cap1630has not been removed, indicating that a sample contained in the sample compartment1614is undisturbed. FIG.16Cdepicts the holder1600after the cap1630has been removed and replaced. The cap1630is configured such that fitment1634separates from the body1632when the cap1630is dislodged from its closure position. The fitment1634is retained in the closure compartment1616when separated from body1632such that a user can see the broken-off fitment1634and verify that the fitment is not in the intact position ofFIG.16B. Further, the fitment1634is not accessible without further damage to the window1622or body1612, thus preventing returning the broken-off fitment1634to the intact position, for example by gluing it to the stub1636of cap1630. If a user attempts to remove the cap1630and replace it with a new undamaged cap (not shown), the new cap will present a fitment in the latched position but the broken off fitment1634ofFIG.16Cwill still be present, thus indicating that the sample has possibly been tampered with. Determination of Sample Location FIGS.17A-17Cdepict an exemplary sample capture device1700, according to certain aspects of the present disclosure. FIG.17Adepicts the sample capture device1700as having a body1710with a user interface1712. In certain embodiments, the user interface1712is a graphic user interface with a touchscreen overlay. In certain embodiments, the user interface1712comprises a keypad having one or more of numeric and alpha keys, wherein the keypad can be virtual or hardware. The body1710further comprises a recess1720configured to accept a material sample holder, for example the sample holder1500shown inFIGS.15A-15Bwherein the body1510fits into the recess1720. In certain embodiments, the device1700is coupled to a secondary device, for example a mobile phone or a laptop (not shown) and the secondary device provides the user interface. FIG.17Bdepicts the sample capture device1700with a sample holder1500disposed in the recess1720. A user is now able to place a sample of a material in the compartment1514. The user then closes the cap1520and secures it to the body1510, thus sealing the sample in the compartment1514. FIG.18depict an exemplary block diagram1800of the sample capture device1700, according to certain aspects of the present disclosure. In certain embodiments, the device1700comprises a scanner1734that scans an identifier1540on the sample holder1500and a sensor1732that detects whether the cap1520has been secured to the body1510of sample holder1500. In certain embodiments, the device1700also includes a camera1736and a GPS module1738and a communication module1742configured to communicate with external devices, for example a server1750, over conventional wired and/or wireless networks and communication systems. The device1700comprises a memory1740is disposed within the body1710. In certain embodiments, the memory is remote. In certain embodiments, memory1740contains instructions that, when loaded into the processor1730and executed, will cause the processor1730to perform one or more of the following actions:receive information associated with latch1530from the sensor1732receive information associated with identifier1540from scanner1734receive the current location of the device1700from the GPS module1738receive information associated with the user and/or sample from the user interface1712receive an image from the camera1736store a portion of the received information in a memory, for example memory1740provide a portion of the received information to an external system, for example server1750, over a known communication system such as Wi-Fi or a cellular network An exemplary use of the sample capture device1700is to take the device1700into a field planted with a plant and collect one or more samples of the plants for later evaluation. The user enters information associated with this field through the user interface1712, for example the user, the field identification, a company farming the field, and the plant. In certain embodiments, the device1700captures the identification of the user through an alternate method, for example facial recognition through a camera, scanning a barcode, or detection of an RFD-enabled badge. The user places an empty sample holder1500in the sample capture device1700. The user takes a picture with the camera1736of a plant from which a sample will be collected. The user collects a portion of a plant and places the portion in the compartment1514of the sample holder1500, then closes the cap1520and secures it in place. The device1700detects the closure of the cap1520, which triggers the processor1730to receive the identifier1540from the scanner1734and the physical location from the GPS module1738. The processor1730stores the entered and received information in the memory1740along with the date and time to create a data record associated with this sample. In certain embodiments, a portion of the data record is copied to the server1750. The user then removes the sample holder1500from the sample capture device1700and places the sample holder1500in a transfer container. The user need not mark the sample holder or provide special handling to maintain a record of where the sample was collected or protect the sample. In certain embodiments, the sealed sample holder1700provides a traceable and tamper-evident container that can be transferred to an analysis device or a testing service. In certain embodiments, the sample holder is configured to provide a stable environment in order to reduce changes in the sample over time. In certain embodiments, the sample holder1500is archived as a long-term record of the sample and measurement. In certain embodiments, the instructions in memory1740, or software running on the server1750or another device such as a tablet or personal computer, uses the data record to provide a map showing the location of the sample collection. Absorbance Spectrometry In absorbance spectrometry, a material sample is illuminated with a beam of incident light. The material absorbs the light and re-emits a portion of the absorbed energy as light of a frequency determined by the molecular structure of the material. The spectral response of the emitted light will have peaks a resonant frequencies associated with specific types of bonds and structures. These peaks can be measured and compared to reference spectral responses of known reference materials to determine the contact of a reference material in a sample, much as discussed with respect toFIG.14A-14C. FIG.19Adepicts an exploded view of an exemplary sample holder1900configured for analysis of a sample based on absorbance of light, according to certain aspects of the present disclosure. The holder1900has a body1910that is similar to the body912ofFIG.10D. The optical assembly1050ofFIG.10Dis replaced by an optical assembly1950comprising a cover slip1920, a slit array1930, an array of collimating lenses1932, and optionally a grating1934. In certain embodiments, the cover slip1920has a transparent body1922with a reflective layer1924covering a portion of the body1922. FIG.19Bdepicts an exemplary illustration of how light emitted by the sample1901passes through the elements of optical assembly1950. A sample of material1901is placed into compartment1912that is covered by the optical assembly1950. In the exploded view ofFIG.19B, the sample1901is shown as located such that the underside of the sample1901is in contact with the upper surface of the cover slip1922, as it would be in actual use. Although the incident light is omitted fromFIG.19for clarity, a beam of excitation light is directed upward (in the orientation ofFIG.19A) to illuminate the underside of cover slip1920over both reflective layer1924and the remaining unobstructed portion of cover slip1920. In certain embodiments, the excitation light has a specific wavelength, for example having passed through a selectable filter or generated by a light source that emits light having a narrow bandwidth of light about that wavelength. An example source light is light from a laser that emits light with a nominal wavelength of 532 nm that is passed through a 527-537 nm bandpass filter to produce a narrow-band beam of excitation light that is directed to the sample. In certain embodiments, the sample is serially observed using a plurality of wavelengths of excitation light, for example using a filter wheel comprising a plurality of optical filters. The light1950that is emitted by the sample1901is radiated (shown as thick arrows) through the coverslip over a solid angle. A portion of the incident light is reflected by the reflective layer1924to produce reference light1952(thin arrows). The light1950and1952both pass through the slit array1930, whereupon each slit illuminated by light1950emits light1960and each slit illuminated by light1952emits light1962, which are then respectively focused by the array of collimating lenses1932into collimated beams of light1970from the sample1901and light1972from the reflective surface1924. The collimated beams1970,1972are optionally passed through a grating1934to spatially separate the light by wavelength as discussed with respect toFIG.6. The light beams1970and1972are spatially separated such that beam1970can be measured separately from beam1972, whereupon the measured characteristics of the reference beam1972can be used to calibrate the sensor used for light1970or to interpret the measurement of light1970. In certain embodiments, the beams1970,1972may be alternately directed to a common sensor (not shown inFIG.19B). In certain embodiments, a sliding mask (not shown inFIG.19) or functional equivalent is provided at a point along the optical path from the cover slip1920to the grating1934to enable a user to direct the collimated light from either the sample or the reflective surface to the detector such that the detector observes only the reflective surface rays, which provides a reference signal suitable for calibration of the system, or only the sample emission rays. Embodiments A1. An apparatus for analysis of a sample, comprising: a frame having a first axis; a sample holder coupled to the frame and disposed on the first axis; a transmissive diffraction grating coupled to the frame and disposed along the first axis such that light traveling along the first axis from the sample holder passes through the grating in a first direction; and a source coupled to the frame and configured to emit a first light to pass through the grating in a second direction that is opposite the first direction. A2. The apparatus of A1, further comprising: a lens coupled to the frame; and a spatial filter coupled to the frame; wherein the lens and spatial filter are disposed along the first optical axis. A3. The apparatus of A1, wherein a portion of the first light emitted by the source is diffracted by the grating to travel parallel to the first optical axis. A4. The apparatus of A3, wherein: the light emitted by the source is monochromatic; the diffracted portion of the first light comprises a mode; the light emitted by the source travels to the grating along a second optical axis that is not parallel to the first optical axis; and an angle between the first and second optical axes determines the mode of the diffracted portion of the first light. A5. The apparatus of A4, wherein: the light source comprises a plurality of sources each emitting light at a plurality of unique frequencies; the second optical axis comprises a plurality of secondary optical axes that are respectively associated with the plurality of unique frequencies and respectively disposed at a plurality of unique angles to the first optical axis. A6. The apparatus of A3, wherein: the light emitted by the source is white light; the diffracted portion of the white light comprises a color; the light emitted by the source travels to the grating along a second optical axis that is not parallel to the first optical axis; and an angle between the first and second optical axes determines the color of the diffracted portion of the light. A7. The apparatus of A1, wherein: the sample holder is configured to accept the sample such that the sample is disposed on the first optical axis; the first light illuminates the sample, whereupon the sample emits a second light that enters the grating in the first direction; and a portion of the second light exits the grating as diffracted second light; the apparatus further comprises: a lens coupled to the frame and configured to focus the diffracted second light to form a Raman spectrum; a detector coupled to the frame and configured to sense the Raman spectrum and provide data related to the Raman spectrum; a processor communicatively coupled to the detector; and a non-volatile memory communicatively coupled to the processor and comprising: a reference file associated with a material; and an instruction file that, when executed by the processor, causes the processor to receive the data from the detector, compare the received data with a portion of the reference file, and determine an attribute of the sample. A8. The apparatus of A7, wherein the attribute of the sample comprises an amount of a material component in the sample. A9. The apparatus of A1, wherein the light passes from the source to the grating without being reflected. B1. A method of obtaining a Raman spectrum of a sample, the method comprising the steps of: illuminating the sample with a first light, whereupon the sample emits a second light that passes through a transmissive diffraction grating in a first direction and exits the grating as diffracted second light, wherein the first light passed through the grating in a second direction opposite the first direction prior to illuminating the sample; focusing the diffracted second light to form a Raman spectrum. B2. The method of B1, further comprising the steps of: coupling a disposable element to an apparatus, wherein the disposable element comprises a sample holder and the grating and the apparatus comprises a light source configured to emit the first light; and placing the sample on the sample holder. B3. The method of B1, wherein the first light is coherent. B4. The method of B1, wherein the first light is monochromatic. B5. The method of B1, further comprising the step of filtering the second light to remove a portion of the first light. B6. The method of B1, further comprising the step of evaluating the Raman spectra to determine an attribute of the sample. B7. The method of B6, wherein the attribute of the sample comprises an amount of a material component in the sample. C1. An apparatus for analysis of a sample of a material, comprising a holder configured to accept the sample, the holder comprising a sample plate comprising a first surface configured to contact the accepted sample; and a sample lens array coupled to the sample plate, the sample lens array comprising a plurality of focusing elements. C2. The apparatus of C1, wherein the holder further comprises a slit array coupled to the sample lens array, the slit array comprising a plurality of slits; and a collimating lens array coupled to the slit array, the collimating lens array comprising a plurality of collimating lenses. C3. The apparatus of C1, wherein the plurality of focusing elements are configured to collect light from a respective plurality of regions of the surface of the sample and produce a respective plurality of beams of light. C4. The apparatus of C2, wherein a portion of the plurality of focusing elements are arranged in a first straight row that is parallel to a first slit of the plurality of slits of the slit array; and the focusing elements of the first row are configured to focus their respective beams of light on the first slit. C5. The apparatus of C4, wherein the plurality of collimating lenses are configured to receive a portion of the plurality of beams of light that pass through the plurality of slits; and modify each of the plurality of beams of light such that all of the modified plurality of beams of light are collimated in a common direction. C6. The apparatus of C1, wherein the holder further comprises a compartment configured to accept the sample, wherein the sample plate forms a portion of the compartment; and a lid that is coupled to the holder and configured to selectably close over the compartment and permanently prevent removal of an accepted sample from the holder. C7. The apparatus of C1, wherein the focusing elements are holographic lenses. C8. The apparatus of C2, wherein the collimating lenses are holographic lenses. C9. The apparatus of C1, wherein the sample plate further comprises a channel configured to accept a liquid sample. C10. The apparatus of C1, wherein the sample plate further comprises an actuator selected from the group of a temperature control element, a filtering element, and a stimulation element. C11. The apparatus of C1, wherein the holder is configured to accept a beam of illuminating light and guide the accepted beam of illuminating light to a side of the sample plate that is not the first surface. C12. The apparatus of C1, further comprising a frame configured to removably accept the holder; a detector coupled to the frame; a focusing lens coupled to the frame; and a transmissive diffraction grating coupled to the frame. C13. The apparatus of C12, further comprising an optical filter coupled to the frame; and a spatial filter coupled to the frame. C14. The apparatus of C12, wherein the grating comprises a first surface and a second surface that is opposite the first surface; a portion of a beam of light emitted by the accepted sample passes through the grating from the first surface to the second surface; and the frame is further configured to accept a beam of illuminating light and guide the accepted beam of illuminating light to the second surface of the grating such that a refracted portion of the beam of illuminating light is directed through the grating and exits the first surface toward the accepted sample. C15. The apparatus of C1, wherein the holder is configured for use with only a single sample. Headings and subheadings, if any, are used for convenience only and do not limit the invention. Reference to an element in the singular is not intended to mean “one and only one” unless specifically so stated, but rather “one or more.” Use of the articles “a” and “an” is to be interpreted as equivalent to the phrase “at least one.” Unless specifically stated otherwise, the terms “a set” and “some” refer to one or more. Terms such as “top,” “bottom,” “upper,” “lower,” “left,” “right,” “front,” “rear” and the like as used in this disclosure should be understood as referring to an arbitrary frame of reference, rather than to the ordinary gravitational frame of reference. Thus, a top surface, a bottom surface, a front surface, and a rear surface may extend upwardly, downwardly, diagonally, or horizontally in a gravitational frame of reference. Although the relationships among various components are described herein and/or are illustrated as being orthogonal or perpendicular, those components can be arranged in other configurations in some embodiments. For example, the angles formed between the referenced components can be greater or less than 90 degrees in some embodiments. Although various components are illustrated as being flat and/or straight, those components can have other configurations, such as curved or tapered for example, in some embodiments. Pronouns in the masculine (e.g., his) include the feminine and neuter gender (e.g., her and its) and vice versa. All structural and functional equivalents to the elements of the various aspects described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. No claim element is to be construed under the provisions of 35 U.S.C. § 112, sixth paragraph, unless the element is expressly recited using the phrase “means for” or, in the case of a method claim, the element is recited using the phrase “operation for.” A phrase such as an “aspect” does not imply that such aspect is essential to the subject technology or that such aspect applies to all configurations of the subject technology. A disclosure relating to an aspect may apply to all configurations, or one or more configurations. A phrase such as an aspect may refer to one or more aspects and vice versa. A phrase such as an “embodiment” does not imply that such embodiment is essential to the subject technology or that such embodiment applies to all configurations of the subject technology. A disclosure relating to an embodiment may apply to all embodiments, or one or more embodiments. A phrase such as an embodiment may refer to one or more embodiments and vice versa. The word “exemplary” is used herein to mean “serving as an example or illustration.” Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. All structural and functional equivalents to the elements of the various aspects described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. No claim element is to be construed under the provisions of 35 U.S.C. § 112, sixth paragraph, unless the element is expressly recited using the phrase “means for” or, in the case of a method claim, the element is recited using the phrase “step for.” Furthermore, to the extent that the term “include,” “have,” or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term “comprise” as “comprise” is interpreted when employed as a transitional word in a claim. Although embodiments of the present disclosure have been described and illustrated in detail, it is to be clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, the scope of the present invention being limited only by the terms of the appended claims. | 66,084 |
11860104 | DETAILED DESCRIPTION OF THE DRAWINGS In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention. The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings. It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. Any reference in the specification to either one of a system, a method and a non-transitory computer readable medium should be applied mutatis mutandis to any other of the system, a method and a non-transitory computer readable medium. For example—any reference to a system should be applied mutatis mutandis to a method that can be executed by the system and to a non-transitory computer readable medium that may stores instructions executable by the system. Because the illustrated at least one embodiment of the present invention may for the most part, be implemented using electronic components and circuits known to those skilled in the art, details will not be explained in any greater extent than that considered necessary as illustrated above, for the understanding and appreciation of the underlying concepts of the present invention and in order not to obfuscate or distract from the teachings of the present invention. Any number, or value illustrated below should be regarded as a non-limiting example. There may be provided a system, a method, and a non-transitory computer readable medium that stores instructions for accurate Raman spectroscopy. There may be provided a system, a method, and a non-transitory computer readable medium that stores instructions for collection and interpretation of angle resolved Raman scattered light from Raman-active materials, micro-structures and nano-structures. There may be provided a system, a method, and a non-transitory computer readable medium that stores instructions that may obtain Raman spectra at different scattering angles. There may be provided a system, a method, and a non-transitory computer readable medium that stores instructions for illuminating the sample from different illumination angles, creating additional independent spectra. There may be provided a system, a method, and a non-transitory computer readable medium that stores instructions for using polarization optics, enabling control of illumination polarization continuously and extraction of specific Raman spectra at different polarizations. There may be provided a system, a method, and a non-transitory computer readable medium that stores instructions for performing further analysis of information retrieved from the illumination of the sample for unraveling critical quantities about the nano-structure materials and dimensions. The terms “micro-scale” and “nano-scale” are used in an interchangeable manner. Any reference to a nano-scale may be applied mutatis mutandis to a micro-scale. Nano-scale means having at least one dimension that may range between one tenth of a nanometer till one hundred nanometers. Micro-scale means having at least one dimension that may range between one tenth of a micron till one hundred microns. FIG.1illustrates an example of some elements of the optical measurement system. FIG.1illustrates an example of a part200of an optical measurement system. Part200allows to illumination sample300by a laser or other light source (or several light sources) from various angles and polarizations and the collection of Raman scattered light from the sample from various angles and polarizations. Part200includes an illumination path that includes laser201, field imaging path202, entrance aperture stop203, mirror204, aperture imaging path205, first illumination lens206, illumination field stop207, second illumination lens208, illumination polarizer209, beam splitter210, illumination half wavelength plate (HWP) such as a illumination rotating HWP211, objective aperture stop/back focal plane212, and objective lens213. The collection path (for collecting radiation from sample214) includes objective lens213, objective aperture stop/back focal plane212, beam splitter210, collection quarter wavelength plate (QWP) such as collection rotating QWP215, collection rotating polarizer216, collection filter217, first collection lens218, first collection field stop219, rotating dove prism220, second collection lens221, collection aperture stop222, cylindrical lens223, second field stop224, slit lens225, slit226and optical unit235. The optical unit235may be an optical spectrometer. InFIG.1the optical unit235is illustrated as including a grid (grating)231, first mirror/lens232for directing radiation that passed through the region of interest onto the grid231, second lens/mirror233for directing light from grid231towards a detector234. The Illumination Path The optical scheme inFIG.1describes two main imaging paths. First, the central imaging path follows a path of a collimated light beam exiting from a laser201. The beam goes through entrance aperture stop203, and is reflected by mirror204. The mirror204position may be controlled by a linear motor or any other mechanical manipulator, causing a lateral shift in the reflected laser beam. The laser beam is then focused by first illumination lens206onto illumination field stop207and recollimated again by second illumination lens208. It than passes through illumination polarizer209and is partially reflected by beam splitter210. The reflected light then goes through illumination rotating HWP211which rotates the polarization. After passing the waveplate, the light goes through a high NA (numerical aperture) objective lens213and is imaged on the plane of sample214. The second imaging path is wider that the first and illustrates the imaging of the entrance aperture stop203onto the objective aperture stop/back focal plane212. This is done using the relay lenses (first illumination lens206and second illumination lens208). The magnification between entrance aperture stop203and objective aperture stop/back focal plane212is a function of the focal lengths of first illumination lens206and second illumination lens208: M=f2/f1. The Collection Path After reaching the sample, light is scattered from it and goes through the objective and waveplate (thus, the polarization of Reighley scattered light is rotated back to its original state). From there, it reaches the beam splitter210, where part of the scattered light is transmitted and reaches collection rotating QWP215, collection rotating polarizer216collection filter217that may be a long-pass filter which filters the Reighley scattered light and transmits only the Raman scattered light. Then, using first collection lens218, the light is focused onto a conjugate plane where first collection field stop219is located. Close to the field stop there is a rotating dove prism220, responsible for rotating the image of the aperture stop. After that, second collection lens221collimates the light again. The image of the objective aperture stop/back focal plane212(rotated by the dove prism) is formed at the focal plane of second collection lens221on the collection aperture stop222. Following that, cylindrical lens223focuses the light in one direction onto second field stop224. At this surface, the rays originating from the aperture stop are collimated in one direction. A slit lens225creates an image of the Raman scattered light on a slit226, where one direction (x) holds information on different field points and the other direction (y) holds information on different angular (NA) points. Angle of Illumination (AOI) Control. By use of mirror (denoted204inFIG.1—may be a motorized mirror), the suggested system has the capability to change the angle of illumination on the sample (seeFIG.2). Thus, different information can be extracted from the sample. For example, in most crystal materials different phonons are sensitive to the direction of the incident electric field. By illuminating light mainly in normal direction (NA0272inFIG.2), the electric field of linearly polarized light will be mostly in the direction parallel to the sample's surface (e.g. Ex), exciting one phonon (in Silicon that will be LO—Longitudinal Optic phonon). However, illuminating from a large angle (NA+1or NA−1for example the NAs denotes271and273inFIG.2), some of the electric field is in the z direction (Ez), i.e. parallel to the surface's normal, which excites a different phonon (in Silicon [100]: TO—Transverse optic) in addition to the LO phonon. Exciting these different phonons can contribute, in case the outcome Raman spectra in different angles is uncorrelated, i.e. each spectrum is correlated to different parameters in the sample, or in case one parameter requires information from both angles. We suggest two methods to displace a beam by amount Δlight(seeFIG.3): A. In reflection: using a linear motor to displace mirror204(FIG.3) such that: Δlight=xmirror[1] B. In refraction: tilting an optical plate of a transparent plate204′ instead of mirror such that a (tplate, nplateand θplateare the plate's thickness, refractive index and tilt angle, respectively): Δlight=tplatesinθplate[1-cosθplatenplate2-sin2θplate][2] Both suggested methods A and B allow scanning only in one direction. scanning in the perpendicular direction can be done by:a. In A: using another mirror rotated by 90 degrees relative to the current mirrorb. In B: using another tilted plate or tilting the same plate in two orthogonal anglesc. A composition of A and Bd. Rotating the sample Thus, with AOI control at two axes, the electric field distribution260in illumination is given by Eill(x−rAOIcos θAOI, y−rAOIsin θAOI) (seeFIG.4), where (x,y) is the objective aperture stationary axes and (rAOI, θAOI) are the polar coordinates of the illumination beam center (controlled via AOI such that Δlight(x)=rAOIcos θAOIand Δlight(y)=rAOIsin θAOI). Illumination Polarization Control As described above and inFIG.1, the combination of three elements: mirror204, illumination polarizer209, and illumination rotating HWP211, allow control of the polarization direction in illumination. The illumination polarizer209could be a fixed element, its task is to increase the contrast of polarized light before it reaches the sample. The direction of polarized light should be either ‘s’ or ‘p’ relative to the beam splitter210before it reaches the sample, as any other direction will introduce elliptical polarization in reflection due to difference in Fresnel reflections (magnitude and phase) between ‘s’ and ‘p’ polarized light. Thus, the illumination polarizer209could be adjusted by rotating about the optical axis to achieve maximum contrast in reflection from the beam splitter210. After reflection, the light reaches the illumination rotating HWP211, which is positioned in a manner that its fast axis is at an angle ϕHWPrelative to the incident polarization. After crossing it, the polarization is rotated by twice that amount, achieving an angle 2ϕHWPrelative the original polarization. Thus, with AOI and polarization control, the illumination beam field distribution, upon the objective's aperture stop is described by the following formula (seeFIG.4): E→illumination(12)=E0(x-rAOIcosθAOI,y-rAOIsinθAOI)[cos(2ϕHWP)sin(2ϕHWP)][3] Where E0(x, y) is the field distribution at the aperture plane before the AOI transformations and the superscript “12” indicates that this is the field on the aperture stop at illumination. Upon crossing the objective, each (x-y) point on the aperture is refracted in a different angle, resulting in variations in polarization state when hitting the sample. These variations affect the intensity distribution and polarization state of the incoherent scattered light from the sample. This can be formulated as a general two-dimensional Stokes vector: S→scattered(12)=[S0(12)S1(12)S2(12)S3(12)](x,y,rAOI,θAOI,ϕHWP)[4] Where S0-3(x, y, rAOI, θAOI, ϕHWP) are real functions of x, y, rAOI, θAOIand ϕHWP. Collection polarization measurement: In collection, there are three participating elements in evaluating polarization state (in the following order, for an incoherent beam exiting the sample, neglecting the effect of the objective and beam-splitter on the scattered light): the illumination rotating HWP211, collection rotating QWP215and collection rotating polarizer216. For simplicity, the beam splitter's210effect on polarization is neglected. When the scattered light passes through the illumination rotating HWP211it undergoes a phase difference between polarization components (same as in illumination). Thus, for the simple case of the scattered light polarization being equal to illumination polarization (the latter was rotated by angle 2ϕHWPafter crossing the ½ waveplate (HWP)), than in the return direction it is rotated in the opposite direction by an angle −2ϕHWP, retrieving the original polarization above the ½ waveplate (HWP). Using Muller calculus, the effect of the illumination rotating HWP211on the scattered light is as follows: S→scattered(11)=[10000cos(4ϕHWP)sin(4ϕHWP)00sin(4ϕHWP)-cos(4ϕHWP)0000-1]S→scattered(12)=[S0(12)cos(4ϕHWP)S1(12)+sin(4ϕHWP)S2(12)sin(4ϕHWP)S1(12)-cos(4ϕHWP)S2(12)-S3(12)][5] After crossing illumination rotating HWP211, the scattered light is partially transmitted through the beam-splitter. Above it, two options are suggested for implementing polarization measurement/ A collection rotating polarizer216(angle ϕCLP) only, collection rotating QWP215is removed. In this case, {right arrow over (S)}scattered(16)will be: S→scattered(16)=12[1cos(2ϕCLP)sin(2ϕCLP)0cos(2ϕCLP)cos2(2ϕCLP)12sin(4ϕCLP)0sin(2ϕCLP)12sin(4ϕCLP)sin2(2ϕCLP)00000]S→scattered(11)[6] Which allows extraction of all stokes parameters but S3(12). The stokes vectors for ϕCLP=0andπ2 are (using equations [4]-[6] and dropping (x, y, rAOI, θAOI, ϕHWP)): S→scattered(16)=12[1±100](S0(11)±S1(11))=12[1±100]Iscattered(16)[7] Where (+) and (−) signs are for ϕCLP=0 and ϕCLP=π2, respectively. The intensity on the detector is: Iscattered(16)=S0(12)(x,y,rAOI,θAOI,ϕHWP)±(cos(4ϕHWP)S1(12)(x,y,rAOI,θAOI,ϕHWP)+sin(4ϕHWP)S2(12)(x,y,rAOI,θAOI,ϕHWP)) [8] Thus, by measuring the intensity on the detector for ϕCLP=0andπ2, this configuration allows interpreting partial information on the Raman scattered light (enabling to extract S0(12)and the summation cos(4ϕHWP)S1(12)+sin(4ϕHWP)S2(12)). B—In this option, the combination of the ¼ waveplate (QWP) and the collection polarizer allows to receive the full Stokes vector of the scattered light. The waveplate is allowed to rotate with rotation angle ϕQWPwhereas the collection polarizer is stationary in either ‘p’ or ‘s’ polarizations. After crossing both elements, {right arrow over (S)}scattered(16)is (for horizontal polarizer): S→scattered(16)=12[1cos2(2ϕQWP)12sin(4ϕQWP)sin(2ϕQWP)1cos2(2ϕQWP)12sin(4ϕQWP)sin(2ϕQWP)00000000]S→scattered(11)[9] Substituting [5] in [9] yields the intensity on the detector: Iscattered(16)=S0(12)+cos2(2ϕQWP)(cos(4ϕHWP)S1(12)+sin(4ϕHWP)S2(12))+12sin(4ϕQWP)(sin(4ϕHWP)S1(12)-cos(4ϕHWP)S2(12))-sin(2ϕQWP)S3(12)[10] Angular Resolved Raman (ARS) The suggested system enables one to retrieve information on the Raman scattering distribution from different angles of the sample. In that sense, the scattered light can be decomposed to a series of plane waves, where each of these plane waves is focused onto a distinct point on the objective aperture stop/back focal plane212(seeFIG.5with different plane waves274,275and276—at the center and edges of the numerical aperture of the objective lens). Using second collection lens221and slit lens225, an image of the aperture plane is formed on collection aperture stop222. By using a dove prism220, located between these lenses, and rotating it by angle θDPthe collection aperture image on collection aperture stop222will be rotated by 2θDP(seeFIG.6). Neglecting non-ideal imaging effects (such as optical aberrations), the aperture-stop images after passing through an unrotated (θDP=0°) and rotated (θDP=22.5°) dove prism261and262are shown inFIG.7—first and right sides of the figure, respectively. The cylindrical lens collects light from all adjacent horizontal points and focuses them onto a single geometric point. Thus, a thin line is formed along the vertical direction of the slit (y). For example, at θDP=0° points5through9are imaged onto a single point on the slit center, whereas at θDP=22.5° (aperture rotates by 45°) points4,7and10are summed at the center. Thus, if the intensity on the non-rotated aperture212is I(12)(x, y) (where (x, y) are the non-rotated coordinates) then the rotated aperture image222is: I(22)(x′,y′,θDP)=I(12)(x′ cos 2θDP−y′ sin 2θDP,x′ sin 2θDP+y′ cos 2θDP) [11] On the slit226, after passing through both cylindrical and slit lenses, light rays along the ‘x’ axis are focused onto a single point. Therefore, the intensity distribution on the spectrometer's slit (I(26)) is the accumulated intensity after integrating along the ‘x’ axis (the spectral direction): I(26)(y′,θDP)=∫I(12)(x′ cos 2θDP−y′ sin 2θDP,x′ sin 2θDP+y′ cos 2θDP)*P(x′,y′,θDP)dx′[12] Where P(x, y, θDP) is a known transfer function of the optical system and (*) states convolution. By taking several measurements at various rotation angles and using inversion formulas, one can reconstruct the original intensity on the aperture I(12)(x, y). For the simple case in which systematic effects are negligible, P(x′, y′, θDP)=δ(x′, y′) (where δ(x, y) is delta function); thus, equation [12] is reduced to the known Radon transform and I(12)(x, y) is found through an inverse Radon transform: I(12)(x,y)=12π∫0πI(26)(y′(x,y,θDP),θDP)*g(x,y,θDP)dθDP[13] where y′(x, y, θDP)=y cos 2θDP−x sin 2θDPand g(x, y, θDP)=∫−∞∞|r|e−iry′(x,y,θDP)dr. As an example to the benefit of the ARS technic,FIG.9shows the angular Raman scattering from Silicon substrate for Co-pol-0 configuration (where Co-pol refers to the case in which ϕCLPequals the illumination polarization state on the sample and the ‘0’ means that ϕHWP=0). In that case, equation [8] is reduced to (rAOI=θAOI=0): Iscattered(16)=S0(12)(x,y,0,0,0)+S1(12)(x,y,0,0,0) [14] FIG.9illustrates examples of a silicon substrate has three different phonons (two Transverse modes (TO) and one longitudinal mode (LO)) with a very distinctive and singular NA map for each phonon. On the detector, information from all three phonons will be collected simultaneously. TO1 phonons have more intensity at the edges—radiation pattern281of TO1 has regions of interest282at its edges centered at angles 45 degrees, 135 degrees, 225 degrees and 315 degrees. TO2 phonons have more intensity at the edges—radiation pattern282of TO2 has regions of interest285at its edges centered at angles 90 degrees and 270 degrees. LO phonon is more evident at the aperture center—radiation pattern283of LO has region of interest288at its center. It should be noted that changes in one or more acquisition parameters may change the radiation pattern. Which radiation pattern is expected to obtain when illumination a certain location of a wafer and at certain acquisition parameters may be represented by a model. FIG.9illustrates that spatial filters (masks)283,286and289may be are applied on the NA/aperture plane to mask irrelevant signals in each one of TO0, TO1 and LO. Thus, by measuring at two dome configurations, at θDP=0 and π8, one can decouple the information of the TO phonons from that of the masking LO phonon. Mask on the NA/Aperture Plain An additional implementation of ARS is the use of spatial masks in the aperture plain222instead of the rotating dove prism220and the cylindrical lens223. Using Raman modeling capabilities, we can simulate the angular dependence of each vibrational mode. Different vibrational modes have different distributions along the aperture plain. For example, inFIG.9the angular distribution of the aperture plain of the 2 Transverse modes (TO) and the longitudinal mode (LO) of bare Si in co-0 configuration is presented. Using a mask will allow to block/pass specific modes of interest. There are several use cases for such a technique. For example to increase the extinction ratio (super nulling configuration) and increase the sensitivity to specific layers on top of the Si substrate. An additional application is using these masks to couple to each mode (LO or TO) for strain decomposition or to couple to different materials in the stack of interest. FIG.10is an example of an optical measurement system200. Optical measurement unit200includes an illumination path, a collection path, a control unit and a mechanical movement unit303for supporting sample300and for moving the sample300in relation to the collection and illumination paths. It should be noted that the sample300may be static while the illumination and/or collection paths may move. It should be noted that both the sample300and at least one path of the collection and/or illumination may move in relation to each other. InFIG.10the collection path and the illumination path share an objective lens213, and a half wavelength plate (HWP)109. It should be noted that the illumination path and the collection path may share more components, may share other components, or may not share any component. InFIG.10the illumination angle and the collection angle are perpendicular to the sample. It should be noted that any other illumination angles and/or collection angles may be provided. The illumination path is configured to control various parameters of an illumination beam such as but not limited to polarization, frequency spectrum, shape, size, coherency, path, intensity, and the like. Various elements illustrated in the figure assist in the control of said parameters. Elements that control polarizations are referred as polarization control elements. Elements that control other parameters of the beam are referred to as additional control elements. It should be noted that a single element may control one or more parameters of the beam. Non-limiting examples of elements include polarizers, half waveplates, quarter waveplates, analyzers, lenses, grids, apertures, and the like. The collection path is configured to control various parameters of the impinging beam such as but not limited to polarization, frequency spectrum, shape, size, coherency, path, intensity, and the like. Various elements illustrated in the figure assist in the control of said parameters. The illumination path is illustrated as including (a) laser102, (b) illumination optics103that include illumination polarization control element103(1) and additional illumination control element103(2), (c) a beam splitter such as dichroic beam splitter210, (d) HWP109, and (e) objective lens213. The additional illumination control element may control one or more parameters that differ from polarization—for example shape, size, angle of propagation, and the like. The collection path is illustrated as including (a) a beam splitter such as dichroic beam splitter210, (b) HWP109, (c) objective lens213, (d) collection optics105that include adjustable optics105(1) for changing the collection path thereby compensating for misalignments, additional collection control element105(2), and collection polarization control element105(3), (e) spatial filter223, and (f) optical unit235(e.g. an optical spectrometer) that includes a grid/grating231, first lens/mirror232for directing radiation that passed through the region of interest onto the grid231, second lens/mirror233for directing light from grid231towards detector234. The optical unit235is configurable in the sense that the spatial relationship between the grid231and at least the second lens233may be altered to direct different radiation lobes from the grid231towards the second lens233.FIG.4(as wellFIGS.10and11) illustrates a rotating unit238that may rotate the grid231in relation to the first and second lenses. Movements other than rotations may be used to change the spatial relationship between the elements of optical unit235. Detector234is configured to generate Raman spectra. The detector234is coupled to control unit239that is configured to control various components/units/elements of the optical measurement system and may be configured to control the calibration process. FIG.11is an example of an optical measurement system200′. Measurement system200′ differs from measurement unit200by (a) not including HWP109, (b) including multiple lasers102′, and (c) including a processing unit234′ for processing detection signals. The illumination optics103may be configured to combine or select radiation from the multiple lasers. In some cases only one laser may be activated at a time. FIG.12is an example of an optical measurement system200″. Measurement system200″ differs from measurement unit200by (a) including multiple lasers102′. The illumination optics103may be configured to combine or select radiation from the multiple lasers. In some cases only one laser may be activated at a time. FIG.13illustrates an example of a method400. Method400may include one or multiple iterations of steps410,420and430. Method400may start by step410of determining current acquisition parameters of a Raman spectroscope to provide a current acquisition set-up. The determining is based on at least one expected radiation pattern to be detected by a sensor of the Raman spectroscope as a result of an illumination of a first area of a sample. The first area may include a first nano-scale structure. At least a part of the at least one expected radiation pattern is indicative of at least one property of interest of the first nano-scale structure of the sample. The current acquisition parameters belong to a group of acquisition parameters. Step410may be based on a model that maps different acquisition set-ups, to different expected radiation patterns. The model is merely a non-limiting example for predicting maps the relationship between different acquisition set-ups and different expected radiation patterns. The group of acquisition parameters may include an illumination angle, an illumination polarization, a collection angle, a collection polarization, an illumination spatial masking and a collection spatial masking. At least one acquisition parameter may be changed between one iteration of steps410,420and430—to another iteration of steps410,420and430. Current acquisition parameters may include an illumination angle, wherein the setting comprises determining a position of a mirror of an illumination path of the Raman spectroscope Current acquisition parameters may include an angular position of a dove prism of a collection path of the Raman spectroscope. Current acquisition parameters may include a polarization set (provided, generated) by an optical module that may include an illumination polarizer, a rotating illumination half waveplate, and a rotating collection one fourth waveplate. Only a beam splitter may be positioned (a) in an optical path between the illumination polarizer and the rotating illumination half waveplate, and (b) in another optical path between the rotating collection one fourth waveplate and the rotating illumination half waveplate. Current acquisition parameters may include a location of a spatial mask. Step410may be followed by step420of setting the Raman spectroscope according to the current acquisition set-up. Step420may be followed by step430acquiring at least one first Raman spectrum of the first nano-scale structure of the sample, while being set according to the current acquisition set-up. Step430may include extracting different information regarding different phonon modes. Steps410,420, and430may be repeated multiple times. A current iteration of steps410,420and430may be repeated by a next iteration. This is illustrated by the dashed arrow from step430to step410. It should be noted that the analysis of radiation and/or a generating of a Raman spectrum from detection signals of a detector and/or an analysis of a Raman spectrum to determine features of the objects may be executed, at least in part, by a controller and/or a processing circuit that does not belong to the optical measurement system and/or may be remotely positioned from the illumination and/or collection paths. There may be provided a method for Raman spectroscopy, the method may include determining first acquisition parameters of a Raman spectroscope to provide a first acquisition set-up, the determining may be based on at least one expected radiation pattern to be detected by a sensor of the Raman spectroscope as a result of an illumination of a first area of a sample, the first area may include a first nano-scale structure, wherein at least a part of the at least one expected radiation pattern may be indicative of at least one property of interest of the first nano-scale structure of the sample; wherein the first acquisition parameters belong to a group of acquisition parameters; setting the Raman spectroscope according to the first acquisition set-up; and acquiring at least one first Raman spectrum of the first nano-scale structure of the sample, while being set according to the first acquisition set-up. The group may include an illumination angle, an illumination polarization, a collection angle, a collection polarization, an illumination spatial masking and a collection spatial masking. The first acquisition parameters may include an illumination angle, wherein the setting may include determining a position of a mirror of an illumination path of the Raman spectroscope. The first acquisition parameters may include an angular position of a dove prism of a collection path of the Raman spectroscope. The first acquisition parameters may include a polarization set by an optical module that may include an illumination polarizer, a rotating illumination half waveplate, and a rotating collection one fourth waveplate; wherein only a beam splitter may be positioned (a) in an optical path between the illumination polarizer and the rotating illumination half waveplate, and (b) in another optical path between the rotating collection one fourth waveplate and the rotating illumination half waveplate. The first acquisition parameters may include a location of a spatial mask. The acquiring may include extracting different information regarding different phonon modes. The determining may be based on a model that maps different acquisition set-ups, to different expected radiation patterns. The method may include determining second acquisition parameters of the Raman spectroscope to provide a second acquisition set-up, the determining may be based on at least one expected radiation pattern to be detected by the sensor of the Raman spectroscope as a result of an illumination of a second nano-scale area of the sample, wherein at least a part of the at least one expected radiation pattern may be indicative of at least one property of interest of the second nano-scale area of the sample, the second acquisition parameters belong to the group; setting the Raman spectroscope according to the second acquisition set-up; and acquiring at least one second Raman spectrum of the second nano-scale area of the sample, while being set according to the second acquisition set-up. There may be provided an optical measurement system that may include optics, the optics may include an illumination path and a collection path; a Raman spectroscope; a controller that may be configured to determine first acquisition parameters of a Raman spectroscope to provide a first acquisition set-up, the determining may be based on at least one expected radiation pattern to be detected by a sensor of the Raman spectroscope as a result of an illumination of a first area of a sample, the first area may include a first nano-scale structure, wherein at least a part of the at least one expected radiation pattern may be indicative of at least one property of interest of the first nano-scale structure of the sample; wherein the first acquisition parameters belong to a group of acquisition parameters; wherein the Raman spectroscope may be arranged to be configured according to the first acquisition set-up; and wherein the optics may be configured to acquire at least one first Raman spectrum of the first nano-scale structure of the sample, while being set according to the first acquisition set-up. The group may include an illumination angle, an illumination polarization, a collection angle, a collection polarization, an illumination spatial masking and a collection spatial masking. The first acquisition parameters may include an illumination angle, wherein the setting may include determining a position of a mirror of an illumination path of the Raman spectroscope. The first acquisition parameters may include an angular position of a dove prism of a collection path of the Raman spectroscope. The first acquisition parameters may include a polarization set by an optical module that may include an illumination polarizer, a rotating illumination half waveplate, and a rotating collection one fourth waveplate; wherein only a beam splitter may be positioned (a) in an optical path between the illumination polarizer and the rotating illumination half waveplate, and (b) in another optical path between the rotating collection one fourth waveplate and the rotating illumination half waveplate. The first acquisition parameters may include a location of a spatial mask. The optics may be configured to extract different information regarding different phonon modes. The controller may be configured to determine based on a model that maps different acquisition set-ups, to different expected radiation patterns. The controller may be configured to determine second acquisition parameters of the Raman spectroscope to provide a second acquisition set-up, the determining may be based on at least one expected radiation pattern to be detected by the sensor of the Raman spectroscope as a result of an illumination of a second nano-scale area of the sample, wherein at least a part of the at least one expected radiation pattern may be indicative of at least one property of interest of the second nano-scale area of the sample, the second acquisition parameters belong to the group; setting the Raman spectroscope according to the second acquisition set-up; and wherein the optics may be configured to acquire at least one second Raman spectrum of the second nano-scale area of the sample, while being set according to the second acquisition set-up. There may be provided a non-transitory computer readable medium that stores instructions for: determining first acquisition parameters of a Raman spectroscope to provide a first acquisition set-up, the determining may be based on at least one expected radiation pattern to be detected by a sensor of the Raman spectroscope as a result of an illumination of a first area of a sample, the first area may include a first nano-scale structure, wherein at least a part of the at least one expected radiation pattern may be indicative of at least one property of interest of the first nano-scale structure of the sample; wherein the first acquisition parameters belong to a group of acquisition parameters; setting the Raman spectroscope according to the first acquisition set-up; and acquiring at least one first Raman spectrum of the first nano-scale structure of the sample, while being set according to the first acquisition set-up. Any arrangement of components to achieve the same functionality is effectively “associated” such that the desired functionality is achieved. Hence, any two components herein combined to achieve a particular functionality may be seen as “associated with” each other such that the desired functionality is achieved, irrespective of architectures or intermedial components. Likewise, any two components so associated can also be viewed as being “operably connected,” or “operably coupled,” to each other to achieve the desired functionality. Furthermore, those skilled in the art will recognize that boundaries between the above described operations merely illustrative. The multiple operations may be combined into a single operation; a single operation may be distributed in additional operations and operations may be executed at least partially overlapping in time. Moreover, alternative embodiments may include multiple instances of an operation, and the order of operations may be altered in various other embodiments. Also for example, in one embodiment, the illustrated examples may be implemented as circuitry located on a single integrated circuit or within a same device. Alternatively, the examples may be implemented as any number of separate integrated circuits or separate devices interconnected with each other in a suitable manner. Also for example, the examples, or portions thereof, may implemented as soft or code representations of physical circuitry or of logical representations convertible into physical circuitry, such as in a hardware description language of any appropriate type. However, other modifications, variations and alternatives are also possible. The specifications and drawings are, accordingly, to be regarded in an illustrative rather than in a restrictive sense. In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word ‘comprising’ does not exclude the presence of other elements or steps then those listed in a claim. Furthermore, the terms “a” or “an,” as used herein, are defined as one or more than one. Also, the use of introductory phrases such as “at least one” and “one or more” in the claims should not be construed to imply that the introduction of another claim element by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim element to inventions containing only one such element, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an.” The same holds true for the use of definite articles. Unless stated otherwise, terms such as “first” and “second” are used to arbitrarily distinguish between the elements such terms describe. Thus, these terms are not necessarily intended to indicate temporal or other prioritization of such elements. The mere fact that certain measures are recited in mutually different claims does not indicate that a combination of these measures cannot be used to advantage. While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention. The terms “including”, “comprising”, “having”, “consisting” and “consisting essentially of” are used in an interchangeable manner. For example—any method may include at least the steps included in the figures and/or in the specification, only the steps included in the figures and/or the specification. | 40,286 |
11860105 | DETAILED DESCRIPTION The following description discloses embodiments of a Raman spectrometer that is particularly suited to be carried into the field for use. The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The appended drawings are incorporated herein and constitute a part of the detailed description. The detailed description includes specific details for the purpose of providing a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. In some instances, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology. Like, or substantially similar, components are labeled with identical element numbers for ease of understanding. FIG.1illustrates diffraction of a coherent monochromatic light beam100at a slit110in a plate112, according to certain aspects of the present disclosure. The white lines in the incident beam100represent the “valleys” of the sinusoidal wave of the light, shown to the right for reference. The light that passes through the slit110is diffracted, also referred to as “scattered,” with the diffracted light102propagating as a spherical wave toward a surface120as seen inFIG.1. FIG.2depicts the intensity distribution200of diffracted light102on surface120, according to certain aspects of the present disclosure. The central peak210is centered under the incident beam100and flanked on both sides by secondary lobes (maxima)220and dark lines (minima)230, with the intensity of each succeeding lobe222decreasing as the lateral distance from the center increases. The pattern250formed on the surface120is governed by equation 1: sin(θ1)=1.22(λ/d) Equation 1:wherein:θ1is the angular position of the first order diffraction minima (the first dark ring)λ is the wavelength of the incident lightd is the width of the slit FIG.3depicts the refractive and reflective scattering of modes of an incident beam of monochromatic light, according to certain aspects of the present disclosure. A beam100of coherent light strikes the grating300at an angle θincrelative to an axis302that is perpendicular to the plane of the grating300. A portion of the incident beam100is transmissively diffracted into various modes at various angles θmtrnrelative to the incident beam100, wherein “M” is the mode number. For example, beam320is the 0th-order mode (primary) transmitted mode, beams322A,322B are the 1st-order transmitted modes, and beams324A,324B are the 2nd-order transmitted modes. The 0th-order mode is at an angle θ0trnwhile the 1st-order modes322A,322B are at ±θ1trnrelative to beam100. The angles θmtrnare dependent upon the frequency of the incident light, the mode order, the geometry and the index of refraction of the material of the grating300as shown in Equation 2. This equation presumes air on both sides of the grating300. sinθm=sinθinc-mλΛEquation2wherein:λ is the wavelength of the incident lightΛ is the spacing of the lines of the gratingm is the order of the refracted rayθ is the angle from perpendicular to the grating, the subscript “inc” indicates the incident ray and the subscript “m” indicates the mthrefracted ray A portion of the incident beam100may be reflectively diffracted into various modes at various angles θmrefrelative to axis302. FIG.4illustrates Rayleigh scattering, according to certain aspects of the present disclosure. Raman spectroscopy relies upon inelastic scattering of photons. An incident beam of monochromatic light introduces energy into the molecules of a sample material and excites the system. The material emits the absorbed energy at frequencies associated with the various energy states of its molecules. The shift in energy gives information about the vibrational modes in the system. FIG.5illustrates an example Raman spectrum, according to certain aspects of the present disclosure. The light emitted by a material is plotted as intensity vs. frequency shift relative to the frequency of the source light, referred to as the “Raman shift,” traditionally measured in a unit called the wavenumber, which is the number of waves per cm (cm−1). The spikes of the plot are associated with vibrational modes of chemical bonds in one of the component materials. These spikes are referred to as “Raman bands” and the frequency and relative intensities of the bands allow us to identify the material by comparison of their spectral “signature” with a library of reference signatures of known materials. Heavy atoms and weak bonds have low Raman shifts. Light atoms and strong bonds have high Raman shifts. The plot ofFIG.5is the Raman spectrum of polystyrene. The high frequency carbon-hydrogen (C—H) bonds have a resonant frequency that creates the Raman band at about 3000 cm−1. The carbon-carbon (C—C) bonds create the small Raman band at around 800 cm−1. The C—H vibrations have a higher frequency than the C—C vibrations because hydrogen is lighter than carbon. The vibrations of a complex molecule partly consist of many simple diatomic vibrations while also showing the vibrational modes of larger groups of atoms, such as the expanding/contracting “breathing mode” of the aromatic carbon rings in polystyrene that appears at 1000 cm−1. FIG.6depicts a schematic representative of a conventional Raman spectrometer600, according to certain aspects of the present disclosure. A sample602to be characterized is placed against a surface of a sample holder610. A partially reflective mirror630deflects a portion of beam of source light634emitted by a light source632to travel toward the sample602as illumination beam636. The scattered light emitted by the sample602passes through the sample holder610and the sample lens620to the mirror630, where a portion604of the scattered light continues through a spatial filter, e.g. a slit,642. The sample lens310focuses the light604on the spatial filter642. The light606that has passed through the spatial filter642is formed into a unidirectional beam608by the collimating lens650. An excitation filter660blocks the transmission of the light from source632. The beam of filtered light608strikes the transmissive diffraction grating670. The diffracted light609coming out of the grating670is focused by the final focus lens680onto surface692of a detector690, which may be a charge-coupled device (CCD) sensor or camera. FIG.7depicts a block diagram700of an exemplary Raman spectrometer, according to certain aspects of the present disclosure. An instrument body702is configured to accept a holder710that has a compartment712configured to accept a sample of a material. In certain embodiments, the holder710includes a specimen plate720, a sample lens array722, a slit array724, and a collimating lens array726. In certain embodiments, the specimen plate forms a portion of the compartment712. In certain embodiments, the holder710comprises a lid714configured to selectably close over the accepted sample and permanently prevent removal of the sample from the holder compartment712. In certain embodiments, closure of the lid714is a non-reversable event wherein the lid714cannot be opened again without damage to the lid714or holder710, i.e. evidence of tampering. This feature makes the holder710into a tamper-resistant sample container that can be archived for later retrieval and re-examination. In certain embodiments, the lid714is hingedly attached to the body of the holder710. The instrument body702is coupled to a transmissive grating730, a focusing lens732, and a detector740. The detector740is communicatively coupled to a processor750and configured to provide information about the Raman spectrum of this sample, i.e. the sample signature, to the processor750. The processor750can store the sample signature and associated data, e.g. a date, a sample ID, a location such as where the sample was collected, a field designator, a user name, etc., in the memory760. The processor750is coupled to a comm module752that is communicatively coupled to a server790that may be at a remote location or implemented as a virtual device on a “cloud” server. In certain embodiments, the server790is implemented as a software service. The body702is also coupled to a user interface754, for example a color graphics display with an overlaid touchscreen. The body702is also coupled to a power supply770that provides power to all of the electronic components of the apparatus and, in certain embodiments, received information from one of more of the components. In certain embodiments, the body702is also coupled to a GPS module756that provides location information to the processor750. Light782from the light source780is guided to the sample, which is shown as contained in compartment712having a lid714in this example. The sample is stimulated by the light782to emit light, a portion of which passes through the sample plate720, the sample lens array722, the slit array724, and the collimating lens array726to the grating730. The light is refracted by the grating726and a portion of the refracted light passes through the final focus lens732to the detector740. Detector740creates data that is provided to the processor750. The processor850is communicatively coupled a memory760via a bidirectional path. In certain embodiments, the memory760contains instructions that, when transferred to the processor750and executed by the processor750, cause the processor750to receive the data from the detector740, compare the received data with a portion of one or more reference files, and determine an attribute of the sample. In certain embodiments, memory760also contains the one or more reference files that are respectively associated with one or more materials and the instructions comprise instructions to transfer a portion of the files to the processor750. In certain embodiments, the one of more reference files are stored on the server790that is communicatively connected to the processor750through the comm module752, for example over a wired and/or wireless network. In certain embodiments, the light source780emitting light at a determined frequency. In certain embodiments, the frequency is in the infrared band. In certain embodiments, the frequency is in the visible band. In certain embodiments, the frequency is in the ultraviolet band. In certain embodiments, the light source780comprises an optical filter (not shown inFIG.7) that passes light only in a selected band having a frequency bandwidth. In certain embodiments, the source780emits light in a band having a bandwidth that is less than or equal to 5 nm. In certain embodiments, the source780emits light in a band having a bandwidth that is less than or equal to 2 nm. In certain embodiments, the light source780comprises a plurality of sources each emitting light at a different frequency. FIG.8depicts a flow chart800of an exemplary method of use, according to certain aspects of the present disclosure. In the first step810, a user places a sample of a material in the holder. This step may include closing a lid over the sample and, in certain embodiments, closing of the lid may be a one-time action, i.e. a non-reversable event. In step820, the user places the holder in the instrument body. In step822, the user performs set-up and data entry operations. In certain embodiments, one of steps820and822includes the instrument retrieving a unique identifier (ID) from the holder that was placed in the body and storing this ID. In certain embodiments, one of steps820and822includes determination of the current physical location of the instrument using a locating system, e.g. an internal global position system (GPB) module and storage of the location. Once the instrument is fully configured for this sample, the user initiates the analysis of the sample in step830. In certain embodiments, step830comprises one of more of collection of multiple spectral signatures using different frequencies of source light and collection of multiple spectral signatures using different optical filters to modify the light emitted by the sample. In certain embodiments, step830comprises stimulation of the sample, for example by exposure of the sample to one or more of a magnetic field, an electrostatic field, and a radio frequency (RF) field. In certain embodiments, step830comprises introduction of a fluid into the sample. After the spectral signatures are collected, the instrument transfers a portion of the data, which includes one or more of the spectral signatures and information entered by the user and determined by the instrument, to a server that may be remote. Software on the server analyzes the data in step840, compares the results of the analysis to a library of signatures in step842, and identifies a match between the sample and the materials of the library in step844. In step846, the software on the server analyzes the spectral signatures and determines an amount of the matched material in the sample. In certain embodiments, the analysis determines an amount present only for a pre-determined material. In certain embodiments, the analysis may calculate a ratio of the amount of one material to the amount of another material. Step850stores the results of the analysis and the data in a memory on the server. In certain embodiments, the memory is located separate from the server. In certain embodiments, the results are sent to the instrument and stored in a memory in the instrument or a removable drive, e.g. a thumb drive, attached to the instrument. The results are sent to the instrument in step860and provided to the user on the user interface. In certain embodiments, step860includes providing the information on one of a personal computer, a laptop, a tablet, a smart phone, or other display. FIG.9Adepicts an exemplary embodiment of an optical system900of the apparatus, according to certain aspects of the present disclosure. In this embodiment, the system900comprises a holder910, an optical filter920, a grating930, a focusing lens940, and a detector950. The optical filter920is configured to block the wavelength of the excitation light. The grating930separates the light emitted by the sample into its various wavelengths. The focusing lens940focuses each separated wavelength onto the detector950in a spatially separated position. FIG.9Bdepicts the passage of light emitted by the sample (not visible as it is located within the holder910) through the optical system900, according to certain aspects of the present disclosure. A single beam960of collimated light being emitted from the holder910is shown for clarity, although there are a plurality of adjacent beams of light coming from the holder910that are collimated and parallel to each other. After passing through the filter920, the filtered light962strikes the grating930and a portion is transmissively refracted into a refracted beam964. Each of the matching-wavelength spectral sub-component beams964of the plurality of adjacent beams of light that are exiting the grating930are still collimated and parallel to each other, i.e. all sub-components at the same wavelength will enter the focusing lens940at the same angle. For example, the green portions from the multiple beams are all collimated and parallel to each other as they enter the focusing lens940. The focusing lens940focuses the spectral sub-component beams964into converging sub-component beams966that have foci on a plurality of spatially separate locations on the detector950. In certain embodiments, the focusing lens940comprises multiple elements for focusing and beam shaping. In certain embodiments, the focusing lens940comprises one or more of a curved mirror and a flat mirror. In certain embodiments, the detector950comprises one or more of a linear 1D array of sensing elements, e.g. pixels, and a 2D array of sensing elements. FIG.10Adepicts an exemplary embodiment of a single-use holder910, according to certain aspects of the present disclosure. The holder910has a frame912on which is printed a unique identifier914, e.g. a matrix code. In certain embodiments, the identifier914comprises a human-readable code. In certain embodiments, the identifier914comprises an electronic device, e.g. a ROM chip or an RFID chip, that stores the identifier. FIG.10Bdepicts an optical assembly1000removed from the cavity916of the holder. The cavity916is adjacent to a compartment (not visible inFIG.10B) of the frame912that is configured to accept a sample of a material such that the sample is pressed against the optical assembly1000. FIG.10Cdepicts an exploded view of an exemplary optical assembly1000, according to certain aspects of the present disclosure. In this embodiment, optical assembly1000comprises a sample plate1010, a sample lens array1020, a slit array1030, and a collimating lens array1040, each coupled to at least one of the adjacent component and to the frame912. In this embodiment, the sample plate1010is an optically clear planar sheet that is disposed, when the optical assembly1000is mounted in the frame912, proximate to the sample compartment such that the sample is in contact with a surface of the sample plate1010. Other embodiments of the sample plate are discussed with respect toFIGS.12A and12B. The sample lens array1020comprises a plurality of focusing elements1022that are mounted in a frame1024with a set-back1026that provides clearance for the height of the focusing elements1022as well as a portion of a separation of the focusing elements1022from the next component. In certain embodiments, the focusing elements1022comprise one or more of spherical, aspherical, and diffractive optical components. In certain embodiments, the plurality of focusing elements1022are configured to collect light from a respective plurality of regions of the surface of the sample and produce a respective plurality of beams of light. The slit array1030comprises one or more slits each having a width. In certain embodiments, a portion of the plurality of focusing elements1022is arranged in a straight row that is parallel to a slit of the slit array1030and the focusing elements of the row are configured to focus the respective beams of light on the slit. In certain embodiments, the plurality of focusing elements and the plurality of slits are arranged in a non-rectilinear pattern, e.g. concentric circles. The collimating lens array1040comprises a plurality of collimating lenses1042mounted in a frame1044with a set-back1046that provides clearance for the height of the collimating lenses1042as well as a portion of a separation of the collimating lenses1042from the next component. In certain embodiments, a portion of the plurality of collimating lenses1042is arranged in a straight row that is parallel to a slit of the slit array1030. Each collimating lens1042is configured to accept the refracted light emanating from one of the slits and modify the light to form a collimated beam of light. All of the modified plurality of beams of light are collimated in a common direction. In certain embodiments, the diameter of the individual focusing elements1022and/or the collimating lenses1042is less than 125 nm. In certain embodiments, the focusing elements1022and/or the collimating lenses1042are holographic lenses. In certain embodiments, the use of holographic lenses in place of conventional lenses provides a 10× improvement in light capture. In certain embodiments, the use of holographic lenses in place of conventional lenses provides a 50× improvement in light capture. In certain embodiments, the use of holographic lenses in place of conventional lenses provides a 100× improvement in light capture. In certain embodiments, the separation of the sample plate1010from the sample lens array1020is less than 5 mm. In certain embodiments, the separation of the sample plate1010from the sample lens array1020is less than 2 mm. In certain embodiments, the separation of the sample lens array1020and the slit array1030is less than 5 mm. In certain embodiments, the separation of the sample lens array1020and the slit array1030is less than 2 mm. In certain embodiments, the separation of the slit array1030and the collimating lens array is less than 5 mm. In certain embodiments, the separation of the slit array1030and the collimating lens array is less than 2 mm. FIG.11Adepicts an exemplary embodiment of a sample plate1100, according to certain aspects of the present disclosure. The sample plate1100comprises a channel1102that is configured to accept a liquid sample (not shown inFIG.11A). In certain embodiments, the channel1102passes through the width of the sample plate1100so as to form a passage through which a liquid sample can flow, thus enabling continuous monitoring of a stream to be periodically tested. In certain embodiments, the channel1102is a sealed compartment with entrance and exit ports (not shown inFIG.11A) so as to facilitate introduction of a liquid sample into the channel1102and removal of air. FIG.11Bdepicts an exemplary embodiment of a sample plate1110, according to certain aspects of the present disclosure. In certain embodiments, the sample plate1110comprises an actuator1112at least partially embedded in the sample plate1110. In certain embodiments, the actuator1112is selected from the group of a temperature-control element, a filtering element, and a stimulation element. In certain embodiments, the temperature-control element can perform at least one of heating or cooling the sample. In certain embodiments, the filtering element can selectively allow or block selected frequencies of light. In certain embodiments, the stimulation element generates one of a magnetic field, an electrostatic field, and a dynamically oscillating electric field, e.g. a radiofrequency (RF) field. In certain embodiments, the sample plate1110comprises a coating1114on one or more surfaces. In certain embodiments, the coating1114functions as one or more of an optical filter, an electric shield, an antenna, and an electric conductor that may be patterned. FIG.11Cdepicts an exemplary embodiment of a sample plate1120, according to certain aspects of the present disclosure. In certain embodiments, the sample plate1120comprises a reservoir1124embedded within the body1122of the sample plate1120and configured to contain a fluid1126and a pump1128fluidically coupled between the reservoir1124and a surface of the sample plate1120and configured to selectably expel a portion of the fluid1126from the sample plate. FIG.12A-12Bdepicts exemplary means of providing illumination to the sample, according to certain aspects of the present disclosure. FIG.12Adepicts a schematic of certain aspects of a novel Raman spectrometer1200, according to certain aspects of the present disclosure. In this exemplary embodiment, an incident ray1210of coherent, monochromatic, unidirectional illuminating light strikes a transmissive diffraction grating930at an angle1230. In certain embodiments, angle1230is selected such that one of the mode rays1220is directed along the optical axis1202of the spectrometer1200. In certain embodiments, the angle1230is selected to direct a higher-order mode ray, for example a 1st-order ray1222, along the optical axis1202. In certain embodiments, the angle1230is selected to direct the primary ray along the optical axis1202. One advantage of the novel arrangement of the light source (not shown inFIG.12A) is the elimination of the partially reflective mirror630shown inFIG.6. As the light in a conventional spectrometer must be first reflected and then transmitted by the mirror630, there is a loss of energy, normally about 50%, of the scattered light coming from the sample. Although the selected mode with have only a portion of the energy of the incident beam1210, there is no energy loss in the optical path from the sample to the grating930. A second advantage of the spectrometer1200is the more compact arrangement of components, as the light source is now generally aligned with the long dimension of the device, while a conventional spectrometer600has a laser light source632, which may be large and heavy, positioned on one side. Repositioning the source632in a conventional design requires additional optical elements, for example folding mirrors and rigid supporting structure, that add weight and cost. Light passing through grating920from a first surface to a second surface on the opposite side of the grating920from the first surface is described as passing through the grating920in a first direction, regardless of the angle of the path of the light to a perpendicular reference axis, such as axis1202. Similarly, light passing through grating920from the second surface to the first surface is described as passing through the grating830in a second direction regardless of whether the path of the light traveling in the second direction is parallel to the path of the light traveling in the first direction. The use of “first direction” and “second direction” are meant only to convey the general direction of transmission from one surface to another. FIG.12Bdepicts another exemplary embodiment of means of providing illumination of the sample, according the certain aspects of the present disclosure. In certain embodiments, a beam of illuminating light is provided via a fiber optic cable1280, or functional equivalent, that passes through openings1262in the frame1260of holder1250and then into a receiving port1272of the sample plate1270. This type of side illumination is known in optics and provides light output across the planar surface of the sample plate1272. In certain embodiments, the beam of illuminating light is provided via a fiber optic cable1290, or functional equivalent, that passes through the holder1250from a backside and mates with a diffuser (not visible inFIG.12B) within the frame1260and disperses the light across the planar surface of the sample plate1270. In certain embodiments, the illumination light is modulated, for example by driving the light source with a square wave, thereby producing periods of illumination of the sample, i.e. when the source is on, separated by intervals of dark, i.e. when the source is off. Sensing of the output of the detector is synchronized with the square wave, for example by recording the output only while the source is off and adding the recordings of multiple dark intervals. In certain embodiments, sensing of the output of the detector occurs during portions of both the illuminated periods and the dark periods and the respective sets of measurements are compared during analysis. SUMMARY Certain embodiments of the disclosed Raman spectrometer incorporate a novel arrangement of a light source that introduces the light into the optical path of the apparatus by passing the light through the transmissive diffraction grating in direction opposite the direction of the light passing from the sample to the detector. This novel arrangement beneficially reduces the size and complexity of the optical path by eliminating components that are critical in conventional spectrometers. Certain embodiments of the disclosed Raman spectrometer consolidate critical elements of the optical path into a single-use holder. Miniaturization of the optical elements and the use of arrays of lenses in place of single lenses enables precise alignment without requiring complex alignment techniques during manufacturing. EMBODIMENTS A1. An apparatus for analysis of a sample, comprising: a frame having a first axis; a sample holder coupled to the frame and disposed on the first axis; a transmissive diffraction grating coupled to the frame and disposed along the first axis such that light traveling along the first axis from the sample holder passes through the grating in a first direction; and a source coupled to the frame and configured to emit a first light to pass through the grating in a second direction that is opposite the first direction. A2. The apparatus of A1, further comprising: a lens coupled to the frame; and a spatial filter coupled to the frame; wherein the lens and spatial filter are disposed along the first optical axis. A3. The apparatus of A1, wherein a portion of the first light emitted by the source is diffracted by the grating to travel parallel to the first optical axis. A4. The apparatus of A3, wherein: the light emitted by the source is monochromatic; the diffracted portion of the first light comprises a mode; the light emitted by the source travels to the grating along a second optical axis that is not parallel to the first optical axis; and an angle between the first and second optical axes determines the mode of the diffracted portion of the first light. A5. The apparatus of A4, wherein: the light source comprises a plurality of sources each emitting light at a plurality of unique frequencies; the second optical axis comprises a plurality of secondary optical axes that are respectively associated with the plurality of unique frequencies and respectively disposed at a plurality of unique angles to the first optical axis. A6. The apparatus of A3, wherein: the light emitted by the source is white light; the diffracted portion of the white light comprises a color; the light emitted by the source travels to the grating along a second optical axis that is not parallel to the first optical axis; and an angle between the first and second optical axes determines the color of the diffracted portion of the light. A7. The apparatus of A1, wherein: the sample holder is configured to accept the sample such that the sample is disposed on the first optical axis; the first light illuminates the sample, whereupon the sample emits a second light that enters the grating in the first direction; and a portion of the second light exits the grating as diffracted second light; the apparatus further comprises: a lens coupled to the frame and configured to focus the diffracted second light to form a Raman spectrum; a detector coupled to the frame and configured to sense the Raman spectrum and provide data related to the Raman spectrum; a processor communicatively coupled to the detector; and a non-volatile memory communicatively coupled to the processor and comprising: a reference file associated with a material; and an instruction file that, when executed by the processor, causes the processor to receive the data from the detector, compare the received data with a portion of the reference file, and determine an attribute of the sample. A8. The apparatus of A7, wherein the attribute of the sample comprises an amount of a material component in the sample. A9. The apparatus of A1, wherein the light passes from the source to the grating without being reflected. B1. A method of obtaining a Raman spectrum of a sample, the method comprising the steps of: illuminating the sample with a first light, whereupon the sample emits a second light that passes through a transmissive diffraction grating in a first direction and exits the grating as diffracted second light, wherein the first light passed through the grating in a second direction opposite the first direction prior to illuminating the sample; focusing the diffracted second light to form a Raman spectrum. B2. The method of B1, further comprising the steps of: coupling a disposable element to an apparatus, wherein the disposable element comprises a sample holder and the grating and the apparatus comprises a light source configured to emit the first light; and placing the sample on the sample holder. B3. The method of B1, wherein the first light is coherent. B4. The method of B1, wherein the first light is monochromatic. B5. The method of B1, further comprising the step of filtering the second light to remove a portion of the first light. B6. The method of B1, further comprising the step of evaluating the Raman spectra to determine an attribute of the sample. B7. The method of B6, wherein the attribute of the sample comprises an amount of a material component in the sample. C1. An apparatus for analysis of a sample of a material, comprising a holder configured to accept the sample, the holder comprising a sample plate comprising a first surface configured to contact the accepted sample; and a sample lens array coupled to the sample plate, the sample lens array comprising a plurality of focusing elements. C2. The apparatus of C1, wherein the holder further comprises a slit array coupled to the sample lens array, the slit array comprising a plurality of slits; and a collimating lens array coupled to the slit array, the collimating lens array comprising a plurality of collimating lenses. C3. The apparatus of C1, wherein the plurality of focusing elements are configured to collect light from a respective plurality of regions of the surface of the sample and produce a respective plurality of beams of light. C4. The apparatus of C2, wherein a portion of the plurality of focusing elements are arranged in a first straight row that is parallel to a first slit of the plurality of slits of the slit array; and the focusing elements of the first row are configured to focus their respective beams of light on the first slit. C5. The apparatus of C4, wherein the plurality of collimating lenses are configured to receive a portion of the plurality of beams of light that pass through the plurality of slits; and modify each of the plurality of beams of light such that all of the modified plurality of beams of light are collimated in a common direction. C6. The apparatus of C1, wherein the holder further comprises a compartment configured to accept the sample, wherein the sample plate forms a portion of the compartment; and a lid that is coupled to the holder and configured to selectably close over the compartment and permanently prevent removal of an accepted sample from the holder. C7. The apparatus of C1, wherein the focusing elements are holographic lenses. C8. The apparatus of C2, wherein the collimating lenses are holographic lenses. C9. The apparatus of C1, wherein the sample plate further comprises a channel configured to accept a liquid sample. C10. The apparatus of C1, wherein the sample plate further comprises an actuator selected from the group of a temperature control element, a filtering element, and a stimulation element. C11. The apparatus of C1, wherein the holder is configured to accept a beam of illuminating light and guide the accepted beam of illuminating light to a side of the sample plate that is not the first surface. C12. The apparatus of C1, further comprising a frame configured to removably accept the holder; a detector coupled to the frame; a focusing lens coupled to the frame; and a transmissive diffraction grating coupled to the frame. C13. The apparatus of C12, further comprising an optical filter coupled to the frame; and a spatial filter coupled to the frame. C14. The apparatus of C12, wherein the grating comprises a first surface and a second surface that is opposite the first surface; a portion of a beam of light emitted by the accepted sample passes through the grating from the first surface to the second surface; and the frame is further configured to accept a beam of illuminating light and guide the accepted beam of illuminating light to the second surface of the grating such that a refracted portion of the beam of illuminating light is directed through the grating and exits the first surface toward the accepted sample. C15. The apparatus of C1, wherein the holder is configured for use with only a single sample. Headings and subheadings, if any, are used for convenience only and do not limit the invention. Reference to an element in the singular is not intended to mean “one and only one” unless specifically so stated, but rather “one or more.” Use of the articles “a” and “an” is to be interpreted as equivalent to the phrase “at least one.” Unless specifically stated otherwise, the terms “a set” and “some” refer to one or more. Terms such as “top,” “bottom,” “upper,” “lower,” “left,” “right,” “front,” “rear” and the like as used in this disclosure should be understood as referring to an arbitrary frame of reference, rather than to the ordinary gravitational frame of reference. Thus, a top surface, a bottom surface, a front surface, and a rear surface may extend upwardly, downwardly, diagonally, or horizontally in a gravitational frame of reference. Although the relationships among various components are described herein and/or are illustrated as being orthogonal or perpendicular, those components can be arranged in other configurations in some embodiments. For example, the angles formed between the referenced components can be greater or less than 90 degrees in some embodiments. Although various components are illustrated as being flat and/or straight, those components can have other configurations, such as curved or tapered for example, in some embodiments. Pronouns in the masculine (e.g., his) include the feminine and neuter gender (e.g., her and its) and vice versa. All structural and functional equivalents to the elements of the various aspects described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. No claim element is to be construed under the provisions of 35 U.S.C. § 112, sixth paragraph, unless the element is expressly recited using the phrase “means for” or, in the case of a method claim, the element is recited using the phrase “operation for.” A phrase such as an “aspect” does not imply that such aspect is essential to the subject technology or that such aspect applies to all configurations of the subject technology. A disclosure relating to an aspect may apply to all configurations, or one or more configurations. A phrase such as an aspect may refer to one or more aspects and vice versa. A phrase such as an “embodiment” does not imply that such embodiment is essential to the subject technology or that such embodiment applies to all configurations of the subject technology. A disclosure relating to an embodiment may apply to all embodiments, or one or more embodiments. A phrase such as an embodiment may refer to one or more embodiments and vice versa. The word “exemplary” is used herein to mean “serving as an example or illustration.” Any aspect or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. All structural and functional equivalents to the elements of the various aspects described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. No claim element is to be construed under the provisions of 35 U.S.C. § 112, sixth paragraph, unless the element is expressly recited using the phrase “means for” or, in the case of a method claim, the element is recited using the phrase “step for.” Furthermore, to the extent that the term “include,” “have,” or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term “comprise” as “comprise” is interpreted when employed as a transitional word in a claim. Although embodiments of the present disclosure have been described and illustrated in detail, it is to be clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, the scope of the present invention being limited only by the terms of the appended claims. | 40,210 |
11860106 | DESCRIPTION OF CERTAIN EMBODIMENTS OF THE DISCLOSURE The systems and methods described herein differ from the above-described attempts at SRS with CW lasers, in that they recover much of the gain that would normally be lost with CW lasers when performing SRS spectroscopy. Embodiments disclosed herein employ two CW lasers, at least one of which is optically tunable, that are each modulated with custom radio frequency (RF) patterns and run at higher powers than are usually encountered in spontaneous Raman spectrometry. Embodiments of the system include a high-throughput dual-beam confocal rasterizing probe and a tuned differential photodiode sensor system having a large capture area and solid angle, along with specialized detection hardware and algorithms. The disclosed systems and methods make feasible the acquisition of Raman spectra with high signal-to-noise ratios and resolutions. Robust measurements are obtained when working with inhomogeneous samples, and the high risk of tissue damage that would normally result from the extreme peak pulse powers associated with picosecond and femtosecond systems is ameliorated. Signal-to-noise ratio is vastly improved by eliminating interference from fluorescence (except for shot noise), and the impact of sample heating and photobleaching. SRS gains far greater than that which would normally be achievable with CW lasers running at low power are achieved, bringing the effective SRS sensitivity to a level approaching that achieved with ultrafast lasers. The systems and methods described herein may be implemented with inexpensive CW lasers and ancillary instrumentation. FIG.1is a schematic diagram of a system100for high gain SRS Raman Spectroscopy according to an embodiment of the present disclosure. The system includes two custom RF-modulated CW lasers, a first laser apparatus110and a second laser apparatus120. Laser device includes a first laser driver112, a first laser device114, referred to as the “Pump” or excitation laser, and a fiber coupler116that receives and transfers the pump laser114to a first optical fiber118. The second laser apparatus120includes a second laser driver122, a second laser device124referred to as the “Probe” or stokes laser, and a second laser coupler126that receives and transfers the probe laser to a second optical fiber128. Each of laser devices114,124can comprise a standard laser diode mounted on a cold plate that is attached to a thermoelectric cooling element (TEC). The TEC is, in turn, is clamped to a heat sink. This arrangement allows the temperature of both the Pump laser114and the Probe Laser124to be stabilized and precisely controlled. At least one of the Pump or Probe lasers114,124are optically tunable in terms of frequency. Typically, the Pump laser114operates at a shorter optical wavelength than the Probe laser124. Both lasers114,124are continuous wave (CW), coherent light sources. Laser drivers112,122can be implemented using custom electronics that provide both low-frequency modulation (from DC to about 5 KHz) on one input, and high-frequency modulation (50 KHz to over 20 MHz) on the other input. Laser driver112has a low-frequency input132, and a high-frequency input134. Likewise, laser driver122has a low-frequency input142and a high-frequency input144. In some embodiments, the low-frequency inputs132,142are used for switching the laser on and off, for applying slow pulse modulation, and for controlling power. The high-frequency inputs134,144are used for RF modulation. The low-frequency inputs132,142to each laser driver112,122can be generated by a single microcontroller150that receives commands from the host computer155. The high-frequency inputs134,144used for RF modulation are provided with the required signals by a dual-channel, crystal-controlled function generator160which is coupled to the host computer155. The host computer155can configure the modulation frequencies and waveforms of the high-frequency inputs134,144by sending commands to the function generator160. The actual laser modulations may be examined with an oscilloscope connected to a small, properly terminated, reverse-biased photodiode. This measure can be useful to verify that the lasers are being modulated as required by the exemplary implementation of the systems and methods described herein. The output of laser apparatus110is coupled via optical fiber118to a first input of a dual-beam rasterizing Probe device200(“R-Probe) which is discussed in greater detail below. The output of laser apparatus120is coupled via optical fiber128to a second input of the R-Probe200. A third “rasterizing” input is further coupled to the R-Probe200. At least one of the laser devices114,124(i.e., either or both) is optically tunable over a small range. In some embodiments tuning is accomplished by varying the temperatures of the laser diodes which can be accomplished by changing the setpoints of the TEC drivers which are coupled to and controlled by the host computer155. Is well-known that the output wavelength of a typical laser diode is affected by temperature (approximately 0.3 nm/° C.), with higher temperatures resulting in longer wavelengths. However, there are many other ways to implement a tunable laser, many with a wider wavelength range and faster tuning than that provided by thermal methods. In an exemplary implementation that was used to demonstrate the effectiveness of the systems and methods disclosed herein, the TEC-based (thermal) method was employed for tuning the Pump laser114over a range of wavelengths between 635.6 and 639.6 nm. This wavelength range was selected because lasers operating at such wavelengths are readily available and this wavelength range and returned Raman signals penetrate human skin. The Probe laser124was fixed at a wavelength of 685 nm. This wavelength, when used with a Pump laser114operating in the range between 635.6 and 639.6 nm, allows Raman peaks in the range from 1036 cm−1to 1134 cm−1to be detected. This range includes and enables detection of the large glucose signals at 1054 cm−1and at 1126 cm−1. In other implementations, by tuning both the Pump and Probe lasers114,124and applying suitable temperature settings, a Raman coverage much greater than twice this range can be achieved. With multiple switched lasers, all preferably, but not necessarily on a common TEC plate and coupled to a single multimode fiber, the full Raman fingerprint range and beyond can be captured. As noted above, both the Pump and Probe lasers114,124are RF modulated. In the exemplary implementation, the shorter wavelength Pump laser was modulated at f1 (4.8 MHz), while the longer wavelength Probe laser was modulated at f2 (5.9 MHz). Consequently, the beat notes generated by nonlinear interactions between the laser beams and the molecules in the sample will be found at the difference and sums of the two frequencies, i.e., 1.1 MHz (|f1−f2|) and 10.7 MHz (|f1+f2|). The modulation frequencies f1 and f2 were chosen so that they, as well as any beat notes, would fall above the 1/f noise “knee” of the lasers and sensors. This choice of frequencies dramatically reduces the impact of high levels of “1/f” or flicker noise that is exhibited by many lasers at frequencies up to about 1 MHz, as well as the lower levels of flicker noise occurring in the sensors, allowing performance approaching the shot noise limit to be achieved. In addition, well-chosen modulation frequencies reduce the influence of stray light sources, switching power supplies, Compact Fluorescent (CFL) lamps, and even local AM radio stations. The modulation frequencies can be easily adjusted to avoid any local interference sources. In addition, the effect of slow baseline drift is eliminated. Beyond noise reduction, modulating the lasers at frequencies such as those selected in the exemplary implementation enables discrimination between the strong unwanted signals derived from Rayleigh scattering and sample fluorescence, on the one hand, and the relatively weak beat note resulting from stimulated Raman scattering, on the other. This is because the beat note occurs at a frequency far removed from the laser modulation frequencies. Given that the desired beat note appears, in the exemplary setup, at 1.1 MHz, with the strong unwanted signals at DC, and at 4.8 MHz and 5.9 MHz, a large blocking dynamic range can be achieved in the resonant sensor system. To summarize, the use of RF modulated lasers provides several advantageous benefits. First, the two modulation frequencies (f1 and f2) result in a heterodyne or “beat note” (|f1−f2|) due to the non-linear interaction in the sample molecules when the optical frequency difference (|ω1−ω2|) between the lasers corresponds to a Raman-active band of those molecules. Hence a beat note is only generated by the non-linear SRS process when the lasers differ in optical frequency by amounts that correspond to Raman active molecular resonances. No beat notes are generated by fluorescence, Raleigh scattering, and other types of noise since these processes do not involve nonlinear interactions between the two modulated laser signals. Accordingly, the beat notes reflect primarily stimulated Raman signals, free of interference from sample fluorescence and other sources. The reduction of noise allows a clean Raman spectrum to be obtained by detecting and measuring the intensity of the beat note(s) (at |f1−f2| or at |f1+f2|) that reflect stimulated Raman scattering as the optical frequency difference (|ω1−ω2|) between the lasers is swept over the desired spectral range. Additionally, as noted, keeping the RF laser modulations and the RF beat note over 1 MHz dramatically lowers the impact of “1/f” noise as it moves all significant signals well above the 1/f noise “knee” of both the lasers and the sensors (described below), minimizing this important source of noise. Preferred embodiments of the systems and methods herein employ Pump and Probe lasers114,124that have moderately higher output powers than those usually found in the context of spontaneous Raman instruments. Normally, higher laser powers would result in excessive sample heating and photobleaching. However, this problem is solved by the R-Probe200which dramatically reduces the average power density at the sample. The use of moderately higher laser power adds to the SRS enhancement achieved by the system. Additional gain, in the sense of increased total SRS signal levels, results from the 100% duty cycle in which SRS scattered photons are continuously collected. By way of contrast, in ultrafast pulsed laser systems that operate at low duty cycles, photons are only collected for a small fraction of the time during which a spectrum is being acquired. Increased laser powers are highly desirable for SRS because signal levels scale roughly as the product of the laser powers. Doubling the power of each laser results in a quadrupling of the SRS signals. The laser powers employed are only about 2 to 10 times greater than the laser powers commonly used for spontaneous Raman. In an exemplary implementation, the Pump laser114can have a 250-1000 mW output compared to the typical 20-100 mW Pump lasers used in spontaneous Raman without any rasterizing probe. A similar power range may be used for the Probe laser in the context of the systems and methods described herein. As no Probe lasers are used in spontaneous Raman instruments, no comparison of power can be made for the Probe laser, but it can be seen that the power level is much lower than the peak power levels encountered in ultrafast regimes. The spectral resolution of the system is limited primarily by the quality of the lasers. The more stable and narrowband the optical output from the lasers, the higher the resolution. It is noted that there are no slits, lenses, or dispersive components in the light paths to limit the ultimate resolution of the system. One key to improving gain when using CW lasers is to moderately increase laser power, and hence peak power, at any single point on the sample, yet keep the average power relatively low. Typically, the SRS effect is obtained using ultrafast lasers operating at extremely high peak powers, even when the average power may be only 10 or so milliwatts. In embodiments of the systems and methods disclosed herein, CW lasers with power range from around 200 milliwatts to a few watts are employed. As noted, this power level is far lower than the high peak powers encountered with ultrafast lasers. Even at these reduced levels, the average power density that impinges upon a given sample (usually measured in W/cm2) is minimized by use of the R-probe200. A schematic diagram of an embodiment of an R-probe according to the present disclosure is shown inFIG.2. R-Probe At the R-Probe200, the optical fiber carrying the Pump laser118is fed to a first fiber collimator205and the optical fiber carrying the Probe Laser128is fed to a second fiber collimator210. The fiber collimators205,210are used to transform the light output from optical fibers118,128into respective free-space collimated beams. The output from the first fiber collimator205(collimated Pump laser) is fed through a first filter (F1) which filters out long wavelengths. The output from the second fiber collimator (collimated Probe laser) is fed through a second filter (F2) which filters out short wavelengths. The outputs from F1 and F2 are both fed to a dichroic beam combiner215. The output of the beam combiner215includes both the Pump and Probe lasers. The combined lasers output from the combiner are fed to a 50% silvered mirror220. There are two outputs from the mirror220. A first output is directed toward a beam dump225. A second, output from the mirror220is directed to a rasterizer230. The rasterizer230is a component that rapidly moves or scans the confocal beam which it receives from the mirror220. The purpose of the rasterization is to reduce the average power of the lasers as they impinge upon a sample, such as live human tissue. A number of rasterization techniques can be employed to move the beam. For example, some implementations can employ mirrors attached and actuated by one or more voice coil actuators, piezo stacks, simple motors, or MEMS devices. The rasterizer outputs the moving beam, which includes combined Pump laser and the Probe laser photons, through an objective lens235onto a sample240at the focal plane of the lens. Regarding the rasterization, the beam can be scanned across the sample240in a pattern (e.g., a circular or other shape), which can yield nearly a 70-fold decrease in average power density. With this magnitude of average power reduction, in the exemplary implementation, the lasers run at 25 times the power that would normally be used with a standard fixed-beam Raman probe. Increasing the laser power by a factor of 25 provides a significant benefit in the context of spontaneous Raman spectroscopy, where the signal is proportional to I (the laser flux or power) and the signal-to-noise is proportional to √I. In the case of spontaneous Raman, the signal strength is increased by a factor of 25, and the S/N by a factor of 5, whereas in the case of SRS, as a result of the nonlinear interactions between the lasers and sample, the benefit from the higher laser power is a gain of roughly 625 (25 times 25). Similarly, the S/N would be improved by a factor of 625/√25 which is 125. This is because with SRS the signal scales roughly with I2and the S/N with I2/√I. The increase in laser power that can be achieved safely, without excessive sample heating. Thus, the R-Probe (and the higher laser power it permits) provides a heightened benefit in the case of SRS vis a vis standard spontaneous Raman, further increasing the sensitivity of the systems and methods described herein. Returning toFIG.2, Raman signals and other radiation output from the sample240travels backwards through the objective lens235to the rasterizer230. The rasterizer reflects light coming from the objective lens235backwards to the 50-percent mirror220. In the case of the returning beam, a first output from mirror220is directed toward dichroic beam-splitter250. This beam includes the original Pump and Probe laser signals. A portion of the returning beam is transmitted back through the mirror toward the beam combiner215, where they have no adverse effect. The signal reaching the dichroic beam splitter includes the Raman signals received from the sample240. At the beam-splitter250, the beam is split into first and second beams which travel in perpendicular directions. The first beam is fed to filter (F3) which passes long wavelengths and filters out short wavelengths. The second beam is fed to filter (F4) which, inversely, passes short wavelengths and filters out long wavelengths. The result is that the first beam, which includes long wavelengths, is fed to a first sensor260(“Probe” sensor) while the second beam, which includes short wavelengths is fed to a second sensor270(“Pump” sensor). Outputs of the Probe sensor260and Pump sensor270are fed via a coaxial cable to a differential amplifier (not shown inFIG.2, reference number300inFIG.1). The two-sensor arrangement makes possible differential measurements. Differential measurements are beneficial since the SRS effect involves both gain and loss. Any gain at the Probe wavelength is accompanied by a corresponding loss at the Pump wavelength, and vice-versa. Differential measurements can exploit this effect and thereby obtain improved performance over a single sensor system. The R-probe shown inFIG.2should be considered as one embodiment, and variations on the implementation would be understood by those of skill in the art. As one example, it is possible to include only a single sensor. Another variation is to use fiber coupling for the sensors. Alternatively, both the sensors and lasers can be integrated into the R-Probe for free-space coupling. This implementation could minimize photon loss, but at the cost of reduced modularity. In the exemplary implementation shown inFIG.2, the sensors are integrated in the R-probe while the laser apparatus, being large and complex assemblies that benefit from a modular design, are coupled to the R-probe by optical fibers. There are other filter arrangements that might prove beneficial, and there are also designs that can be adapted for various modalities such as Offset Raman and Transmission Raman. For transmission Raman, the beam dump shown inFIG.2can be eliminated, significantly improving the throughput. In addition, the large area low noise sensors make Transmission Raman far more practical in terms of photon collection and acquisition speed than it would otherwise be. The beam dump can also be eliminated and replaced with multiple mirrors in another embodiment of the R-probe. In this embodiment, the mirrors can direct the beams that would otherwise have been removed by the beam dump to the objective lens at a slightly different angle than the primary beam so as to converge at a slightly offset spot on the sample. This technique avoids loss of photons to the beam dump while acquiring additional Raman-scattered photons from the sample, increasing the throughput and the effective sampling area. This embodiment can also eliminate problems with interference from light absorbing materials located in the beam dump that are Raman active. The combination of the R-Probe and higher power CW lasers brings the sensitivity of the system closer to the sensitivity achieved using the more common picosecond and femtosecond pulse laser systems. Because the pulses of light hitting any single spot on the sample are far longer, and the peak powers far lower, than with ultrafast regimes, there is far less risk of tissue damage or other unwanted effects on the sample. The R-probe of the exemplary implementation is coupled to a tuned photodiode sensor device having high etendue as a result of large area detectors having large solid angles. These sensors, as well as two-port differential detection, also contribute to the CW-SRS sensitivity that can be achieved by the system and method described herein. Tuned Photodiode Sensor Equations (1) and (2) below describe the light intensity levels received at the sensors from SRS stimulation: S1≈a1I1+b1I2+c1I1I2(1) and S2≈a2I1+b2I2−c2I1I2(2) In equations (1) and (2), S1and S2represent the total signal intensities at the Probe and Pump sensors, respectively, I1represents the intensity of the Pump laser, I2represents the intensity of the Probe laser, a1I1represents the signal received due to fluorescence (and some spontaneous Raman) induced by the Pump laser, b1I2represents the signal received due to Rayleigh scattering of Probe laser photons, a2I1represents the signal generated by Rayleigh scattering of Pump laser photons, b2I2represents the total spontaneous anti-stokes Raman signal (which is generally negligible), and c1I1I2and c2I1I2represent the stimulated Raman signals, both gain and loss, generated by the nonlinear SRS interaction. The coefficients a1, a2, b1, b2, c1and c2are constants associated with various kinds of scattering and fluorescence in the sample, with c1and c2being roughly proportional to stimulated Raman scattering. This stimulated Raman signal is generated when the difference in laser optical frequencies, |ω1−ω2|, corresponds to a Raman active band of one or more of the substances in the sample. When the laser optical frequencies differ by an amount that does not correspond to any Raman active band, c1and c2are generally negligible since they reflect stimulated Raman scattering. It is noted that equations (1) and (2) are low power approximations applicable for the lasers used in the context of the systems and methods disclosed herein. At extremely high laser powers, there are typically additional exponential gains. Those of skill in the art would appreciate that if I1and I2are modulated at radio frequencies f1and f2, respectively, beat notes will be generated only by the product terms in equations (1) and (2), but not by the first two linear terms in each equation. The mathematics is virtually identical to that which describes a mixer or product detector in a communications receiver. The beat notes reflect only Raman signals generated by the nonlinear SRS interactions approximated by the product terms in equations (1) and (2) and they do not reflect the linear terms generated by Raleigh scattering and fluorescence. From equations (1) and (2), it can be seen that stimulated Raman signals and beat notes scale with the product of the laser powers, but that shot noise increases with the square root of the weighted sum of those laser powers. Consequently, in shot-noise limited systems, S/N improves as the product of the laser powers divided by the square root of a weighted sum these powers. If the power of each laser is quadrupled, a 16-fold gain in the SRS signal results, and an 8-fold improvement in S/N. The beat notes mentioned above occur at |f1−f2| and |f1+f2| and are detected by the sensors. In addition to these informative beat notes, which may be fairly weak for weakly scattering analytes at low laser powers, the sensors also receive overwhelmingly strong unwanted signals at f1and f2(the laser RF modulation frequencies) as well as at DC. The Pump sensor receives the most overwhelming signal at f1, as a result of Raleigh scattering of Pump laser photons; the Probe sensor receives the most overwhelming signal at f2, as a result of Rayleigh scattering of Probe laser photons. The probe sensor also receives a fairly intense signal at mainly due to sample fluorescence induced by the Pump laser. The strong, but unwanted, signals at f1and f2can be well over 150 dB greater than the weak, but informative, beat notes at |f1−f2| and |f1+f2|. Consequently, if maximum sensitivity is to be attained without saturation of the sensors or front-end electronics, a sensor system with an exceptional blocking dynamic range (another term familiar to the radio design engineer) would be required. It is almost impossible to obtain a sufficient blocking dynamic range with the standard configuration consisting of a photodiode directly followed by a transconductance amplifier. However, this can be achieved with the tuned photodiode sensor system. In the exemplary implementation, the Pump and Probe sensors comprise large area silicon photodiodes. One example is the Mounted Photodiode SM1PD1A (9 mm clear aperature, 375 pf @5 v reverse bias) provided by Thorlabs Inc. of Newton, NJ. These photodiodes normally work with 50Ω loads. A 50Ω load yields a rolloff frequency near 8 MHz given the high capacitance of the photodiodes. However, while this frequency response might be acceptable, a 50Ω load would reduce the voltage of the desired signal to an extremely low level. Great amplification would be needed and the desired signal would be close to the noise floor of the amplifier and load resistor. To resolve this difficulty, a differential photodiode amplifier as depicted inFIG.3is employed. The differential amplifier300is able to detect weak signals from a photodiode at a selected frequency, while avoiding interference and front-end overload from extremely strong signals at other frequencies. The input signals from the Probe and Pump sensors260,270(FIG.2) are coupled to inputs305,310of the differential amplifier300. The amplifier300contain two critically coupled High-Q resonant circuits315,320coupled to inputs305,310, a cascode amplifier consisting of two transistors325,330in a middle section, and a moderate-Q resonant transformer340at the output. The tuned critically-coupled resonant circuits315,320perform several important functions. First, the coupled high-Q tuned circuits315,320act as short circuit inputs for DC, and as very low impedance inputs at both the laser modulation frequencies f1and f2, providing a high level of photodiode linearity and dynamic range. As the critical-coupled high-Q tuned circuits315,320completely block DC, and substantially attenuate the unwanted signals at f1and f2, they protect the front-end cascode elements325,330from overload and preserve the dynamic range for the desired weak signals. In addition, the high-Q tuned circuits315,320can provide both for a differential input when a pair of photodiodes are used, as is the case in the exemplary implementation, as well as a single-ended input when only a single photodiode sensor is employed. The high capacitance of the large area photodiodes is also incorporated into the first High-Q resonant circuit315. The first resonant circuit315employs a very tightly coupled toroidal transformer317that makes it possible for high levels of photodiode capacitance to be “tuned out” since such capacitance becomes part of the resonant tank circuit formed by C1318and the secondary of the transformer. The transformer reduces the impact of capacitive loading and obviates the need for a resistive load (e.g., a 50-a load) to achieve the desired frequency response. The tightly coupled tuned transformer317also supplies some noiseless gain. The transformer-derived voltage gain can be considered noiseless, since it does not involve any resistors (that produce Johnson noise) or active components (that produce shot noise, 1/f noise, and/or thermal noise). Together, these characteristics yield increased sensitivity to weak signals at the resonant frequency set at the informative beat note at |f1−f2|. The cascode amplifier stage including transistors325,330that follows the double tuned input network provides some additional gain as well as a high impedance input. The high input impedance found at the gate of a field effect transistor330, the first transistor in the cascode, helps maintain the high Q and low loss of the resonant circuits and thus their selectivity. A tuned transformer340at the output of the tuned differential photodiode amplifier provides impedance matching to a standard 50-Ω load connected via coaxial cable350. It also furnishes additional selectivity for the desired signals and further attenuation of the undesired ones. Attenuation of the signals at f1and f2(in the exemplary system, 4.8 MHz and 5.9 MHz, respectively) is greater than 110 dB. The output at resonance is about 0.8 volts with a 1 ma input signal (when properly loaded). Signal Processing Stage The output of the tuned photodiode amplifier300is fed to a software-defined dual channel radio component (SDR)170(shown inFIG.1) that provides additional selectivity and amplification. The SDR170further attenuates the unwanted signals generated by fluorescence and Rayleigh scattering, and also allows the far weaker SRS Raman signals to be amplified and detected. The SDR170also digitizes these signals so that the host computer155can perform further signal processing in software, including sophisticated lock-in or matched filter detection. A coherent dual-channel SDR170such as that used by the author in the exemplary system can, with appropriate software, serve as an excellent lock-in detector. A 130 dB dynamic range can be obtained for the SDR170alone, acting as a simple lock-in detector with a 5 Hz bandwidth, without any selective front-end amplifier. The SDR170comprises a high-speed analog-to-digital converter (ADC) and also incorporates down-conversion capabilities implemented using a field-programmable gate array (FPGA). An 80 MHz sampling rate followed by digital down-conversion was used in one implementation. Since the oscillators used in the laser modulators and in the sampling clock of the SDR are exceedingly stable, a lock-in style homodyne detector with a very slow phase-locked loop local oscillator, implemented in software on the host computer, provides very sensitive weak signal detection. In the exemplary system, a simple 5 Hz bandwidth filter, implemented using software, provides an accurate measurement of the beat note generated by the sample. The SDR170and software allows a 5 uv signal at the SDR input to be easily seen in a spectral waterfall display when the bandwidth is set to 100 Hz (wide for a typical lock-in detector) and the programmable gain is set to its minimum value. A 5 uv signal occurs at the tuned differential photodiode amplifier's output with about 6 na of signal from the photodiodes at its inputs. At the lowest SDR gain setting, a 6 nanoampere RF signal at the beat note frequency of 1.1 MHz can be seen in the waterfall display. The tuned photodiode amplifier300together with the SDR170solves many problems with a Raman spectrometer employing CW-SRS methods. The combination provides a very high blocking dynamic range (greater than 200 dB) at the laser modulation frequencies and a high signal dynamic range (greater than 120 dB) at the beat notes. Due to these dynamic ranges, intense signals at the laser modulation frequencies do not interfere with sensitive detection of weak beat notes generated by the stimulated Raman process. The exemplary implementation described above exploits lasers and sensors operating in the visible part of the spectrum. Coherent light sources and detectors that operate in other parts of the spectrum, for example, the near infrared (NIR) region from 900 to 1700 nm, could also be used. Tuned laser systems are available for this spectral region. For example, large area Indium Galium Arsinide (InGaAs) photodiodes are readily available that can be used as replacements for large area Silicon (Si) photodiodes that are used as sensors in the exemplary system. It should be noted that standard (spontaneous) Raman spectrometry is often carried out with NIR excitation as a means of reducing fluorescence. A disadvantage using NIR is that, due to the 1/λ4dependence of Raman scattering on excitation wavelength, the Raman signals are weaker than what is typically obtained at shorter visible wavelengths. However, in the context of the method and system described herein, the large SRS gain can overcome the problem of weaker Raman signals allowing sensitive NIR Raman spectrometry to be performed. In the exemplary implementation described above, tuned photodiodes are employed as the sensors. However, it is also possible to use other kinds of sensors with appropriate adjustments, such as, but not limited to photomultiplier tubes (PMTs), fast CCD “streak” cameras, avalanche photodiodes, and bolometers. The considerations with the sensors are ensuring sufficient blocking dynamic range and having the ability to reduce or avoid 1/f laser intensity noise. The 1/f noise can be handled by operating the sensor at frequencies greater than 1 MHz, as was done in the context of the exemplary system described herein. For sensors that do not operate well at such high frequencies, 1/f noise can also be reduced at the lasers, for example, by the use of negative feedback of laser intensity. If the 1/f intensity noise is sufficiently reduced at the lasers, a very low frequency beat note, e.g., 0.5 Hz, could be used, rather than the 1.1 MHz beat note described in the exemplary system. Reduction of the beat note frequency would allow even bolometers and other slow sensors to operate effectively. With sufficiently reduced 1/f intensity noise in the light sources, the system could alternatively be implemented with a broadband “Probe” source, such as a high-power LED, a fixed wavelength “Pump” laser, and a fast, low readout noise CCD imager in a dispersive spectrometer arrangement. A low frequency beat note of, say, 0.5 Hz can be detected with such a CCD imager simply by obtaining a sequence of, for example, 0.2 second integrations. Lock-in detection can be performed on the images, after-the-fact, by software in the host computer. This would result in a low-fluorescence and clean Raman spectrum. Improving dynamic range with the alternative sensor embodiments would likely be more challenging, but there are some techniques that can be employed to achieve this; in one exemplary technique, multiple large area photomultiplier tubes together with a diffuser can be used. In an alternative embodiment, the R-probe can be removed from the system. Instead, a more complex laser modulation scheme together with a beam-spreading or “fat beam” probe is employed. With higher peak power lasers, rounded pulses measured in milliseconds, and RF modulations such as those already discussed, the energy is spread over a larger area of the sample by the beam spreader or as a result of the probe design (non-rasterizing). Given the large capture areas of the sensors, the photons returning from a larger area on the sample are efficiently captured. In addition, instead of using a single laser with a large tuning range it is possible to use several lasers with smaller tuning ranges to cover the relevant regions of the Raman spectrum. In a more drastic simplification of the device, several fixed (not tuned) lasers may be employed. This embodiment can work well for many specific applications once the optimal wavelengths are determined. The laser beams can be combined with custom interference filters (which can be miniaturized), or with a fiber combiner in which small multimode fibers are butted with optical cement to a large multimode fiber. This embodiment can enable small, high-performance devices to be constructed. Furthermore, the systems described herein can be miniaturized since there is no need for dispersive elements or long optical paths. Chip-based implementations or other miniature devices are feasible for specific applications because photosensors and lasers can be fabricated as arrays at the chip level and optical components such as interference filters and microlenses can be fabricated at this scale as well. To obtain the effect of the R-Probe on a chip, one can pulse an array of miniature lasers in a scanning pattern, with each miniature laser emitting a relatively high-power pulse. As with other embodiments and implementations, the pulses in this case are on the order of millisecond or microseconds and would employ only moderately higher powers than used for spontaneous Raman. It is to be further understood that like numerals in the drawings represent like elements through the several figures, and that not all components and/or steps described and illustrated with reference to the figures are required for all embodiments or arrangements. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising”, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Terms of orientation are used herein merely for purposes of convention and referencing and are not to be construed as limiting. However, it is recognized these terms could be used with reference to a viewer. Accordingly, no limitations are implied or to be inferred. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. While the invention has been described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications will be appreciated by those skilled in the art to adapt a particular instrument, situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the disclosure as understood by a person having ordinary skill in the art. | 38,445 |
11860107 | BEST MODE According to an aspect of the present disclosure, there is provided a device of detecting a concentration of a sample, the device including a power supply unit configured to supply power to generate plasma, a plasma generation unit connected to the power supply unit and including a pair of electrodes facing each other, a plurality of signal detection units arranged on the electrodes and configured to sense light emitted from the sample because of the plasma, and a controller configured to control a driving start point of the signal detection unit so that the signal detection unit is driven after a preset period of time after oscillation from the electrodes is terminated. The plasma generation unit may have a space between the electrodes and through which the sample passes. The signal detection unit may include a filter configured to filter light in a preset wavelength band and a photodiode configured to electrically convert a signal from the filter. The controller may be configured to control the driving start point to enable the plurality of signal detection units are respectively driven in different delay times. The controller may be configured to integrate signals, which are transmitted from the plurality of signal detection units, into identical periods of time. The device may further include a reference data unit configured to store reference data including information regarding a concentration of a heavy metal, wherein the controller may be configured to integrate the signals from the plurality of signal detection units into a preset period of time, compare integration values with the reference data, and predict the concentration of the heavy metal in the sample. The plurality of signal detection units may sense light in different wavelength bands, respectively. The plasma generation unit may be provided in plural, and the plurality of plasma generation units are connected in parallel and arranged at different locations. According to another aspect of the present disclosure, there is provided a method of detecting concentration of a sample, the method including generating, by a plasma generation unit, plasma in a space between a pair of electrodes facing each other, the plasma generation unit being connected to a power supply unit, terminating oscillation from the pair of electrodes of the plasma generation unit, driving a signal detection unit after a preset period of time, after the oscillation from the pair of electrodes is terminated, sensing, by the signal detection unit, light emitted from the sample, according to a wavelength band, and calculating, by a controller, the concentration of the heavy metal in the sample, from a signal sensed by the signal detection unit. In the driving of the signal detection unit, the controller may be configured to control a driving start point of the signal detection unit. In the sensing of the light by the signal detection unit, a plurality of filters and a plurality of photodiodes may sense light in different wavelength bands, respectively. In the driving of the signal detection unit, the plurality of filters and the plurality of photodiodes may be driven at different driving start points, respectively. The method may further include obtaining reference data, which is information regarding the concentration of the heavy metal, before the sample is detected, wherein the calculating of the concentration of the heavy metal may include integrating a signal from the signal detection unit for a preset period of time, comparing an integration value with the reference data, and predicting the concentration of the heavy metal in the sample. Other aspects, features, and advantages other than those described above will become apparent from the following detailed description, claims and drawings for carrying out the disclosure. MODE OF DISCLOSURE Hereinafter, various embodiments of the present disclosure are described in relation to the attached drawings. As the disclosure allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present disclosure to particular modes of practice, and it is to be appreciated that all changes, equivalents, and substitutes that do not depart from the spirit and technical scope are encompassed in the present disclosure. Like reference numerals in the drawings denote like elements. In various embodiments of the present disclosure, it is to be understood that expressions such as “including” and “comprising” indicate the existence of the functions, actions, or components and are not intended to limit one or more other functions, actions, or components. Also, in the present specification, it is to be understood that the terms such as “including,” “having,” and “comprising” are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof disclosed in the specification, and are not intended to preclude the possibility that one or more other features, numbers, steps, actions, components, parts, or combinations thereof may exist or may be added. In various embodiments of the present disclosure, the term “or” includes any or all combinations of words listed together. For example, “A or B” may include either A or B or both A and B. In various embodiments of the present disclosure, the terms “first,” “second,” or the like may modify various components in the embodiments, but such components are not limited by the above terms. For example, such expressions do not limit the order and/or importance of the components. The above terms are used only to distinguish one component from another. For example, both a first user device and a second user device are user devices and indicate different user devices. For example, without departing from the scope of the disclosure, a first component may be referred to as a second component, and similarly, the second component may be referred to as the first component. It is understood that when a component is referred to as being “coupled” or “connected” to another component, it should be understood that the component can be directly connected or coupled to the other component, or intervening components may exist therebetween. On the other hand, when a component is referred to as being “directly coupled” or “directly connected” to another component, it should be understood that no other components are present between the component and the other component. The terms used in one or more embodiments of the present disclosure are merely used to describe a certain embodiment, and are not intended to limit the embodiments of the disclosure. An expression used in the singular encompasses the expression of the plural, unless it has a clearly different meaning in the context. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. FIG.1is a circuit diagram schematically illustrating a device1for detecting a concentration of a sample, according to an embodiment.FIG.2is a diagram illustrating an enlarged region A ofFIG.1.FIG.3is a block diagram of a controller40of the device1ofFIG.1for detecting a concentration of a sample. Referring toFIGS.1to3, the device1may include a power supply unit10, a plasma generation unit20, a signal detection unit30, the controller40, a reference data unit50, and a display60. Samples may be of various types, and may be variously applied in science investigation, aerospace, chemistry, and a medical field. For example, a sample may be a space rock, and a device for detecting a concentration may detect a concentration of atoms or molecules included in the space rock. Also, the sample may be a tissue cell of a living body, and the device for detecting a concentration may measure blood sugar, electrolytes, or microelements and thus diagnose patients based on the measurement. Also, the sample may be an exhaust gas, and the device for detecting a concentration may measure traffic fumes and aircraft exhaust gases. The sample may also be a nuclear reactor material or an explosive including high-energy materials, and a device for detecting a concentration may detect concentrations of atoms or molecules included in the nuclear reactor material or the explosive. Hereinafter, however, the sample includes air including pollutants, in particular, air including heavy metals, and the device for detecting a concentration may be a device for measuring a concentration of heavy metals included in the air. The device1for detecting a concentration of a sample changes a sample into plasma in an ionized state, electrically analyzes light, which is emitted when the plasma drops to a ground state after a certain period of time, and measures a concentration of atoms or molecules included in the sample. The power supply unit10may supply power to generate the plasma. The power supply unit10may include a voltage V, capacitors C1and C2, resistors R1and R2, a power controller11, and a switch12. The power supply unit10charges a current provided from the voltage V in the capacitors C1and C2. The resistors C1and C2may adjust a current or a charging voltage of the power supply unit10. The number or capacity of capacitors C1and C2and resistors R1and R2and the voltage V is not limited to a certain number and may vary according to a design of the device1. The power controller11may control whether the capacitors C1and C2are charged or discharged. For example, the power controller11may be an MCU controller. For example, the power controller11may control a current to make the capacitors C1and C2be charged. Also, the power controller11may enable plasma P to be generated by the plasma generation unit20as the capacitors C1and C2are discharged. The switch12may control a current flowing to the signal detection unit30. After the discharging is terminated in the plasma generation unit20, the switch12may be used to control a driving time so that the signal detection unit30may function. That is, the switch12may adjust the driving time of the signal detection unit30. The plasma generation unit20may be connected to the power supply unit, and a pair of electrodes of the plasma generation unit20may be arranged to face each other. A first electrode21and a second electrode22of the plasma generation unit20have different polarities and are apart from each other with a certain space therebetween. Air that is a sample may pass through a space A between the pair of electrodes, and thus, the plasma may be generated. A gas G may flow to the plasma generation unit20. The gas G including heavy metals flows between the first electrode21and the second electrode22, and when a capacitor is discharged, the gas G may become ionized because of the plasma. Especially, the heavy metals included in the gas may be ionized, and when the discharging of the capacitor is terminated, the heavy metals are stabilized in a ground state, and light is emitted. The plasma generation unit20may be provided in plural. Referring toFIG.1, the plasma generation units20may be connected in parallel and arranged at different locations. Because the plasma generation units20are spatially arranged at different locations, a centration of heavy metals may be measured in each space. The signal detection unit30may be adjacent to the plasma generation unit20. The signal detection unit30may be adjacent to the first electrode21and the second electrode22and may sense the sample and the emitted light because of the plasma. When the heavy metals are stabilized from the ionized state to the ground state, the signal detection unit30may sense the emitted light and measure the same as an electrical signal. The signal detection unit30may include a sensor or sensors. The number of sensors is not limited to a certain number and may be set according to types of heavy metals for measurement. However, for convenience of explanation, an example in which the number of sensors is four will be mainly described. Also, each sensor may include a filter for filtering light in a preset wavelength band, and a photodiode for converting a signal from the filter into an electrical signal. A first sensor31may detect chromium (Cr) and include a first filter31aand a first photodiode31b. The first sensor31may measure the amount of light emitted in a wavelength band of Cr and may convert the light into an electrical signal based on the measured amount of light. The first filter31amay allow the light from the sample to pass the wavelength band of Cr, and the first photodiode31bmay convert the light passing through the first filter31ainto an electrical signal. A second sensor32detects lead (Pb) and includes a second filter32aand a second photodiode32b. The second sensor32may measure the amount of light emitted in a wavelength band of Pb and may convert the light into an electrical signal based on the measured amount of light. The second filter32amay allow the light from the sample to pass the wavelength band of Pb, and the second photodiode32bmay convert the light passing through the second filter32ainto an electrical signal. A third sensor33detects copper (Cu) and includes a third filter33aand a third photodiode33b. The third sensor33may measure the amount of light emitted in a wavelength band of Cu and may convert the light into an electrical signal based on the measured amount of light. The third filter33amay allow the light from the sample to pass the wavelength band of Cu, and the third photodiode33bmay convert the light passing through the third filter33ainto an electrical signal. A fourth sensor34detects zirconium (Zr) and includes a fourth filter34aand a fourth photodiode34b. The fourth sensor34may measure the amount of light emitted in a wavelength band of Zr and may convert the light into an electrical signal based on the measured amount of light. The fourth filter34amay allow the light from the sample to pass the wavelength band of Zr, and the fourth photodiode34bmay convert the light passing through the fourth filter34ainto an electrical signal. The sensors of the signal detection unit30may have different wavelength bands, respectively. Because the first filter31ato the fourth filter34ahave different wavelength bands, the first filter31ato the fourth filter34amay sense light emitted from certain heavy metals. Depending on the types of heavy metals, times taken for the heavy metals to be ionized in the plasma and return to the ground state are different. For example, Pb is stabilized to the ground state after one microsecond (μs) after the plasma is generated, and Cr is stabilized to the ground state after 1.2 μs. A time taken for each heavy metal to be stabilized in the ground state is associated with a unique characteristic thereof. Therefore, for accurate measurement of the concentration of heavy metals, it is necessary to detect light from each sensor after a certain delay time. The signal detection unit30is provided in plural, and the signal detection units30are driven in different delay times. After the capacitors C1and C2stop being discharged, the signal detection units30are driven in different delay times, respectively. The signal detection units30may be driven in different delay times according to unique characteristics of the heavy metals and may accurately measure the amount of light emitted from each heavy metal, thereby accurately measuring the concentration of the heavy metals included in the air. Therefore, the first sensor31to the fourth sensor34may have different driving start points in time. FIG.4is a graph showing a control relationship of the controller40ofFIG.4. Referring toFIGS.3and4, the controller40may be connected to the power supply unit10, the signal detection unit30, a reference data unit50, and the display60and may control each of the listed components. The controller40may be connected to the power controller11and may control the charging and discharging of the capacitors C1and C2. Also, the controller40may be connected to the switch12and control the driving of the signal detection unit30. The switch12may be used to control a current flowing to the signal detection unit30to control the driving thereof. After the oscillation is terminated between electrodes, the controller40may generate a trigger signal to make the signal detection unit30be driven after a preset period of time. The signal generation unit41may generate trigger signals having different delay times, according to characteristics of heavy metals that are respectively measured by the sensors. In response to the trigger signals generated by the signal generation unit41, the signal detection units30may be driven in different delay times. Referring toFIG.4, the power controller11discharges the capacitor so that the electrodes are oscillated for the plasma generation in a point in time t0. The gas G is changed to a plasma state as the discharging of the capacitor continues until a point in time t1, and the heavy metals included in the gas G are ionized. When the oscillation is terminated between the point in time t1, the ionized heavy metals return to the ground state and emit light. However, as described above, it takes some time for the heavy metals to return to the ground state from the ionized state, according to unique properties of respective metals. Referring toFIG.4, a period of time from t1to t2indicates a time taken for the metals to return to the ground state from the ionized state, and such a time differs according to metal types. When the amount of light emitted when the metals return to the ground state is detected, the concentration of the heavy metals may be accurately measured. In a point in time t2, the controller40may generate a trigger signal to make the switch12be driven. That is, the signal generation unit41of the controller40may adjust a delay time (the period of time from t1to t2) and may be driven in a different period of time in each sensor, according to the trigger signal. Therefore, the period of time from t1to t2may differ according to the metals measured. When the trigger signal is transmitted to the switch12, the signal detection unit30is driven and measures emitted light. Each filter of the signal detection unit30may filter light in a wavelength band corresponding to a characteristic of each metal, and each photodiode of the signal detection unit30may convert the amount or intensity of light into an electrical signal. The controller40may integrate the signal from the signal detection unit30for a certain period of time and may predict the concentration of the heavy metals included in the sample by comparing an integration value with reference data. In detail, based on data transmitted from the signal detection unit30, a calculation unit42of the controller40may transform the data to predict the concentration of the heavy metals. The calculation unit42of the controller40may calculate an integration value of an electrical signal in a period of time from t2and t3when the signal detection unit30is driven. Referring toFIG.5A, a size of the electrical signal detected by the signal detection unit30may be indicated as a voltage. The integration value of the electrical signal may be used as data for predicting the concentration of the heavy metals. As shown inFIG.5B, as an integrated area increases, the concentration increases. Therefore, the integration value in the period of time from t2to t3may be calculated and may be compared with the reference data. Thus, the concentration of the heavy metals may be predicted. In an embodiment, the period of time between t2and t3may be identical for each sensor. The signal detection unit30includes sensors having different delay times (t1to t2), but a time taken for each sensor to measure the light is identical as the period of time from t2to t3. For example, the period of time from t2and t3may be 1 μs. Therefore, the light, which is emitted from each heavy metal from the ionized state to the ground state, is measured by the signal detection unit30for identical periods of time. By comparing the amounts of light measured during the identical periods of time, the amount of heavy metals included in the air may be compared. FIG.5Ais a graph showing an electrical signal detected by the signal detection unit30, andFIG.5Bis a graph showing a concentration calculated by integrating the graph ofFIG.5A. Referring toFIGS.5A and5B, the controller40may predict the concentration of the heavy metals by comparing the integration value calculated by the calculation unit42with the reference data of the reference data unit50. The reference data unit50may store reference data associated with an integration value of an optical signal, according to the concentration of the heavy metals. Pieces of the reference data are stored in the reference data unit50in advance to measure the concentration of the heavy metals, before the device1operates. Referring toFIG.5A, the reference data includes a graph regarding a voltage according to a time when the heavy metals emit the light. Each piece of the reference data differs according to the concentration of the heavy metals. For example, according to the Pb concentration, the graphs are shown differently. Also, the pieces of reference data include different graphs varying according to the types of the heavy metals. The graph ofFIG.5Bis changed to a calibration function, based on the graph ofFIG.5A. For example, the concentration is displayed according to the integration value of the graph ofFIG.5Ain the period of time from t2to t3, and is predicted with a function to generate a calibration curve. Because the reference data unit50has a calibration curve used to predict the concentration according to the integration value of each heavy metal, when the calculation unit42of the controller40calculates the integration value in the period of time from t2to t3, a concentration prediction unit43may predict the concentration of each heavy metal through the graph ofFIG.5B. The display60may be connected to the controller40and may display the concentration of the heavy metals that is interpreted by the concentration prediction unit43. The amount of heavy metals included in the gas G may be identified by displaying the concentration of each heavy metal. FIG.6is a flowchart of a method of detecting a concentration of a sample, according to another embodiment. Referring toFIG.6, the method of detecting a concentration of a sample may include: operation S10in which reference data regarding the concentration of heavy metals is obtained; operation S20in which plasma is generated by a plasma generation unit; operation S30in which the oscillation is terminated between electrodes of the plasma generation unit; operation S40in which a signal detection unit is driven after a preset period of time after the oscillation is terminated; operation S50in which the signal detection unit senses light emitted from the sample in each wavelength band; and operation S60in which a controller calculates the concentration of heavy metals according to a sensed signal. Operation S10, in which the reference data regarding the concentration of the heavy metals is obtained, is an operation including the reference data for comparison before the concentration of the heavy metals included in the sample is measured. The reference data unit50may convert light emitted from each heavy metal into a voltage value that is an electrical signal as shown inFIG.5A, and may store the reference data, which is the calibration curve regarding the concentration of the heavy metals, based on the integration value as shown inFIG.5B. Also, each heavy metal may have the reference data. In operation S20, in which the plasma generation unit generates the plasma, the plasma is generated in a space between a pair of electrodes of the plasma generation unit20that are connected to the power supply unit10. When the capacitor is discharged, the plasma is generated in the space between the first electrode21and the second electrode22because of strong electrical sparks (t0). In this case, the heavy metals, which are included in the gas flowing in the space, are ionized. In operation S30, in which the oscillation stops between electrodes of the plasma generation unit, the discharging is terminated on the capacitor (t1). When the plasma generation is terminated, the ionized heavy metals start being stabilized to the ground state. After the oscillation is terminated, in operation S40, in which the signal detection unit is driven after a preset period of time, when the oscillation is terminated between the electrodes, the signal detection unit30is driven after the preset period of time (t2). Because times taken for the heavy metals to return to the ground states are different, the signal generation unit41of the controller40transmits a trigger signal to the switch12to make each heavy metal have a different delay time, and the signal detection unit30is driven according to each delay time. In operation S50in which the signal detection unit senses the light emitted from the sample in each wavelength band, the signal detection unit30senses the light from the sample in each wavelength band because of the plasma. Respective sensors of the signal detection unit30sense the light in different wavelength bands, according to types of the heavy metals. A filter filters the light in each wavelength band, and a photodiode converts the filtered amount of light into an electrical signal, that is, a voltage value. In operation S60in which the controller40calculates the concentration of the heavy metals according to a sensed signal, the voltage value in the period of time from t2to t3may be integrated, and an integrated area is compared with the reference data. Thus, the concentration may be predicted. According to a device and a method of detecting a concentration of a sample according to the one or more embodiments, the concentration of heavy metals included in the sample may be accurately and quickly calculated by detecting a plasma emission spectrum. The amount of heavy metals in the air may be measured by detecting the amount of light emitted when the heavy metals are ionized and return to a ground state. According to a device and a method of detecting a concentration of a sample according to the one or more embodiments, because signal detection units have different signal start points in time depending on types of the heavy metals, the amount of light emitted from the heavy metals may be accurately measured. Also, the amount of heavy metals may be accurately measured by filtering the light in respective wavelength bands according to the types of the heavy metals. According to a device and a method of detecting a concentration of a sample according to the one or more embodiments, because an electrical spectrum is used without chemical preconditioning, the concentration of the heavy metals may be accurately calculated in real time. While this disclosure has been particularly shown and described with reference to embodiments thereof, it will be understood by one of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the appended claims. INDUSTRIAL APPLICABILITY The present disclosure relates to a device and a method for detecting a concentration of a sample, which are used to accurately and quickly measure a concentration of heavy metals included in the sample. | 28,098 |
11860108 | DETAILED DESCRIPTION OF THE INVENTION Referring to the figures, wherein like numerals indicate like or corresponding parts throughout the several views, an optofluidic diagnostic system according to one exemplary embodiment of the invention is generally shown at20inFIG.1. The automated optofluidic diagnostic system20is designed for rapid biological and chemical analysis. Its many advantages include using less analyte and reagent solutions than the amount used in traditional protocol. Furthermore, the system20is able to perform high-throughput detections because of its capability to automatically load and unload the solutions. Generally stated, the optofluidic diagnostic system20is composed of three primary parts or modules: a sensor array22, a test plate24and an optical detection cartridge26. Each module is independent of the other two modules, in the sense that each module is capable of stand-alone use independent of the unique attributes found in the other modules. However, all three of these modules find their greatest fulfillment when used in the combination which comprises the optofluidic diagnostic system20. The sensor array22comprises a plurality of sensor units28arranged in formation. A single sensor unit28is shown inFIG.2. The invention may be practiced using only a single sensor unit28, as inFIG.2, however greater efficiencies can be gained by multiplying the number of sensor units28into a monolithic array22like that shown inFIG.3so that multiple diagnostic tests can be conducted concurrently. The array22ofFIG.3depicts an exemplary arrangement in which twenty-four sensor units28are joined in a formation of two columns, each column containing twelve sensor units28. In an array22configuration, the distance between the center of one sensor unit28to the center of the next adjacent sensor unit28can be 0.002 to 0.354 inches or even greater. From the vantage ofFIG.3, one of the columns of sensor units28can be described as the first or left-hand column and the other a second or right-hand column. In use, the first column trails the second column as the sensor array22is moved in hopping-like fashion through the sequential steps of a diagnostic test. That is, the second column is always a leading column. Although the array22is shown in several Figures as a 2×12 matrix, the actual number of sensor units28in an array22can be any desired number. Indeed, an array22may consist of only one column of any plural number of sensor units28. Or in some cases, it might be desirable to construct a sensor array22having more three or more columns of sensor units28. And to reiterate, a single sensor unit28operating solo can also be used to accomplish the methods of this invention without joining to other sensor units28into an array22. Many configurations of one or more sensor units28are possible. Each sensor unit28can be seen to extend between a top end30and a bottom end32. The sensor units28are normally oriented in an upright posture so that its bottom end32is spaced vertically from its top end30. Each sensor unit28has a dedicated fluid duct34that extends continuously therethrough from its top end30to its bottom end32. The full length of the fluid duct34can be seen in the images ofFIGS.6A-D. Embodiments are contemplated where the fluid duct34is tapered and/or non-circular in cross-section. Preferably, however, the fluid duct34has a continuous cross-sectional area that is generally cylindrical, i.e., it has a constant circular cross-section along its entire length. In some contemplated embodiments of this invention, the internal diameter of the fluid duct34is between about 0.0003 and 0.06 inches. However, measurements outside this range are certainly possible. In fact, all dimensions and dimensional ranges provided throughout this description are mentioned for illustrative purposes only and are not to be construed as limiting the scope of the invention. Returning toFIGS.2and3, it can be seen that each sensor unit28includes a reactor section36adjacent its bottom end32. In the illustrated examples, the reactor section36comprises the lower half (approximately) of the sensor unit28. The upper half (approximately) of the sensor unit28comprises a coupler section38. A frame40is disposed between each reactor section36and its associated coupler section38. The frame40can serve as a mounting platform or feature in the case of a sensor unit28operating solo (FIG.2). The frames4can also be a convenient point of attachment for integrally joining one sensor unit28to the next adjacent sensor unit28when forming a monolithic array22as in the example ofFIG.3. The fluid duct34may include a reactive coating agent A that has been applied, i.e., immobilized, over at least a portion thereof within the reactor section36. The reactive coating agent A can be any suitable diagnostic substance, including but not limited to, assays used to assess the presence, amount or functional activity of a target entity (i.e., the analyte). The reactive coating agent A contemplated for use in this invention specifically includes, but is not limited to, solid-phase enzyme immunoassays such as those used in typical ELISA test procedures. The reactive coating agent A may either be applied by a manufacturer, by an intermediate vendor, or by the end-user as a preparatory step prior to actual use in the system20. It is also contemplated that the reactive coating agent A could be immobilized inside the fluid duct34using the system20of this invention but prior to the start of an actual diagnostic test. The reactor section36may be partially or entirely fabricated from an optically transmissive material, including materials that can be characterized as fully transparent, semitransparent and/or translucent. More specifically, an optically transmissive material will be selected that has an index of refraction that closely approximates that of water or some other analyte liquid. By matching (or at least approximating) the index of refraction of the reactor section36with the refractive index of the liquid analyte to be used, light will pass from one to the other with minimal reflection or refraction losses. Two of the many suitable materials include glasses and plastics. The sensor units28can be manufactured by injection molding when a transparent plastic material (e.g., clear transparent polystyrene) is chosen. The reactor section36has at least one planar observation face42, at least a portion of which is fabricated from the optically transmissive material. That is to say, at the very least, the portion of the reactor section36that compromises the observation face42must have some optically-transmissive properties. In the illustrated examples, the entire reactor section including the observation face42is made from an optically-transmissive material. When the sensor unit28is ganged with other sensor units28into an array22, the observation face42will face outwardly, i.e., in a direction away from all of the other sensor units28in the array22. The reactor section36has a predetermined outer geometric shape that is preferably, but not necessarily, generally centered about the fluid duct34. Contemplated geometric shapes include rectangles, triangles, hexagons and D-shapes to name a few. In the illustrated examples, the predetermined outer geometric shape of the reactor section36is generally square. The square shape produces four distinct flat exterior faces, one of which is the aforementioned observation face42. In cases such as this where a sensor array22is composed of reactor sections36having more than one planar exterior face, the observation face42will be distinguished as the one facing away from the other sensor units28, as shown inFIG.3. Preferably, but perhaps not necessarily, the observation face42is oriented vertically, and thus generally parallel to the fluid duct34. In this manner, the cross-sectional thickness of optically transmissive material remains generally consistent along the length of the reactor section. In cases where the fluid duct34has a circular cross-section and extends parallel to the observation face42, this configuration produces a generally plano-concave lens as shown by the cross-sections inFIGS.9and10. A plano-concave lens will have beneficial divergent light-handling properties in cases where the refraction indexes between the material of the reactor section36and analyte solutions contained within the fluid duct34do not match. The reactor section36has a leading tip44formed directly adjacent the bottom end32of the fluid duct34. In the examples ofFIGS.2and3, the leading tip44is formed with a lofting square-to-round converging bended surface. In the example ofFIGS.6A-D, the leading tip44is formed as a flat truncated surface. Other shapes are contemplated, including but not limited to semi-spherical. Some specific advantages are attained when the leading tip44is formed with a square to circle tapered lofted blend tip, as explained below in connection withFIGS.12-15. The coupler section38of each sensor unit28is designed to connect with a supply of—positive or/and negative generator. The medium is described at various points below as being air, but other gasses and fluids could be used instead. In the highly-simplified example ofFIG.11, individual feed tubes46are connected to the coupler sections38in a sensor array22configuration. Instead of the individual tubes46, a manifold could be used to connect to the coupler sections38. Or perhaps the atmosphere above the entire sensor array22could be controlled to cause pressure/vacuum fluctuations at the bottom ends32of the fluid ducts34. For convenient connection to individual feed tubes46or a manifold (not shown), the coupler sections38may comprises a conically-tapered exterior surface that is centered about the fluid duct34to accomplish a friction fit. Of course, may other shapes and connection strategies may be used for the coupler section38to effectively connect with a supply of pressurized (positive and/or negative) air or other suitable fluid medium. Each sensor unit28can thus be viewed as an open-ended tubular (i.e., hollow) structure whose fluid duct34is used as an inlet and outlet for reagents/analytes at a bottom end32thereof. Pressure differentials, if necessary, are introduced to the fluid duct32via its top end30. The reactor and coupler sections36,38are connected internally and smoothly via the internal fluid duct34. The preferred outer shape of the tubular reactor section36is square, and the preferred outer shape of the coupler section38is tapered (frusto-conical) for easy insertion of connecting tubes46that link to the pressure differential device(s). Although these shapes can, of course, be modified to suit different applications and manufacturability. That is to say, other geometric shapes may be considered, including but not limited to oval, elliptical, triangular, hexagonal and octagonal tubular configurations to name but a few of the many possible forms. In the context of this optofluidic diagnostic system20, each sensor unit28is configured for sequential movement into and out of registry with a plurality of discrete wells48in the test plate24. In this manner, it can be said that the test plate24is adapted to receive the sensor array22in mating registry, as indicated byFIG.4. However, unlike the wells of a traditional multi-well microplate (e.g., a Microtiter™ plate), it is not intended that any chemical reactions take place in any of the wells48of the test plate24. Rather, in this present system, all chemical reactions of relevance will take place inside the rector sections36of the sensor units28. Thus, the wells48may be seen more as holders or storage centers for various elements used in the process of conducting biological and/or chemical analysis inside the reactor sections36. Each well48is formed as a discrete comb-like cavity having a mouth50at its upper end and a closed base52at its lower end. The vertical distance between mouth50and base52is a well depth. In the illustrated examples, each well48in the test plate24has a generally equal well depth. However, since not all wells48have the same function or job, it is conceivable that the wells48could have different depths and/or different configurations for the base52. The wells48each have a predetermined inner geometric shape that generally corresponds to the predetermined outer geometric shape of the reactor sections36. In other words, if the outer cross-section of the reactor section36is square, then the inner cross-section of the well48is also square. This is perhaps best shown inFIG.9where a cross-section is taken through a well48with a reactor section36poised therein. Preferably a generous sliding fit clearance is maintained between the OD of the reactor sections36and the ID of the wells48so that the reactor sections36can be easily inserted into and withdrawn from the wells48along a vertical path during the several steps in a diagnostic process. The test plate24inFIG.4is shown in the exemplary form having twelve rows corresponding to the number of rows of the sensor array22. In this illustration, each row has a trajectory extending toward the lower right-hand corner of the image, whereas each column has a trajectory extending toward the lower left-hand corner of the image. In an X-Y coordinate system as viewed from above (e.g.,FIG.5), the rows may be said to extend in a horizontal X-direction and the columns in a vertical Y-direction. In most contemplated embodiments, the test plate24will have at least as many rows as the sensor array22. The test plate24could easily have more rows than the sensor array22, however it is unlikely that the test plate24will have fewer rows than the sensor array. The test plate24inFIG.4is shown in the exemplary form having twenty-four columns corresponding (or proportionally-corresponding) to the discrete steps needed to accomplish a diagnostic test. In this example, twelve discrete steps are possible. This is because the sensor array22shown here has two columns of sensor units28. Thus, the twenty-four columns of the test plate24must be shared by the two columns of sensor units28. (24÷2=12.) It will be understood that to complete a diagnostic analysis using the present system20, the sensor units28are moved (relative to the test plate24) along the rows of wells48. Using the previously suggested X-Y coordinate system, it would be said that the sensor units28are moved (relative to the test plate24) along the X-direction. A most efficient, but not exclusive, movement scenario is diagrammed inFIG.11where the sensor array22is sequenced along the test plate24in a straight line hopping fashion. It may be helpful to think of the plurality of wells48as being arranged in respective sequence clusters. Each sensor unit28is associated with a respective one sequence cluster. Thus, in the examples ofFIGS.4and9, there are twenty-four sensor units28in the sensor array22so that the test plate24is configured to provide twenty-four distinct sequence clusters. Each reactor section36is constrained to interact with wells48in one designated sequence cluster. Or to say it another way, no reaction section36is permitted to stray outside its designated sequence cluster throughout the duration of a diagnostic test carried out with the system20. Preferably, but perhaps not necessarily, the wells48in each sequence cluster will be arranged in a linear array or linear pattern. However, when the sensor array22has multiple columns of sensor units28, the wells48in a sequence cluster will not be contiguous with one another. To graphically illustrate, attention is directed toFIG.4were a select one of the twenty-four sequence clusters is indicated by bold edging around the mouths50of the corresponding wells48. The indicated sequence cluster inFIG.4corresponds to the top-most sensor unit28in the second or right-hand column of the sensor array22. (Every other well48in that same top row of the test plate24is associated with a different sequence cluster for the top-most sensor unit28in the first or left-hand column of the sensor array22.) Throughout a diagnostic test, the reactor section36of the top-right sensor unit28will only descend into a well48of its designated sequence cluster. No other reactor section36in the array22will enter one of the wells48in the sequence cluster set aside for the top-right sensor unit28. Thus, the relationship between a sensor unit28and its designated sequence cluster is exclusive throughout a diagnostic test, to avoid contamination. Generally stated, the number of sequence clusters in each row of the test plate24will correspond to the number of columns of sensor units28in a sensor array22. If a sensor array22has only one column of sensor units28(and when a solitary sensor unit28is operating solo), a row of wells48may contain only one active sequence cluster. Or alternatively, if a sensor array22were to have four columns of sensor units28, a row of wells48must contain at least four distinct sequence clusters. And so forth. FIG.5is a fragmentary top view showing a portion of four rows of wells48along the bottom edge of the test plate24, as taken generally along the section line5-5inFIG.2. This view helps to illustrate the different roles, or jobs, that the wells48in any given sequence cluster are required to fulfill. There are at least three jobs that must be fulfilled by the wells48in any sequence cluster, and therefor at a minimum a sequence cluster must have at least three wells48. It will be helpful to keep in mind that each row in this example contains two distinct sequence clusters that occupy alternating wells48. And that for each sequence cluster of wells48, one sensor unit28is dedicated. For these reasons, different types of wells48will appear in matched pairs—one well48for each sensor unit28in the two columns. At least one well48in each sequence cluster comprises a sample reservoir54, indicated inFIG.5by diagonal cross-hatch marks. A sample reservoir54is a well48that has a particular type of use or function. Not all wells48in a sequence cluster are sample reservoirs54. In this example, three sets or pairs of sample reservoirs54are visible in the fragmented section ofFIG.5. The function or job of a sample reservoir54is to contain liquid reagents or analytes that are required to perform the desired diagnostic test. When the reactor section36of a sensor unit28is placed into a sample reservoir54, the liquid reagents or analytes in that sample reservoir54are drawn up into the fluid duct34of the reactor section36, either by capillary action or under the influence of a pressure differential or combination of both. A more detailed explanation of this procedure will be described below in connection withFIGS.6A-D. Typically, the first well48in each sequence cluster will be used as a sample reservoir54specifically to hold a sample taken from a patient (or other source to be tested). As such, it may be useful to configure the test plate24so that the first, or at least one, sample reservoir54in a sequence cluster is detachable from the other wells48in that sequence cluster. In the example of a 12×24 test plate array24like that shown inFIG.4, a person of ordinary skill in this art can envision the first two columns of wells48made separable from the remaining wells48of the test plate24. Depending on the type of fixture used to support the test plate24in a diagnostic system20(e.g.,FIG.1), it may not even be necessary that the detachable columns of wells24be formally joinable or fastenable to the remainder of the test plate24. In other words, the detachable column(s) of sample reservoir(s)54could be a permanently loose-piece component that is brought into proximity with the other wells48in the test plate24within the system20at the time of testing. InFIG.16, the separable concept is illustrated via a separation line84. Such an arrangement, where the sample reservoirs54used as a repository for the patient sample(s) are detachable from the remainder of the test plate24, could make the system20more flexible and more convenient for users. Another type of well48is drainage chamber56. At least one well48in each sequence cluster will be a drainage chamber56. Drain chambers56are dedicated to the drainage of liquid reagents/analytes from the reactor sections36. Each drainage chamber56preferably includes an absorbent pad that is capable of wicking liquid reagent from a reactor section36. Returning to the example ofFIG.5, two sets or pairs of drainage chamber56are seen, and can be identified by stippling, i.e., two drainage chambers56for each of the two sequence clusters visible in the fragmentary section ofFIG.5. In this view, there would very likely be at least one additional (but unseen) pair of drainage chambers56in each row to accommodate the third set of sample reservoirs54. Preferably for purposes of motion economy, but not necessarily, one drainage chamber56will follow each sample reservoir54in a sequence cluster. One can therefore image that a reactor section36descends into a sample reservoir54to uptake liquid reagents or analytes, and then after a suitable incubation period moves to a nearby drainage chamber56so that its liquid contents can be emptied. Then on to another sample reservoir54, incubation, another drainage chamber56, and so on (uptake-incubate-drain) until the required number of steps has been completed. For this reason, one drainage chamber56will typically follow each sample reservoir54within any sequence cluster, and furthermore that the sample reservoirs54in each sequence cluster will tend to be disposed in alternating fashion with the drainage chambers56, like this:54-56-54-56-54-56. . . . As shown inFIG.16, it may be possible in some applications to gang-together one or more drainage chambers in a common column. For example, reference number56′ illustrates how all twelve drainage chambers56′ in a single column can be merged. And in cases where the sensor array22has two (or more) columns of sensor units28, adjacent columns can be merged into a large common drain chamber56″. Of course, many other variations of this idea are possible. A third type of well48in each sequence cluster is a colorant reservoir58. Each sequence cluster includes at least one, typically only one, colorant reservoir58at or near the end of the row. The purpose of the colorant reservoir is to contain a liquid color development reagent. After a sensor unit28has finished its prescribed course of uptake-incubate-drain events, its reactor section36is plunged into the dedicated colorant reservoir58in its sequence cluster. After a suitable period of time has been allotted for the color development reagent to have its effect, the sensor unit28moves to optical detection. After that, the sensor array22can be trashed with or without performing a final drainage step. In situations where a final drainage step is performed, either a fresh drainage chamber56or a previously-used drainage chamber56in the same sequence cluster can be used. (A previously-used drainage chamber56can be used because contamination will no longer be a significant concern at this stage.) The test plate24can thus be viewed as an array of wells48for reagents/analytes and absorbent pads. The array format of the test plate24aligns with the format of sensor array22and has at least the same number of columns as the sensor array22. The reagents/analytes (sample reservoirs54) and absorbent pads (drainage chambers56) are arranged alternately starting with the reagents/analytes. The type of reagents and sequence of various reagents can be determined and pre-programmed based on the analyte(s) to test and the type(s) of diagnostic protocol to perform. The last columns are designated for color development reagent. Optionally, the wells48can be made as individual pieces, or column sub-sets, or row sub-sets, that are combined like building blocks to form a unitary structure of the desired size. The internal shape of each well48will be an outside offset of the outer shape of the reactor sections36so that a loose mated fit is achieved. The offset distance or clearance can, for example, be in the range of about 0.008 to 0.08 inches. The test plate24can be made any color with transparent, translucent, or opaque material. However, the preferred material is mechanically stable (not easily deformed) and inert to all anticipated reagents/analytes. The test plate24can be manufactured by injection molding if a plastic material (e.g., polypropylene) is chosen. Wells48can be manufactured all at once into a fully-formed test plate24or can be assembled by placing different components (e.g., rows or columns) together. FIGS.6A-Dschematically illustrate the flow mechanism of reagents/analytes into and out of the reactor section36of a single optofluidic sensor unit28in the aforementioned uptake-incubate-drain course of events. For clarity, mating wells48are not shown in any ofFIGS.6A-D. It should again be mentioned that the leading tip of the reactor sections36are shown in an optional flat (non-tapered) configuration in theseFIGS.6A-D. FIG.6Arepresents a reactor section36that is inserted or loaded into a well48configured to function as a sample reservoir54. A directional arrow at the bottom end32of the fluid duct34shows the flow direction of the reagents/analytes at the open bottom end32of the single optofluidic sensor unit28. When the reactor section36of the sensor unit28is immersed into a sample reservoir54containing a reagent/analyte solution, the solution flows up into the reactor section36, because of the capillary force or because of a pressure differential induced at the top end30or combination of both. In one example, a pressure differential is accomplished by gently pulling a vacuum through a feed tube46. This corresponds to the “uptake” part of the uptake-incubate-drain process. FIG.6Bcorresponds to the “incubate” part of the uptake-incubate-drain subroutine. The solution drawn into the reactor section36is incubated in the fluid duct34for a certain amount of time to allow the interaction between the solution and reactive coating agent A (FIGS.9and10) pre-applied to the interior hollow surface within the reactor section36. Or as mentioned previously, the reactive coating agent A could alternatively be immobilized using the system20of this invention in a pre-test preparation phase. FIG.6Cportrays the “drain” part of the uptake-incubate-drain cycle. After incubation, the solution contained within the reactor section36is drained out through the bottom end32, as indicated by the downwardly-pointing directional arrow. Typically, the solution is wicked away using an absorbent pad located inside a drainage chamber56, or alternatively using a pressure differential induced through the top end30of the fluid duct34or combination of both. In one example, a pressure differential is accomplished by gently pushing air through a feed tube46. After draining the solution, biochemical molecules60(FIGS.9and10) are attached on the hollow surface within the reactor section36. The processes of injecting the solution (FIG.6A), incubating the solution (FIG.6B), and draining the solution (FIG.6C) can be repeated sequentially as per requirements of the diagnostic protocol. In the last step associated with the test plate24, portrayed inFIG.6D, the reactor section36of the optofluidic sensor unit28is immersed into color development reagent held in a colorant reservoir58located at or near the last columns of the test plate24. Via capillary action or pressure-assist or combination of both, the color development reagent colorant fills the reactor section36and then is subsequently drained after a suitable incubation period or remains inside sensor unit28after a suitable incubation period. Some protocols require that the colorant does not need to be drained out. For example, in chemiluminescence measurement, the color development reagent remains inside the reactor section36. The colorant prepares the biochemical molecules60for optical detection. The processes of coloring the biochemical molecules60can be repeated as per requirements of the diagnostic protocol. After that, the fully prepared sensor unit28is ready for optical detection. To facilitate the optical detection process, the system20of this invention may, optionally, include an optical detection cartridge26. Perhaps best seen inFIGS.7-9, the optical detection cartridge26includes a plurality of light confinement isolation booths62. The number and arrangement of isolation booths62correspond to the number and arrangement of sensor units28. That is to say, the array format of the optical detection cartridge26must be capable of aligning with the format of the sensor array22, and therefore it is desirable that the detection cartridge26have the same number of columns as the sensor array22. Each isolation booth62has a booth height defined by an open ceiling64and a closed floor66. Within the cartridge26, each isolation booth62will typically have the same, i.e., generally equal, booth height. Each isolation booth62is characterized by having an open viewport68surrounded by optically-opaque sides. Similar to the loose mating fit between sensor array22and test plate24, the fit between the sensor array22and the optical detection cartridge26must also be of a somewhat slack male-female relationship. The internal shape of each isolation booth62will be an outside offset of the outer shape of the reactor sections36so that the desired loose mated fit is achieved. The offset distance or clearance can, for example, be in the range of about 0.008 to 0.08 inches. Each isolation booth62is adapted to receive therein a respective reactor section36, so that the observation face42of the reactor section36is oriented toward the viewport68. In particular, each isolation booth62is configured to receive the reactor section36of a sensor unit28through its open ceiling64. When fully inserted, the observation face42of the reactor section36is exposed, i.e., visible, through the viewport68, as shown inFIG.9. In this manner, the observation face42is presented for optical detection. To avoid optical cross talk, the optical detection cartridge26is made with an opaque (preferably black) and mechanically stable material. The optical detection cartridge26can be manufactured by injection molding if a plastic material (e.g., back opaque polystyrene) is chosen. An optical detector70has at least one (typically only one) detection lens72associated with each isolation booth62. In the example ofFIGS.8and11, two optical detectors70are provided, each having twelve lenses72. One optical detector70is provided for capturing the optical conditions of the sensor units located along the first (left-hand) column of the sensor array22. Conversely, the other optical detector70is provided for capturing the optical conditions of the sensor units located along the second (right-hand) column of the sensor array22. Each detection lens72of the optical detector70is arranged opposite the viewport68of a respective the isolation booth62or is otherwise moveable into such a position. Of course, another possible variation is that a single optical detector70is configured with only one lens72for recording optical signals from all twelve sensor units28, either sequentially or concurrently or one snapshot using large field of view lens. Whether plural or singular detection lenses72are employed, the opaque detection cartridge26makes optical cross-talk preventable among individual optofluidic sensor units28, thus enabling improved accuracy in chemiluminescence or fluorescence detection schemes if desired. By fashioning the observation face42as a flat planar surface oriented orthogonally toward the lens72, an ideal imaging condition is established with which to acquire a uniform, relatively evenly distributed optical color representation of the biochemical molecules60. As previously mentioned, the cross-sectional thickness of optically transmissive material directly behind the observation face42may be configured in the form of a plano-concave lens as shown by the cross-sections inFIGS.9and10. Plano-concave lens are naturally divergent, which has the benefit of helping to spread the color-affected light across the observation face42, thus increasing the efficiency, sensitivity and effectiveness of the optical detector70. FIG.11illustrates the relative movement of the sensor array22over the test plate24and finally to the optical detection cartridge26. As also described in the legend provided withFIG.11, solid arrows74represent relative moving directions of the sensor array22into sample reservoirs54containing liquid reagents. Evenly dotted arrows76represent relative moving directions of the sensor array22from sample reservoirs54into drainage chambers56. Typically, an absorbent pad will be located at the base of each drainage chamber56. This process may be repeated through multiple sample reservoirs54based on the required diagnostic protocol. A dot-dash arrow78represents relative movement of the sensor array22into the color development reagent contained with the final colorant reservoirs58. Optionally, not shown, the sensor array22may be drained after incubating in the color development reagent. Evenly dashed arrow80represents final movement of the sensor array22into the optical detection cartridge26, where the isolation booths62confining light contamination between the sensor units28(i.e., undesirable optical cross-talk). Optical detectors70are poised to take readings from each observation face42, which reading are transmitted to an appropriate computerized processing device (not shown) for analysis and reporting. FIG.1demonstrates, in simplified fashion, an exemplary automated optofluidic diagnostics system20combining the three main assembled components: the sensor array22, the test plate24, and the optical detection cartridge26. A suitable transfer mechanism82is operatively disposed between the sensor array22and the test plate24and the optical detection cartridge26for moving the sensor array22relative to the test plate24and the optical detection cartridge26in response to a pre-programmed pattern. In this example, the sensor array22is gripped by a robotic arm attached to a stepper or servo motor. Feed tubes46connect to computer-controlled pressure differential device(s). The robotic arm can be moved vertically using the motor, while the entire module of the robotic arm, the feed tubes46, the motor and sensor array22can be moved horizontally using another stepper motor. In this example, the test plate24and the optical detection cartridge26are fixed on a stationary fixture. For simplicity the optical detectors70are not shown inFIG.1but could of course be mounted on flanking sides of the optical detection cartridge26on the fixture as inFIG.11, or else supported on a separate robotic arm and moved into position when needed. All these parts and modules may be enclosed in an enclosure. A touch screen user access interface (not shown) connected to a suitable microcontroller can be located at any convenient location on or around the enclosure. In other contemplated embodiments, a robotic arm moves the plate24while the sensor array22remains stationary. Naturally,FIG.1represents but a simple desk-top configuration of the system20. Those of skill in the art will readily appreciate that the system20described herein can be scaled-up to include other parts such as automated sample additions to the test plate24, stacking modules for automated insertions, automated ejections and automated re-loadings of sensor arrays22, test plates24and/or optical detection cartridges26. Likewise, the system20could also be scaled-down to a partially or fully manual process with only one or a small number of sensor units28processed at a time. As mentioned previously, the shape of the leading tip of the reactor section36can take different forms. Similarly, the shape of the base52of the wells48, and in particular the bases52of the sample reservoirs54, can also vary. InFIGS.6A-Dand12, the leading tip is presented as a flat, squared-off shape. A flat tip is adequately functional within the system20but has one slight disadvantage—a flat tip naturally forms a relatively large hanging droplet of reagent solution as shown inFIG.12. As the hanging droplet does not enter the fluid duct34, it does not contribute to the diagnostic test and therefore represents an unproductive quantity of reagent solution. Often, the quantity of reagent solution may be limited, and it is necessary to economize usage. ComparingFIGS.12and13, it can be seen that a larger droplet size of reagent solution (as collected from a sample reservoir54or colorant reservoir58) will be greater for the flat tip than for the conical tip. Thus, in some applications it may be preferable to form the leading tips44of the reactor sections36with a generally frustoconical converging shape like that exemplified inFIGS.2,3and13which naturally forms a relatively small hanging droplet of reagent solution. Further economies can be achieved by optimizing the shape of the base52of each well48, or at least those wells48serving as sample reservoirs54, to closely match a conical leading tip44.FIG.14depicts a flat tipped reactor section36like that ofFIG.12. The base52of the well48in this example is matched with a complementary flat shape. As a result of these mating flat shapes and exacerbated by the relatively large size hanging drop carried by the flat leading tip of its reactor section36, a pronounced meniscus is formed by the molecules of the liquid that are attracted to climb the container walls. The quantity of unproductive solution would be even worse if the base52were to have a conical shape while the leading tip of the reactor section36remained flat. However, the situation can be vastly improved by tapering the base52with a complementary conical shape to the tapered leading tip44of the reactor section36as shown inFIG.15. The shaded area shows a minimum amount of solution required for capillary uptake in this case. For maximum efficiency, the base52has a diverging square-to-round shape that exactly complements the generally frustoconical converging shape of the leading tip44of the reactor section36. In other words, the lofted boss square-to-round shape of the leading tip44is matched by the lofted cut square-to-round shape of the base52, resulting is a very small quantity of unproductive reagent solution being trapped at the interface. Consequently, the minimum amount of reagent solution will be required for capillary uptake when both the leading tip44and base52have matched conical configurations like that shown inFIG.15. The present invention describes a complete automated optofluidic diagnostic system20and accompanying methods designed for rapid analyte detections without using a conventional microplate reader or conventional well-plate. The system20comprises three independently usable components: an optofluidic sensor array22, a test plate24having pre-populated sample reservoirs54and drainage chamber56, and an optical detection cartridge26. In one embodiment described, the sensor array22is attachable to and detachable from a robotic arm with two degrees of freedom, movable vertically and horizontally, while the test plate24and optical detection cartridge26are residing at stationary positions. In addition, the system20is able to integrate the user's desired optical detection module (e.g., chemiluminescence, fluorescence, etc.) with or without the stacking modules for high-throughput testing. The envisioned overall system20volume can be designed to occupy less than 1 cubic foot, making it conveniently portable. The user is able to access and control the system20, while also being able to see the status of the system via a touch screen interface (not shown). The alternative 12×24 matrix test plate24shown inFIG.16illustrates an optional set-up in which the first two columns of sample reservoir(s)54are formed as a loose-piece component that is brought into proximity with the other wells48in the test plate24along a separation line84. This type of an arrangement makes it convenient for the initial column(s) of sample reservoirs54to be used for patient sample gathering. As such, it is potentially beneficial that these leading columns be disconnected, at least initially, from remainder of the test plate24. While a portable system20(i.e., smaller than 1 cubic foot) may be desirable for many users, in other contemplated embodiments the system20can be scaled up to include other parts such as automated sample additions to the test plate24, and stacking modules of automated insertion, ejection, and re-loading of sensor array22, test plate24, and optical detection cartridge26to name but a few. The system20has many advantages, including: (1) It does not require bulky standard well plate readers as with the prior art; (2) It does not require adding reagents manually; (3) The optionally small diameter of the optofluidic sensor improves analyte capture efficiency, and reduces assay time that allows for rapid diagnosis; (4) The optofluidic design with two open ends allows for addition and withdrawal of the analytes (solution) which uses capillary force or pressure differential induced by external device(s) or combination of both; (5) Predefined and prepopulated reagents in the reagents/analytes reservoirs and absorbent pads provide efficient means for reagents/analytes delivery and draining; (6) An opaque light confinement cartridge26makes optical cross-talk preventable among individual optofluidic sensor units28. Therefore, chemiluminescence or fluorescence detection schemes can be adopted; (7) It can be deployed at bedside of patients, doctors' offices, and in space-limited laboratories due to the optional compact-size of the system20; (8) It facilitates high-throughput screening due to the nature of the automated system. These as well as other advantages will become apparent to those of skill in this art through the following description and accompanying illustrations. The foregoing invention has been described in accordance with the relevant legal standards, thus the description is exemplary rather than limiting in nature. Variations and modifications to the disclosed embodiment may become apparent to those skilled in the art and fall within the scope of the invention. | 42,476 |
11860109 | DETAILED DESCRIPTION Hereinafter, various embodiments of this disclosure will be described with reference to the accompanying drawings. FIG.1is a perspective view schematically illustrating a GOI device100according to various embodiments.FIG.2is a perspective view schematically illustrating a GOI structure110ofFIG.1.FIG.3is a perspective view exemplifying a light source element130ofFIG.1.FIGS.4A and4Bare views for explaining the performance of the light source element130ofFIG.3.FIGS.5,6A and6Bare views for explaining the performance of a waveguide region120ofFIG.1.FIG.7is a perspective view schematically illustrating the waveguide region120ofFIG.1.FIGS.8and9are views for explaining the performance of the waveguide region120ofFIG.7.FIG.10is a perspective view schematically illustrating an optical detection element150ofFIG.1.FIG.11is a view for explaining the performance of the optical detection element150ofFIG.10. Referring toFIG.1, the GOI device100is for ultra-small on-chip optical sensing, and may include at least one of a GOI structure110, a light source element130, a spectrometer140, or at least one optical detection element150. In this instance, the GOI device100may detect gases by measuring optical absorption spectra of gases on mid-infrared light (mid-IR). Here, the mid-infrared light may represent infrared light in the wavelength range of 3 μm to 8 μm and the frequency range of 38 THz to 100 THz. The GOI structure110may be constructed as a platform for ultra-small on-chip optical sensing. The GOI structure110may support at least one of the light source element130, the spectrometer140, or the optical detection element150. Also, the GOI structure110may be implemented to propagate light generated from the light source element130. To this end, the GOI structure110may have a waveguide region120. That is, some part of the GOI structure110may be formed as the waveguide region120. As illustrated inFIG.2, the GOI structure110may include an insulating substrate111and a germanium (Ge) layer115. The insulating substrate111may support the germanium layer115. In this instance, the insulating substrate111may include a base layer112, and an oxide layer113stacked on top of the base layer112. For example, the base layer112may be made of silicon (Si). The germanium layer115may be disposed on the insulating substrate111, specifically, on the oxide layer113, and may be made of germanium (Ge). The light source element130may be configured to generate light for the waveguide region120. To this end, the light source element130may be disposed on one side of the waveguide region120. Here, the light source element130may be integrated on the GOI structure110and disposed on one side of the waveguide region120. In this instance, the light source element130may generate mid-infrared light. The light source element130may generate light so as to be coupled to the germanium layer115in the waveguide region120. For example, the light source element may include a quantum cascade laser (QCL) as illustrated inFIG.3. The quantum cascade laser may produce infrared light with an output power of several tens of mW to several hundreds of mW. Here, the coupling efficiency versus wavelength of mid-infrared light from the quantum cascade laser to the germanium layer115may be high that is around 60% to 80%, as illustrated inFIG.4A. Also, the mid-infrared light coupled to the germanium layer115from the quantum cascade laser may exhibit a field distribution as illustrated inFIG.4B. The spectrometer140may be configured to obtain a spectrum of light from the light source element130. To this end, the spectrometer140may be disposed between the light source element130and the waveguide region120. Here, the spectrometer140may be integrated on the GOI structure110and disposed between the light source element130and the waveguide region120. Also, the spectrometer140may decompose the light from the light source element130by wavelength and propagate it to the waveguide region120. Through this, the germanium layer115of the waveguide region120may propagate the light from the light source element130while confining it. In this instance, due to the material properties of the germanium layer115, the waveguide region120with the germanium layer115may be better than the existing silicon-based waveguide, in terms of propagation loss, as illustrated inFIGS.5,6A and6B. That is, as illustrated inFIG.5, the waveguide region120with the germanium layer115might be better than the existing silicon-based waveguide, in terms of propagation loss caused by the material properties. Moreover, given the uneven shape of sidewalls due to grains as illustrated inFIG.6A, the waveguide region120with the germanium layer115may be better than the existing silicon-based waveguide in terms of propagation loss caused by the material properties and propagation loss caused by the uneven shape of sidewalls, as illustrated inFIG.6B. Meanwhile, because the germanium layer115has high field confinement capability, the waveguide region120with the germanium layer115may confine light with higher field confinement capability than the existing silicon-based waveguide. In some embodiments, the germanium layer115of the waveguide region120may have at least one slot121, as illustrated inFIG.7. The slot121may be configured in such a way as to collect and propagate the light from the light source element130. Due to this, as illustrated inFIG.8andFIG.9, the germanium layer115(inFIG.8and (b) ofFIG.9) with the slot121may confine light with higher field confinement capability than the germanium layer115(inFIG.8and (a) ofFIG.9) with no slot121. At least one optical detection element150may be configured to detect light coming from the waveguide region120. In some embodiments, a plurality of optical detection elements150may be formed in an array. In some embodiments, the optical detection element150may convert the light from the waveguide region120into heat and detect the heat. The optical detection element150may be implemented to detect heat and therefore detect light, which makes it easy to accomplish advantages such as wavelength independence and room temperature operation, compared to existing photon detection elements which directly detect light, and which furthermore maximizes the extensibility of optical sensing. To this end, the optical detection element150may be disposed on one side of the waveguide region120, that is, on the opposite side of the light source element130, with the waveguide region120in between. In this case, the optical detection element150may be integrated on the GOI structure110and implemented to be integrated with some part of the GOI structure110. As such, the optical detection element140may detect heat that is generated as light is propagated from a heavily-doped germanium layer115of the GOI structure110, and, as a result, may detect light based on the heat. According to an embodiment, the optical detection element150may be a bolometer, which is a type of thermal detector, for detecting heat, and may be implemented using a waveguide-based bolometer structure, in particular. For example, the optical detection element150may include an insulating layer151, a bolometric material layer153, and at least one electrode155, as illustrated inFIG.10. The insulating layer151may prevent electrical connection between the heavily-doped germanium layer115and the bolometric material layer153. To this end, the insulating layer151may be formed on one side of the waveguide region120so as to cover the heavily-doped germanium layer115on the insulating substrate111. For example, the insulating layer151may be made of oxide. The heavily-doped germanium layer115may convert the light from the waveguide region120into heat. In this instance, the heavily-doped germanium layer115may absorb the light propagated from the heavily-doped germanium layer115by a free-carrier absorption (FCA) effect, and therefore the heat generated from the heavily-doped germanium layer115may be transmitted to the bolometric material layer153, causing a change in the resistance of the bolometric material layer153. To this end, the bolometric material layer153may be stacked on top of the insulating layer151. In other words, the bolometric material layer153may be stacked on top of the heavily-doped germanium layer115, with the insulating layer151in between. For example, the bolometric material layer153may be made of a bolometric material such as vanadium oxide, titanium oxide, amorphous silicon, and silicon germanium oxide. Here, the bolometric material layer153may exhibit heat generation efficiency as illustrated inFIG.11. For example, if light with a wavelength of 4.23 μm and an intensity of 10 mW enters the heavily-doped germanium layer115of the optical detection element150with such design variables as illustrated inFIG.11from the waveguide region120, the bolometric material layer153may generate heat with a thermal efficiency of about 9.312 K/mW. The electrode155may be used to detect a change in the resistance of the bolometric material layer153. To this end, the electrode155may be integrated on the bolometric material layer153. In this instance, heat may be detected from the detected resistance, and furthermore light may be detected. According to various embodiments, the GOI device100may be used to detect gases. For example, the GOI device100may detect carbon dioxide (CO2) using mid-infrared light. In this instance, the limit of detection (LoD) of the GOI device100may be calculated by the following [Equation 1]. That is, the limit of detection of the GOI device100may have a high correlation with the noise equivalent temperature difference (NETD) in the bolometric material layer153of the optical detection element150. Accordingly, the noise equivalent temperature difference (NETD) in the bolometric material layer153of the optical detection element150may be improved, thereby greatly enhancing the performances of the optical detection element150and the GOI device100. Cmin=-ln(1-PminP0e-αpropL)ηεgasL[Equation1]Pmin=SNR×NETD(K)Thermalefficiency(K/W) where C is the concentration (mol·L−1) of a gas, Cminis the minimum concentration (mol·L−1) of the gas, Pminis minimum detectable power (W), η is a confinement factor (%), εgasis molar absorption coefficient (mol·L−1·cm), L is the length (cm) of an optical path, P0is the intensity (W) of incoming light, and αpropis optical loss (cm−1). For example, if η is 58.89%, P0is 10 mW, αpropis 1.97 dB/cm (0.4536 cm−1), L is 1.396 cm, εgasis 9200 mol·L−1·cm, the SNR (signal-to-noise ratio) is 3, the thermal efficiency is 9.312 K/mW, and the noise equivalent temperature difference (NETD) is 11.64 mK, Cminwill be 1.1507×10−7mol/L, and the limit of detection of the GOI device100will be about 2.25 ppm (the background temperature is assumed to be 20° C.). FIG.12is a flowchart illustrating a manufacturing method of a GOI device100according to various embodiments.FIGS.13athrough13gare perspective views for explaining in details manufacturing steps of a GOI structure110ofFIG.12. Referring toFIG.12, in the step210, the GOI structure110may be manufactured. The GOI structure110may be constructed as a platform for for ultra-small on-chip optical sensing. As illustrated inFIG.2, the GOI structure110may include an insulating substrate111and a germanium layer115. Here, the insulating substrate111may include a base layer112, and an oxide layer113stacked on top of the base layer112. Also, the GOI structure110may have a waveguide region120. That is, some part of the GOI structure110may be formed as the waveguide region120. A manufacturing procedure of the GOI structure110will be described below in more details with reference toFIGS.13A through13G. First of all, as illustrated inFIG.13A, an epitaxial wafer310may be prepared. The epitaxial wafer310may include a bottom layer311and a germanium layer115on the bottom layer311. That is, the epitaxial wafer310may be prepared as the germanium layer115grows on the bottom layer311. For example, the bottom layer311may include a support layer312, a strain-relaxed buffer layer313, and a base growth layer314. The support layer312may support the strain-relaxed buffer layer313and the base growth layer314. For example, the support layer312may be made of silicon. The strain-relaxed layer313may be disposed on the support layer312. In this instance, the strain-relaxed layer313may be a lattice mismatch with respect to the germanium layer115. In other words, the lattice constant of the strain-relaxed layer313may be different from the lattice constant of the germanium layer115. As such, strain may be applied to the germanium layer115through the strain-relaxed layer313. The base growth layer314may be disposed on the strain-relaxed layer313. For example, the base growth layer314may be made of silicon and germanium. The germanium layer115may be disposed on the base growth layer314. Afterwards, as illustrated inFIG.13B, an oxide layer113may be formed on the epitaxial wafer310. In this instance, the oxide layer113may be formed on the germanium layer115. Meanwhile, as illustrated inFIG.13C, an insulating substrate111may be prepared. In this instance, the insulating substrate111may include a base layer112and an oxide layer113stacked on top of the base layer112. For example, the base layer112may be made of silicon. Next, as illustrated inFIG.13D, the epitaxial wafer310may be bonded onto the insulating substrate111through the oxide layer113. In this instance, the oxide layer113on the epitaxial wafer310may be bonded to the oxide layer113on the insulating substrate111, and be therefore integrated with the insulating substrate111. Thus, the germanium layer115may be disposed on the insulating substrate111. Afterwards, as illustrated inFIG.13E, the bottom layer311may be removed. In this instance, the base growth layer314, the strain-relaxed layer313, and the support layer312may be removed at the same time or sequentially. Here, the bottom layer311may be removed by at least one of a chemical technique and a mechanical technique. According to one embodiment, the bottom layer311may be chemically removed by using at least one tetramethylammonium hydroxide (TMAH) and SC-1 (e.g., NH4OH+H2O2+H2O solution). According to another embodiment, the bottom layer311may be mechanically removed by at least one of a grinding technique or a polishing technique. Thus, as illustrated inFIG.13F, only the germanium layer115may remain on the insulating substrate111. Afterwards, as illustrated inFIG.13G, the germanium layer115on the insulating substrate111may be processed. In this instance, part of the germanium layer115may be removed. Consequently, the GOI structure110may be manufactured. In some embodiments, in the waveguide region120of the GOI structure110, at least one slot121may be additionally generated in the germanium layer115. Next, in the step220, at least one element130,140, and150may be integrated on the GOI structure110. In this instance, the element130,140, and150may include at least one of a light source element130, a spectrometer140, or at least one optical detection element150. Accordingly, the GOI device100as illustrated inFIG.1may be manufactured. The light source element130may be configured to generate light for the waveguide region120. To this end, the light source element130may be disposed on one side of the waveguide region120. Here, the light source element130may be integrated on the GOI structure110and disposed on one side of the waveguide region120. In this instance, the light source element130may generate mid-infrared light. The light source element130may generate light so as to be coupled to the germanium layer115of the waveguide region120. The spectrometer140may be configured to obtain a spectrum of light from the light source element130. To this end, the spectrometer140may be disposed between the light source element130and the waveguide region120. Here, the spectrometer140may be integrated on the GOI structure110and disposed between the light source element130and the waveguide region120. Also, the spectrometer140may decompose the light from the light source element130by wavelength and propagate it to the waveguide region120. The optical detection element150may be configured to detect light coming from the waveguide region120. In some embodiments, a plurality of optical detection elements150may be formed in an array. In some embodiments, the optical detection element150may convert the light from the waveguide region120into heat and detect the heat. To this end, the optical detection element150may be disposed on one side of the waveguide region120, that is, on the opposite side of the light source element130, with the waveguide region120in between. In this case, the optical detection element150may be integrated on the GOI structure110and implemented to be integrated with some part of the GOI structure110. As such, the optical detection element140may detect heat, which is generated as light is propagated from a heavily-doped germanium layer115of the GOI structure110, and as a result detect light based on the heat. That is, the optical detection element150may be implemented on the GOI structure110through monolithic integration. Thus, an additional component for absorbing light from the waveguide region120, for example, a nanostructure, may not be needed to implement the optical detection element150. Consequently, the optical detection element150may detect heat that is generated as light is propagated from the germanium layer115of the GOI structure110, and, as a result, may detect light based on the heat. According to an embodiment, the optical detection element150may be a bolometer for detecting heat. For example, the optical detection element150may include an insulating layer151, a bolometric material layer153, and at least one electrode155, as illustrated inFIG.10. A manufacturing procedure of this optical detection element150is as follows. First of all, the insulating layer151may be formed to cover the germanium layer115on the insulating substrate111, on one side of the waveguide region120. For example, the insulating layer151may be made of oxide. Afterwards, the bolometric material layer153may be stacked on the insulating layer151. In other words, the bolometric material layer153may be stacked on top of the germanium layer115, with the insulating layer151in between. For example, the bolometric material layer153may be made of a bolometric material such as vanadium oxide, titanium oxide, amorphous silicon, and silicon germanium oxide. Next, at least one electrode155may be formed on the bolometric material layer153. According to various embodiments, the optical detection element150and the GOI device100are implemented on a germanium-based GOI structure110, and therefore the optical detection element150and the GOI device100may be implemented in an ultra-small on-chip structure and used for optical sensing. In this instance, the GOI structure110may ensure relatively high field confinement capability through germanium. Thus, the optical detection element150and the GOI device100may be used for optical sensing over a broad band including mid-infrared light. Also, the optical detection element150may detect light by detecting heat into which the light is converted, thereby maximizing the extensibility of optical sensing. Various embodiments may provide an optical detection element150and GOI (Ge-on-insulator) device100for ultra-small on-chip optical sensing, and a manufacturing method of the same. The GOI device100according to various embodiments may include: a GOI structure110with a waveguide region120comprising a germanium layer115; a light source element130configured to generate light for the waveguide region120; and at least one optical detection element150configured to detect light coming from the waveguide region120. According to various embodiments, the light generated from the light source element130may be mid-infrared light. According to various embodiments, at least one slot121configured to collect light from the light source element130may be formed in the germanium layer115in the waveguide region120. According to various embodiments, the light source element130may generate light so as to be coupled to the germanium layer115in the waveguide region120. According to various embodiments, the optical detection element150may be configured to convert the light from the waveguide region120into heat and detect the heat. According to various embodiments, the GOI structure110may include an insulating substrate111and a germanium layer115integrated on the insulating substrate111. Some part of the GOI structure110may be formed as the waveguide region120. According to various embodiments, the optical detection element150may include: an insulating layer151integrated on one side of the waveguide region120on the GOI structure110and formed to cover the germanium layer115on the insulating substrate111; a bolometric material layer153stacked on top of the insulating layer151, whose resistance changes with heat generated as light is propagated from the germanium layer115; and at least one electrode155integrated on the bolometric material layer153and used to detect the resistance after a change in resistance has occurred. According to various embodiments, the GOI device100may further include a spectrometer140disposed between the light source element130and the waveguide region120and configured to decompose the light from the light source element130by wavelength and propagate it to the waveguide region120. A manufacturing method of the GOI device100according to various embodiments may include: a step210of manufacturing a GOI structure110comprising a germanium layer115and provided with a waveguide region120, a step220of integrating, on the GOI structure110, a light source element130configured to generate light for the waveguide region120; and a step220of integrating, on the GOI structure110, at least one optical detection element150configured to detect light coming from the waveguide region120. According to various embodiments, the step210of manufacturing a GOI structure110may include: preparing an epitaxial wafer310with a germanium layer115grown on a bottom layer311; forming an oxide layer113on the germanium layer115; bonding the epitaxial wafer310onto the insulating substrate111through the oxide layer113; and removing the bottom layer311while leaving the germanium layer115on the insulating substrate111. According to various embodiments, the step210of manufacturing a GOI structure110may further include machining the germanium layer115on the insulating substrate111. According to various embodiments, the step210of manufacturing a GOI structure110may further include preparing an insulating substrate111comprising a base layer112and an oxide layer113overlying the base layer112. According to various embodiments, the bonding of the epitaxial wafer310onto the insulating substrate111may include integrating the oxide layer113on the germanium layer115with an oxide layer113of the insulating substrate111. According to various embodiments, at least one slot121configured to collect light from the light source element130may be formed in the germanium layer115in the waveguide region120. According to various embodiments, the light source element130may generate light so as to be coupled to the germanium layer115in the waveguide region120. According to various embodiments, the optical detection element150may be configured to convert the light from the waveguide region120into heat and detect the heat. According to various embodiments, the step220of integrating the optical detection element150may include: forming an insulating layer151on one side of the waveguide region120so as to cover the germanium layer115; stacking a bolometric material layer153on top of the insulating layer151, whose resistance changes with heat generated as light is propagated from the germanium layer115; and forming, on the bolometric material layer153, at least one electrode155used to detect the resistance after a change in resistance has occurred. According to various embodiments, the manufacturing method of the GOI device100may further include integrating a spectrometer140between the light source element130and the waveguide region120on the GOI structure110, which is configured to decompose the light from the light source element130by wavelength and propagate it to the waveguide region120. The optical detection element150according to various embodiments may include: a GOI structure110comprising a germanium layer115configured to propagate light from the outside; and a bolometric material layer153disposed on the GOI structure110, whose resistance changes with heat generated as light is propagated from the germanium layer115; and configured to detect the resistance after a change in resistance has occurred. According to various embodiments, the light may be mid-infrared light. According to various embodiments, the optical detection element150may further include at least one of: an insulating layer151disposed between the germanium layer115and the bolometric material layer153and formed to cover the germanium layer115; or at least one electrode155integrated on the bolometric material layer153and used to detect heat into which light is converted. It should be appreciated that various embodiments of the disclosure and the terms used therein are not intended to limit the technology disclosed herein to specific forms, and should be understood to include various modifications, equivalents, and/or alternatives to the corresponding embodiments. In describing the drawings, similar reference numerals may be used to designate similar constituent elements. As used herein, singular forms may include plural forms as well unless the context clearly indicates otherwise. In the present disclosure, the expression “A or B”, “at least one of A or/and B”, or “one or more of A or/and B” may include all possible combinations of the items listed. The expression “a first”, “a second”, “the first”, or “the second” may modify corresponding elements regardless of the order or importance, and is used only to distinguish one element from another element, but does not limit the corresponding elements. When an element (e.g., first element) is referred to as being “(physically or functionally) connected,” or “coupled” to another element (second element), the element may be connected directly to the another element or connected to the another element through yet another element (e.g., third element). According to various embodiments, each component (e.g., a module or a program) of the above-described components may include a single entity or multiple entities. According to various embodiments, one or more of the above-described components or operations may be omitted, or one or more other components or operations may be added. Alternatively or additionally, a plurality of components (e.g., modules or programs) may be integrated into a single component. In such a case, the integrated component may still perform one or more functions of each of the plurality of components in the same or similar manner as they are performed by a corresponding one of the plurality of components before the integration. According to various embodiments, operations performed by the module, the program, or another component may be carried out sequentially, in parallel, repeatedly, or heuristically, or one or more of the operations may be executed in a different order or omitted, or one or more other operations may be added. | 27,680 |
11860110 | BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, an article inspection apparatus according to the embodiment of the present invention will be described in detail with reference to the drawings. InFIG.1, an article inspection apparatus1according to the embodiment of the present invention irradiates light at a predetermined article inspection position while arranging the articles to be inspected and transporting the articles individually, and inspects the quality of the article based on the spectral characteristics of the light irradiated and then transmitted through the article. The article to be inspected is an article having a small diameter whose light irradiation region is relatively close to the diameter of the irradiation port for irradiating the article to be inspected with light, and includes an article having a predetermined shape and a molded article manufactured by an existing manufacturing facility or a manufacturing facility having no inspection function, and particularly an article whose shape does not change during the transportation process, in addition to an article having an outer diameter cp: several mm to several tens of mm that can be transported individually without packaging, and a bite-sized article. Examples of the articles include a tablet, a capsule, a troche, a preparation such as a drop, a candy, a chocolate, and the like. Hereinafter, as an article to be inspected, a tablet W having a circular shape in a plan view and a substantially columnar in a side view having a small height (thickness) compared to the diameter will be described as an example. The tablet W of the present embodiment constitutes an article. As illustrated inFIG.1, the article inspection apparatus1is provided with an article transport device2that transports the tablet W along a circumferential transport course, and an article inspection unit3that inspects the tablets W at an article inspection position P5in the middle of the transport course. As illustrated inFIG.2, a supply container10is formed in a circular shape in which the upper surface is opened and the cross-sectional area gradually decreases from the upper surface to the lower side. The supply container10is a horizontal annular flange provided in the opening portion10aon the upper surface with a predetermined width over the entire circumference, and is provided with an outer transport unit12serving as a transport path for the tablet W, a circular bottom wall13having a through-hole13bin a part and having a diameter smaller than that of the outer transport unit12, and a cylindrical side wall14connecting the outer transport unit12and the bottom wall13. An inner transport unit11of the present embodiment is provided inward in the radial direction of the outer transport unit12. In other words, the outer transport unit12is provided on the outer side in the radial direction of the inner transport unit11so as to surround the periphery of the inner transport unit11in the top view of the article transport device2. The supply container10is rotatably attached to a main body frame (not illustrated). The axis of rotation of the supply container10passes through the center of the bottom wall13and coincides with a line perpendicular to the bottom wall13. An annular internal tooth gear10bis fixed to the lower surface of the bottom wall13of the supply container10with the center aligned with the bottom wall13. A first motor21is attached to the main body frame, and a pinion22provided on a drive shaft21aof the first motor21meshes with the internal tooth gear10b. When the first motor21is driven, the supply container10(outer transport unit12) rotates around the rotation center axis in the rotation direction A (refer toFIG.1). In the present embodiment, the rotation direction A of the supply container10is the counterclockwise rotation direction in a plan view of the article transport device2. As illustrated inFIGS.1and2, the inner transport unit11is inclined and disposed inside the supply container10. Since the diameter of the inner transport unit11is larger than the diameter of the bottom wall13of the supply container10, the inner transport unit11can be inclined and stored in the supply container10. That is, since the supply container10is formed in a circular shape in which the upper surface is open and the cross-sectional area decreases from the upper surface toward the bottom wall13, the inner transport unit11can be inclined and stored in the supply container10. As illustrated inFIG.2, an upper end portion11bof the inner transport unit11in the inclined direction faces an upper edge of the side wall14of the supply container10, and a lower end portion11cof the inner transport unit11in the inclined direction located 180° opposite to the upper end portion11bin the circumferential direction, faces a lower edge of the side wall14of the supply container10. That is, the lower end portion11cof the inner transport unit11in the inclined direction has the lowest portion facing the lower edge of the side wall14of the supply container10, and the upper end portion11bof the inner transport unit11in the inclined direction has the highest portion facing the upper edge of the side wall14of the supply container10. A drive shaft11a(refer toFIG.2) is fixed perpendicularly to the inner transport unit11at the center of the lower surface of the inner transport unit11, and the drive shaft11ais connected to a second motor23(refer toFIG.2) by inserting the through-hole13bof the bottom wall13of the supply container10in an inclined state. The second motor23is attached to the main body frame that supports the supply container10, and when the second motor23is driven, the inner transport unit11rotates around the drive shaft11ain the rotation direction B (refer toFIG.1) inside the supply container10. The rotation direction B of the inner transport unit11and the rotation direction A of the outer transport unit12are the same direction. As illustrated inFIG.1, the article transport device2is provided with an article supply unit24, and the article supply unit24is installed above the article transport device2. For example, when a tablet W of a molded product manufactured by an existing manufacturing facility or a manufacturing facility having no inspection function is loaded, the article supply unit24accumulates the loaded tablet W, discharges the accumulated tablet W from a discharge port24ato an article loading position P1, and supplies the accumulated tablet W to the inner transport unit11at the article loading position P1. The article supply unit24transports the loaded tablet W to the inner transport unit11, and is configured to include, for example, a linear feeder or a conveyor. The article supply unit24has a function of supplying the tablet W according to the capacity of the inner transport unit11and the outer transport unit12to transport the tablet W. In other words, in the article supply unit24, the supply amount of the tablet W to the inner transport unit11is set according to the capacity of the article transport device2to transport the tablet W (for example, the number of tablets W transported per unit time). In the article transport device2, when the first motor21and the second motor23are driven in the same direction at the same speed or at different speeds, while loading the tablet W from the article supply unit24into the inner transport unit11, and each of the inner transport unit11and the supply container10are rotated at the same speed in the rotation direction A and the rotation direction B, the tablet W fallen to the lowest portion (lower end portion11cside in the inclined direction) of the article supply unit24moves to the highest portion (upper end portion11bside in the inclined direction) of the inner transport unit11as the inner transport unit11rotates, and is placed on the outer transport unit12. As illustrated inFIGS.1and3, the outer transport unit12is provided with a groove portion12A and a storage groove12B. As illustrated inFIGS.4and5, the groove portion12A is recessed downward from an upper surface12aof the outer transport unit12and extends in the radial direction of the outer transport unit12. The storage groove12B of the present embodiment constitutes a storage portion. A plurality of groove portions12A are provided side by side at equal intervals in the circumferential direction of the outer transport unit12, and these plurality of groove portions12A are adjacent to each other in the circumferential direction of the outer transport unit12with the upper surface12aof the outer transport unit12interposed therebetween. As illustrated inFIG.3, the groove portion12A is formed so that the width in the circumferential direction gradually narrows from an inner end portion12ntoward an outer end portion12mof the outer transport unit12in the radial direction. The width of the inner end portion12nof the groove portion12A in the circumferential direction is formed to be smaller than the width when the tablets W are arranged in two in the circumferential direction, that is, the width twice the diameter of the tablet W (or smaller than the width when two tablets W are arranged vertically and horizontally in the circumferential direction), and is formed in a shape in which two tablets W are difficult to enter at the same time. The tablet W that rides on the outer transport unit12due to the rotation of the inner transport unit11enters the groove portion12A from the inner end portion12nof the groove portion12A in the radial direction, is guided from the groove portion12A to the storage groove12B by the centrifugal force due to the rotation of the outer transport unit12and an article guide unit27described later, and is stored in the storage groove12B. As illustrated inFIGS.3and4, the storage groove12B is located radially outward of the outer transport unit12with respect to the groove portion12A. The storage groove12B may be formed in a circular shape slightly larger than the tablet W in a plan view, may be recessed below a bottom surface12bof the groove portion12A, or may have the same height as that of the groove portion12A. Specifically, the storage groove12B is provided with a bottom wall12C having an annular mounting surface12dlocated below the bottom surface12bof the groove portion12A. The bottom wall12C is formed with a circular opening portion12clocated inside the mounting surface12dand through which inspection light passes. That is, the mounting surface12dis provided radially outward of the opening portion12cso as to surround the circular opening portion12c, and the tablet W is placed on the annular mounting surface12dso as to close the opening portion12cfrom above. The storage groove12B extends upward from the bottom wall12C and is provided with a peripheral wall12D that surrounds an outer peripheral side surface Wa of the tablet W, and the tablet W is stored in the storage groove12B in a state of being fitted. Specifically, the peripheral wall12D of the storage groove12B is formed by a step between the bottom surface12bof the groove portion12A and the mounting surface12dof the storage groove12B, and a step between the upper surface12aof the groove portion12A and the mounting surface12dof the storage groove12B. Since the opening portion12cis formed to have a diameter smaller than the outer diameter of the tablet W in a plan view of the outer transport unit12, in a state where the tablet W is stored in the storage groove12B, the opening portion12cis hidden from above, and the tablet W can be prevented from falling from the opening portion12c. The distance from the rotation center axis of the outer transport unit12to the central portions of all the storage grooves12B is equal, and in a state where the tablet W is stored in the storage groove12B, the tablet W is aligned in a row in the rotation direction of the outer transport unit12. That is, the line connecting the central portion of the storage groove12B (central portion of the opening portion12c) in the rotation direction is a circle. As a result, when the outer transport unit12rotates, the tablets W stored in the storage groove12B are aligned in a row in the rotation direction of the outer transport unit12. In addition, in a state where the tablet W is stored in the storage groove12B, a gap between the outer peripheral side surface Wa of the tablet W and the peripheral wall12D is significantly small. As a result, it is possible to prevent light from leaking from the gap between the tablet W and the peripheral wall12D and prevent light from entering the gap between the tablet W and the peripheral wall12D. That is, the peripheral wall12D blocks the light incident on a light detection unit32described later from the outer peripheral side surface Wa of the tablet W. The peripheral wall12D of the present embodiment constitutes a light-shielding portion, and the outer peripheral side surface Wa of the tablet W constitutes the outer peripheral side surface of the article. The gap between the outer peripheral side surface Wa and the peripheral wall12D of the tablet W and a gap between the outer peripheral side surface Wa and the bottom wall12C of the tablet W are conditioned on the condition that the peripheral wall12D can prevent light from leaking from the outer peripheral side surface Wa of the tablet W or light from entering from the outer peripheral side surface Wa, and the tablet W is set to a gap in which the tablet W is surely stored in the storage groove12B. As illustrated inFIG.4, in a state where the tablet W is stored in the storage groove12B, the dimension a of the tablet W in the height direction, that is, the dimension a from the mounting surface12dof the storage groove12B to the upper surface of the tablet W, is larger than the dimension b in the height direction from the mounting surface12dof the storage groove12B to the upper surface12aof the groove portion12A. As a result, in the state where the tablet W is stored in the storage groove12B, the upper surface of the tablet W protrudes upward from the upper surface12aof the groove portion12A. In the state where the tablet W is stored in the storage groove12B, the dimension a of the tablet W in the height direction may be smaller than the dimension b in the height direction from the mounting surface12dof the storage groove12B to the upper surface12aof the groove portion12A. In this case, in the state where the tablet W is stored in the storage groove12B, the upper surface of the tablet W is located below the upper surface12aof the groove portion12A. As illustrated inFIG.1, the article transport device2is provided with an article regulation unit25. The article regulation unit25regulates the number of tablets W to be supplied so that only one tablet W is supplied to the groove portion12A, and aligns the tablets W in a row in the rotation direction of the outer transport unit12. The article regulation unit25is configured to include a guide plate that is curved and extended with a certain length in the rotation direction of the outer transport unit12and extends in the vertical direction, and is fixed to a main body frame that supports the supply container10. The article regulation unit25is provided at a regulation position P2on the downstream side in the rotation direction of the outer transport unit12with respect to the article loading position P1. In other words, the article loading position P1is installed on the upstream side of the article regulation unit25in a plan view of the article transport device2. Here, the rotation direction of the outer transport unit12and the transport direction of the tablet W are the same direction. When one tablet W is supplied from the inner transport unit11to the groove portion12A through the radial inner end portion12nof the groove portion12A, the radial outer end portion of the tablet W comes into contact with the article regulation unit25. That is, the radial dimension of the outer transport unit12at the inner end portion12nof the article regulation unit25and the groove portion12A is set to the same dimension as the diameter of the tablet W, a dimension slightly larger than the diameter of the tablet W, or a dimension slightly smaller than the diameter of the tablet W so that the tablet W does not fall from the groove portion12A to the inner transport unit11. As a result, the number of tablets W supplied is regulated so that only one tablet W is supplied to the groove portion12A by the article regulation unit25. In addition, only one tablet W supplied to the groove portion12A is regulated from moving outward in the radial direction of the outer transport unit12by the article regulation unit25, and is transported toward the article inspection position P5in a state of being aligned in a row in the rotation direction of the outer transport unit12. The outer transport unit12of the present embodiment constitutes a transport unit. The article transport device2is provided with an article adjustment unit26. The article adjustment unit26is provided at an article adjusting position P3on the downstream side of the outer transport unit12in the rotation direction with respect to the article regulation unit25, returns the tablet W that is not located in the groove portion12A in the normal posture to the normal posture in the groove portion12A, and removes the tablet W protruding from the groove portion12A from the groove portion12A. Here, the normal posture of the tablet W is, for example, a posture in which the tablet W can smoothly move from the groove portion12A to the storage groove12B and can be stored sideways in the storage groove12B. The article adjustment unit26is configured to include a brush, air blower, an exclusion plate, and the like, and in a case where the tablet W supplied to the groove portion12A rides on the upper surface12aof the outer transport unit12, adjusts the tablet so that the tablet has a normal posture in the groove portion12A, or removes the tablet from the groove portion12A. In addition, when one tablet W has a poor posture (vertical orientation), the tablet W is removed from the groove portion12A. The tablet W removed from the groove portion12A is returned to the inner transport unit11. Specifically, in a case where the tablet W is located above the upper surface of the tablet W in the normal posture in the groove portion12A, the article adjustment unit26adjusts the tablet W having a poor posture (posture correction or removal) by supplying air above the upper surface of the tablet W or by acting the exclusion plate. The article transport device2is provided with an article guide unit27. The article guide unit27is provided at an article guide position P4on the downstream side in the rotation direction of the outer transport unit12with respect to the article adjustment unit26, and guides the tablet W supplied to the groove portion12A to the storage groove12B along the groove portion12A. The article guide unit27is configured to include a guide plate that is curved and extended with a certain length in the rotation direction of the outer transport unit12and extends in the vertical direction, and is fixed to a main body frame that supports the supply container10. The article guide unit27is configured to include a guide plate extending with a certain length from the inner end portion12ntoward the outer end portion12mof the groove portion12A in the rotation direction of the outer transport unit12, and extending in the vertical direction. In the article guide unit27, the upstream end portion27ais located on the inner transport unit11side from the inner end portion12nof the groove portion12A in the radial direction of the outer transport unit12, and the downstream end portion27bis located on the inner end portion12nside of the groove portion12A from the storage groove12B in the radial direction of the outer transport unit12and is located near the outer end portion12mof the groove portion12A. The article guide unit27moves the tablets W arranged in a row in the rotation direction of the outer transport unit12on the inner end portion12nside of the storage groove12B at the regulation position P2toward the storage groove12B along the groove portion12A with the rotation of the outer transport unit12, and stores the tablets W in the storage groove12B. The tablet W stored in the storage groove12B is transported to the article inspection unit3by the rotation of the outer transport unit12. As illustrated inFIG.1, the article inspection unit3is provided at the article inspection position P5on the downstream side of the article guide position P4in the rotation direction of the outer transport unit12. The article transport device2is provided with an article blocking unit28. The article blocking unit28is provided from the middle in the rotation direction of the outer transport unit12of the article guide unit27to the downstream side of the article inspection position P5in the rotation direction of the outer transport unit12. The article blocking unit28is configured to include a guide plate that is curved in the rotation direction of the outer transport unit12, extended with a certain length, and extended in the vertical direction, and is fixed to a main body frame that supports the supply container10. The article blocking unit28prevents the tablet W stored in the storage groove12B by the article guide unit27from moving outward in the radial direction from the storage groove12B due to the centrifugal force due to the rotation of the outer transport unit12. As illustrated inFIG.6, the article inspection unit3is configured to include a light irradiation unit31and a light detection unit32. The article inspection unit3irradiates the tablet W stored in the storage groove12B with a broadband light L from the light irradiation unit31at the article inspection position P5from the opening portion12cside, and the light transmitted through the tablet W is detected by the light detection unit32. The light irradiation unit31is provided with a light source31a, a light guide31b, and a condenser lens31c. The light source31ais configured to include, for example, a halogen lamp in order to irradiate the tablet W to be inspected with a broadband light. The light source31a, the light guide31b, and the condenser lens31cconstitute a light source unit integrally assembled together with a case (not illustrated) in which heat radiation fins (not illustrated) are formed, and the light source unit is attached to the main body frame that supports the supply container10. The light source unit is provided with an optical sensor (not illustrated) that measures the amount of light of the light source31a, and a temperature sensor (not illustrated) that measures the temperature of the light source31a. The light guide31bis configured by bundling multiple glass optical fibers and guides the light from the light source31ato the condenser lens31cthat collects the light. The condenser lens31ccollects the light from the light guide31bonto the tablet W at the article inspection position P5. The light irradiation unit31emits a broadband light from the light source31ato the condenser lens31cvia the light guide31b, adjusts the magnification of the condenser lens31c(position of the condenser lens31c, the light source31a, and the tablet W) so that the light irradiation unit31covers the entire upper surface of the tablet W at the article inspection position P5by the condenser lens31c, and efficiently irradiates the tablet W with the light from the light source31aat the article inspection position P5. The light detection unit32constitutes a light detection unit integrally assembled with an optical fiber32aand a spectroscope32b, and is integrally held by a holding means (not illustrated) and attached to the main body frame that support the supply container10. In the light detection unit32, the light transmitted through the tablet W at the article inspection position P5enters from the end surface (incident opening portion) of the incident surface as the light receiving unit of the optical fiber32a. When the light entering from the end surface of the incident surface passes through the optical fiber32aand reaches the spectroscope32b, the spectroscope32bperforms spectroscopy by glazing using the difference in the diffraction angle depending on the wavelength of the light. The article inspection unit3processes the signal of the spectral characteristics obtained by the light detection unit32, and determines the quality of the tablet W from the result of processing the signal. At the article inspection position P5, a light-shielding plate39having an opening portion39athrough which light can pass may be installed between the storage groove12B and the light detection unit32(the light-shielding plate39is illustrated by a virtual line inFIG.6). In this manner, the detoured light from the outer peripheral side surface Wa (refer toFIG.4) of the tablet W toward the light detection unit32can be more reliably blocked by the peripheral wall12D and the light-shielding plate39, and appropriate light analysis can be more reliably performed by the light detection unit32. As illustrated inFIG.1, an NG product collection unit35, an all product collection unit36, and an OK product collection unit37are provided on the downstream side in the rotation direction of the outer transport unit12from the article inspection position P5at predetermined intervals in the rotation direction of the outer transport unit12. When the article inspection unit3determines that the tablet W is a defective product (NG product), and the tablet W determined to be the defective product is transported to an NG product selection position P6on the downstream side in the rotation direction of the outer transport unit12with respect to the article inspection unit3, for example, the article transport device2blows air onto the tablet W from the inside of the outer transport unit12in the radial direction and discharges the tablet W from the storage groove12B to the NG product collection unit35. The tablet W may be picked up from the outside of the outer transport unit12in the radial direction at the NG product selection position P6and discharged from the storage groove12B to the NG product collection unit35. On the other hand, when the article inspection unit3determines that the tablet W is a normal product (OK product), and the tablet W determined to be the normal product is transported from the NG product selection position P6to an OK product selection position P7, for example, air is blown onto the tablet W from the inside of the outer transport unit12in the radial direction and the tablet W is discharged from the storage groove12B to the OK product collection unit37. The tablet W may be picked up from the outside of the outer transport unit12in the radial direction at the OK product selection position P7and discharged from the storage groove12B to the OK product collection unit37. The all product collection unit36is provided at a total discharge position P8between the NG product selection position P6and the OK product selection position P7in the rotation direction of the outer transport unit12. The all product collection unit36collects a tablet W that could not be inspected normally or failed to be discharged, such as a tablet W failed to discharge NG, at the start of operation of the article inspection apparatus1, after the return operation after an emergency stop, and the like, and for example, blows air onto the tablet W from the inside of the outer transport unit12in the radial direction and discharges the tablet W from the storage groove12B to the all product collection unit36. The tablet W may be picked up from the outside of the outer transport unit12in the radial direction at the total discharge position P8and discharged from the storage groove12B to the all product collection unit36. In addition, a confirmation sensor38may be provided at a confirmation position P9on the downstream side of the NG product collection unit35in the rotation direction of the outer transport unit12, and the confirmation sensor38may be used to confirm whether or not a defective product is discharged at the NG product selection position P6. In this manner, the reliability of selection the defective product can be improved. The spectrum of the spectral characteristics obtained by the light detection unit32of the article inspection unit3is displayed on a display device (not illustrated) and is illustrated in the upper part ofFIG.7. InFIG.7, the spectrum of a plurality of tablets W is superimposed and graphed. In this spectrum, a black-painted spectrum indicated by ZA is a spectrum of the defective product, and a blank portion indicated by ZB is a spectrum of the normal product. In the present embodiment, the article inspection unit3standardizes the measured values for each wavelength of the measured spectrum, and determines whether the article is a normal product or a defective product based on the standardized values. The article inspection unit3calculates, for example, statistical values such as an average value, a maximum value, a minimum value, and a standard deviation for each wavelength of the measured values of the wavelengths in a measurement range at the wavelengths at predetermined intervals, and standardizes by calculating (measured value−average value)/standard deviation and the like, as illustrated in the lower part ofFIG.7. There are various methods for standardization, but when the statistical value is calculated using the measurement result using the OK product and the standardization is calculated from the statistical value, a method that does not increase the fluctuation of the values when the measured values of OK products not used in the calculation are standardized is selected. The standardization calculation may be (measured value−average value)/(maximum value−minimum value), (measured value−maximum value)/standard deviation, or the like. At this time, the statistical value is obtained from the result of measurement using a normal product known to be normal. In addition, the statistical value of the normal product may be used as the statistical value, and in a case where the spectral spectrum of the normal product has changed due to a change in lot or the like, the statistical value may be calculated by adding the latest measured value of the normal product to the measured value of the same tablet W inspected in the past. In addition, in a case where a numerical value that can be used for standardization such as spectral characteristics and the variation is known as a known value instead of a statistical value, the value may be used for standardization. By performing such standardization, the spectrum illustrated in the upper part ofFIG.7is converted as illustrated in the lower part. In the graph, the black part indicated by ZA′ is the standardized value of the defective product, the blank part indicated by ZB′ is the standardized value of the normal product, and compared with the case of the spectrum, it is possible to easily determine whether the article is a normal product or a defective product. The graph display format illustrated inFIG.7is an example, and can be expressed in various display formats such as a radar chart and expression by shades of color. The radar chart displays the results of the calibration curve, standardized values for each spectral wavelength, and for example, thickness values measured by other measuring instruments. In addition, the color, line thickness, and display style (for example, solid line, broken line, dashed line, circle, square, and the like) may be changed and displayed depending on the case where the determination is OK, the case where the determination is NG, or the level of NG. In addition, the number of display data (for example, the latest 1 item, the latest 100 items, and the like) and the type of data to be displayed (for example, OK only, NG only, all, and the like) may be combined and displayed. For example, the article inspection unit3sets one predetermined threshold value in the entire wavelength range of the measurement range, and in a case where the standardized value of the measured value exceeds the threshold value at any wavelength, it is determined that the article is a defective product. For example, the predetermined threshold value is determined from the value by calculating a standardized value from a spectrum of a plurality of normal products as illustrated in the lower part ofFIG.7. For example, the predetermined threshold value is set to twice the maximum value, twice the minimum value of the value of the normal product after standardization, an intermediate value between the maximum value of the normal product and the minimum value of the defective product, or a value capable of identifying a defective product. In addition, these values may be automatically set. In addition, the display data to be determined to be OK may be selected, and the threshold value may be automatically readjusted so that the determination is OK. At this time, it may be selected whether to recalculate the statistical values used for standardization. In addition, the display data determined to be NG may be selected, and the threshold value may be automatically readjusted so that the determination is OK. As the spectrum of a normal product for determining a predetermined threshold value, spectral information or the result of using spectrum conversion processing such as absorbance calculation, smoothing processing, or differential calculation (may be multiple stages) may be used. The absorbance calculation is processing of calculating the attenuation of the work result from the reference when the spectral characteristics of the light used to measure the tablet W and the spectral characteristics when the prototype is measured are used as references, and the spectral characteristics when the tablet W is measured are used as the work result. The smoothing processing is processing of smoothing data of each of the wavelengths adjacent to each other by averaging, filtering processing (load moving average method), or the like. The differential calculation is difference processing between data at fixed intervals by taking over a specific rule such as adjacent wavelength data and wavelength data at specific intervals. A plurality of spectra of the result of such conversion processing may be used to determine whether the article is a normal product or a defective product. In this case, the predetermined threshold value may be different depending on the conversion processing. In addition, since the spectral information changes due to environmental changes such as the ambient temperature, the temperature of the spectroscope32b, the temperature of the device, and the amount of light of the light source31a, a predetermined threshold value may be switched depending on information other than the spectrum, for example, the ambient temperature, the temperature of the spectroscope32b, the temperature of the device, the amount of light of the light source31a, and the like. For example, in a case of switching by temperature, the normalization value and limit at 20° C. to 30° C. and the normalization value and limit at 30° C. to 35° C. are measured and calculated in advance, the temperature change is observed during the inspection, the normalization value and the limit are switched depending on the value, and it is determined whether the article is a normal product or a defective product. In addition, as illustrated inFIG.8, the predetermined threshold value is provided with an upper limit threshold value and a lower limit threshold value, and in a case where the standardized value of the measured value for any wavelength exceeds the upper limit threshold value, or in a case where the standardized value of the measured value for any wavelength is below the lower limit threshold value, the article inspection unit3may determine that the article is a defective product. The upper limit threshold value and the lower limit threshold value may be, for example, twice the maximum value of the normal product after standardization as the upper limit threshold value, twice the minimum value as the lower limit threshold value, or may be set by another method. In addition, these values may be automatically set. In addition, as illustrated inFIG.9, by making the standardized value an absolute value, the lower limit threshold value may be eliminated and the threshold value may be set to one. In addition, a predetermined threshold value may be set for each divided range of wavelengths by dividing the wavelength direction. For example, as illustrated in FIG.10, the wavelength is divided by the boundary wavelength illustrated by C in the figure, the threshold value of the shorter wavelength is set as the first upper limit threshold value and the first lower limit threshold value, and the threshold value of the longer wavelength is set as the second upper limit threshold value and the second lower limit threshold value. The number of divisions may be two or more. In this manner, for example, even in a normal product, in a case where there is a region where the influence value is large and a region where the influence value is small, the threshold value is set to be large in the region where the influence value is large, the threshold value is set to be small in the region where the influence value is small, and it is possible to accurately determine whether the article is a normal product or a defective product. In addition, a range where the determination by the threshold value is not performed may be provided. For example, as illustrated inFIG.11, in a range of wavelengths illustrated by D in the figure, for example, a spectrum measured in a specific range of wavelengths illustrates a waveform having a different tendency from a spectrum measured in another range of wavelengths without performing determination by a threshold value. When the operator determines that a value for distinguishing and judging between a normal product and a defective product cannot be obtained or it may not be possible to correctly distinguish between a normal product and a defective product, the standardized values of the measured values in such range are set not to be used for the determination. As described above, in the above-described embodiment, the article inspection unit3standardizes the measured value for each wavelength of the measured spectrum, and determines whether the article is a normal product or a defective product based on the standardized values. As a result, the measured value for each wavelength of the measured spectrum is standardized, and the standardized value is used to determine whether the article is a normal product or a defective product. Therefore, when an unspecified foreign substance is contained, the change in the spectrum can be sensitively and stably detected, and the article can be determined as a defective product. In addition, the article inspection unit3calculates the statistical value of the measured value for each wavelength of the spectrum of the spectral characteristics of the light detected by the light detection unit32, and standardizes the measured value for each wavelength based on the statistical value. As a result, the statistical value of the measured value for each wavelength of the spectrum of the spectral characteristics of the light detected by the light detection unit32is calculated, and the measured value for each wavelength is standardized based on the statistical value. Therefore, when an unspecified foreign substance is contained, the change in the spectrum can be sensitively and stably detected, and the article can be determined as a defective product. In addition, the article inspection unit3determines whether the tablet W is a normal product or a defective product based on the threshold value for the standardized value. As a result, it is determined whether the tablet W is a normal product or a defective product based on the threshold value for the standardized value. Therefore, it is possible to easily determine whether the tablet W is a normal product or a defective product. In addition, as the threshold value for the standardized value, the article inspection unit3is provided with an upper limit threshold value which is an upper limit value for determining that the standardized value is normal, and a lower limit threshold value which is a lower limit value for determining that the standardized value is normal. As a result, it is determined whether the tablet W is a normal product or a defective product based on the upper limit threshold value and the lower limit threshold value. Therefore, it is possible to easily and accurately determine whether the tablet W is a normal product or a defective product. In addition, the article inspection unit3determines whether the tablet W is a normal product or a defective product based on the threshold value for the absolute value of the standardized value. As a result, it is determined whether the tablet W is a normal product or a defective product based on the threshold value for the absolute value of the standardized value. Therefore, it is possible to easily determine whether the tablet W is a normal product or a defective product by combining the upper limit and lower limit threshold values into one. In addition, the article inspection unit3sets a threshold value for the standardized value for each range divided in the wavelength direction. As a result, the threshold value is set for each range divided in the wavelength direction. Therefore, depending on the characteristics of the tablet W, a normal range can be narrowed or widened for each range of wavelengths, and it is possible to accurately determine whether the tablet W is a normal product or a defective product. In addition, the article inspection unit3is provided with a range of wavelengths in which it is not determined whether the tablet W is a normal product or a defective product by using the threshold value. As a result, the range of wavelengths is provided in which it is not determined whether the tablet W is a normal product or a defective product based on the threshold value. Therefore, a range of wavelengths that is not related to the determination of whether the tablet W is a normal product or a defective product can be set as a range in which it is not determined whether the tablet W is a normal product or a defective product, and it is possible to accurately determine whether the tablet W is a normal product or a defective product by suppressing the influence of the value in the range of wavelengths that is not related to the determination. In addition, the threshold value is set based on the standardized value of the measured value of the normal product of the tablet W. As a result, the threshold value is set based on the standardized value of the measured value of the normal product of the tablet W. Therefore, the threshold value can be set by reflecting the characteristics of the normal product, and it is possible to accurately determine whether the tablet W is a normal product or a defective product. In addition, the article inspection unit3standardizes the value of the result obtained by performing the spectrum conversion processing on the spectrum of the spectral characteristics of the light detected by the light detection unit32for each wavelength, and determines whether the tablet W is a normal product or a defective product based on the standardized value. As a result, the value of the result obtained by performing the spectrum conversion processing on the spectrum of the spectral characteristics of the light detected by the light detection unit32is standardized for each wavelength, and it is determined whether the tablet W is a normal product or a defective product based on the standardized value. Therefore, when an unspecified foreign substance is contained, the change in the spectrum can be sensitively and stably detected, and the tablet W can be determined as a defective product. In the present embodiment, the case where the light of the light irradiation unit31is transmitted to the tablet W and the quality of the tablet W is inspected based on the spectral characteristics of the transmitted light is illustrated, and the same can be applied even in a case where the light of the light irradiation unit31is reflected on the tablet W and the quality of the tablet W is inspected based on the spectral characteristics of the reflected light. In addition, the measured spectral information can be stored, the spectral conversion processing such as absorbance calculation, smoothing processing, and differential processing can be performed from the spectral information after the measurement, calculation processing to create a calibration curve can be performed by the magnitude of a specific peak, machine learning, and the like, and complex discrimination processing such as a discrimination method using a calibration curve can be performed. In addition, the configuration of the article transport device2illustrated inFIGS.1to5is an example, and it goes without saying that another configuration may be used. The tablet W may be sequentially transported to the article inspection position P5of the article inspection unit3. Although embodiments of the invention have been disclosed, it will be apparent to those skilled in the art that modifications may be made without departing from the scope of the invention. All such modifications and equivalents are intended to be included in the following aspects. DESCRIPTION OF REFERENCE NUMERALS AND SIGNS 1Article inspection apparatus2Article transport device3Article inspection unit12Outer transport unit (transport unit)12B Storage groove (storage portion)31Light irradiation unit31aLight source31bLight guide31cCondenser lens32Light detection unit32aOptical fiber32bSpectroscopeP5Article inspection position | 46,143 |
11860111 | DESCRIPTION OF EMBODIMENTS First Embodiment Referring to the drawings, an X-ray device according to the first embodiment of the present invention will be explained. The X-ray device irradiates an X-rays to a measurement object and detects the X-rays that passed through the measurement object to acquire information about the inside (for instance, inside structure) of the measurement object or the like non-destructively. In case where the target of measurement, i.e., the measurement object is a part for industrial use, for instance, a machine part or an electronic component, the X-ray device is called an industrial X-ray CT (Computed Tomography) inspection device that inspects the part for industrial use. Note that the inspection target of the measurement object may be an animate being such as a human body, an animal, a plant, etc. Also, the target may be one of tissues of a living organism. Also, the target may be a combination of an animate being and a non-living material such as a part for industrial use. The present embodiment is presented in order to concretely explain the gist of the present invention and unless indicated specifically, it does not limit the present invention. FIG.1is a diagram showing an example of the configuration of an X-ray device100according to the present embodiment. Note that for convenience of explanation, a coordinate system consisting of an X-axis, a Y-axis and a Z-axis is set as shown inFIG.1. The X-ray device100includes a housing1, an X-ray source2, a mounting unit3, a detector4, a control unit5, a display monitor6, and a frame8. The housing1is disposed on a floor surface of factory or the like so as to be substantially parallel (horizontal) to an XZ-plane and in the inside thereof are housed the X-ray source2, the mounting unit3, the detector4, and the frame8. To prevent the X-rays from leaking to the outside, the housing1is made of a material that contains lead. The X-ray source2radiates X-rays extending along an optical axis Zr that is parallel to the Z axis, spreading in the form of a cone (so-called cone beam) toward the positive direction of the Z axis with an output point Q shown inFIG.1being at the top of the cone. The output point Q corresponds to a focal spot of the X-ray source2. That is, the optical axis Zr connects the output point Q, which is the focal spot of the X-ray source2, with the center of an imaging area of the detector4described later. Note that the X-ray source2that radiates X-rays in a fan-like form (so-called fan beam) and one that radiates X-rays in a form of a line (so-called pencil beam) instead of one that emits X-rays in a cone form are also included as the aspects of the present invention. The X-ray source2can radiate at least one of, for instance, an ultrasoft X-ray of about 50 eV, a soft X-ray of about 0.1 to 2 keV, an X-ray of about 2 to 20 keV, and a hard X-ray of about 20 to 100 keV. Furthermore, the X-ray source2may radiate an X-ray of, for instance, 1 to 10 Mev. The mounting unit3includes a mount stage30on which a measurement object S is mounted and a manipulator unit36that includes a rotary drive unit32, a Y-axis movement unit33, an X-axis movement unit34and a Z-axis movement unit35and is provided on the positive (+) side along the Z-axis relative to the X-ray source2. The mount stage30is provided rotatable by the rotary drive unit32and moves together with the manipulator unit36when the manipulator unit36moves in the direction along the X axis, Y axis, and Z axis respectively. The rotary drive unit32is structured so as to include, for instance, an electric motor that rotates the mount stage30by rotative force generated by the electric motor being driven under control of the control unit5as described later such that the mount stage30rotates about an axis that is parallel to the Y-axis and that passes the center of the mount stage30as a rotation axis Yr. That is, the rotary drive unit32changes relative direction of the mount stage30and the measurement object S thereon, with respect to the X-rays radiated from the X-ray source2by rotating the mount stage30. The Y-axis movement unit33, the X-axis movement unit34, and the Z-axis movement unit35, under control by the control unit5, move the mount stage30along the X-axis direction, along the Y-axis direction, and along the Z-axis direction, respectively, so that the measurement object S can be positioned within the area in which the X-rays outputted from the X-ray source2is irradiated. The Z-axis movement unit35, under control by the control unit5, moves the mount stage30along the Z-axis direction to a position at which the distance of the measurement object S from the X-ray source2is a distance such that a projection image of the measurement object S has a desired magnification ratio. A Y position detector331, an X position detector341, and a Z position detector351are encoders that detect the positional movements of the mount stage30along the X-axis direction, along the Y-axis direction, and along the Z-axis direction, respectively, by the Y-axis movement unit33, the X-axis movement unit34, and the Z-axis movement unit35, respectively, and output signals that indicate the detected position (hereafter, referred to as detected positional movements) to the control unit5. The rotational position detector321is an encoder that detects the rotational position of the mount stage30that is rotated about the rotation axis Yr by the rotary drive unit32and outputs a signal that indicates the detected rotational position (hereafter, referred to as detected rotational position) to the control unit5. That is, the detected rotational position indicates a relative direction of the measurement object S on the mount stage30with respect to the X-rays radiated from the X-ray source2. The detector4is provided on the positive (+) side of Z-axis relative to the X-ray source2and the mount stage30. That is, the mount stage30is provided between the X-ray source2and the detector4along the Z-axis direction. The detector4has an incident surface41that is parallel to the XY-plane. At the incident surface41enter the X-rays which include the X-rays that have been irradiated from the X-ray source2and passed through the measurement object S mounted on the mount stage30. The detector4is constituted by a scintillator unit that contains a scintillation material that is known in the art, a photomultiplier tube, a light receiving unit, and so on. The detector4converts energy of the X-rays incident to the incident surface41of the scintillator unit into light energy of visible light or ultraviolet light, amplifies the light energy with the photomultiplier tube, converts the amplified light energy into electric energy with the light receiving unit, and outputs the electric energy as electric signals to the control unit5. In the detector4, the scintillator unit, the photomultiplier tube and the light receiving unit are respectively have structures in which divided into a plurality of pixels. These pixels are arranged in a two-dimensional array. Due to this, it is possible to obtain, at one time, the intensity distribution of the X-rays that has passed through the measurement object S after radiating from the X-ray source2. Note that the detector4may be one that converts the energy of the incident X-rays into electric energy and then outputs the converted electric energy in the form of electric signal without converting the energy of the incident X rays into light energy. The detector4is not limited to one in which the pixels are arranged in a two-dimensional array. The detector4has an incident surface41that extends on a plane parallel to the XY-plane, for instance, extends to the X direction. However, the incident surface41may be constituted by a line sensor that includes each of pixels disposed one after another in the Y direction. The direction along which the pixels of the line sensor are disposed is not limited to the Y direction but they may be arranged in the X direction or in the Z direction. The detector4may have a structure in which no photomultiplier tube is provided and the scintillator unit is directly formed on the light receiving unit (photoelectric conversion unit). The frame8supports the X-ray source2, the mounting unit3, and the detector4. The frame8is produced to have sufficient rigidity. Due to this, it is possible to stably support the X-ray source2, the mounting unit3, and the detector4while a projection image of the measurement object S is being acquired. The frame8is supported by a vibration isolation mechanism81to prevent transmitting the vibration that is generated in the outside to the frame8directly without being attenuated. The control unit5, which includes a microprocessor and its peripheral circuits and so on, reads in a control program that is stored in advance in a storage medium (for instance, a flash memory or the like) (not shown) and executes the program to thereby control each of units of the X-ray device100. The control unit5includes an X-ray control unit51, a movement control unit52, an image generation unit53, and a work memory55. The X-ray control unit51controls the operation of the X-ray source2. The movement control unit52controls the movement operation of the manipulator unit36. The image generation unit53performs image reconstruction processing based on electric signals that are outputted from the detector4in correspondence to the intensity distribution of the X-rays that passed through the measurement object S at every predetermined angle as the measurement object S is being rotated according to the rotation of the mount stage30to generate a three-dimensional image of the measurement object S. The image generation unit53performs, as the image reconstruction processing, processing of generating X-ray projection image data (detection data) of the measurement object S based on the electric signals outputted from the detector4, and processing a three-dimensional image showing an inside structure (cross-section structure) of the measurement object S by generating a back projection image based on the X-ray projection image data of the measurement object S under the different directions of projection, that is, different detected rotational positions. Examples of the processing of generating a back projection image include a back projection method, a filtered back projection method, a successive approximation method, and so on. The work memory55is constituted by, for instance, a volatile storage medium, at which the X-ray projection image data generated by the image generation unit53is temporarily stored. Hereafter, the processing of generating a three-dimensional image of the measurement object S performed by the image generation unit53will be explained in detail. The following explanation will be done in separate parts, one on a concept of generating a three-dimensional image according to the present embodiment and another on the processing performed by the image generation unit53based on this concept. Concept of Generating a Three-Dimensional Image Referring toFIG.2andFIG.3, the concept of generating a three-dimensional image will be explained.FIG.2is a conceptual diagram schematically showing the processing of generating a three-dimensional image according to the present embodiment.FIG.2(a)schematically shows a stereoimage of the measurement object S generated based on the detection data, which is generated based on the electric signals outputted from the detector4. The stereoimage of the measurement object S can be obtained using plurality of sets of detection data which are obtained based on the X-rays that passed through the measurement object S by irradiation the X-rays from different directions as the mount stage is rotating. The stereoimage of the measurement object S is based on the transmission intensity distribution of the X-rays that passed through the measurement object S. Accordingly, if the measurement object S contains any defect, such as a cavity in the inside thereof, the stereoimage of the measurement object S contains information about the defect such as a cavity in the inside thereof. As a result, an error between the measurement object S and the predetermined design information, so that the stereoimage of the measurement object S occurs.FIG.2(a)shows a stereoimage of a detect S1in the inside of the measurement object S as an example of the error.FIG.3(a)schematically shows the transmission intensity of X-ray that is used for generating the stereoimage of the measurement object S, that is, a specified single set of detection data D1among plurality of sets of detection data.FIG.3(a)shows the transmission intensity of the X-ray at a cross-sectional surface (a plane P indicated in broken line inFIG.2(a)) that passes through the defect S1in the inside of the measurement object S when the mount stage30is in one of a moved and rotated position for detection. To simplify the explanation, the intensity in the vertical axis ofFIG.3(a)shows values calculated by dividing the transmission intensity of the X-ray by the transmission length (distance) of the X-ray. The same is true forFIG.3(b)andFIG.3(c). Since the detect S1and the measurement object S have different absorption coefficients of an X-ray therebetween, there occurs a difference between the transmission intensity of the X-ray that passed through the defect S1and the intensity of the X-ray that did not passed through the defect S1.FIG.3(a)shows an example in which the transmission intensity of the X-ray that passed through the defect S1is higher than the intensity of the X-ray that did not passed through the measurement object S, because a ratio of X-ray absorbed at the defect S1is smaller than the ratio of X-ray absorbed at the surrounding of the defect S1. FIG.2(b)schematically shows a stereoimage of an imaginary estimated structure S2that is based on a shape information such as CAD or the like as design information and a material information of the measurement object S. The stereoimage of the estimated structure S2corresponds to an ideal state in which the measurement object S is manufactured according to the design values. Therefore, the estimated structure S2does not contain the defect S1.FIG.3(b)schematically shows the transmission intensity of the X-ray which is estimated to be detected if the X-rays passes through the estimated structure S2, that is, estimated data D2. The estimated data D2is an information about the intensity of the X-ray that is assumed to be passed through the estimated structure S2that is acquired by simulation for the case in which the X-rays are irradiated to the estimated structure S2, which corresponds to that the measurement object S is manufactured according to design values (in an ideal state). Since the estimated structure S2does not contain the defect S1, the transmission intensity of the X-ray which is estimated to be passed through the estimated structure S2is influenced only by the absorption coefficient of the estimated structure S2as shown inFIG.3(b). Note that the estimated data D2is generated under the same condition as the condition under which the detection data D1is obtained. That is, the estimated data D2is generated on the presumption that X-rays are irradiated to the estimated structure S2at the moved and rotated position for detection of the mount stage30as that are the same as the moved and rotated position for detection of the mount stage30when the detection data D1is generated. Moreover, the estimated data D2is generated on the presumption that X-rays are irradiated to the estimated structure S2at an output that is the same as the output of the X-rays that are outputted from the X-ray source2when the detection data D1is generated. Therefore, each of the estimated data D2corresponds to each of the detection data D1, that are detected for respective different irradiation directions of the X-ray according to the measurement object S. Then, as shown inFIG.2(c), a difference between the stereoimage of the measurement object S and the stereoimage of the estimated structure S2is extracted. InFIG.2(c), the detect S1, which is the difference between the measurement object S and the estimated structure S2, is extracted. In this case, as shown inFIG.3(c), the differential data D3, which is the difference between the detection data D1and the estimated data D2, is calculated. Note that, the differential data D3inFIG.3(c)indicates an absolute value of intensity. The differential data D3contains the defect S1, such as the cavity, or the like, inside the measurement object S and an error in shape between the measurement object S and the estimated structure S2. The differential data D3is obtained by extracting the difference between each of the detection data D1and each of the estimated data D2, which are in correspondence to each other. Therefore, each of the differential data D3that correspond to the detection data D1, respectively, detected for respective irradiation directions of the X-ray according to the measurement object S. As shown inFIG.2(d), the extracted stereoimage of the defect S1and the extracted stereoimage of the estimated stricture S2are combined with each other. As mentioned above, the stereoimage of the estimated structure S2corresponds to the shape manufactured according to the design values and to the ideal state that does not contain any defect or the like. As a result, a stereoimage, which is a combination of the stereoimage of the estimated structure S2in which generation of artifacts is prevented with the defect S1that is extracted from the stereoimage of the measurement object S. That is, the stereoimage of the defect S1and the stereoimage that has substantially the same shape as the measurement object S and of which generation off artifacts is prevented or reduced are generated. In this case, plurality of calculated differential data D3are subjected to back projection to generate a back projection image Im1relative to the difference between the measurement object S and the estimated structure S2. Then, the back projection image Im1and an image Im2that corresponds to the estimated structure S2are combined with each other to generate an image Im3. That is, the image Im3is a three-dimensional image in which the defect or the like that is present inside the actual measurement object S is reproduced in the image Im2of the estimated structure S2, which has substantially the same shape as the measurement object S and of which generation of artifacts is prevented or reduced. Therefore, in the image Im3, generation of artifacts by beam hardening is prevented or reduced. Note that although, for instance, in the image Im1or the image Im3, generation of artifacts by beam hardening is prevented ore reduced, sometimes there may be demanded further precision in its size, shape, for example, in case of a spherical shape, position such as position of the center of the spherical shape. Accordingly, the estimated data D2is calculated as supposing that the image obtained as the image Im3is treated as the image of the estimated structure S2. Based on the calculated estimated data D2and the detection data D1, differential data D3is extracted. Using the extracted differential data D3, the stereoimage of the defect S1is generated. By combining the thus-generated stereoimage of the defect S1with the image that is once obtained as the image Im3, a new image Im3is generated. In this manner, by performing again the step ofFIG.2(c), the generation of artifacts by beam hardening in the secondly obtained image Im3is more prevented or reduced than in the firstly obtained image Im3. This is because the difference between the estimated data D2of the estimated structure S2and the detection data D1becomes smaller. Note that by repeatedly using the image Im3obtained in the step ofFIG.2(c)as the estimated structure S2in this manner, the influence of beam hardening can be prevented or reduced. The step of repetition is not limited to once but the step may be repeated a plurality of times. The number of times of repetition may be determined based on the differential data D3that is extracted. Note that in case a line sensor is used as the detector4, back projection may be performed using X-ray intensity data groups in correspondence to different positions of the measurement object S along the Y direction to generate a three-dimensional image showing the inside structure of the overall measurement object S. Processing by the Image Generation Unit53 The processing that is performed by the image generation unit53for generating the above-mentioned three-dimensional image Im3will be explained. The image generation unit53generates the estimated data D2using the expressions (1) and (1)′ and extracts the differential data D3from the detection data D1based on the output from the detector4. yi=Biexp{−dlδμ(X,Y,Z)}+ri(1) Bi=∫dεIi(ε)exp{−dl μ0(X,Y,Z,ε)} (1)′ In the expression (1)′, μ0(X,Y,Z,ε) is an attenuation coefficient of the X-ray at a position (X,Y,Z) when supposing the X-ray having a photon energy ε passes through the inside of the imaginary estimated structure S2that has been estimated based on the design information of the measurement object S. On the other hand, μ(X,Y,Z,ε) in the expression (1) is a attenuation coefficient of the X-ray having a photon energy ε at a position (X,Y,Z) when the X-ray passes through the inside of the measurement object S. The relationship between these attenuation coefficients is represented by expression (2). μ(X,Y,Z,ε)=μ0(X,Y,Z,ε)+δμ(X,Y,Z) (2) Here, δμ(X,Y,Z) is an difference between both attenuation coefficients. The expressions (1) and (1)′ are derived from expression (2) as follows. The attenuation amount of the X-ray when the X-ray travels over a minute portion having a distance Δl in the measurement object S is represented by expression (3) as below. μ(X,Y,Z,ε)Δl=μ0(X,Y,Z,ε)Δl+δμ(X,Y,Z)Δl(3) Therefore, the X-ray having an intensity Ii (ε) that has entered the minute portion having a distance Δl has an intensity represented by expression (4) at a point that the X-ray has been passed over a minute portion having a distance Δl. Ii(ε)exp{−μ(X,Y,Z,ε)Δl}=Ii(ε)exp{−μ0(X,Y,Z,ε)Δl+δμ(X,Y,Z)Δl}(4) Therefore, the intensity yi of the X-ray that enters the detector4after passed through the inside of the measurement object S along its traveling direction is represented by expression (5). yi=∫Ii(ε)exp{-∮ldlμ(X,Y,Z,ε)}=∫Ii(ε)exp{-∮l(dlμ(X,Y,Z,ε)+δμ(X,Y,Z))}=∫dεIi(ε)exp{-∮ldlμ0(X,Y,Z,ε)}exp{-∮ldIδμ(X,Y,Z)}+ri(5) Note that i indicates the position of one detection pixel among a plurality of detection pixels included in the detector4. For instance, when a plurality of detection pixels are arranged in a line form in the detector4, yi represents the intensity of the X-ray that enters an i-th detection pixel from a beginning. Moreover, ri represents a noise component such as dark current contained in the outputted from the detection pixel arranged at i-th position in the detector4. That is, according to expression (5), intensities yi in numbers in correspondence to the number of detection pixels in the detector4are generated for each detected rotational position. Here, in expression (5), by substituting ∫dεIi(ε)exp{−dlμ0(X,Y,Z,ε)}=Bi (that is, expression (1)′), expression (1) is obtained. As mentioned above, the attenuation coefficient μ0(X,Y,Z,ε) is a attenuation coefficient of the X-ray at the position (X,Y,Z) of the inside of the estimated structure S2and it depends on the design information of the measurement object S. As the design information, for instance, the material information about the material that constitutes the measurement object S may be used. The material information of the measurement object S may be, for instance, information about the ratio of materials contained in the measurement object S. Also, the material information of the measurement object S may be, for instance, information as to whether the material contained in the measurement object S is metal or nonmetal. The material information of the measurement object S may be, for instance, information about elements or compounds contained in respective members that constitute the measurement object S. As the design information, for instance, information about an external shape and/or an internal shape of the measurement object S may be used. The intensity Ii (ε) depends on the intensities and spectra of the X-rays irradiated to the measurement object S, which are measuring conditions under which the measuring device is operated. Therefore, Bi represented by the expression (1)′ corresponds to the estimated detection intensity estimated to be detected by i-th detection pixel of the detector4that is calculated based on the design information, the material information, and the measuring conditions. That is, Bi corresponds to the transmission intensity of the X-ray that has passed through the estimated structure S2when the X-rays are irradiated to the estimated structure S2according to the measuring conditions. By arranging this Bi in numbers corresponding to the number of the detection pixels in the detector4, data that corresponds to the above-mentioned estimated data D2is obtained. Namely, the image generation unit53generates the estimated data D2by applying the design information, the material information, and the measuring conditions to expression (1). Then, Bi and ri are substituted into the right-hand side of the expression (1). Into yi of the left-hand side of the expression (1) is substituted the intensity of the X-ray that is actually detected by the detector4. As mentioned above, since it is assumed that δμ(X,Y,Z) is a difference between the attenuation coefficient μ(X,Y,Z,ε) and the attenuation coefficient μ0(X,Y,Z,ε) and because supposing that it does not depend on the photon energy ε, the error term δμ(X,Y,Z) can be calculated. Here, δμ(X,Y,Z) is a value that is based on a difference between a distance along which the X-ray that has passed through the inside of the measurement object S travels and a distance along which the X-ray that has passed through the inside of the estimated structure S2travels at each of the plurality of detection pixels of the detector4. That is, δμ(X,Y,Z) corresponds to a difference between the detection data D1and the estimated data D2. Therefore, the image generation unit53calculates δμ(X,Y,Z) for a detected intensity that is outputted from each of the plurality of detection pixels arrayed in the detector4at each irradiation position of the X-ray irradiated to the measurement object S. By so doing, the image generation unit53extracts a plurality of sets of differential data D3at each irradiation position of the X-ray irradiated to the measurement object S. Then, the image generation unit53evaluates each of the δμ(X,Y,Z) values, whose number corresponds to the number of the detection pixels of the detector4, calculated by expression (1) for any irradiation position of the X-ray The image generation unit53, when all the calculated δμ(X,Y,Z) values do not exceed a first predetermined value, judges that the differential data D3corresponds to the difference caused by a cavity or the like in the inside of the measurement object S (for instance, the defect S1inFIG.2). The image generation unit53, when at least one of the calculated δμ(X,Y,Z) values is equal to or exceeds the first predetermined value, judges that the error in shape between the measurement object S1and the estimated structure S2is considerably large, that is, the size of the measurement object S differs from the design value considerably. When all the calculated δμ(X,Y,Z) values do not exceed the first predetermined value, the image generation unit53performs back projection of the differential data D3to generate a back projection image Im1and combines it with the image Im2of the estimated structure S2to generate the image Im3. This enables one to evaluate location, shape, size and so on of defects, if any, in the inside of the measurement object S. On the other hand, when at least one of the calculated δμ(X,Y,Z) values is equal to or exceeds the first predetermined value, the image generation unit53performs, based on the δμ(X,Y,Z) value, correction of the estimated structure S2so that it becomes closer to the shape of the measurement object S. Then, the image generation unit53, based on the corrected size of the estimated structure S2, obtains δμ(X,Y,Z) in the same procedure as described above and evaluates its value. This procedure is repeated until all the calculated δμ(X,Y,Z) values will be below the first predetermined value. FIG.4schematically illustrates the concept of correction of the estimated structure S2. InFIG.4, in the same manner as in the case shown inFIG.3(a), a solid line indicates a detected intensity distribution L1of the X-ray detected by the detector4, that is, a detected intensity distribution of the X-ray that corresponds to the detection data D1, when the X-rays, that are outputted from the X-ray source2, is irradiated to the measurement object S having the defect S1in the inside thereof. Assuming that the X-rays are irradiated to the estimated structure S2that corresponds to the measurement object S, estimated detection intensity distributions L2, L3, and L4of the X-ray that are estimated to be detected by the detector4, that is, estimated detection intensity distributions of the X-ray that correspond to the estimated data D2are indicated in broken line. L2indicates the estimated intensity distribution of the X-ray to which the first processing was performed but the correction of the shape of the estimated structure S2was not performed yet. InFIG.4, it is shown that L1is higher than L2in the transmission intensity distribution of the X-ray. This indicates that the transmission distance of the X-ray that transmits through the measurement object S is shorter than that of the designed size. That is, the size of the measurement object S is smaller than the design value. L3and L4indicate each an estimated intensity distribution of the X-ray that is estimated after the correction of the size of the estimated structure S2in the second and subsequent processing are performed. InFIG.4, the estimated transmission intensity of the X-ray for detection pixels included in the area ir among the plurality of the detection pixels included in the detector4is higher than the estimated transmission intensity of the X-ray for any one of detection pixels of the detector4other than those included in the area ir. This indicates that the defect S1in the inside of the measurement object S gives sonic influence on the estimated intensity of the X-ray for pixels in the area ir. Each of the differences between the intensity distribution of L1and the intensity distributions of L2, L3, or L4shown inFIG.4correspond to δμ(X,Y,Z). The image generation unit53changes the estimated transmission intensity distribution of the X-ray from L2in the direction indicated by an arrow A, that is, in the direction in which the intensity increases inFIG.4by performing correction of the shape of the estimated structure S2. Correcting the shape of the estimated structure S2corresponds to changing the distance in which the X-ray transmits through the inside of the estimated structure S. The image generation unit53changes the shape of the estimated structure S2in a direction in which it becomes closer to the shape of the measurement object S In the case of this example, the intensities in correspondence to the area ir for L3and L4become higher than the intensities in correspondence to a remaining area other than the area ir. Of course, in the case of this example, the transmission distance of the X-ray that transmits through the measurement object is larger than that of the designed size Note that the size of the measurement object is larger than the design value, the relationship of the magnitude of L2to that of L1is in reverse to the above relationship. When the shape of the estimated structure S2is corrected to change the transmission intensity distribution of the X-ray that corresponds to the newly generated estimated data D2from L2to L3, new δμ(X,Y,Z) is calculated as a difference in transmission intensity distributions of the X-ray between L1and L3. When this δμ(X,Y,Z) value is larger than the first predetermined value, the shape of the estimated structure S2is further corrected based on δμ(X,Y,Z) value. When the estimated transmission intensity distribution of the X-ray changes from L2or L3to L4, the intensity distribution of the X-ray transmits through the measurement object S and the intensity distribution of the X-ray that is estimated to be passed through the estimated structure S2after the correction of its shape are deemed identical to each other. That is, the value of δμ(X,Y,Z) that is newly calculated as a difference in transmission intensity between L1and L4is equal to or lower than the first predetermined value. This means that the estimated data D2reproduces the transmission intensity distribution of the X-ray that has been passed through the measurement object S. Note that the image generation unit53may be configured to align on data the measurement object S represented by the detection data D1and the estimated structure S2represented by the estimated data D2. For instance, the image generation unit53detects the position of a boundary (edge) (pixel position i) between the measurement object S represented by the detection data D1and the background. The image generation unit53detects the pixel position i+a in correspondence to the boundary on the estimated data D2. The image generation unit53performs calculation by expression (1) using the detection data D1at pixel i (that is, yi) and the estimated data D2at pixel i±a (that is, Bi+a). As a result, at time of the first processing, the value of δμ(X,Y,Z) can be calculated as a relatively small value and hence the number of times of processing can be decreased even when subsequently correction of δμ(X,Y,Z) may become necessary. The image generation unit53performs the above-mentioned processing on each of the plurality sets of the detection data D1and each of the plurality of sets of the estimated data D2, each are based on different projection directions of X-ray toward the measurement object S, that is, each are based on different detected rotational position to effect alignment and subsequently synthesizes the back projection image Im1with the image Im2relating to the estimated structure S2to generate the image Im3. The image generation unit53commands the display monitor6to display the generated image Im3in a three-dimensional image. Referring to the flowchart shown inFIG.5, the processing of generating a three-dimensional image of the measurement object S by the image generation unit53will be explained. The processing shown inFIG.5is performed by executing a program by the image processing unit53. The program is stored in a memory (not shown) in the control unit5and is activated and executed by the image generation unit53. In Step S1, the shape information and the material information, which constitute design information of the measurement object S, and the measuring conditions for measuring the measurement object S, which is measuring device information, are acquired, and the procedure proceeds to Step S2. In Step S2, Bi for each of the pixels is calculated based on the shape information, the material information, and the measuring information using the above-mentioned expression (1)′ (that is, estimated data D2is generated) and the procedure proceeds to Step S3. In Step S3, transmission images for all the irradiation directions are acquired and the procedure proceeds to Step S4. Note that in Step S3, the transmission image in all measuring directions is acquired for using in image reconfiguration. For instance, inFIG.1, the transmission images that are acquired when the measurement object S is rotated by one turn of 360° with the stage30. In Step S4, a detected value detected at each of the pixels by causing X-rays to be actually passed through the measurement object S and calculated Bi are substituted into expression (1) and δμ(X,Y,Z) value is calculated according to the back projection method (that is, differential data D3is extracted) and the procedure proceeds to Step S5. In Step S5, it is determined whether all the position-dependent δμ(X,Y,Z) values that are calculated for the detection pixels, respectively, are equal to or smaller than the first predetermined value. When all the δμ(X,Y,Z) values are equal to or smaller than the first predetermined value, Step S5is determined to be affirmative and the procedure proceeds to Step S7. When at least one of δμ(X,Y,Z) values exceeds the first predetermined value, Step S5is determined to be negative and the procedure proceeds to Step S6. In Step S6, the shape of the estimated structure S2is corrected and the procedure proceeds to Step S2. In this case, in Step S2, the shape of the estimated structure S2that has been corrected in Step S6is used as design to information (shape information) of the measurement object S. In Step S7, since the back projection image Im1of the differential data D3is 0, the shape of the estimated structure S2, correction of which has thus far been continued, is deemed to match the shape of the measurement object S (structure). The image of the estimated structure S2is displayed on the display monitor6as a three-dimensional image of the measurement object S and the processing is terminated. Note that the image to be displayed on the display monitor6is not limited to the image Im3of the estimated structure S2. According to the first embodiment described above, the following operations and advantageous effects are obtained.) (1) The image generation unit53generates a plurality of sets of detection data D1relating to the transmission intensity of the X-ray that has been passed through the measurement object S and a plurality of sets of estimated data D2relating to the estimated transmission intensity of the X-ray on the assumption that the X-rays have been passed through an imaginary estimated structure S2that is configured based on design information under the same irradiation condition as that under which the X-rays have been passed through the measurement object S. Using the detection data D1and the estimated data D2that correspond to each other with respect to the irradiation direction of the X-ray, the image generation unit53extracts differential data D3, which indicates the difference between the detection data D1and the estimated data D2. Therefore, it is possible to acquire, from the detection data D1of the measurement object S, information about defect S1such as a cavity or the like in the inside of the measurement object S, which is not included in the estimated structure S2that is estimated based on the design information and information about an error in shape between the measurement object S and the estimated structure S2. (2) The image generation unit53performs back projection of the extracted differential data D3to generate a back projection image Im1relating to the difference and combines the back projection image Im1with the estimated structure S2to generate the image Im3relating to the inside structure of the measurement object S. Therefore, the image Im3, of which generation of artifacts due to beam hardening is prevented or more reduced as compared with the back projection image obtained by performing back projection of the detection data D1, is generated. As a result, it becomes easier to grasp the inside defect of the measurement object S by the image Im3. In particular, even in the case of a detect such as a small cavity, generation of a trouble that it is difficult to grasp the shape and size of the measurement object S due to artifacts on its image can be prevented or reduced. That is, by preventing or reducing artifacts from being contained in the generated image, a decrease in inspection accuracy can be prevented or reduced. Furthermore, an image of which generation of artifacts due to beam hardening is prevented can be generated using expression (1) in contrast to the case in which artifacts due to beam hardening are reduced by correction. This contributes to a reduction in load of processing and to shortening of processing time. Note that according to the present embodiment, the differential data D3is generated and reconstruction processing is performed using only the differential data D3and thus it is possible to reduce the load of processing and shorten the time of processing. (3) The image generation unit53generates estimated data D2by estimating the transmission intensity of the X-ray when the X-rays are irradiated to the estimated structure S2based on the material information of the measurement object S and spectrum information of the X-ray as the measuring condition of the measurement object S. Therefore, the estimated data D2can be generated without requiring a large load of processing. (4) The attenuation coefficient μ(X,Y,Z,ε) of the X-ray is expressed using the attenuation coefficient μ0(X,Y,Z,ε), which is a first component that depends on the photon energy ε, and the error term δμ(X,Y,Z), which is a second component. The image generation unit53calculates the value of δμ(X,Y,Z) using the detection data D1and the estimated data D2, thereby extracting differential data D3. Therefore, in contrast to the case of solving an approximation expression that depends on the photon energy ε by the conventional technology, in which an influence of artifacts due to beam hardening remains in the generated back projection image, it is possible that the influence of artifacts due to beam hardening in the generated image Im3is to be reduced and an image Im3having a high image quality is to be provided. (5) The image generation unit53performs back projection of the differential data D3to generate a back projection image Im1when all the values of δμ(X,Y,Z) that are calculated with respect to a certain irradiation direction of the X-ray do not exceed the first predetermined value. Therefore, a back projection image Im1can be generated so that a small cavity or the like included in the measurement object S can be observed. (6) The image generation unit53, when a plurality of δμ(X,Y,Z) values calculated for a certain irradiation direction of an X-ray exceeds the first predetermined value, corrects the estimated data D2based on the δμ(X,Y,Z) values to generate new estimated data D2. Therefore, the defect such as a small cavity or the like included in the measurement object S can be correctly evaluated in condition that the error in shape between the measurement object S and the estimated structure S2is sufficiently small. (7) The image generation unit53, when it generates new estimated data D2, extracts new differential data D3using the detection data D1and the new estimated data D2. The image generation unit53, when the new differential data D3does not exceed the first predetermined value, performs back projection of the new differential data D3to generate a back projection image Im1. Thereafter, the image generation unit53combines the back projection image Im1with the image Im2relating to the estimated structure S2to generate an image Im3that relates to the inside structure of the measurement object S. Therefore, since the image of the estimated structure S2, whose shape is substantially identical to that of the measurement object S, is combined with the back projection image Im1that shows a defect or the like, the shape that may be deemed to be identical to that of the measurement object S that is actually measured can be reproduced on an image of which occurrence of artifacts due to beam hardening is reduced. That is, a defect or the like in the inside of the actually measured measurement object S can be understood with ease. Second Embodiment Referring to the drawings, a second embodiment of the present invention will be explained. In the following explanation, the same components as those in the first embodiment are assigned the same reference signs and explanation thereof is focused mainly on differences with the first embodiment. What is not particularly explained is the same as that in the first embodiment. In the second embodiment, the image generation unit53generates estimated data D2based on the concept that is explained in the first embodiment using the following expressions (7) and (7)′ and extracts differential data D3from the detection data D1that is based on the output from the detector4. As described later, the image generation unit53generates estimated data D2by calculating Ai using expression (7)′ and calculates δμ(X,Y,Z) by substituting the calculated Ai into expression (7), thereby extracting the differential data D3. Aiexp{−dlδμ(X,Y,Z)}+ri(7) Ai=∫dεIi(ε)exp{−fPE(ε)dlα1(X,Y,Z)−fKN(ε)dlα2(X,Y,Z)} (7)′ The expressions (7) and (7)′ are derived as follows. In the present embodiment, the attenuation coefficient μ(X,Y,Z,ε) of the X-ray having a photon energy ε at a position (X,Y,Z) where the X-rays pass through the measurement object S is assumed to be represented by expression (8) as follows. μ(X,Y,Z,ε)=α1(X,Y,Z)fPE(ε)+α2(X,Y,Z)fKN(ε)+δμ(X,Y,Z) (8) α1(X,Y,Z) indicates a spatial distribution of reduction of the X-ray by photoelectric absorption in the measurement object S and α2(X,Y,Z) indicates a spatial distribution of reduction of the X-ray due to the Compton effect in the measurement object S. Both α1(X,Y,Z) and α2(X,Y,Z) are known values that depend on the material of the measurement object S and are stored in a memory (not shown) in advance. Both fPE(ε) and fKN(ε), which are functions that depend on the photon energy ε, are represented by the following expressions (9) to (11), respectively. fPE(ε)=1ε1(9)fKN(ε0)=1+ε0ε02[2(1+ε0)1+2ε0-1ε0ln(1+2ε0)]+12ε0ln(1+2ε0)-1+3ε0(1+2ε0)2(10)ε0=ε510.975keV(11) Note that 510.975 keV in expression (11) corresponds to static energy of an electron and ε0 represents photon energy of the X-ray that is normalized with the static energy of the electron. Therefore, α1(X,Y,Z)fPE(ε)+α2(X,Y,Z)fKN(ε) in expression (8) is information relating to a attenuation of the X-ray when the X-ray having a photon energy ε passes through the inside of the estimated structure S2, which is estimated as the measurement object S in an ideal state based on the material information of the measurement object S. δμ(X,Y,Z) is a term indicating an error between the attenuation coefficient μ(X,Y,Z,ε) of the X-ray having a photon energy ε at a position (X,Y,Z) when the X-ray passes through the inside of the measurement object S and the information relating to the attenuation described above. Note that the attenuation coefficient μ(X,Y,Z,ε), the functions fPE(ε) and fKN(ε) are elements that depend on the photon energy ε. δμ(X,Y,Z) is an element that is assumed not to depend on the photon energy ε. Assuming the X-ray travels along a minute portion having a distance Δl through the measurement object S, the attenuation coefficient of the X-ray is represented by the following expression (12) using expression (8). μ(X,Y,Z,ε)Δl={α1(X,Y,Z)fPE(ε)+α2(X,Y,Z)fKN(ε)}+δμ(X,Y,Z)Δl(12) The expression (12) is changed using the spectrum Ii (ε) of the X-ray that enters the pixels of the detector4in the same manner as the case in the first embodiment in which using expression (4) and then derives expression (5), the intensity yi of the X-ray that enters the detector4according to the present embodiment is substituted to obtain expression (13). yi=∫Ii(ε)exp{-∮ldlμ(X,Y,Z,ε)}=∫Ii(ε)exp{-∮ldl(α1(X,Y,Z)fPE(ε)+α2(X,Y,Z)fKN(ε)+δμ(X,Y,Z)}}=∫dεIi(ε)exp{-fPE(ε)∮ldlα1(X,Y,Z)-fKN(ε)∮ldlα2(X,Y,Z)-∮ldlδμ(X,Y,Z)}+ri=exp{-∮ldlδμ(X,Y,Z)}∫dεIi(ε)exp{-fPE(ε)∮ldlα1(X,Y,Z)-fKN(ε)∮ldlα2(X,Y,Z)}+ri(13) Note that also in expression (13), i indicates the position of one pixel among a plurality of pixels included in the detector4. Also, ri represents a noise component such as dark current contained in the output from the i-th pixel arranged in the detector4. In expression (13), by defining ∫dεIi(ε)exp{−fPE(ε)dlα1(X,Y,Z)−fKN(ε)dlα2(X,Y,Z)} as Ai, expression (7)′ is derived. As mentioned above, α1(X,Y,Z) and α2(X,Y,Z) are values that depend on the design information of the measurement object S. The intensity Ii (ε) depends on the intensity and spectrum of the X-ray, as the measuring conditions of the measuring device, which is irradiated to the measurement object S. Therefore, Ai represented by expression (7)′ corresponds to the calculated detection intensity assuming to be detected by the i-th detection pixel of the detector4based on the design information and the measuring conditions. That is, Ai, like Bi in the first embodiment, corresponds to the transmission intensity of the X-ray that is assumed to being passed through the estimated structure S2when the X-ray assumed to be irradiated to the estimated structure S2according to the measuring conditions. That is, it corresponds to the estimated data D2. Therefore, the image generation unit53generates the estimated data D2by applying the design information and the measuring conditions to expression (7)′. Then, Ai and ri are substituted into the right-hand side of expression (7). Into the left-hand side of expression (7) is substituted the intensity of the X-ray that is actually detected by the detector4. δμ(X,Y,Z) represents an error between the sum of the attenuation coefficient due to the photoelectric effect and the attenuation coefficient due to the Compton effect and the attenuation coefficient μ(X,Y,Z,ε), and δε(X,Y,Z) does not depend on the photon energy ε, so that the error term δμ(X,Y,Z) can be calculated. Hereafter, the image generation unit53performs the same processing as that in the first embodiment. That is, the image generation unit53evaluates the magnitude relationship between the calculated δμ(X,Y,Z) value and the first predetermined value and depending on the result of the evaluation, performs a generation of a back projection image and displaying a three-dimensional image, or performs a generation of a back projection image and displaying a three-dimensional image after correcting the estimated. data D2. Also, in the second embodiment, the image generation unit53performs series of processing shown in the flowchart ofFIG.5in the first embodiment to generate a three-dimensional image. However, in Step S3, the image generation unit53calculates Ai using expression (7)′ and generates the estimated data D2. Also, in Step S4, the image generation unit53calculates δμ(X,Y,Z) using expression (7). Note that in the above explanation, the image generation unit53uses α1(X,Y,Z), which is information relating to the photoelectric absorption, and α2(X,Y,Z), which is information relating to the Compton effect for expressing the attenuation coefficient μ(X,Y,Z,ε). However, the attenuation coefficient μ(X,Y,Z,ε) may be expressed using either one of α1(X,Y,Z) or α2(X,Y,Z). For instance, for expressing the attenuation coefficient μ(X,Y,Z,ε), when the photon energy ε of the X-rays that are irradiated to the measurement object S is relatively small, the image generation unit53uses the information relating to the photoelectric absorption, α1(X,Y,Z), and when the photon energy ε is relatively large, the image generation unit53uses the information relating to the Compton effect, α2(X,Y,Z). According to the second embodiment as described above, in addition to the operations and advantageous effects (1), (2), and (4) to (7) obtained according to first embodiment, the following operations and advantageous effects can be obtained. The image generation unit53generates the estimated data D2based on the spectrum information of the X-ray that enters the measurement object S and at least one of the information of, α1(X,Y,Z), which is information relating to the photoelectric absorption of the X-ray that is passed through the measurement object S, and α2(X,Y,Z), which is information relating to the Compton effect of the X-ray that is passed through the measurement object S. That is, the image generation unit53calculates Ai using the to expression (7)′. Therefore, the estimated data D2can be generated without requiring a large load of processing. Third Embodiment Referring to the drawings, a third embodiment of the present invention will be explained. In the following explanation, the same components as those in the first embodiment are assigned the same reference signs and explanation thereof is focused mainly on differences between this embodiment and the first embodiment. What is not explained particularly is the same as that in the first embodiment. The present embodiment is different from the first embodiment in that conforming product determination on the measurement object is performed using extracted differential data. The image generation unit53calculates δμ(X,Y,Z), that is, extracts differential data D3using expression (1) in the same manner as that in the first embodiment. The image generation unit53determines the magnitude relationship between the calculated δμ(X,Y,Z) and the second predetermined value. Note that the second predetermined value may be, for instance, an acceptable tolerance value of the measurement object S. When δμ(X,Y,Z) is equal to or smaller than the second predetermined value, it is determined that the shape of the measurement object S is within an acceptable tolerance range with respect to the shape of the estimated structure S2. That is, in this case the image generation unit53determines that the measurement object S does not have a large error in shape with respect to the design dimensions and determines that this measurement object S is a conforming product. If the measurement object S is determined to be a conforming product, the image generation unit53performs generation of a back projection image Im1, combination of the back projection image Im1with the image Im2relating to the estimated structure S2, and generation of the image Im3in the same manner as that in the first embodiment. The image generation unit53generates a three-dimensional image using the image Im3and displays it on the monitor6. That is, the image generation unit53deems that the estimated structure S2obtained by repeating corrections until the back projection image Im1of the differential data D3becomes 0 matches the shape of the actual measurement object S and the image generation unit53generates a three-dimensional image using the image Im3. Of course, the image Im3may be displayed in comparison with the design image, or only a differential image between the image Im3and the design image may be displayed. When δμ(X,Y,Z) exceeds the second predetermined value, the image generation unit53determines that the shape of the measurement object S exceeds an acceptable tolerance range with respect to the shape of the estimated structure S2. That is, it determines that the measurement object S has a significantly large error in shape with respect to the design dimensions. In this case the image generation unit53determines that the measurement object S is a defective product and does not perform subsequent processing. Note that the image generation unit53may not display the three-dimensional image of the measurement object S on the display monitor6and may, for instance, display a warning notifying that the measurement object S is a defective product on the monitor6. Note that even when the image generation unit53determines that the measurement object S is a defective product, it may generate a three-dimensional image of the measurement object S and displays it on the display monitor6. In this case, the image generation unit53may display a warning notifying that the measurement object S is a defective product in superposition to the three-dimensional image of the measurement object S displayed on the display monitor6. Note that although in the above explanation, the image generation unit53calculates δμ(X,Y,Z) in the same manner as that in the first embodiment, it may be configured to calculate δμ(X,Y,Z) in the same manner as that in the second embodiment. Referring to the flowchart shown inFIG.6, the generation processing of a three-dimensional image of the measurement object S by the image generation unit53will be explained. The processing illustrated inFIG.5is performed by executing a program by the image processing unit53. This program is stored in a memory (not shown) in the control unit5and booted and executed by the image generation unit53. Each processing of from Step S11(acquisition of design information and measuring conditions) to Step S14(extraction of differential data) is similar to each processing in Step S1(acquisition of design information and measuring condition) to Step S4(extraction of differential data) inFIG.5. In Step S15, it is determined whether all the position-dependent δμ(X,Y,Z) values that are calculated for respective pixels are equal to or smaller than the second predetermined value. When all the δμ(X,Y,Z) values are equal to or smaller than the second predetermined value, Step S15is determined to be affirmative and the procedure proceeds to Step S17. When at least one of the δμ(X,Y,Z) values exceeds the second predetermined value, Step S15is determined to be negative and the procedure proceeds to Step S16. In Step S16, the measurement object S is determined to be a defective product and the processing is terminated. Note that in Step S16, a message that the measurement object S is a defective product or the like may be displayed on the display monitor6. Each processing of from Step S17(determination of magnitude relationships between each of the δμ(X,Y,Z) values and the first predetermined value) to Step S19(deeming the shape of the corrected estimated structure as the shape of the actual measurement object (structure)) is similar to each processing of from Step S5(determination of magnitude relationships between each of the δμ(X,Y,Z) values and the first predetermined value) to Step S7(deeming the shape of the corrected estimated structure as the shape of the actual measurement object (structure) inFIG.5. However, when the processing in Step S13is performed again via Step S18, the determination in Step S15is skipped and the procedure proceeds to Step S17. According to the third embodiment as explained above, the following operations and advantageous effects can be obtained in addition to the operations and advantageous effects obtained in the first embodiment and/or in the second embodiment. (1) The image generation unit53, when δμ(X,Y,Z) values that correspond to the differential data D3do not exceed the second predetermined value, determines the measurement object S to be a conforming product. Therefore, it becomes possible to use δμ(X,Y,Z) values in processing other than the generation of images, so that convenience can be increased. (2) The image generation unit53, when it determines the measurement object S as a conforming product based on the differential data D3, performs back projection of the differential data D3to generate a back projection image Im1and combines it with the image Im2relating to the estimated structure S2to generate an image Im3of the inside structure of the measurement object S. Therefore, in case where the measurement object S does not have a large error in shape, the Im3relating to the inside structure of the measurement object S can be generated. This enables prevention of any possible increase in load to be caused by generation of images of low necessity for the measurement object S that has been determined to be a defective product can be prevented. Fourth Embodiment Referring to the drawings, a structure manufacturing system according to an embodiment of the present invention will be explained. The structure manufacturing system according to the present embodiment manufactures molding products, for instance, door parts, engine parts, or gear parts for automotive use, and electronic components provided with circuit boards, and the like. FIG.7is a block diagram showing an example of the configuration of a structure manufacturing system600according to the present embodiment. The structure manufacturing system600includes the X-ray device100explained in any one of the first to third embodiments and variation examples thereof, a design device610, a molding device620, a control system630, and a repairing device640. The design device610, which is a device that is used by the user for generating design information relating to the shape of a structure, performs design processing to generate design information and store it. The design information is information that indicates respective positions of the structure on the coordinate. The design information is outputted to the molding device620and the control system630described later. The molding device620performs molding processing to manufacture the structure based on the design information generated by the design device610. The molding device620that performs at least one of a lamination processing represented by 3D printing processing, a casting processing, a forging processing, and a cutting processing is included as an aspect of the present invention. The X-ray device100performs measurement processing in which the shape of the structure molded by the molding device620is measured. The X-ray device100outputs information that indicates coordinate information (hereafter, referred to as “shape information”) of the structure, which information is a result of measurement of the structure to the control system630. The control system630includes a coordinate storage unit631and an inspection unit632. The coordinate storage unit631stores the design information generated by the design device610. The inspection unit632determines whether the structure molded by the molding device620has been manufactured according to the design information generated by the design device610. In other words, the inspection unit632determines whether the manufactured structure is a conforming product. In this case, the inspection unit632reads out the design information stored at the coordinate storage unit631and performs inspection processing, in which the design information is compared with the shape information that is inputted from the image measuring device100. The inspection unit632performs, as inspection processing, comparison of, for instance, coordinates indicated by the design information with corresponding coordinates indicated by the shape information and determines, when the coordinates of the design information matches the coordinates of the shape information as a result of the inspection processing, that the structure is a conforming product that has been manufactured according to the design information. When the coordinates of the design information does not match the coordinates of the shape information, the inspection unit632determines whether a difference in coordinates is within a predetermined range. If the difference is within the predetermined range, the inspection unit632determines that the structure is a repairable defective product. When the structure is determined to be a repairable defective product, the inspection unit632outputs repair information that indicates a defective region and an amount of correction to a repairing device640. The defective region indicates the region in which the coordinates of the shape information does not match the coordinates of the design information, and the amount of correction is a difference between the coordinates of the design information and the coordinates of the shape information in the defective region. The repairing device640performs repair processing in which the defective region of the structure is reprocessed based on the inputted repair information. The repairing device640in repair processing performs again processing similar to the molding processing performed by the molding device620. Referring to the flowchart illustrated inFIG.8, the processing that the structure manufacturing system600performs will be explained. In Step S111, the design device610, which is used by the user for designing a structure, performs design processing to generate design information relating to the shape of the structure and stores it. Then the procedure proceeds to Step S112. Note that the design information is not limited to the one that is generated by the design device610. According to an aspect of the present invention, the design device610may, when there is the design information that has already been prepared, acquire such design information by inputting it therein. In Step S112, the molding device620performs molding processing to manufacture a structure based on the design information and the procedure proceeds to Step S113. In Step S113, the image measuring device100performs measurement processing to measure the shape of the structure and outputs shape information, and then the procedure proceeds to Step S114. In Step S114, the inspection unit632performs inspection processing in which the design information generated by the design device610is compared with the shape information acquired by measurement and outputted by the image measuring device100, and then the procedure proceeds to Step S115. In Step S115, the inspection unit632determines whether the structure manufactured by the molding device620is a conforming product based on the result of the inspection processing. When the structure is a conforming product, that is, when the coordinates of the design information matches the coordinates of the shape information, Step S115is determined to be affirmative and the processing is terminated. When the structure is not a conforming product, that is, the coordinates of the design information does not match the coordinates of the shape information, or coordinate information that are not contained in the design information are detected, Step S115is determined to be negative, and then the procedure proceeds to Step S116. In Step S116, the inspection unit632determines whether the defective region of the structure is repairable. When the defective region is not repairable, that is, a difference between the coordinates of the design information and the coordinates of the shape information in the defective region exceeds a predetermined range, Step S116is determined to be negative, and then the processing is terminated. When the defective region is repairable, that is, a difference between the coordinates of the design information and the coordinates of the shape information in the defective region is within the predetermined range, Step S116is determined to be affirmative, and then the procedure proceeds to Step S117. In this case, the inspection unit632outputs repair information to the repairing device640. In Step S117, the repairing device640performs repair processing to the structure based on the inputted repair information, and then the procedure returns back to Step S113. Note that as mentioned above, the repairing device640performs again processing similar to the molding processing performed by the molding device620in the repair processing. The structure manufacturing system according to the fourth embodiment described above, the following operations and advantageous effects are obtained. (1) The X-ray device100in the structure manufacturing system600performs measurement processing, in which the shape information of the structure generated by the molding device620by the design processing performed by the design device610is acquired and the inspection unit632in the control system630performs inspection processing, in which the shape information acquired by the measurement processing is compared with the design information generated in the design processing. Therefore, it is possible to inspect the defect of the structure or acquire the information of the inside of the structure by a non-destructive inspection and determine whether the structure is a conforming product that has been manufactured according to the design information. This contributes quality control of the structure. (2) The repairing device640is configured to perform repair processing in which the molding processing is performed again to the structure depending on the result of comparison in the inspection processing. Therefore, when the defective region of the structure is repairable, processing similar to the molding processing can be performed to the structure again. This contributes to manufacture of a high quality structure that is approximate to the design information. As explained above, according to the present invention, data showing a difference between the measurement object and an estimated structure obtained based on the shape information of the measurement object can be extracted. Also, variation examples as described below are within the scope of the present invention and one or more variation examples may be combined with the above-mentioned embodiments. (1) In the X-ray device100according to any one of the first to fourth embodiments, the image generation unit53has been explained as estimating the shape of the estimated structure S2using the shape information contained in the design information. However, the X-ray device100may be one that generates the estimated data D2based on the detection data D1of the measurement object S. In this case, the image generation unit53performs back projection of the detection data D1of the measurement object S using, for instance, filtered back projection (FBP) to generate an image. Since this image is expressed by a contrasting density in correspondence to the intensity of the X-ray that has been passed through the measurement object S, the image generation unit53estimates a substance that constitutes the measurement object S, that is, a material based on this contrasting density. For instance, the image generation unit53is configured to have data in which the density of the image is in relation to the material and estimate the material based on the density of the generated image, that is, the intensity of the X-ray. The image generation unit53may use the estimated material as the material information and calculate Bi according to expression (1)′ to generate the estimated data D2. In this case, the material information of the measurement object S is acquired from projection image of the measurement object S in Step S1ofFIG.5or Step S11ofFIG.6. Therefore, when no material information is obtained as the design information of the measurement object S, the estimated data D2can be generated. (2) The image generation unit53may be configured to extract shape information of the measurement object S by performing edge detection or the like using the back projection image of the measurement object S. In this case, by setting an irradiation direction of the X-ray for each small rotation angle to increase the number of detection data to be generated, thereby increasing the number of edges, that is, the number of profile shape of the measurement object S, the shape of the measurement object S can be obtained with high precision. In particular, when the measurement object S has a complicated profile shape, it is advantageous to increase the number of irradiation directions so that the number of detection data increases. The image generation unit53may be configured to extract the profile shape of the measurement object S using an image of the measurement object S captured by an image-capturing device such as a camera and to use the extracted external contour as the shape of the estimated structure S2. In this case, the X-ray device100includes an image-capturing device (not shown) provided with image sensors that are constituted by CMOS, CCD or the like. The image-capturing device, which is provided on a part of the ceiling of the housing1(on the internal wall surface on the positive side of Y-axis), captures an image of the external appearance of the measurement object S mounted on the mount stage30along the Y-axis direction that is substantially orthogonal to the direction of projection of the X-ray (Z-axis) and the image-capturing device outputs the generated image signal to the control unit5. The image generation unit53performs known (edge) detection processing and the like to the inputted image signal to extract a contour of the measurement object S on the image signal. Note that the image-capturing device is preferably one that is capable of capturing images over a wide range so that the measurement object S in whole can be captured regardless of the position of the measurement object S that may be varied by the Y-axis movement unit33and the X-axis movement unit34. Alternatively, the image-capturing device may be provided so that it can be moved in synchronization with the movements of the Y-axis movement unit33and the X-axis movement unit34. Note that the shape information is not limited to one that is obtained by the image-capturing device. For instance, the shape information may include information obtained by measuring the measurement object S using a profile projector that projects the optical image of the measurement object S on a screen, or a shape measuring device, which is a contact type three-dimensional measuring device using a touch probe, or which is a non-contact type three-dimensional measuring device, such as a scanning laser probe type or an optical type. (3) The mount stage30on which the measurement object S is mounted is not limited to one that is moved along X-axis direction, Y-axis direction, and Z-axis direction by the X-axis movement unit34, the Y-axis movement unit33, and the Z-axis movement unit35, respectively. In one aspect of the present invention, the mount stage30may be one that does not move along the X-axis, the e Y-axis, and the Z-axis direction but instead causes the X-ray source2and the detector4move along the X-axis direction, the Y-axis direction, and the Z-axis direction, respectively, relative to the measurement object S. In one aspect of the present invention, the X-ray device100may be structured so that the mount stage30does not rotate but the X-ray source2and the detector4rotate about the rotation axis Yr instead of the mount stage30that rotates about the rotation axis Yr. Note that in the above-mentioned embodiments, for instance, in Step S3ofFIG.5, it may be configured that after transmission images are acquired for a plurality of irradiation directions of the X-ray, the differential data D3may be generated using only a part of the transmission images. (4) The function of an interface through which the detection data D1, the design information of the measurement object S, and the measuring conditions are inputted and the function of the image generation unit53may be implemented by a computer. In this case the implementation may be achieved by storing a program for implementing the function of the image generation processing into a computer-readable recording medium and causing a computer system to read in the above-mentioned program relating to image generation that is stored in the recording medium and execute it. Note that the “computer system” as used herein includes hardware such as an OS (Operating System) and peripheral devices. The “computer-readable recording medium” as used herein includes portable recording media such as a flexible disk, a magneto-optical disk, an optical disk, and a memory card and so on, and a storage device such as a hard disk incorporated in the computer system. Furthermore, the “computer-readable recording medium” as used herein may include a thing that can hold a program for a short time and dynamically such as a communication wire in case a program is transmitted through a network such as an internet or a telephone line, and a thing that holds the program for a certain period of time such as a volatile memory in the inside of the computer system serving as a server or a client in such a case. The program may be one that implements a part of the above-mentioned function or that implements the above-mentioned function in combination with the program that has already been stored in the computer system. In case the above-mentioned program relating to control is applied to a personal computer or the like, it may be provided by a recording medium such as a CD-ROM or data signal such through the internet or the like.FIG.9is a diagram illustrating this situation. A personal computer950receives a program via a CD-ROM953. Also, the personal computer950has a connection function to be connected with a communication line951. A computer952is a server-computer that stores above described program into a recording medium such as a hard disk. A communication line951is a communication line such as the internet, a personal computer communication, or the like, or a dedicated communication line. A computer952reads out the program from the hard disk and transmits the read out program to the personal computer950through the communication line951. That is, it carries the program as data signal with a carrier wave to transmit it through the communication line951. In this manner, the program can be provided as a computer-readable computer program product in one of various forms such as a recording medium having it stored therein or a carrier wave. So far as one or more of the features of the present invention are not damaged, the present invention is not limited to the above-mentioned embodiments and/or variation examples and other forms conceivable within the technical idea of the present invention are included within the scope of the present invention. REFERENCE SIGNS LIST 2. . . X-ray source,4. . . detection device,5. . . control unit,53. . . image generation unit,100. . . X-ray device,600. . . structure manufacturing system,610. . . design device,620. . . molding device,630. . . control system,632. . . inspection unit,640. . . repairing device | 78,228 |
11860112 | DESCRIPTION OF EMBODIMENTS Hereinafter, example embodiments of the present invention will be described with reference to the drawings. Note that, in all of the drawings, a similar component has a similar reference sign, and description thereof will be appropriately omitted. First Example Embodiment “Outline” First, an outline of an inspection system according to the present example embodiment will be described by usingFIG.1. The inspection system is used in any facility that requires a belongings inspection. As the facility, a building, a company, an amusement facility, an airport, a station, and the like are exemplified, which are not limited thereto. The inspection system first performs a belongings inspection at a first inspection site. Then, the inspection system decides a path in which an inspection target person advances after the first inspection site, based on a result of the belongings inspection, and guides the inspection target person to the decided path. For an inspection target person from which a detection target object is not detected, the inspection system decides a path different from a path leading to a secondary inspection, for example, a path leading to the inside of a facility. On the other hand, for an inspection target person from which a detection target object is detected, the inspection system decides whether to perform the secondary inspection at a place (a second inspection site close to the first inspection site) or perform the secondary inspection later, depending on a type of the detection target object being detected, and decides a path in response to the decided content. For example, for an inspection target person from which a detection target object (for example: a gun, a knife, and the like) having a high degree of urgency is detected, the inspection system decides that the secondary inspection is performed at that place, and decides a path leading to the second inspection site. On the other hand, for an inspection target person from which a detection target object (for example: a plastic bottle, and the like) having a low degree of urgency is detected, the inspection system decides that the secondary inspection is performed later, and decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. In this way, the inspection system for deciding a path in which an inspection target person advances after the first inspection site, based on a result of a belongings inspection at the first inspection site, and guiding the inspection target person to the decided path can appropriately and efficiently guide the inspection target person to the path after the first inspection site. As a result, an operation at the first inspection site can be made more efficient, and a waiting time for an inspection at the first inspection site can be shortened. Further, the inspection system capable of sorting an inspection target person into the secondary inspection performed at that place (second inspection site) and the secondary inspection performed later, depending on a type of a detection target object being detected, instead of performing the secondary inspection at that place on all inspection target persons from which a detection target object is detected, can reduce congestion of the secondary inspection at that place. As a result, a waiting time for an inspection at the second inspection site can be shortened. “Hardware Configuration” Next, one example of a hardware configuration of the inspection system will be described.FIG.2is a diagram illustrating a hardware configuration example of the inspection system. Each functional unit included in the inspection system is achieved by any combination of hardware and software concentrating on as a central processing unit (CPU) of any computer, a memory, a program loaded into the memory, a storage unit such as a hard disc that stores the program (that can also store a program downloaded from a storage medium such as a compact disc (CD), a server on the Internet, and the like in addition to a program previously stored at a stage of shipping of an apparatus), and a network connection interface. Then, various modification examples of an achievement method and an apparatus thereof are understood by a person skilled in the art. As illustrated inFIG.2, the inspection system includes a processor1A, a memory2A, an input/output interface3A, a peripheral circuit4A, and a bus5A. Various modules are included in the peripheral circuit4A. The inspection system may not include the peripheral circuit4A. Note that, the inspection system may be formed of a plurality of apparatuses separated physically and/or logically, or may be formed of one apparatus integrated physically and logically. When each apparatus is formed of a plurality of apparatuses separated physically and/or logically, each of the plurality of apparatuses can include the hardware configuration described above. The bus5A is a data transmission path for the processor1A, the memory2A, the peripheral circuit4A, and the input/output interface3A to transmit and receive data to and from one another. The processor1A is an arithmetic processing apparatus such as a CPU and a graphics processing unit (GPU), for example. The memory2A is a memory such as a random access memory (RAM) and a read only memory (ROM), for example. The input/output interface3A includes an interface for acquiring information from an input apparatus, an external apparatus, an external server, an external sensor, an electromagnetic wave transmission/reception apparatus, and the like, an interface for outputting information to an output apparatus, an external apparatus, an external server, an electromagnetic wave transmission/reception apparatus, and the like, and the like. The input apparatus is, for example, a keyboard, a mouse, a microphone, and the like. The output apparatus is, for example, a display, a speaker, a printer, a mailer, and the like. The processor1A can output an instruction to each of modules, and perform an arithmetic operation, based on an arithmetic result of the modules. “Functional Configuration” Next, a functional configuration of the inspection system will be described.FIG.3illustrates one example of a functional block diagram of an inspection system10. As illustrated, the inspection system10includes an electromagnetic wave transmission/reception unit11, a detection unit12, a decision unit13, a guide unit14, and a storage unit15. The electromagnetic wave transmission/reception unit11irradiates an electromagnetic wave (for example: a microwave, a millimeter wave, a terahertz wave, and the like) with a wavelength of equal to or more than 30 micrometers and equal to or less than one meter toward a person present in a predetermined region, and receives a reflection wave. The electromagnetic wave transmission/reception unit11is, for example, a radar. The electromagnetic wave transmission/reception unit11can be formed by adopting various techniques. For example, as in an example inFIG.4, the electromagnetic wave transmission/reception unit11may be a sensor panel formed of a radar in which a plurality of antenna elements are aligned. Note that, a panel is one example, and the electromagnetic wave transmission/reception unit11may be formed by another technique such as a gate through which a person passes, and a booth that a person enters. The detection unit12performs detection processing of detecting an abnormal state, based on a signal of a reflection wave being received by the electromagnetic wave transmission/reception unit11. The abnormal state in the present example embodiment is a state where a person present in a predetermined region carries a preset detection target object. The detection target object is, for example, an object prohibited from being brought, and a gun, a knife, a camera, a plastic bottle, and the like are exemplified, which are not limited thereto. Hereinafter, one example of determination processing by the detection unit12will be described. First Processing Example In the example, the detection unit12creates a transmission image, based on a signal of a reflection wave being received by the electromagnetic wave transmission/reception unit11. Then, the detection unit12detects a detection target object from the transmission image, based on a shape of an object appearing in the transmission image. When the detection target object is detected from the transmission image, it is determined that a person present in a predetermined region carries the detection target object being detected. A feature value of a shape of each of a plurality of detection target objects is generated by an advance preparation, and is stored in the storage unit15. The detection unit12detects a detection target object from a transmission image, based on a comparison result between a feature value of a shape of the detection target object stored in the storage unit15and a feature value of a shape appearing in the transmission image. The processing by the detection unit12may be achieved by using an estimation model generated by machine learning based on training data formed of a transmission image and a label of a plurality of objects, or may be achieved by template matching. Second Processing Example In the example, the detection unit12determines whether a person present in a predetermined region carries a preset detection target object, based on a feature value (reflection wave feature value) appearing in a signal of a reflection wave being received by the electromagnetic wave transmission/reception unit11. When a reflection wave feature value peculiar to a detection target object is detected from a signal of a reflection wave, it is determined that a person present in a predetermined region carries the detection target object being detected. A reflection wave feature value of each of a plurality of objects is generated by an advance preparation, and is stored in the storage unit15. The detection unit12detects a reflection wave feature value peculiar to a detection target object from a signal of a reflection wave, based on a comparison result between a reflection wave feature value of the detection target object stored in the storage unit15and a feature value appearing in the signal of the reflection wave. The processing by the detection unit12may be achieved by using an estimation model generated by machine learning based on training data formed of a signal of a reflection wave and a label of a plurality of objects, or may be achieved by template matching. The decision unit13decides a path in which an inspection target person advances, based on a detection result by the detection unit12. For an inspection target person from which an abnormal state is not detected, i.e., an inspection target person from which a detection target object is not detected, the decision unit13decides a path (another path) different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. On the other hand, for an inspection target person from which an abnormal state is detected, i.e., an inspection target person from which a detection target object is detected, the decision unit13decides whether to perform the secondary inspection at a place or perform the secondary inspection later, depending on a type of the detection target object being detected, and decides a path in response to the decided content. For example, for an inspection target person from which a detection target object (for example: a gun, a knife, and the like) having a high degree of urgency is detected, the decision unit13decides that the secondary inspection is performed at that place, and decides a path (first path) leading to the second inspection site. On the other hand, for an inspection target person from which a detection target object (for example: a plastic bottle, and the like) having a low degree of urgency is detected, the decision unit13decides that the secondary inspection is performed later, and decides a path (another path) different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. In the present example embodiment, as illustrated inFIG.5, at least a part of a detectable detection target object is registered as a first detection target object. For example, a detection target object having a high degree of urgency is registered as the first detection target object. Then, the decision unit13decides that the secondary inspection is performed at that place, for an inspection target person from which the first detection target object is detected, and decides that the secondary inspection is performed later, for an inspection target person from which a detection target object that does not correspond to the first detection target object is detected. The information as illustrated inFIG.5is stored in advance in the storage unit15. The secondary inspection is an inspection different from a primary inspection performed at the first inspection site. A more detailed inspection is performed on an inspection target person determined to carry a detection target object in the primary inspection. The secondary inspection may be, for example, a belongings inspection by a visual inspection, a body touch, and the like by a person in charge, or may be another inspection. The guide unit14performs processing of guiding an inspection target person to a decided path. For example, as illustrated inFIG.4, a gate G may be installed on each of a plurality of paths. Then, the guide unit14may perform processing of opening the gate G on a path in which the inspection target person advances, and closing the gate G on another path. Further, the guide unit14may perform processing of notifying a predetermined person of a path in which an inspection target person advances in addition to or instead of control of the gate G. The notification is achieved via various output apparatuses such as a display, a speaker, a lamp, and a projection apparatus installed at an inspection site. The notified person may be an inspection target person, may be a person who is present at an inspection site and takes charge of managing an inspection, or may be both. Next, one example of a flow of processing of the inspection system10will be described by using a flowchart inFIG.6. First, the inspection system10performs processing of detecting a detection target object from an inspection target person (S10). Specifically, the inspection system10irradiates an inspection target person with an electromagnetic wave having a wavelength of equal to or more than 30 micrometers and equal to or less than one meter, and receives a reflection wave. Then, the inspection system10performs the processing of detecting a detection target object, based on a signal of the reflection wave. Next, the inspection system10decides a path in which the inspection target person advances, based on a detection result in S10(S11). Specifically, for an inspection target person from which a detection target object is not detected, the inspection system10decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Further, for an inspection target person from which the first detection target object (seeFIG.5) being predefined is detected, the inspection system10decides that the secondary inspection is performed at that place, and decides a path leading to the second inspection site. Further, for an inspection target person from which a detection target object different from the first detection target object (seeFIG.5) is detected, the inspection system10decides that the secondary inspection is performed later, and decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Next, the inspection system10performs processing of guiding the inspection target person to the path decided in S11(S12). Specifically, opening and closing of a gate on each path are controlled, and a decided path is notified to an inspection target person and a person in charge of an inspection. Advantageous Effect As described above, the inspection system10for deciding a path in which an inspection target person advances after the first inspection site, based on a result of a belongings inspection at the first inspection site, and guiding the inspection target person to the decided path can appropriately and efficiently guide the inspection target person to the path after the first inspection site. As a result, an operation at the first inspection site can be made more efficient, and a waiting time for an inspection at the first inspection site can be shortened. Further, the inspection system capable of sorting an inspection target person into the secondary inspection performed at that place (second inspection site) and the secondary inspection performed later, depending on a type of a detection target object being detected, instead of performing the secondary inspection at that place on all inspection target persons from which a detection target object is detected, can reduce congestion of the secondary inspection at that place. As a result, a waiting time for an inspection at the second inspection site can be shortened. Second Example Embodiment An inspection system10according to the present example embodiment can dynamically change the first detection target object (seeFIG.5) described in the first example embodiment according to a situation. Details will be described below. As described in the first example embodiment, a decision unit13decides that a secondary inspection is performed at a place, for an inspection target person from which the first detection target object is detected, and decides a path leading to a second inspection site. On the other hand, the decision unit13decides that the secondary inspection is performed later, for an inspection target person from which a detection target object that does not correspond to the first detection target object is detected, and decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Then, the decision unit13changes a detection target object included in the first detection target object according to a situation. The decision unit13can perform any of the following first to third changing processing. —First Changing Processing— In the processing, the decision unit13changes a detection target object included in the first detection target object according to a situation at the second inspection site. The situation at the second inspection site includes at least one of a congestion situation of the secondary inspection performed at the second inspection site, the number of persons in charge of the secondary inspection performed at the second inspection site, and a skill of a person in charge of the secondary inspection performed at the second inspection site (a skill level of the secondary inspection). For example, the first detection target object may be defined in advance for each congestion situation (for example: congested, slightly congested, normal, less crowded, least crowded) of the secondary inspection performed at the second inspection site, and may be stored in a storage unit15. The decision unit13can determine the first detection target object according to the congestion situation of the secondary inspection performed at the second inspection site, based on the information indicating the definition. Note that, the definition is made in such a way that the number of detection target objects included in the first detection target object decreases as the secondary inspection performed at the second inspection site is more congested. A means for determining a congestion situation of the secondary inspection performed at the second inspection site is not particularly limited. For example, a congestion situation may be determined based on the number of persons who are waiting for an inspection by analyzing an image in which the second inspection site is captured and counting the number. In addition, the first detection target object may be defined in advance for each number of persons in charge of the secondary inspection performed at the second inspection site, and may be stored in the storage unit15. The decision unit13can determine the first detection target object according to the number of persons in charge of the secondary inspection performed at the second inspection site, based on the information indicating the definition. Note that, the definition is made in such a way that the number of detection target objects included in the first detection target object decreases as the number of persons in charge decreases. A means for determining the number of persons in charge of the secondary inspection performed at the second inspection site is not particularly limited. For example, the number of persons in charge may be counted by analyzing an image in which the second inspection site is captured. In addition, the number of persons in charge may be determined based on work shift information (information indicating a person in charge of the second inspection site for each date and time) registered in a server, information indicating a person who is working at a point in time of an input to a system installed at the second inspection site, and the like. In addition, the first detection target object may be defined in advance for each skill of a person in charge of the secondary inspection performed at the second inspection site, and may be stored in the storage unit15. The decision unit13determines the first detection target object according to a skill of a person in charge of the secondary inspection performed at the second inspection site, based on the information indicating the definition. Note that, the definition is made in such a way that the number of detection target objects included in the first detection target object increases as the number of persons in charge with a high skill increases. A means for determining a skill of a person in charge of the secondary inspection performed at the second inspection site is not particularly limited. For example, a person in charge of the secondary inspection may be determined based on work shift information (information indicating a person in charge of the second inspection site for each date and time) registered in a server, information indicating a person who is working at a point in time of an input to a system installed at the second inspection site, and the like. Then, a skill of the person in charge of the secondary inspection may be determined by referring to a skill of each person in charge being registered in the server in advance. Note that, herein, an example in which cases are classified by using each of a congestion situation of the secondary inspection performed at the second inspection site, the number of persons in charge of the secondary inspection performed at the second inspection site, and a skill of a person in charge of the secondary inspection performed at the second inspection site, and the first detection target object according to each case is defined in advance is described. In addition, cases may be classified by using a plurality of a congestion situation of the secondary inspection performed at the second inspection site, the number of persons in charge of the secondary inspection performed at the second inspection site, and a skill of a person in charge of the secondary inspection performed at the second inspection site, and the first detection target object according to each case may be defined in advance. As a case in this case, for example, “congested, six or more persons in charge”, “congested, five or less persons in charge”, or the like, and “congested, five or less persons in charge, presence of a person in charge at a skill level or more”, “congested, five or less persons in charge, absence of a person in charge at a skill level 5 or more”, or the like are exemplified. —Second Changing Processing— In the processing, the decision unit13changes a detection target object included in the first detection target object according to at least one of a date and time, a day of a week, weather, a temperature, a congestion situation of a facility in which the inspection system10is installed, and a content of an event performed at the facility. Cases are classified in advance by using at least one of the items, and the first detection target object according to each case is defined and stored in the storage unit15. The decision unit13determines the first detection target object, based on the information about the definition. For example, the definition is made in such a way that the number of detection target objects included in the first detection target object decreases for a case where a facility is congested. On the other hand, the definition is made in such a way that the number of detection target objects included in the first detection target object increases for a case where a facility is not crowded. A means for determining a congestion situation of a facility in which the inspection system10is installed is not particularly limited. For example, the congestion situation may be determined by analyzing an image in which the inside of the facility is captured. Further, a means for determining a content of an event performed at a facility is also not particularly limited. For example, an operator may register the content in advance in the inspection system10. —Third Changing Processing— In the processing, the decision unit13acquires attribute information about an inspection target person, and changes a detection target object included in the first detection target object, based on the attribute information. The attribute information is gender, age, and the like. Cases are classified in advance by using at least one of the items, and the first detection target object according to each case is defined and stored in the storage unit15. The decision unit13determines the first detection target object, based on the information about the definition. The definition is made in such a way that the number of detection target objects included in the first detection target object decreases as a degree of urgency decreases such as a case where an inspection target person is a child. A means for acquiring attribute information about an inspection target person is not particularly limited. For example, gender and age may be estimated by analyzing an image in which an inspection target person is captured. In addition, an input of identification information (such as face information, fingerprint information, voiceprint information, iris information, gait information, and a string of numbers and characters) about an inspection target person may be received via any input apparatus, and attribute information about the inspection target person associated with the acquired identification information may be acquired from a server. In the latter case, the identification information and the attribute information about the inspection target person need to be registered in advance in the server. Another configuration of the inspection system10according to the present example embodiment is similar to that in the first example embodiment. The inspection system10according to the present example embodiment achieves an advantageous effect similar to that in the first example embodiment. Further, the inspection system10according to the present example embodiment can dynamically change, according to a situation, a definition of the first detection target object (seeFIG.5) used for deciding whether to guide to the second inspection site where the secondary inspection is performed. Since a reference for guiding to the second inspection site can be dynamically changed according to a situation, congestion at the inspection site can be effectively reduced, and a waiting time for an inspection at the inspection site can be effectively shortened. Third Example Embodiment An inspection system10according to the present example embodiment includes a means for achieving a secondary inspection performed on an inspection target person decided that the secondary inspection is performed later. FIG.7illustrates one example of a functional block diagram of the inspection system10according to the present example embodiment. As illustrated, the inspection system10includes an electromagnetic wave transmission/reception unit11, a detection unit12, a decision unit13, a guide unit14, a storage unit15, a registration unit16, and a postprocessing unit17. The registration unit16registers, in association with a result of detection, identification information about an inspection target person decided that the secondary inspection is performed later. In the present example embodiment, a facility using the inspection system10includes a plurality of seats provided to a user. The plurality of seats are purchased in advance by a user. A seat purchased by a user A can be used only by the user A. Then, in the present example embodiment, identification information about the seat purchased by the user is used as “identification information about an inspection target person”. In other words, the registration unit16registers, in association with a result of detection, identification information about a seat of an inspection target person decided that the secondary inspection is performed later. At an inspection site, identification information about a seat (seat purchased by an inspection target person) provided to an inspection target person is input to the inspection system by any means. For example, usage of an operation of reading a code (such as a bar code and a two-dimensional code) indicating identification information about a seat via a code reader, an operation of transmitting and receiving information by bringing a portable storage apparatus (such as a smartphone, a cellular phone, a smartwatch, an IC card, and an IC tag) that stores identification information about a seat into a short-range wireless communicable state with (close to) a reader, and the like is exemplified. In addition, an input of identification information (such as face information, fingerprint information, voiceprint information, iris information, gait information, and a string of numbers and characters) about an inspection target person may be received via any input apparatus, and identification information about a seat associated with the acquired identification information may be acquired from a server. In this case, the identification information about the inspection target person and the identification information about the seat need to be registered in advance in the server. FIG.8schematically illustrates one example of information registered by the registration unit16. The information is stored in the storage unit15. In the illustrated example, identification information about a seat and a detection target object being detected are registered in association with each other. Note that, in addition, an inspection target person may be captured at an inspection site. Then, the registration unit16may register an image generated by the capturing in association with identification information about an inspection target person decided that the secondary inspection is performed later. The postprocessing unit17notifies a predetermined person in charge of identification information about a seat provided to an inspection target person decided that the secondary inspection is performed later. For example, the postprocessing unit17notifies a predetermined person in charge of a list as illustrated inFIG.8. The person in charge goes to a seat on the list, and performs the secondary inspection on a person sitting in the seat. The secondary inspection may be performed in the seat, or may be performed after moving to another place. Further, when an image of an inspection target person captured at an inspection site is registered, the postprocessing unit17may provide the image to a predetermined person in charge. The person in charge can confirm, based on the image, whether a person sitting in a seat is the same person as an inspection target person decided that the secondary inspection is performed later. A means for providing the list and an image to a predetermined person in charge is not particularly limited. For example, the list and an image may be transmitted to a preregistered e-mail address, may be displayed on a predetermined screen after a login to an application or a system, or may be acquired by using a push notification function of an application. Next, one example of a flow of processing of the inspection system10will be described by using a flowchart inFIG.9. First, the inspection system10performs processing of detecting a detection target object from an inspection target person (S20). Specifically, the inspection system10irradiates an inspection target person with an electromagnetic wave having a wavelength of equal to or more than 30 micrometers and equal to or less than one meter, and receives a reflection wave. Then, the inspection system10performs the processing of detecting a detection target object, based on a signal of the reflection wave. Next, the inspection system10decides a path in which the inspection target person advances, based on a detection result in S20(S21). Specifically, for an inspection target person from which a detection target object is not detected, the inspection system10decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Further, for an inspection target person from which the first detection target object (seeFIG.5) being predefined is detected, the inspection system10decides that the secondary inspection is performed at that place, and decides a path leading to the second inspection site. Further, for an inspection target person from which a detection target object different from the first detection target object (seeFIG.5) is detected, the inspection system10decides that the secondary inspection is performed later, and decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Next, the inspection system10registers, in association with the result of the detection in S20, identification information about the inspection target person decided in S21that the secondary inspection is performed later (S22). Specifically, the inspection system10acquires identification information about a seat provided to the inspection target person by any means. Then, the inspection system10registers, in association with the result of the detection in S20, the identification information about the seat. Next, the inspection system10performs processing of guiding the inspection target person to the path decided in S21(S22). Specifically, opening and closing of a gate on each path are controlled, and a decided path is notified to an inspection target person and a person in charge of an inspection. Subsequently, the inspection system10performs processing for performing the secondary inspection on the inspection target person decided that the secondary inspection is performed later (S24). Specifically, the inspection system10notifies a predetermined person in charge of the identification information about the seat provided to the inspection target person decided that the secondary inspection is performed later. Another configuration of the inspection system10according to the present example embodiment is similar to that in the first and second example embodiments. The inspection system10according to the present example embodiment achieves an advantageous effect similar to that in the first and second example embodiments. Further, the inspection system10according to the present example embodiment can register a detection result in advance in association with identification information about a seat provided to an inspection target person decided that the secondary inspection is performed later. Then, the inspection target person decided that the secondary inspection is performed later can be tracked based on the identification information about the seat, and the secondary inspection can be performed. Fourth Example Embodiment An inspection system10according to the present example embodiment includes a means for achieving a secondary inspection performed on an inspection target person decided that the secondary inspection is performed later, which is a means different from that in the third example embodiment. FIG.7illustrates one example of a functional block diagram of the inspection system10according to the present example embodiment. As illustrated, the inspection system10includes an electromagnetic wave transmission/reception unit11, a detection unit12, a decision unit13, a guide unit14, a storage unit15, a registration unit16, and a postprocessing unit17. The registration unit16registers, in association with a result of detection, identification information about an inspection target person decided that the secondary inspection is performed later. In the present example embodiment, “identification information about an inspection target person” associated with a result of detection is a feature value of an appearance of the inspection target person. An inspection target person is captured at an inspection site, and a feature value of an appearance of the inspection target person is extracted from an image generated by the capturing. As the feature value of the appearance, a feature value of a face, a feature value of a build, a feature value of clothing, and the like are exemplified, which are not limited thereto. FIG.10schematically illustrates one example of information registered by the registration unit16. The information is stored in the storage unit15. In the illustrated example, a feature value of an appearance of an inspection target person and a detection target object being detected are registered in association with each other. Note that, as illustrated, an image generated by capturing an inspection target person at an inspection site may be registered in association with identification information about an inspection target person decided that the secondary inspection is performed later. The postprocessing unit17detects an inspection target person from an image generated by a capturing apparatus that captures the inside of a facility, based on a feature value of an appearance of an inspection target person decided that the secondary inspection is performed later. Then, the postprocessing unit17notifies a predetermined person in charge of a location in which the inspection target person is detected. At a time of the notification, an image (at least one of the image captured in the facility and an image captured at an inspection site) of the detected inspection target person may be provided to the predetermined person in charge. A means for achieving a notification to a predetermined person in charge is not particularly limited. For example, the notification may be transmitted to a preregistered e-mail address, may be displayed on a predetermined screen after a login to an application or a system, or may be acquired by using a push notification function of an application. Herein, a means for determining a location in which an inspection target person is detected will be described. For example, an installation location and a capturing direction of the capturing apparatus may be fixed. Then, information indicating a capturing area (such as an “area A of a seat on a second floor”, a “room B”, and “in front of a bathroom on a third floor”) of the capturing apparatus may be stored in advance in the storage unit15. In this case, the postprocessing unit17determines, as a location in which an inspection target person is detected, a capturing area of the capturing apparatus that generates an image in which the inspection target person is detected. In addition, a feature value of an appearance of a landmark being a guideline for each of a plurality of locations in a facility may be stored in advance in the storage unit15. In this case, the postprocessing unit17detects the landmark from an image in which an inspection target person is detected, and determines, as a location in which the inspection target person is detected, an installation location of the detected landmark. In this case, the capturing apparatus may capture the inside of the facility while moving in the facility by any movement means. Further, a capturing direction of the capturing apparatus may be changed by any mechanism. A person in charge who receives the notification goes to a notified location, and finds the inspection target person decided that the secondary inspection is performed later. Then, the person in charge performs the secondary inspection on the found inspection target person. The secondary inspection may be performed in the seat, or may be performed after moving to another place. Next, one example of a flow of processing of the inspection system10will be described by using the flowchart inFIG.9. First, the inspection system10performs processing of detecting a detection target object from an inspection target person (S20). Specifically, the inspection system10irradiates an inspection target person with an electromagnetic wave having a wavelength of equal to or more than 30 micrometers and equal to or less than one meter, and receives a reflection wave. Then, the inspection system10performs the processing of detecting a detection target object, based on a signal of the reflection wave. Next, the inspection system10decides a path in which the inspection target person advances, based on a detection result in S20(S21). Specifically, for an inspection target person from which a detection target object is not detected, the inspection system10decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Further, for an inspection target person from which the first detection target object (seeFIG.5) being predefined is detected, the inspection system10decides that the secondary inspection is performed at that place, and decides a path leading to the second inspection site. Further, for an inspection target person from which a detection target object different from the first detection target object (seeFIG.5) is detected, the inspection system10decides that the secondary inspection is performed later, and decides a path different from a path leading to the secondary inspection, for example, a path leading to the inside of a facility. Next, the inspection system10registers, in association with the result of the detection in S20, identification information about the inspection target person decided in S21that the secondary inspection is performed later (S22). Specifically, the inspection target person decided that the secondary inspection is performed later is captured at the inspection site, and an image is generated. The inspection system10analyzes the image, and extracts a feature value of an appearance of the inspection target person decided that the secondary inspection is performed later. Then, the inspection system10registers, in association with the result of the detection in S20, the feature value of the appearance of the inspection target person. Next, the inspection system10performs processing of guiding the inspection target person to the path decided in S21(S22). Specifically, opening and closing of a gate on each path are controlled, and a decided path is notified to an inspection target person and a person in charge of an inspection. Subsequently, the inspection system10performs processing for performing the secondary inspection on the inspection target person decided that the secondary inspection is performed later (S24). Specifically, the inspection system10detects the inspection target person decided that the secondary inspection is performed later from an image generated by a capturing apparatus that captures the inside of a facility, based on the feature value of the appearance of the inspection target person decided that the secondary inspection is performed later, and notifies a predetermined person in charge of a detected location. Another configuration of the inspection system10according to the present example embodiment is similar to that in the first and second example embodiments. The inspection system10according to the present example embodiment achieves an advantageous effect similar to that in the first and second example embodiments. Further, the inspection system10according to the present example embodiment can register a detection result in advance in association with a feature value of an appearance of an inspection target person decided that the secondary inspection is performed later. Then, the inspection target person decided that the secondary inspection is performed later can be tracked based on the feature value of the appearance, and the secondary inspection can be performed. Modification Example First Modification Example In the example embodiments described above, two paths after the first inspection site are prepared in advance, and a subsequent path is decided for each inspection target person according to a detection result at the first inspection site. As a modification example, three or more paths after the first inspection site may be prepared in advance, and a subsequent path may be decided for each inspection target person according to a detection result (whether a detection target object is detected, a type of a detection target object being detected, or the like) at the first inspection site. Second Modification Example The abnormal state in the example embodiments described above is a state where a person present in a predetermined region carries a preset detection target object. Then, the detection unit12detects, from a signal of a reflection wave, abnormal data (a feature value of a detection target object) that are not preferable to be included in the signal of the reflection wave. In the modification example, the detection unit12performs detection processing of referring normal data that are preferable to be included, and detecting an abnormal state (a state different from a state indicated by the normal data) from a signal of a reflection wave. Then, the decision unit13decides whether to perform the secondary inspection at that place or perform the secondary inspection later, based on a content of the detected abnormal state. Third Modification Example In the example embodiments described above, a target object prohibited from being brought is set as a detection target object. Then, a state where a person present in a predetermined region carries a preset detection target object is detected as an abnormal state. In the modification example, a target object needed to be carried by a user is set as a detection target object. For example, a badge of a police officer, an object required to be carried by a person who participates in an event, and the like are a detection target object in the modification example. Then, in the modification example, a state where a person present in a predetermined region does not carry a preset detection target object is detected as an abnormal state. In this case, whether to perform the secondary inspection at that place or perform the secondary inspection later may be decided based on an attribute of a user from which the abnormal state is detected. The attribute of a user may be an attribute estimated from an image, such as gender and age, or may be another attribute. Note that, a target object prohibited from being brought may be set as a detection target object A, and a target object needed to be carried by a user may be set as a detection target object B. In this case, a detection result is classified into a plurality of cases, such as a “case where the detection target object A is detected and the detection target object B is not detected”, a “case where the detection target object B is detected and the detection target object A is not detected”, a “case where both of the detection target object A and the detection target object B are detected”, and a “case where both of the detection target object A and the detection target object B are not detected”. Which case is detected as the abnormal state is a design matter. An advantageous effect similar to that in the example embodiments described above is also achieved in the modification examples. Note that, in the present specification, “acquisition” includes at least any one of “acquisition of data stored in another apparatus or a storage medium by its own apparatus (active acquisition)”, based on a user input or an instruction of a program, such as reception by making a request or an inquiry to another apparatus and reading by accessing to another apparatus or a storage medium, “inputting of data output to its own apparatus from another apparatus (passive acquisition)”, based on a user input or an instruction of a program, such as reception of data to be distributed (transmitted, push-notified, or the like) and acquisition by selection from among received data or received information, and “generation of new data by editing data (such as texting, sorting of data, extraction of a part of data, and change of a file format) and the like, and acquisition of the new data”. A part or the whole of the above-described example embodiment may also be described in supplementary notes below, which is not limited thereto.1. An inspection system, including:an electromagnetic wave transmission/reception means for irradiating an electromagnetic wave having a wavelength of equal to or more than 30 micrometers and equal to or less than one meter, and receiving a reflection wave;a detection means for performing detection processing of detecting an abnormal state, based on a signal of the reflection wave;a decision means for deciding, for an inspection target person from which the abnormal state is detected, whether to perform a secondary inspection at a place or perform a secondary inspection later; anda registration means for registering, in association with a result of the detection processing, identification information about the inspection target person decided that a secondary inspection is performed later.2. The inspection system according to supplementary note 1, whereinthe detection means detects a detection target object, based on a signal of the reflection wave, andthe decision means decides, for the inspection target person from which the detection target object is detected, whether to perform a secondary inspection at that place or perform a secondary inspection later.3. The inspection system according to supplementary note 2, whereinthe decision meansdecides that a secondary inspection is performed at that place, for the inspection target person from which a first detection target object being predetermined is detected, anddecides that a secondary inspection is performed later, for the inspection target person from which the detection target object other than the first detection target object is detected.4. The inspection system according to supplementary note 3, whereinthe decision means changes the detection target object included in the first detection target object according to at least one of a congestion situation of a secondary inspection performed at that place, a number of persons in charge of a secondary inspection performed at that place, and a skill of a person in charge of a secondary inspection performed at that place.5. The inspection system according to supplementary note 3 or 4, whereinthe decision means changes the detection target object included in the first detection target object according to at least one of a date and time, a day of a week, weather, a temperature, a congestion situation of a facility, and a content of an event performed at a facility.6. The inspection system according to any one of supplementary notes 2 to 5, whereinthe decision means acquires attribute information about the inspection target person from which the detection target object is detected, and changes the detection target object included in the first detection target object, based on the attribute information.7. The inspection system according to any one of supplementary notes 1 to 6, whereinidentification information about the inspection target person is identification information about a seat provided to the inspection target person.8. The inspection system according to supplementary note 7, further includinga postprocessing means for notifying a predetermined person in charge of identification information about a seat provided to the inspection target person decided that a secondary inspection is performed later.9. The inspection system according to any one of supplementary notes 1 to 6, whereinidentification information about the inspection target person is a feature value of an appearance of the inspection target person.10. The inspection system according to supplementary note 9, further includinga postprocessing means for detecting the inspection target person decided that a secondary inspection is performed later from an image generated by a capturing apparatus that captures an inside of a facility, based on a feature value of an appearance of the inspection target person decided that a secondary inspection is performed later, and notifying a predetermined person in charge of a detected location.11. An inspection method, including:by a computer,irradiating an electromagnetic wave having a wavelength of equal to or more than 30 micrometers and equal to or less than one meter, and receiving a reflection wave;performing detection processing of detecting an abnormal state, based on a signal of the reflection wave;deciding, for an inspection target person from which the abnormal state is detected, whether to perform a secondary inspection at a place or perform a secondary inspection later; andregistering, in association with a result of the detection processing, identification information about the inspection target person decided that a secondary inspection is performed later.10Inspection system11Electromagnetic wave transmission/reception unit12Detection unit13Decision unit14Guide unit15Storage unit16Registration unit17Postprocessing unit1A Processor2A Memory3A Input/output interface (I/F)4A Peripheral circuit5A Bus | 54,890 |
11860113 | DETAILED DESCRIPTION OF THE INVENTION The present disclosure may be modified in various ways and implemented by various embodiments, so that specific embodiments are shown in the drawings and will be described in detail. However, the present disclosure is not limited thereto, and the exemplary embodiments can be construed as including all modifications, equivalents, or substitutes in a technical concept and a technical scope of the present disclosure. It will be understood that when an element is referred to as being coupled or connected to another element, it can be directly coupled or connected to the other element or intervening elements may be present therebetween. In contrast, it will be understood that when an element is referred to as being “directly coupled” or “directly connected” to another element, there are no intervening elements present. The terms used in the present specification are merely used to describe particular embodiments, and are not intended to limit the present disclosure. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. In the present specification, it is to be understood that terms such as “including”, “having”, etc. are intended to indicate the existence of the features, numbers, processes, actions, elements, parts, or combinations thereof disclosed in the specification, and are not intended to preclude the possibility that one or more other features, numbers, processes, actions, elements, parts, or combinations thereof may exist or may be added. Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Hereinafter, the present disclosure will be described in more detail with reference to the accompanying drawings. Prior to offering the description, the terms or words used in the present specification and claims should not be interpreted as being limited to typical meanings or dictionary definitions, but should be interpreted as having meanings and concepts relevant to the technical scope of the present disclosure based on the rule according to which an inventor can appropriately define the concept of the term to describe most appropriately the best method he or she knows for carrying out the disclosure. In addition, unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains. In the following description and the accompanying drawings, descriptions of known functions and components that make the gist of the present disclosure unclear will be omitted. The drawings exemplified below are provided as examples so that the idea of the present disclosure can be sufficiently transferred to those skilled in the art to which the present disclosure pertains. Therefore, the present disclosure is not limited to the accompanying drawings and may be embodied in other forms. In addition, the same reference numerals refer to the same elements throughout the specification. It is noted that the same elements in the drawings are denoted by the same reference numerals throughout the drawings, if possible. FIG.1is a conceptual diagram illustrating a tube weld X-ray inspection device according to an embodiment of the present disclosure.FIG.2is a conceptual diagram illustrating a tube weld X-ray inspection device according to another embodiment of the present disclosure.FIG.3is a conceptual diagram illustrating a tube weld X-ray inspection device according to still another embodiment of the present disclosure.FIG.4is a conceptual diagram illustrating a tube weld X-ray inspection device according to yet still another embodiment of the present disclosure. FIG.1is a conceptual diagram illustrating a tube weld X-ray inspection device according to an embodiment of the present disclosure.FIG.1shows that an X-ray source is inserted into a tube so that the tube weld X-ray inspection device according to the embodiment of the present disclosure inspects a welding part located at an inlet side of the tube. As shown inFIG.1, the tube weld X-ray inspection device according to the embodiment of the present disclosure includes an X-ray support100, an X-ray source200, and an IP (Image Plate) fixing part300. The X-ray support100is formed in a length direction so that the X-ray support100is inserted into the tube10. The X-ray support100is for inserting the X-ray source200into the tube10, and any shape capable of being inserted into the tube10may be applied, for example, the X-ray support100is formed in a rod shape. The X-ray source200is provided at a side of the X-ray support100in the length direction thereof, and emits X-rays. The X-ray source200is for being inserted into the tube10and emitting X-rays to the inlet side of the tube10(the direction from which the X-ray source200enters), that is, a welding part30between the tube10and a tube sheet20. As long as X-rays can be emitted to the welding part30located at the inlet side of the tube10, various applications, such as omnidirectional radiation and directional radiation, are possible. The IP fixing part300is for fixing an image plate310in a direction perpendicular to the axis of the length direction of the X-ray support100while being spaced a predetermined distance apart from the X-ray source200. The image plate310absorbs X-rays and stores the X-rays in the form of energy. The image plate310is a plate coated with a photo-stimulable fluorescent material. The image plate310absorbs the X-rays with which the image plate310is irradiated and stores the X-rays in the form of energy, and then when the image plate310is irradiated with irradiation light such as red light, signal light, such as blue light, in a particular color diverges. That is, the image plate310stores information obtained by an X-ray photographing device, in the form of energy in the image plate310. Herein, when the image plate310is irradiated with the irradiation light in a particular color, such as red light, the signal light, such as blue light, diffuses and diverges from the irradiated point in all directions. Herein, the diverging particular color may transfer image information, so is called signal light. The IP fixing part300may be realized in various ways as long as the IP fixing part300can fix the image plate310to a predetermined location. For example, if the image plate310itself can be fixed at a user-desiring location on the axis of the length direction of the X-ray support100, the image plate310itself may be the IP fixing part300. The IP fixing part300may be realized in various ways, for example, the image plate310of a cartridge type is made to be replaceable in a fixed state, and only the image plate310is made to be replaceable. The reason why the IP fixing part300is replaceable is that the image plate310is a consumable material, and in order to enable various applications, such as the size of the image plate310being changed according to the size of the tube. As shown inFIG.1, the IP fixing part300of the tube weld X-ray inspection device according to the embodiment of the present disclosure is movable in the length direction of the X-ray support100along the X-ray support100. This is to adjust the location of the IP fixing part300when necessary, for example, adjusting the location of the IP fixing part300depending on the depth of insertion of the X-ray source200into the tube10, so that more various measurement targets are measured. As shown inFIG.1, the IP fixing part300of the tube weld X-ray inspection device according to the embodiment of the present disclosure enables the image plate310to be attached and detached. The IP fixing part300may be formed in a shape supporting the image plate310from the rear (seeFIG.1). Alternatively, the IP fixing part300may be formed in a shape (a casing shape) surrounding the image plate310(not shown). Herein, it is preferable that the portion surrounding the front of the image plate310(the X-ray source200side) is made of a material, for example, a transparent material, which does not seriously interrupt the progress of X-rays. As shown inFIG.1, the image plate310of the tube weld X-ray inspection device according to the embodiment of the present disclosure is formed in a shape of a plate with a hollow center. The image plate310may have a hollow so that the axis portion of the length direction of the X-ray support100is inserted through the hollow portion of the image plate310and the image plate310is fixed. This is to facilitate the movement along the X-ray support100because the image plate310is a consumable material, and also to store the X-rays that have passed through the welding part30of the tube10, in the form of energy in the image plate310formed in a single integrated piece. When the image plate310is not formed as a single integrated piece and several attached plates are used as the image plate310, it is undesirable because image loss or image distortion may occur at the seam portion. Therefore, it is preferable that the image plate310is formed in a shape corresponding to the outer diameter of the tube and that the hollow in the image plate310is formed in a shape corresponding to the outer diameter of the X-ray support100. For example, when the outer diameter of the tube is circular and the outer diameter of the X-ray support100is circular, the image plate310is formed in a donut shape. FIG.2is a conceptual diagram illustrating a tube weld X-ray inspection device according to another embodiment of the present disclosure.FIG.2shows the embodiment in which a reader, an initialization module, or both a reader and an initialization module are added to the form inFIG.1. As shown inFIG.2, the tube weld X-ray inspection device according to the embodiment of the present disclosure may include a reader800that irradiates the image plate310with irradiation light, and receives signal light diverging from the image plate310to convert the signal light into information in the form of an image. The reader800irradiates the image plate310with irradiation light required to obtain image information stored in the image plate310, a light receiving element receives the signal light diverging from the image plate310, and the light receiving element converts the received signal light into an electrical signal to generate image information. When the irradiation light and the signal light have different wavelength ranges, a filter that passes only the signal light is provided between the image plate310and the light receiving element. For example, as the irradiation light, infrared light or light in a red light wavelength range may be used, and as the signal light, ultraviolet light or light in a blue light wavelength range may be used. The signal light diverging from the image plate310laminated with a barium-based mixture and resin is blue light, so when a blue color filter is used, the signal light passes through the filter, and the irradiation light and the ambient light coming from outside are blocked by the filter, thereby reducing image distortion caused by the irradiation light or the ambient light other than the signal light in obtaining an image. The filter is for obtaining light, mainly, excitation light. When the signal light is blue light and a blue color filter is used, excitation light in a wavelength range from ultraviolet light to blue light (about 500 nm) or lower is absorbed and irradiation light in a higher range is blocked. The light receiving element is at least one selected from the group of a photodiode (PD) or avalanche photodiode (APD), a multi-pixel photon counter (MPPC), and a photomultiplier tube (PMD) that convert signal light into electrical signals. Any other light receiving elements having a function of converting light into an electrical signal may be used. The multi-pixel photon counter (MPPC) is also called a silicon photomultiplier (SiPM), and Geiger-mode avalanche photodiodes are provided in an array. A process for obtaining an X-ray image by the tube weld X-ray inspection device according to the embodiment of the present disclosure is as follows. An image plate is mounted and X-rays are emitted>>the emitted X-rays are stored as energy in the image plate>>the image plate is irradiated with irradiation light (e.g., laser beams) by the reader>>the energy stored in the image plate is emitted as signal light>>the obtained signal light signal is converted into a digital signal by the reader>>through image processing, one digital X-ray image is completed After the X-ray image is completed in that order, the X-ray energy remaining in the image plate is removed by irradiating the image plate with light, so that the image plate is used repeatedly. That is, compared to an analog film that cannot be reused, waste is reduced in terms of environment and cost. The above-described method is called computed radiography (CR). According to the CR, an image plate is reusable, and since information is obtained using X-rays with which the image plate is directly irradiated, if an appropriate imaging process is performed, a clearer image can be obtained than when a film is used. In addition, a darkroom required for an existing film-screen detector is not required, so that an inspector is able to conduct inspection more conveniently. In the tube weld X-ray inspection device according to the embodiment of the present disclosure, as the image plate310is rotated and the reader800is fixed, or as the reader800is rotated and the image plate310is fixed, the reader800obtains information in the form of an image. In the case in which the image plate310is rotated and the reader800is fixed, when the reader800is fixed at the X-ray support100, the X-ray support100is fixed and thus the reader800is fixed, and the image plate310is rotated on the X-ray support100. In the case in which the reader800is rotated and the image plate310is fixed, when the image plate310is fixed at the X-ray support100, the X-ray support100is fixed and thus the image plate310is fixed, and the reader800is rotated around the X-ray support100. The case in which the X-ray support100is fixed has been described above as an example, but the present disclosure is not limited thereto and various applications, such as the X-ray support100being rotatable, are possible. As shown inFIG.2, the reader800of the tube weld X-ray inspection device according to the embodiment of the present disclosure moves to the outside of the region for the image plate310before X-ray photography, and moves to the position at which image information of the image plate310is extractable, after X-ray photography. The region for the image plate310refers to a region in which the X-rays emitted from the X-ray source200reach the image plate310. InFIG.2, the region for the image plate310is an inside region of the dotted line. That is, the outside of the region for the image plate310refers to a region (the outside of the dotted line) (which does not intercept the X-rays) in which the X-rays emitted from the X-ray source200are not interrupted until the X-rays reach the image plate310. The example inFIG.2shows that the reader800is movable only in an upward-downward direction, but the present disclosure is not limited thereto. Various applications are possible as long as the reader800can move so as not to interrupt photographing during X-ray photography, for example, move in an upward-downward direction and then in a forward-backward direction, and can move to the position at which image information of the image plate310is extractable so as to obtain an image after X-ray photography. The example inFIG.2shows that ahead of the image plate310, the reader800moves to the position at which image information of the image plate310is extractable, but the present disclosure is not limited thereto. Various applications are possible as long as the image information can be extracted from the image plate310, for example, behind the image plate310, the reader800moves to the position at which the image information of the image plate310is extractable. As shown inFIG.2, the tube weld X-ray inspection device according to the embodiment of the present disclosure may include an initialization module900that irradiates the image plate310with light to make the image plate310be in a re-photographable state. The initialization module900is for initializing the energy stored in the image plate310, and removes the energy remaining in the image plate310by irradiating the image plate310with light. In the tube weld X-ray inspection device according to the embodiment of the present disclosure, as the image plate310is rotated and the initialization module900is fixed, or as the initialization module900is rotated and the image plate310is fixed, the initialization module900makes the image plate310be in the re-photographable state. In the case in which the image plate310is rotated and the initialization module900is fixed, when the initialization module900is fixed at the X-ray support100, the X-ray support100is fixed and thus the initialization module900is fixed, and the image plate310is rotated on the X-ray support100. In the case in which the initialization module900is rotated and the image plate310is fixed, when the image plate310is fixed at the X-ray support100, the X-ray support100is fixed and thus the image plate310is fixed, and the initialization module900is rotated around the X-ray support100. The case in which the X-ray support100is fixed has been described above as an example, but the present disclosure is not limited thereto and various applications, such as the X-ray support100being rotatable, are possible. As shown inFIG.2, the initialization module900of the tube weld X-ray inspection device according to the embodiment of the present disclosure moves to the outside of the region for the image plate310before X-ray photography, and moves to the position at which the image plate310can be made to be in the re-photographable state after X-ray photography. The region for the image plate310refers to a region in which the X-rays emitted from the X-ray source200reach the image plate310. InFIG.2, the region for the image plate310is an inside region of the dotted line. That is, the outside of the region for the image plate310refers to a region (the outside of the dotted line) (which does not intercept the X-rays) in which the X-rays emitted from the X-ray source200are not interrupted until the X-rays reach the image plate310. The example inFIG.2shows that the initialization module900is movable only in an upward-downward direction, but the present disclosure is not limited thereto. Various applications are possible as long as the initialization module900can move so as not to interrupt photographing during X-ray photography, for example, move in an upward-downward direction and then in a forward-backward direction, and can move to the position at which the image plate310can be made to be in the re-photographable state so as to initialize the image plate310. The example inFIG.2shows that ahead of the image plate310, the initialization module900moves to the position at which the image plate310can be made to be in the re-photographable state, but the present disclosure is not limited thereto. Various applications are possible as long as the image plate310can be made to be in the re-photographable state, for example, behind the image plate310, the initialization module900moves to the position at which the image plate310can be made to be in the re-photographable state. FIG.3is a conceptual diagram illustrating a tube weld X-ray inspection device according to an embodiment of the present disclosure.FIG.3shows that an X-ray source is inserted into a tube so that the tube weld X-ray inspection device according to the embodiment of the present disclosure inspects a welding part located at an inlet side of the tube. As shown inFIG.3, the tube weld X-ray inspection device according to the embodiment of the present disclosure includes an X-ray support100, an X-ray source200, and IP fixing parts300. The X-ray support100is formed in a length direction so that the X-ray support100is inserted into the tube10. The X-ray support100is for inserting the X-ray source200into the tube, and any shape capable of being inserted into the tube may be applied, for example, the X-ray support100is formed in a rod shape. The X-ray source200is provided at a side of the X-ray support100in the length direction thereof, and emits X-rays. The X-ray source200is for being inserted into the tube and emitting X-rays to the inlet side of the tube (the direction from which the X-ray source200enters), that is, a welding part30between the tube10and a tube sheet20. As long as X-rays can be emitted to the welding part30located at the inlet side of the tube10, various applications, such as omnidirectional radiation and directional radiation, are possible. The IP fixing parts300are for fixing respective image plates310in a direction perpendicular to the axis of the length direction of the X-ray support100while being spaced respective predetermined distances apart from the X-ray source200. The image plates310absorb X-rays and store the X-rays in the form of energy. A plurality of the IP fixing parts300are provided such that the plurality of the IP fixing parts300are spaced apart from each other by a predetermined distance. Each of the image plates310is a plate coated with a photo-stimulable fluorescent material. The image plates310absorb the X-rays with which the image plates310are irradiated and store the X-rays in the form of energy, and then when the image plates310are irradiated with irradiation light such as red light, signal light, such as blue light, in a particular color diverges. That is, the image plates310store information obtained by an X-ray photographing device, in the form of energy in the image plates310. Herein, when the image plates310are irradiated with the irradiation light in a particular color, such as red light, the signal light, such as blue light, diffuses and diverges from the irradiated points in all directions. Herein, the diverging particular color may transfer image information, so is called signal light. The IP fixing parts300may be realized in various ways as long as the IP fixing parts300can fix the image plates310to predetermined locations. For example, if the image plates310themselves can be fixed at user-desiring locations on the axis of the length direction of the X-ray support100, the image plates310themselves may be the IP fixing parts300. The IP fixing parts300may be realized in various ways, for example, the image plates310of a cartridge type are made to be replaceable in a fixed state, and only the image plates310are made to be replaceable. The reason why the IP fixing parts300are replaceable is that the image plates310are a consumable material, and in order to enable various applications, such as the sizes of the image plates310being changed according to the size of the tube. Providing the plurality of the IP fixing parts300is for more accurate measurement. Herein, it is preferable that the IP fixing parts300are made of a material through which some of the X-rays can pass. The tube weld X-ray inspection device according to the embodiment of the present disclosure can take n (n is a natural number equal to or greater than 2) images simultaneously by performing one X-ray photography, and combines the n images obtained from the n image plates310photographed in that way, so that a three-dimensional image highlighting a problem portion can be obtained. As shown inFIG.3, the IP fixing parts300of the tube weld X-ray inspection device according to the embodiment of the present disclosure are movable in the length direction of the X-ray support100along the X-ray support100. This is to adjust the locations of the IP fixing parts300when necessary, for example, adjusting the locations of the IP fixing parts300depending on the depth of insertion of the X-ray source200into the tube10, so that more various measurement targets are measured. As shown inFIG.3, the IP fixing parts300of the tube weld X-ray inspection device according to the embodiment of the present disclosure enable the image plates310to be attached and detached. The IP fixing parts300may be formed in a shape supporting the respective image plates310from the rear (seeFIG.3). Alternatively, the IP fixing parts300may be formed in a shape (a casing shape) surrounding the respective image plates310(not shown). Herein, it is preferable that the portion surrounding the front of each of the image plates310(the X-ray source200side) is made of a material, for example, a transparent material, which does not seriously interrupt the progress of X-rays. As shown inFIG.3, each of the image plates310of the tube weld X-ray inspection device according to the embodiment of the present disclosure is formed in a shape of a plate with a hollow center. Each image plate310may have a hollow so that the axis portion of the length direction of the X-ray support100is inserted through the hollow portion of each image plate310and each image plate310is fixed. This is to facilitate the movement along the X-ray support100because the image plates310are a consumable material, and also to store the X-rays that have passed through the welding part30of the tube10, in the form of energy in the image plates310each formed in a single integrated piece. When each image plate310is not formed as a single integrated piece and several attached plates are used as each image plate310, it is undesirable because image loss or image distortion may occur at the seam portion. Therefore, it is preferable that each image plate310is formed in a shape corresponding to the outer diameter of the tube and that the hollow in each image plate310is formed in a shape corresponding to the outer diameter of the X-ray support100. For example, when the outer diameter of the tube is circular and the outer diameter of the X-ray support100is circular, the image plates310are formed in a donut shape. FIG.4is a conceptual diagram illustrating a tube weld X-ray inspection device according to yet still another embodiment of the present disclosure.FIG.4shows the embodiment in which readers, initialization modules, or readers and initialization modules are added to the form inFIG.3. As shown inFIG.4, the tube weld X-ray inspection device according to the embodiment of the present disclosure may include readers800that irradiate the respective image plates310with irradiation light, and receive signal light diverging from the image plates310to convert the signal light into information in the form of an image. The readers800irradiate the image plates310with irradiation light required to obtain image information stored in the image plates310, light receiving elements receive the signal light diverging from the image plates310, and the light receiving elements convert the received signal light into electrical signals to generate image information. When the irradiation light and the signal light have different wavelength ranges, filters that pass only the signal light are provided between the image plates310and the light receiving elements. For example, as the irradiation light, infrared light or light in a red light wavelength range may be used, and as the signal light, ultraviolet light or light in a blue light wavelength range may be used. The signal light diverging from the image plates310laminated with a barium-based mixture and resin is blue light, so when blue color filters are used, the signal light passes through the filters, and the irradiation light and the ambient light coming from outside are blocked by the filters, thereby reducing image distortion caused by the irradiation light or the ambient light other than the signal light in obtaining an image. Each of the filters is for obtaining light, mainly, excitation light. When the signal light is blue light and blue color filters are used, excitation light in a wavelength range from ultraviolet if) light to blue light (about 500 nm) or lower is absorbed and irradiation light in a higher range is blocked. The light receiving elements are at least one selected from the group of a photodiode (PD) or avalanche photodiode (APD), a multi-pixel photon counter (MPPC), and a photomultiplier tube (PMD) that convert signal light into electrical signals. Any other light receiving elements having a function of converting light into an electrical signal may be used. The multi-pixel photon counter (MPPC) is also called a silicon photomultiplier (SiPM), and Geiger-mode avalanche photodiodes are provided in an array. A process for obtaining an X-ray image by the tube weld X-ray inspection device according to the embodiment of the present disclosure is as follows. Image plates are mounted and X-rays are emitted>>the emitted X-rays are stored as energy in the image plates>>the image plates are irradiated with irradiation light (e.g., laser beams) by the readers>>the energy stored in the image plates is emitted as signal light>>the obtained signal light signals are converted into digital signals by the readers>>through image processing, one digital X-ray image is completed After the X-ray image is completed in that order, the X-ray energy remaining in the image plates is removed by irradiating the image plates with light, so that the image plates are used repeatedly. That is, compared to an analog film that cannot be reused, waste is reduced in terms of environment and cost. The above-described method is called computed radiography (CR). According to the CR, an image plate is reusable, and since information is obtained using X-rays with which the image plate is directly irradiated, if an appropriate imaging process is performed, a clearer image can be obtained than when a film is used. In addition, a darkroom required for an existing film-screen detector is not required, so that an inspector is able to conduct inspection more conveniently. In the tube weld X-ray inspection device according to the embodiment of the present disclosure, as the image plates310are rotated and the readers800are fixed, or as the readers800are rotated and the image plates310are fixed, the readers800obtain information in the form of an image. In the case in which the image plates310are rotated and the readers800are fixed, when the readers800are fixed at the X-ray support100, the X-ray support100is fixed and thus the readers800are fixed, and the image plates310are rotated on the X-ray support100. In the case in which the readers800are rotated and the image plates310are fixed, when the image plates310are fixed at the X-ray support100, the X-ray support100is fixed and thus the image plates310are fixed, and the readers800are rotated around the X-ray support100. The case in which the X-ray support100is fixed has been described above as an example, but the present disclosure is not limited thereto and various applications, such as the X-ray support100being rotatable, are possible. As shown inFIG.4, the readers800of the tube weld X-ray inspection device according to the embodiment of the present disclosure move to the outside of the region for the image plates310before X-ray photography, and move to the positions at which image information of the image plates310is extractable, after X-ray photography. The region for the image plates310refers to a region in which the X-rays emitted from the X-ray source200reach the image plates310. InFIG.4, the region for the image plates310is an inside region of the dotted line. That is, the outside of the region for the image plates310refers to a region (the outside of the dotted line) (which does not intercept the X-rays) in which the X-rays emitted from the X-ray source200are not interrupted until the X-rays reach the image plates310. The example inFIG.4shows that each reader800is movable only in an upward-downward direction, but the present disclosure is not limited thereto. Various applications are possible as long as each reader800can move so as not to interrupt photographing during X-ray photography, for example, move in an upward-downward direction and then in a forward-backward direction, and can move to the position at which image information of the matched image plate310is extractable so as to obtain an image after X-ray photography. The example inFIG.4shows that ahead of the matched image plate310, each reader800moves to the position at which image information of the matched image plate310is extractable, but the present disclosure is not limited thereto. Various applications are possible as long as the image information can be extracted from the matched image plate310, for example, behind the matched image plate310, each reader800moves to the position at which the image information of the matched image plate310is extractable. As shown inFIG.4, the tube weld X-ray inspection device according to the embodiment of the present disclosure may include initialization modules900that irradiate the respective image plates310with light to make the image plates310be in a re-photographable state. The initialization modules900are for initializing the energy stored in the image plates310, and remove the energy remaining in the image plates310by irradiating the image plates310with light. In the tube weld X-ray inspection device according to the embodiment of the present disclosure, as the image plates310are rotated and the initialization modules900are fixed, or as the initialization modules900are rotated and the image plates310are fixed, the initialization modules900make the image plate310be in the re-photographable state. In the case in which the image plates310are rotated and the initialization modules900are fixed, when the initialization modules900are fixed at the X-ray support100, the X-ray support100is fixed and thus the initialization modules900are fixed, and the image plates310are rotated on the X-ray support100. In the case in which the initialization modules900are rotated and the image plates310are fixed, when the image plates310are fixed at the X-ray support100, the X-ray support100is fixed and thus the image plates310are fixed, and the initialization modules900are rotated around the X-ray support100. The case in which the X-ray support100is fixed has been described above as an example, but the present disclosure is not limited thereto and various applications, such as the X-ray support100being rotatable, are possible. As shown inFIG.4, the initialization modules900of the tube weld X-ray inspection device according to the embodiment of the present disclosure move to the outside of the region for the image plates310before X-ray photography, and move to the positions at which the image plates310can be made to be in the re-photographable state after X-ray photography. The region for the image plates310refers to a region in which the X-rays emitted from the X-ray source200reach the image plates310. InFIG.4, the region for the image plates310is an inside region of the dotted line. That is, the outside of the region for the image plates310refers to a region (the outside of the dotted line) (which does not intercept the X-rays) in which the X-rays emitted from the X-ray source200are not interrupted until the X-rays reach the image plates310. The example inFIG.4shows that each initialization module900is movable only in an upward-downward direction, but the present disclosure is not limited thereto. Various applications are possible as long as each initialization module900can move so as not to interrupt photographing during X-ray photography, for example, move in an upward-downward direction and then in a forward-backward direction, and can move to the position at which the matched image plate310can be made to be in the re-photographable state so as to initialize the matched image plate310. The example inFIG.4shows that ahead of the matched image plate310, each initialization module900moves to the position at which the image plate310can be made to be in the re-photographable state, but the present disclosure is not limited thereto. Various applications are possible as long as the matched image plate310can be made to be in the re-photographable state, for example, behind the matched image plate310, each initialization module900moves to the position at which the matched image plate310can be made to be in the re-photographable state. The present disclosure is not limited to the above-described embodiments and has a wide range of application. Various modifications are possible without departing from the substance of the present disclosure set forth in the accompanying claims. | 37,282 |
11860114 | DETAILED DESCRIPTION OF VARIOUS ASPECTS OF THE DISCLOSURE One tool that may be useful to help interpret a diffraction pattern is the Patterson map. The Patterson map is the inverse Fourier transform of the square of the magnitudes of the diffraction data. Whereas a Fourier transform of magnitudes and phases gives molecular structure, the Fourier transform of the square of the magnitudes gives a map of vectors between each pair of atoms in the structure. This may be a more intuitive starting point than diffraction data. To say that the Patterson map is the inverse Fourier transform of the diffraction magnitudes squared is equivalent to saying that the Patterson map is the original electron density map convolved with its inverse. This is a restatement of the Convolution Theorem: multiplication in Fourier space is equivalent to convolution in real space. In mathematical terms, if the electron density may be expressed in the form: ρxyz=∑b∑k∑ℓF→bkℓe-i2π(bx+ky+ℓz) (see, e.g., kinemage.biochem.duke.edu/teaching/BCH681/2013BCH681/elasticScattering/400.ElasticPattersonFunction.html), then the Patterson function (which generates the Patterson map, when considered over the space of the crystal), may be expressed in the form: Pxyz=∑b∑k∑ℓ❘"\[LeftBracketingBar]"Fbkℓ❘"\[RightBracketingBar]"2e-i2π(bx+ky+ℓz) That is, squaring the diffraction magnitudes in Fourier space, then doing an inverse Fourier transform on the product, is equivalent to doing a convolution of the electron density map with itself, i.e., P(x,y,z)=ρ(x,y,z)⊗ρ(−x,−y,−z). P(x,y,z)may be referred to as the “Patterson map function.” It is noted that the above represents a discrete inverse Fourier transform, which may be implemented in the form of an inverse Fast Fourier Transform (IFFT); this may apply to later references to inverse Fourier transformation in this disclosure. Similarly, when Fourier transformation is discussed in this disclosure, it may be implemented in the form of a Fast Fourier Transform (FFT). So, the problem of going from a Patterson map to atomic coordinates may be thought of as a deconvolution. In this light, it may be seen that the problem of going from Patterson maps to atomic coordinates is, in some ways, comparable to other deconvolution problems that have already been examined with neural networks, such as image sharpening, and image “super-resolution.” Neural networks are a shift from traditional rules-based programming. They learn to solve a problem by example, as opposed to solving a problem by following a comprehensive set of logical operations. A neural network is trained to do a task by showing the network, typically, many thousands of training examples, then seeing if the network can generalize for cases not in the training set. A neural network may typically be composed of highly interconnected nodes, arranged in layers, with weights that multiply the connection strengths between nodes.FIG.1shows a conceptual example of a neural network. A neural network may include an input layer I and an output layer O, with one or more so-called “hidden layers” H disposed between the input layer I and the output layer O. Each of the layers I, O, H comprises nodes. The (weighted) connections may be summed in the nodes, and a non-linearity may be applied to each node's output. During training, the weights may be adjusted so that computed neural network outputs match known outputs from the training set. An important advancement in modern neural network methods was the development of learning by back-propagation of errors, for training the networks. FIG.2shows a conceptual example of training by back-propagation, according to aspects of the present disclosure. Input data from a known input/output data pair of a training set may be applied20as input to the neural network. The input data may then be processed21by the neural network to obtain output data. The output data may then be compared22to the known output of known input/output pair, which may measure the error between the output data and the known output data. The error may then be back-propagated through the neural network to adjust weights and biases within the neural network23; this is how the neural network23“learns,” and there are many known algorithms for implementing this process. The process may be repeated until a predetermined acceptable error is achieved (e.g., but not limited to, a percent error or mean-square error from the positions of the atoms in the known output data, which may, e.g., be based on summations of differences from the positions of the atoms in the known output data, but which is not thus limited (another alternative would be to work directly from the values of the “true” electron density map and the electron density map output from neural network23, which provides the position information)). When neural network layers are “fully connected,” the number of weights and biases in the network can be prohibitively large. In a fully connected layer, each node in the layer is connected to each node in the previous layer. For networks where the input to the network is, say, a high-quality image, and that rough image size is propagated through the network, the number of weights in a fully connected layer is the square of the image size, which may be unworkable. For this reason, the development of convolutional neural networks was critically important to developing effective neural networks for imaging applications. In convolutional neural networks, convolution operations are performed on small portions of a layer's nodes at a time. Convolution “kernels” are typically, but are not limited to, 3-7 pixels per dimension; note that in the present case of three-dimensional molecular structures, this may correspond to three-dimensional convolution kernels of size 27-343 voxels (but again, this is an example range, not intended to be limiting). Many kernels can be used at each layer, but still, the number of weights in the network can be quite small because the number of weights is proportional to the size of the kernel, not the square of the number of pixels in an image. The kernels may be applied across an entire image, one patch at a time (and similarly, across blocks of voxels of a three-dimensional image; various three-dimensional convolution methods are known, and for explanations of three-dimensional convolution, see, e.g., towardsdatascience.com/a-comprehensive-introduction-to-different-types-of-convolutions-in-deep-learning-669281e58215 or www.kaggle.com/shivamb/3d-convolutions-understanding-use-case)). As a result, the kernel weights may be effectively shared across the image. Convolutional neural networks have enabled neural networks to be built with a relatively small number of weights at each layer of the neural network, and a large number of layers. This type of network architecture was another important advancement in the deep-learning revolution. Convolutional neural networks may include input and output layers, along with one or more “hidden layers,” similar to multi-layer perceptrons (seeFIG.1). However, the one or more hidden layers of a convolutional neural network may include at least one convolution layer, as described in the preceding paragraph, and may also include other types of layers, known in the art, such as pooling layers, rectified linear unit (ReLU) layers, fully-connected layers and/or loss layers. Each layer of the neural network may have multiple output volumes/images, which may be denoted as “channels.” These channels may correspond to the operation of different convolution kernels on the previous layer's outputs. Convolution kernels may be directed to different aspects of the volume/image. As such, the results obtained from applying such kernels may be combined, e.g., at the output layer of the convolutional neural network, to obtain a final volume/image. According to aspects of the present disclosure, the phase problem may be presented to a convolutional neural network as three-dimensional images, with Patterson maps on the input, and simulated electron density maps on the output, which may thus take advantage of the advances in image processing based on convolutional neural networks. That is, one or more convolutional neural networks may be used to, in essence, perform a deconvolution of the Patterson map to obtain atomic structure. In order to do this, the convolutional neural network may be trained on a known training set. For training the convolutional neural network, given the above-described description of Patterson maps, one may take known atomic structures, e.g., in the form of density maps, and may “work backwards” to obtain their corresponding Patterson maps, by convolving them with their inverses, i.e., by using the Patterson map function. The resulting Patterson map/electron density map pairs may form a training set for a convolutional neural network. FIGS.3and4provide representations of how atomic structure data, known or output by a convolutional neural network, according to various aspects of the present disclosure. Because a molecule or other structure composed of atoms may generally be three-dimensional, the output data/known data may thus be represented in three dimensions. In one example, shown inFIG.3, a 3×3×3 cube30is shown. In one example, the output layer of the convolutional neural network may represent an entire n×n×n three-dimensional space in units of 3×3×3 blocks, and the atoms may fall entirely or partially within one or more of the 3×3×3 blocks. It is noted that the three-dimensional space may not necessarily be n×n×n, but may alternatively be of different numbers of voxels in different dimensions. In one non-limiting example, the atoms of the training data atomic structures may lie entirely within a single cube; but this is not necessary. As shown inFIG.3, a 3×3×3 cube may be viewed, in each of the three dimensions, as having three layers of voxels.FIG.4shows a two-dimensional non-limiting representation of one such layer40. An atom41, which may, as noted above, be reflected by an electron density, may be placed (in training data) or may be output (in an output of the convolutional neural network) completely within cube30and layer40. Given that an atom is three-dimensional, in the conceptual example ofFIG.4, the atom may be represented by a sphere41, which may correspond to a circle41in two dimensions, e.g., in a particular layer40of cube30. The numbers inFIG.4may represent a fraction of a given voxel that is occupied by sphere41. FIG.5shows code based on the Keras Platform (available at keras.io) and used in a demonstration of a non-limiting convolutional neural network according to aspects of the present disclosure.FIG.6shows a schematic representation of this non-limiting example of a convolutional neural network. The example convolutional neural network ofFIGS.5and6uses twelve three-dimensional convolution layers (represented by horizontal arrows inFIG.6), using kernels of 5×5×5 voxels or 7×7×7 pixels. Again, this is intended as a non-limiting example. In this example, each layer other than the output layer has a set of twenty output channels. The input volume size used in a demonstration using this neural network example was 40×40×40 voxels. A max-pooling layer (represented by the downward arrow inFIG.6) was used in this example to shrink this down by a factor of eight, to 20×20×20 voxels, and an up-sampling layer (represented by the upward arrow inFIG.6) was used in this example to expand the dimensions back to 40×40×40 voxels. In this example, the first and last layers used 2,500 weights; two layers used 50,000 weights; and eight layers used 137,200 weights. The result, including 221 bias terms (corresponding to the kernels), was 1,202,821 weights. Again, this is a non-limiting example, and the sizes of the datasets, the numbers and types of layers, etc., may vary in other examples. Returning now toFIG.2, training of the convolutional neural network may require judicious choice of the training set so that the network will be properly trained and will be able to generalize to Patterson maps not included in the training set. In particular, it may generally be necessary to ensure that the network is not trained based on conflicting data. Namely, a given Patterson map of the training set cannot correspond to two or more different atomic output structures. One property of a Patterson map is that it is translation-invariant, i.e., a set of atomic coordinates has the same Patterson map regardless of its position in space. This may give rise to the potential for having a training set with multiple training cases having the same Patterson map but different translations of the atoms for different output cases. To address this, translational freedom may be removed by translating the set of atoms used as desired outputs so that the average atom position is at a center point of the output map (e.g., by summing the respective positional coordinates and dividing each by the number of atoms). A second property of a Patterson map is that it is invariant with respect to centrosymmetric inversion. This means that a set of atoms treated as a desired output may give rise to the same Patterson map as its centrosymmetry-related atoms. To address this ambiguity, the network may be trained using both sets of atoms simultaneously.FIGS.7A-7Cshow a non-limiting example of such a case.FIG.7Ashows a collection of ten, non-overlapping atoms.FIG.7Bshows a centrosymmetric inversion of the ten, non-overlapping atoms ofFIG.7A. Then, a Patterson map corresponding toFIG.7C, which combines the examples ofFIGS.7A and7B, may be used to train the convolutional neural network. That is, one may train the convolutional neural network to output a combined set of atoms that correspond to two centrosymmetrically-related sets of atoms for a given Patterson map. Note that, in contrast withFIG.4, when dealing with simultaneous sets of centrosymmetrically-related atoms, the densities may be scaled for each set, such that the maximum density per set is 0.5, instead of 1.0 (as inFIG.4), and therefore, the maximum density in any one voxel is still limited to 1.0. Further note that, even though the two sets of centrosymmetrically-related atoms may be “tangled” with each other, according to various techniques presented later in this disclosure, it may be possible to untangle the two sets of atoms and separate them. A third issue in the use of Patterson maps as training data is that the atomic coordinates may not be unique, e.g., regarding vector origins in space. This may be demonstrated by the example ofFIGS.8A-8F. The two sets of three atoms shown inFIGS.8B and8Chave different spatial arrangements, as shown when they are superimposed inFIG.8A. However, if vectors between the atoms are drawn, as shown in the examples ofFIGS.8E and8F, the endpoints may be identical, as demonstrated by the example ofFIG.8D. Hence, a given Patterson map may be produced by such different arrangements of atoms. To address this, distances between atoms may be restricted to less than half an edge length of the “output box” of the convolutional neural network. Here, “output box” refers to a three-dimensional structural constraint within which the output atomic structures may be scaled, and which may be predetermined. The restriction of the distances between atoms to less than half an edge length of the output box may be accomplished by adding sufficient empty space around the atoms. This may be done by conceiving of the arrangement of atoms as being contained within a hypothetical inner box contained within the output box. If the size of the inner box is sufficiently small, then the distance between any pair of atoms in the inner box may be less than half the length of an edge distance of the output box, as shown inFIG.9C. As a result, a given peak of the Patterson map data may result in a vector that originates at the vertex of the output box to which it is closest. An illustrative example of this is shown inFIGS.9A-9B. This may serve to eliminate all but one possible vector origin for each Patterson map data peak (corresponding to an atomic position). With the use of the preceding three techniques together, the maximum possible atomic arrangements for a given Patterson map may be limited to two, and those two may be centrosymmetrically-related arrangements. In view of this, one may use a further technique to identify the two sets of atoms corresponding to the two centrosymmetrically-related arrangements.FIGS.10A and10Bprovide a conceptual flow diagram of an example of such a technique, according to various aspects of the present disclosure. The output density map may include peaks in three-dimensional space, and a collection of these peaks may be selected101. From these peaks, atom positions may be estimated102, which may be done, for example, by averaging a voxel position corresponding to a peak with positions of its neighbors, weighted by the densities of the respective voxels (see discussion ofFIGS.3and4, above). The number of atom positions may then be doubled by adding centrosymmetric positions103to the set of atom positions. A subset of the resulting atom positions may then be selected104. A test density map may then be generated105based on the selected subset of atom positions. A test Patterson map may then be generated106from the test density map. In order to determine how close the test Patterson map and the true Patterson map (i.e., the Patterson map corresponding to the known atomic structure) are, a similarity score between the test Patterson map and the true Patterson map may be calculated107; as noted above, this may be done in a number of ways, for example, by using a mean-square error between the test Patterson map and the true Patterson map, which may be done, e.g., on a voxel-by-voxel basis. The similarity score may be tested, e.g., against a predetermined threshold tolerance, to determine if the test Patterson map is sufficiently close to the true Patterson map108. If such criterion is satisfied, the process may end109. If not, one atomic position of the selected subset of atomic positions may be swapped1010for an atomic position not in the subset. A modified test density map based on this modified subset of atomic positions may then be created1011. A new test Patterson map may then be calculated1012based on the modified subset, and a similarity score between the new test Patterson map and the true Patterson map may be calculated1013. The resulting similarity score may then be compared with the previous similarity score (i.e., the similarity score between the test Patterson map resulting from the originally selected subset and the new test Patterson map, resulting from the modified subset), and if the similarity score is improved, the modified subset of atomic positions may be kept, and the process may branch back to block108to determine if the corresponding test Patterson map is sufficiently close to the true Patterson map. If the similarity score was not improved, the swap of atomic positions that created the modified subset may be undone1015; this may also include reverting back to the previous test density map. At this point, the process may branch back to block1010to create a new modified subset of atomic positions. These latter elements of the process (blocks1010-1015) may serve to “untangle” the combined centrosymmetric atomic structures. As an alternative to comparing the similarity score with a predetermined criterion108, the process may simply be permitted to run for a predetermined sufficiently long time and may accept the resulting split of atoms into two groups, one group being a collection of atoms, the other group being their centrosymmetric counterparts, and each group having a Patterson map that closely matches the target Patterson map, based on a similarity criterion, for example, but not limited to, mean-square error (MSE), absolute difference, etc. Once the convolutional neural network has been trained, it may be used in obtaining atomic coordinates/density maps based on x-ray diffraction data derived from unknown samples.FIG.11shows a block diagram of an example of an apparatus that may be used in doing so, andFIG.12describes an example of a method. An x-ray crystallography apparatus110may operate on an unknown sample, and x-ray diffraction data may be obtained120. The x-ray diffraction data from x-ray crystallography apparatus110may be fed into a squarer111, which may square121the x-ray diffraction pattern elements. The results of the squarer111may be fed into an inverse Fourier transform113(e.g., but not limited to, a dedicated hardware component, a portion of a specialized integrated circuit, a programmed processor, etc.) which may perform an inverse Fourier transform121on the squared x-ray diffraction pattern elements, to thus obtain a Patterson map. The Patterson map may then be fed122to a trained convolutional neural network112, e.g., as described above, which may generate atomic coordinate data, which may be in the form of a density map. The atomic coordinates/density map may be output123. The system ofFIGS.5and6was used in trials to show the validity of the approach according to aspects of the present disclosure. The neural network ofFIGS.5and6was trained for more than 4,000 epochs (where an “epoch” denotes a training pass over all the data in a training batch), cumulatively, over 26 runs (“runs” were simply sets of epochs using the same training parameters; while the training parameters were adjusted, between runs, this is not necessarily needed, and was only considered for this particular example, to test the above processes). Hyperparameters were adjusted for each run. Typically, the training batch size for a run was 3,000 and the validation batch size was 100 (the validation set is a set of non-training data that may be used to monitor the progress of the training process and is separate from the training set; a member of the validation set, like a member of the training set, may contain a known Patterson map and corresponding known density map). The validation batch was constructed at the start and used for the entire run, while the training batch was constructed at the start, then remade every few epochs (i.e., the validation batch was used after each epoch to test if the network was properly training on the training batch). As a result of this training schedule, the training loss exhibited a zigzag character. For clarity, rather than display losses after each epoch, the high frequency zigzags of the training loss were removed by displaying losses every few epochs, just prior to remaking the training data. FIG.13shows the training and validation loss over all training runs (the training loss is represented by the lower curve, and the validation loss is represented by the upper curve). The loss was the mean squared error between the known and inferred density maps. This plot indicates that the neural network generalized to cases not in the training set. First, the validation loss decline was in tandem with the training loss. Secondly, new training cases were made every few epochs, and the starting loss for these cases declined in parallel with the validation loss. In other words, it was evident just by swapping in new training cases that the network was generalizing. Trials were also run comparing the cases of adding centrosymmetrically-related atoms, versus without the centrosymmetrically-related atoms. Results showed that the network trained and generalized much more effectively when training on both original atoms and centrosymmetrically-related atoms simultaneously. The trained convolutional neural network was presented with Patterson maps that had not been used during training, but which were based upon known (synthetic) atomic data, in order to test the neural network.FIGS.14A-14Fshow results for six different Patterson maps. These results reflect that the predicted atomic structures (right-hand sides) are substantially similar to the original data (left-hand sides). While the above discussion focuses on the use of Patterson maps as input to the convolutional neural network, for training and for determining atomic structures for unknown data, the present disclosure is not thus limited. The convolutional neural network may, alternatively, operate on training, test, and unknown data corresponding to magnitudes of x-ray diffraction data or squared magnitudes of x-ray diffraction data (without the inverse Fourier transformation to obtain the Patterson map). In these cases, the apparatus ofFIG.11and the method ofFIG.12may be modified to accommodate these different inputs. In the case of using magnitudes of the x-ray diffraction data, inFIG.11, blocks111and113may be omitted and replaced with a magnitude determining block (not shown), which may take the form of a squarer111followed by a square-root block (not shown); correspondingly, inFIG.12, block121may be omitted and replaced with a block of “determining magnitude,” which, again, may be performed by squaring and performing a square-root. In the case of squared magnitudes, inFIG.11, block113may be omitted, and inFIG.12, the inverse Fourier transform may be omitted from block121. Note that each of these operations, in each case, may be applied to each component of the x-ray diffraction data. Training of the convolutional neural network in these cases may proceed as shown inFIG.2. In these cases, however, the generation of the input/output pairs of the training data may be modified in accordance with the type of input data that the convolutional neural network is intended to accept. That is, one may begin with a known output atomic structure/electron density map, which may include a known atomic structure combined with its centrosymmetrically-related atomic structure, but instead of applying the Patterson function, different processes may be used to generate the corresponding input. For either magnitude or magnitude-squared x-ray diffraction data, one may first apply Fourier transformation to the known electron density map (or equivalently, to its centrosymmetric counterpart). For magnitude x-ray diffraction input data, one may then obtain the magnitudes of the results of the Fourier transformation to form magnitude synthetic x-ray diffraction data. For squared-magnitude x-ray diffraction input data, one may obtain squared magnitudes of the results of the Fourier transformation to form squared-magnitude synthetic x-ray diffraction data. As an aside, one could also obtain Patterson map training data by beginning with the squared-magnitude Fourier transform data and applying inverse Fourier transformation to the squared-magnitude Fourier transform data. It is noted that the operations ofFIGS.10A-10Bmay equivalently use magnitude x-ray diffraction data or magnitude-squared x-ray diffraction data in place of Patterson maps. The convolutional neural network, as discussed above, may be simultaneously trained on an atomic structure, along with its centrosymmetrically-placed atomic structure. As also discussed above, the procedure according toFIGS.10A-10Bmay be used to separate the two atomic structures (original and centrosymmetric). In some example demonstrations, this process was applied to neural network outputs resulting from non-training data.FIG.15shows mean-squared error plots, between Patterson maps computed from the neural network outputs and known Patterson maps, for seven test cases. As shown, mean-square error may improve (decrease) as the process proceeds and settles on a set of atomic positions and its centrosymmetric counterpart (for determining accuracy, the closer of the structure and its centrosymmetric counterpart was compared to the known test case output). The various techniques presented according to various aspects of the present disclosure may be implemented in various ways, including in hardware, software, firmware, or combinations thereof.FIG.16shows an example of a system in which at least portions of various aspects of the present disclosure may be implemented. Such a system may include one or more processors160, which may include, but which are not limited to, central processing units (CPUs), graphical processing units (GPUs), computer systems, etc. The one or more processors160may be communicatively coupled to one or more memory units161, which may store system software and/or software/instructions that may cause the system to implement operations according to various techniques discussed above. Memory unit(s)161may include read-only memory (ROM), random-access memory (RAM), flash memory, programmable memory, magnetic memory (e.g., disk memory, tape memory, etc.), optical memory (e.g., compact disk (CD), digital versatile disk (DVD), etc.), etc. The one or more processor(s)160may also be communicatively coupled to input/output (I/O) devices/interfaces162, which may permit the system, or the portion implemented by the system ofFIG.16, to communicate with other portions of an overall system, other systems, a system operator, etc. I/O162may allow data to be provided to processor(s)160and/or to be output from processor(s)160. Programmable or customized devices, such as, but not limited to, programmable logic arrays (PLAs), application-specific integrated circuits (ASICs), systems-on-a-chip (SOCs), etc., may also be used to implement at least portions of a system according to various aspects of the present disclosure. Various aspects of the disclosure have now been discussed in detail; however, the invention should not be understood as being limited to these aspects. It should also be appreciated that various modifications, adaptations, and alternative embodiments thereof may be made within the scope and spirit of the present invention. | 29,913 |
11860115 | DETAILED DESCRIPTION All the parts illustrated in figures are individually assigned a reference numeral and the corresponding terms of these numbers are listed below.1—Measurement mechanism2—Body3—Vacuum chamber4—First sample5—Second sample6—Piston7—Measurement unit8—Cooler9—Air duct10—Connection element11—Central circle12—Circumscribing circle13—Connection point14—Heater15—pressure gauge The measurement mechanism (1) comprises a body (2); a vacuum chamber (3) which is located on the body (2) and in which a measurement process is performed; a first sample (4) and a second sample (5) between which a heat transfer occurs, which are placed in the vacuum chamber (3) and contact each other; a piston (6) which provides the first sample (4) and the second sample (5) to continuously contact each other; a heater (14) located above the first sample; a measurement unit (7) which contacts the first sample (4) and the second sample (5); and a cooler (8) located below the first sample (4) and the second sample (5). Thanks to the piston (6), the first sample (4) and the second sample (5) continuously contact each other. Therefore, it is provided that the measurement unit (7) is able to measure thermal contact resistances of the first sample (4) and the second sample (5). By performing the measurement process in the vacuum chamber (3), external environment factors do not affect the measurement results. Thus, more accurate measurement results are provided. The measurement mechanism (1), which is the subject matter of the present invention, comprises a piston (6) which provides transmitting the pressure directly onto the first sample (4) and the second sample (5) due to a connection element (10) that comprises thereon a plurality of air ducts (9). Thanks to the connection element (10), the piston (6) is centred on the vacuum chamber (3) and provides power transmission. Due to the air ducts, force of the piston (6) proceeds through the connection element (10) without decreasing. In an embodiment of the invention, the measurement mechanism (1) comprises a connection element (10) which has a conical form. Thus, aerodynamics of the piston (6) is provided. Material to be used is reduced, thereby providing ease-of-production. In an embodiment of the invention, the measurement mechanism (1) comprises a connection element (10) comprising a central circle (11) through which the piston (6) passes, a circumscribing circle (12) which encircles the central circle (11), and air ducts which are located between the central circle (11) and the circumscribing circle (12). The air ducts are located between the central circle (11) and the circumscribing circle (12). The central circle (11) and the circumscribing circle (12) provide increasing the endurance of the connection element (10). In an embodiment of the invention, the measurement mechanism (1) comprises a connection element (10) comprising support walls which are located between the air ducts. Thanks to the support walls, it is provided that endurance of the connection element (10) is increased. The connection element (10) makes high piston (6) powers balanced, thereby providing the piston (6) to be centred. In an embodiment of the invention, the measurement mechanism (1) comprises support walls comprising a slope from the central circle (11) towards the circumscribing circle (12). Due to the fact that the support walls comprise slope, pressure transmitted by the piston (6) through the connection element (10) proceeds by sliding along the support walls. Therefore, movement of the piston (6) is facilitated. In an embodiment of the invention, the measurement mechanism (1) comprises a plurality of connection points (13) which are located on the circumscribing circle (12) and provide fixing the connection element (10) onto the vacuum chamber (3). The connection element (10) is fixed onto the vacuum chamber (3) by means of the connection points. The connection points may be removable or irremovable connections. In an embodiment of the invention, the measurement mechanism (1) comprises a connection element (10) which is made of a stainless steel material. Thus, mechanical strength of the connection element (10) is increased. An increase in the quality perception of the user is provided. In an embodiment of the invention, the measurement mechanism (1) comprises a pressure gauge (15) which is located on the piston (6) and provides measuring a pressure applied by the piston (6). Therefore, the pressure applied by the piston (6) onto the samples is able to be measured, and a more accurate measurement result is obtained. With the present invention, there is achieved a measurement mechanism (1) having a connection element (10) which provides centring the piston (6) by balancing the power transmitted onto the vacuum chamber (3). Therefore, it is provided that the power transmitted by the piston (6) is transmitted directly onto the samples. The efficiency is increased. | 4,979 |
11860116 | DETAILED DESCRIPTION The terms used herein may correspond to words selected in consideration of their functions in presented embodiments, and the meanings of the terms may be construed to be different according to ordinary skill in the art to which the embodiments belong. If defined in detail, the terms may be construed according to the definitions. Unless otherwise defined, the terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiments belong. It will be understood that although the terms “first” and “second,” “side,” “top,” and “bottom or lower” may be used herein to describe various devices, these devices should not be limited by these terms. These terms are only used to distinguish one device from another device, but not used to indicate a particular sequence or number of devices. The semiconductor device may include a semiconductor substrate or a structure in which a plurality of semiconductor substrates are stacked. The semiconductor device may refer to a semiconductor package structure in which a structure in which semiconductor substrates are stacked is packaged. The semiconductor substrate may refer to a semiconductor wafer, a semiconductor die, or a semiconductor chip in which electronic components and devices are integrated. The semiconductor chip may refer to a memory chip in which memory integrated circuits, such as dynamic random access memory (DRAM) circuits, static random access memory (SRAM) circuits, NAND-type flash memory circuits, NOR-type flash memory circuits, magnetic random access memory (MRAM) circuits, resistive random access memory (ReRAM) circuits, ferroelectric random access memory (FeRAM) circuits, or phase change random access memory (PcRAM) are integrated, logic dies or ASIC chips in which logic circuits are integrated in a semiconductor substrate, or processors such as application processors (Aps), graphic processing units (GPUs), central processing units (CPUs) or system-on-chips (SoCs). The semiconductor device may be employed in information communication systems such as mobile phones, electronic systems associated with biotechnology or health care, or wearable electronic systems. The semiconductor device may be applicable to internet of things (IoT). Same reference numerals refer to same devices throughout the specification. Even though a reference numeral might not be mentioned or described with reference to a drawing, the reference numeral may be mentioned or described with reference to another drawing. In addition, even though a reference numeral might not be shown in a drawing, it may be shown in another drawing. FIG.1is a schematic plan view illustrating a semiconductor device10W according to an embodiment of the present disclosure.FIG.1may illustrate a shape in the X-Y plane of the semiconductor device10W before being diced into individual semiconductor chips. Referring toFIG.1, the semiconductor device10W may include a wafer or a semiconductor substrate100in which integrated circuits are integrated. The integrated circuits may include memory devices, such as dynamic random access memory (DRAM) devices or NAND flash memory devices. The semiconductor device10W may be diced into individual semiconductor chips along a dicing line150L. The semiconductor device10W may include chip regions101and a scribe lane region102. The chip region101and the scribe lane region102of the semiconductor device10W may correspond to chip regions101and a scribe lane region102of the semiconductor substrate100. The chip regions101may correspond to regions in which integrated circuits are integrated. The chip regions101may include rectangular or square regions in a plan view. A guard wall101G that protects the integrated circuits may be disposed in an inner edge portion of each of the chip regions101. The guard wall101G may be formed in a shape to extend along a boundary between the chip region101and the scribe lane region102. The scribe lane region102may be a region that surrounds the chip region101. The dicing line150L may be set as a certain portion within the scribe lane region102. The semiconductor device10W may include crack sensors200for detecting cracks. The crack sensors20may be configured to detect cracks that may occur when the semiconductor device10W is diced into semiconductor chips. The crack sensors200may be disposed at locations at which a crack or cracks are likely to occur. The crack sensors200may be disposed within the scribe lane region102. A plurality of crack sensors200may be disposed in the scribe lane region102while being spaced apart from each other. The crack sensors200may be disposed in local regions of the scribe lane region102that is adjacent to rectangular-shaped side of each of the chip regions101. The rectangular-shaped side of each of the chip regions101may correspond to a portion that constitutes a boundary101S between the chip region101and the scribe lane region102. Because the dicing lines150L are located in portions of the scribe lane region102, and the dicing proceeds along the dicing lines150L, a crack or cracks may be preferentially generated in the scribe lane region102where the dicing lines150L are located. As the dicing proceeds along the dicing lines150L, a side surface150S of each of the diced semiconductor chips may be exposed. The stress due to dicing may be relatively concentrated on the side surfaces150S of the semiconductor chips so that cracks may be generated on the side surfaces150S of the semiconductor chips, propagating into the semiconductor chips. In order for the crack sensors200to effectively detect the cracks, the crack sensors200may be disposed in the scribe lane region102that is adjacent to the dicing lines150L. The crack sensors200may be disposed in various places in the scribe lane region102while being arranged to surround the chip regions101. FIG.2is a schematic plan view illustrating a semiconductor device10according to an embodiment of the present disclosure.FIG.2may illustrate the semiconductor device10in the form of an individual semiconductor chip separated from the semiconductor device10W ofFIG.1. Referring toFIGS.1and2, a dicing process may be performed along the dicing lines150L to separate the semiconductor device10in the shape of an individual semiconductor chip from the semiconductor device10W. The semiconductor device10may include crack sensors200that are disposed in the scribe lane region102. The crack sensors200may be disposed in corner portions of the semiconductor device10, that is, portions of the scribe lane region102that is adjacent to the rectangular-shaped corners101C of each of the chip regions101. The corner portions of the semiconductor device10may be portions where the dicing lines150L intersect so that stress may be relatively more concentrated on the corner portions of the semiconductor device10in the dicing process. Accordingly, the cracks may be relatively predominantly generated in the corner portions of the semiconductor device10or the portions of the scribe lane region102that is adjacent to the corners101C of the chip regions101. By disposing the crack sensors200in the portions of the scribe lane region102that is adjacent to the corners101C of the chip regions101, it is possible to more effectively detect the cracks that may be generated in the semiconductor device10. FIG.3is a schematic cross-sectional view illustrating a semiconductor device10according to an embodiment of the present disclosure.FIG.3may illustrate the semiconductor device10in the shape of an individual semiconductor chip separated from the semiconductor device10W ofFIG.1. Referring toFIG.3, the semiconductor device10may include a semiconductor substrate100, a target layer110, and a crack sensor200. The target layer110may refer to a target from which cracks are to be detected. The target layer110may be formed on the semiconductor substrate100and refer to a layer in which cracks may be generated or cracks may propagate. The target layer110may refer to a layer including a plurality of layers. The target layer110may be formed over a scribe lane region102. The target layer110may be formed to extend from the scribe lane region102to a chip region101. The target layer110may include a dielectric material layer. The target layer110may further include a conductive layer or a metal layer. The crack sensor200may be configured to detect the cracks that may occur or propagate in the target layer110. Elements constituting the crack sensor200may be disposed substantially over the scribe lane region102of the semiconductor substrate100. Each of the crack sensor200may include a first conductive pattern270, a second conductive pattern260, a plurality of resistors220,230, and240, a first node N1, and a second node N2. The crack sensor200may further include an additional resistor210. The first conductive pattern270and the second conductive pattern260may be configured as electrodes that substantially face each other with the target layer110that is interposed therebetween. Each of the first conductive pattern270and the second conductive pattern260may include a layer of a conductive material. The conductive material may include metal or a semiconductor material that is doped with a dopant. Each of the first conductive pattern270and the second conductive pattern260may include a metal material, such as aluminum (Al), copper (Cu), or tungsten (W). The first conductive pattern270may be formed to be positioned on a bottom surface1106of the target layer110. The bottom surface1106of the target layer110may be a surface of the target layer110that faces the semiconductor substrate100. Another material layer may be further interposed between the bottom surface1106of the target layer110and the semiconductor substrate100. The second conductive pattern260may be formed to be positioned on a top surface110T of the target layer110. The top surface110T of the target layer110may face the bottom surface1106with the target layer110that is interposed therebetween or may be another surface of the target layer100that is opposite to the bottom surface1106. The first and second nodes N1 and N2 may be nodes that are connected to the first conductive pattern270and the second conductive pattern260, respectively. A resistance meter (not shown) may be used to measure the total resistance of the resistors210,220,230, and240. The resistance meter may include a current-voltage meter that measures a current-voltage (I-V) curve. The first and second nodes N1 and N2 may include contact pads271and261, respectively, to which probes for measuring the resistance contact. The first contact pad271may be disposed on the top surface110T of the target layer110to be used as the first node N1. The second contact pad261may be disposed on the top surface110T of the target layer110to be used as the second node N2. The second contact pad261may include an extended portion or an expanded portion of the second conductive pattern260. The first conductive pattern270may be disposed on the bottom surface1106of the target layer110so that the first resistor210may connect the first conductive pattern270to the first node N1 or may connect the first conductive pattern270to the first contact pad271. The first resistor210may refer to an additional resistor that is connected in series to the resistors220,230, and240of the crack sensor200. The first resistor210may include a conductive via that is configured to substantially penetrate the target layer110to connect the first conductive pattern270and the first contact pad271to each other. The first resistor210may include a conductive wiring or a conductive pattern for connecting the first conductive pattern270and the first contact pad271to each other. The resistors220,230, and240of the crack sensor200may be configured as sensing portions that substantially sense cracks. The resistors220,230, and240of the crack sensor200may be configured to vary the total resistance value of the resistors220,230, and240while being broken by the propagation of the crack. When some or all of the resistors220,230, and240of the crack sensor200are broken while the crack propagates, the total resistance value of the resistors220,230, and240or the total resistance value of the crack sensor200may be changed. The change in total resistance value may indicate whether or not cracks have occurred so that the crack sensor200may be used as a means for detecting whether cracks have occurred. The resistors220,230, and240may be configured to substantially vertically penetrate the target layer110. Each of the resistors220,230, and240may be formed in a shape of a conductive wiring or a shape of a conductive via of which one end is connected to the first conductive pattern270and the other opposite end is connected to the second conductive pattern260. The resistors220,230, and240may be configured to be connected in parallel to each other through the first conductive pattern270and the second conductive pattern260. Because the resistors220,230, and240are connected in parallel to each other, different total resistance values may be detected based on the degree to which the resistors220,230, and240are broken by the cracks. Depending on the degree of variation of the total resistance values, it is possible to determine the degree to which the cracks have propagated. On the other hand, if the resistors220,230, and240are connected in series to each other, even if the degree to which the cracks break the resistors220,230,240is different, the total resistance values may be detected to be substantially the same. The resistors220,230, and240may be disposed sequentially away from the boundary101S over the scribe lane region102of the semiconductor substrate100. The resistors220,230, and240may be disposed to be spaced apart from each other while sequentially moving away from the chip region101over the scribe lane region102. The resistors220,230, and240may be disposed while being sequentially spaced apart from each other away from the boundary101S between the chip region101and the scribe lane region102. The resistors220,230, and240may be disposed between the boundary101S of the chip region101and the scribe lane region102and the diced side surface150S of the semiconductor device10. The resistors220,230, and240may be sequentially disposed from the boundary101S of the chip region101and the scribe lane region102toward the diced side surface150S of the semiconductor device10and may be spaced apart from each other. The diced side surface150S of the semiconductor device10may refer to a side surface of the target layer110and a side surface of the semiconductor substrate100and may be a side surface that faces the boundary101S of the chip region101and the scribe lane region102. The resistors220,230, and240may include the second resistor220, the third resistor230, and the fourth resistor240. More resistors may be further disposed between the second resistor220, the third resistor230, and the fourth resistor240. The first resistor210that is connected to the first node271may be disposed between the boundary101S of the chip region101and the scribe lane region102and the resistors220,230, and240. The first resistor210may be disposed at position P1that is spaced apart from the boundary101S of the chip region101and the scribe lane region102by a certain distance. The first resistor210may be disposed at position P1, which is relatively close to the chip region101. The second resistor220may be disposed at position P2closer to the diced side surface150S of the semiconductor device10than the first resistor210. The second resistor220may be disposed at position P2, farthest from the boundary101S of the chip region101and the scribe lane region102. The third resistor230may be disposed at position P3between the first resistor210and the second resistor220, and the fourth resistor240may be positioned at position P4between the second resistor220and the third resistor230. As described above, the first, third, fourth, and second resistors210,230,240, and220may be sequentially disposed, so that as the cracks progress from the divided side surface150S of the semiconductor device10toward the chip region101, the cracks may sequentially break the second, fourth, third, and first resistors220,240,230, and210in the order of the second, fourth, third, and first resistors220,240,230, and210. According to the degree of breakage of the second, fourth, and third resistors220,240, and230, the total resistance values that are measured through the crack sensor200may be measured as different resistance values. The measured total resistance value may be a factor that indicates which of the second, fourth, and third resistors220,240, and230has been broken due to the crack. In this way, it is possible to identify the position of the broken resistor with the measured total resistance value, and thus, it is possible to identify the position at which the propagation of the crack has progressed. The resistors210,220,230, and240may be configured to provide substantially the same resistance values. The resistors210,220,230, and240may be configured to provide different resistance values. Referring toFIGS.1,2, and3, the resistors210,220,230, and240of the crack sensor200may be disposed on the scribe lane region101of the semiconductor substrate100that is adjacent to the guide wall101G. The resistors210,220,230, and240of the crack sensor200may be disposed on a portion of the scribe lane region101of the semiconductor substrate100that is adjacent to the rectangular-shaped side of the chip region101. The resistors210,220,230, and240of the crack sensor200may be disposed on a portion of the scribe lane region101of the semiconductor substrate100that is adjacent to the rectangular-shaped corner101C of the chip region101. FIG.4is a schematic circuit diagram illustrating resistance components R1, R2, R3, and R4 of the crack sensor200of the semiconductor device10ofFIG.3. Referring toFIGS.3and4, the first resistance component R1 of the first resistor210may be connected in series to the first node N1 of the crack sensor200. The second resistance component R2 of the second resistor220, the third resistance component R3 of the third resistor230, and the fourth resistance component R4 of the fourth resistor240may be connected in parallel to each other to configure a crack sensing unit of the crack sensor200that substantially senses a crack. The second, third, and fourth resistance components R2, R3, and R4 may be connected in parallel to form a first sensing resistance component Rs1, and the first sensing resistance component Rs1 may be connected to the first resistance component R1, in series. The first sensing resistance component Rs1 may have a resistance value that is calculated based on the formula 1/Rs1=1/R2+1/R3+1/R4. A resistance meter290may be connected to the first node N1 and the second node N2 of the crack sensor200to measure a first total resistance value R20 of the crack sensor200. The first total resistance value R20 may have a resistance value that is calculated based on the formula R20=Rs1+R1. As the crack progresses, the second resistor220, the fourth resistor240, and the third resistor230may be sequentially broken so that the parallelly connected sensing resistance component Rs may represent different resistance values according to the number of broken resistors, among the resistors220,240, and230. FIGS.5to11are schematic views illustrating crack sensing operations of the crack sensor200ofFIG.3. FIG.5is a schematic cross-sectional view illustrating a state in which a crack C1of a semiconductor device10A breaks the second resistor220.FIG.6is a schematic circuit diagram20A illustrating resistance components R1, R3, and R4 of the crack sensor200ofFIG.5. Referring toFIGS.5and6, as the crack C1propagates inwardly from the diced side surface150S, the second resistor220that is disposed at position P2that is closest to the side surface150S may be broken. When the second resistor220is broken, a current cannot flow through the second resistor220. As described above, in a state in which the second resistor220is broken by the crack C1, a second sensing resistance component Rs2 may be calculated based on the third resistance component R3 and the fourth resistance component R4. The second sensing resistance component Rs2 may have a resistance value that is calculated based on the formula 1/Rs2=1/R3+1/R4. The second sensing resistance component Rs2 may be different from the first sensing resistance component Rs1 in that the second sensing resistance component Rs2 has a greater resistance value than the first sensing resistance component Rs1 when the second resistor220is not broken. Accordingly, a second total resistance value R20A may represent a greater resistance value than the first total resistance value R20. As such, the sensing resistance components Rs1 and Rs2, in a state in which the crack C1is not generated, may represent different resistance values compared to the sensing resistance components Rs1 and Rs2, in a state in which the second resistor220is broken by the crack C1. Because the measured total resistance values R20 and R20A have different resistance values, it is possible to confirm whether the crack C1has occurred at position P2at which the crack C1has been generated by comparing the total resistance values R20 and R20A. FIG.7is a schematic cross-sectional view illustrating a state in which a crack C2of the semiconductor device10B breaks the fourth resistor240.FIG.8is a schematic circuit diagram20B illustrating the resistance components R1 and R3 of the crack sensor200ofFIG.7. Referring toFIGS.7and8, as the crack C2progresses further inwardly from the diced side surface150S, not only the second resistor220, but also the fourth resistor240, may be broken. The fourth resistor240may be disposed at position P4that is farther from the diced side surface150S than position P2of the second resistor220. The fourth resistor240may be disposed closer to the chip region101or the boundary101S than position P2of the second resistor220. When the second resistor220and the fourth resistor240are broken, a current cannot flow through the second and fourth resistors220and240. As such, in a state in which the second and fourth resistors220and240are broken by the crack C2, the third sensing resistance component Rs3 may be calculated as the third resistance component R3. The third sensing resistance component Rs3 may represent a resistance value that is different from the first sensing resistance component Rs1 in a state in which the second resistor220is not broken. The third sensing resistance component Rs3 may represent a resistance value that is different from the second sensing resistance component Rs2 in a state in which the second resistor220is broken and the fourth resistor240is not broken. The third sensing resistance component Rs3 may have a greater resistance value than the first sensing resistance component Rs1 and the second sensing resistance component Rs2. Accordingly, the third total resistance value R20B may be different from the first total resistance value R20 and the second total resistance value R20A and may represent a greater resistance value than the first total resistance value R20 and the second total resistance value R20A. As described above, the sensing resistance component Rs1 in a state in which the crack C1is not generated, the sensing resistance component Rs2 in a state in which the second resistor220is broken by the crack C1, and the sensing resistance component Rs3 in a state in which the second and fourth resistors220and240are broken by the crack C2may represent different resistance values. Because the measured total resistance values R20, R20A, and R20B have different resistance values, it may be possible to confirm whether the cracks C1and C2have occurred at positions P2or P4at which the cracks C1and C2are generated or advanced, respectively, by comparing the total resistance values R20, R20A, R20B. FIG.9is a schematic cross-sectional view illustrating a state in which a crack C3of a semiconductor device10C breaks the third resistor230.FIG.10is a schematic circuit diagram20C illustrating resistance components of the crack sensor200ofFIG.9. Referring toFIGS.9and10, as the crack C3progresses further inwardly from the diced side surface150S, not only the second resistor220and the fourth resistor240, but also the third resistor230, may be broken. The third resistor230may be disposed at position P3farther from the diced side surface150S than position P4of the fourth resistor240. The third resistor230may be disposed closer to the chip region101or the boundary101S than position P4of the fourth resistor240. When the second resistor220, the fourth resistor240, and the third resistor230are broken, a current cannot flow through the second, fourth, and third resistors220,240,230. Even if the crack C3does not break the first resistor210, because the first resistor210is connected in series with the second, fourth, and third resistors220,240, and230, no current flows even through the first resistor210. As such, in a state in which the second, fourth, and third resistors220,240, and230are broken by the crack C3, the fourth sensing resistor Rs4 might not function as a resistor. Unlike the first, second, and third sensing resistance components Rs1, Rs2, and Rs3, the fourth sensing resistance component Rs4 may represent a substantially insulating state that is not measured as a specific resistance value. Accordingly, the fourth total resistance value R20C may be different from the first, second, and third total resistance values R20, R20A, and R20B and may represent a substantially insulative state. As such, the sensing resistance component Rs1 in a state in which the crack C1is not generated, the sensing resistance component Rs2 in a state in which the second resistor220is broken by the crack C1, the sensing resistance component Rs3 in a state in which the second and fourth resistors220and240are broken by the crack C2, and the sensing resistance components Rs4 in a state in which the second, fourth, and third resistors220,240, and230are broken by the crack C3may represent different resistance values from each other. Because the measured total resistance values R20, R20A, R20B, and R20C have different resistance values, it is possible to confirm whether the cracks C1, C2, and C3have occurred at positions P2, P4, and P3at which the cracks C1, C2, and C3are generated or advanced, respectively, by comparing the total resistance values R20, R20A, R20B, and R20C. FIG.11is a schematic view illustrating current-voltage (I-V) curves showing total resistance values R20, R20A, R20B, and R20C that can be sensed by the crack sensor200ofFIG.3. Referring toFIG.11, in a state in which crack C1is not generated as shown inFIG.3, in a state in which the second resistor220is broken by the crack C1as shown inFIG.5, in a state in which the second and fourth resistors220and240are broken by the crack C2as shown inFIG.7, and in a state in which the second, fourth, and third resistors220,240, and230are broken by the crack C3as shown inFIG.9, the total resistance values R20, R20A, R20B, and R20C may have different resistance values or may be measured with different current-voltage curves. The current-voltage curves representing the total resistance values R20, R20A, R20B, and R20C, shown inFIG.11, may be used as a reference for confirming whether cracks occur and the locations of crack propagation. It is possible to confirm the location where the cracks have progressed along with the occurrence of cracks by comparing the measured value measured through the crack sensor200or the measured current-voltage curve with the current-voltage curves representing the total resistance values R20, R20A, R20B, and R20C, shown inFIG.11. FIG.12is a schematic cross-sectional view illustrating a semiconductor device10D according to another embodiment of the present disclosure. InFIG.12, elements indicated by the same reference numerals as inFIG.3may indicate substantially the same elements. Referring toFIG.12, the semiconductor device10D may include a crack sensor200-1. The crack sensor200-1may include a first conductive pattern270, a second conductive pattern260, a plurality of resistors210-1,220-1,230-1, and240-1. The resistors210-1,220-1,230-1, and240-1may be configured to include conductive vias in different numbers. For example, the first resistor210-1and the third resistor230-1may be configured to include substantially the same number of conductive vias, and the second resistor220-1and the fourth resistor240-1may include a greater number of conductive vias than the third resistor230-1. The fourth resistor240-1may include a smaller number of conductive vias than the second resistor220-1. The resistors220-1,230-1, and240-1may be configured to include different numbers of conductive vias so that the resistors220-1,230-1, and240-1may have different resistance values. Because the resistors220-1,230-1, and240-1have different resistance values, the differences between the total resistance values that can be measured by the crack sensor200-1may become greater according to the degree of crack propagation. Meanwhile, as illustrated inFIG.3, the resistors220,230, and240may be configured to include the same number of conductive vias to be configured to have the same resistance value. FIG.13is a schematic cross-sectional view illustrating a semiconductor device10E according to another embodiment of the present disclosure. InFIG.13, elements indicated by the same reference numerals as inFIG.3may indicate substantially the same elements. Referring toFIG.13, the semiconductor device10E may include a crack sensor200-2and a target layer110-1that is a crack detection target. The target layer110-1may include a plurality of target sub-layers111,112, and113. The second target sub-layer112may be stacked on the first target sub-layer111, and the third target sub-layer113may be stacked on the second target sub-layer112. The crack sensor200-2may include a first conductive pattern270, a second conductive pattern260, and a plurality of resistors210-2,220-2,230-2, and240-2. The first, second, third, and fourth resistors210-2,220-2,230-2, and240-2may include structures in which conductive vias201and conductive lands202are combined. The conductive vias201may be formed in conductive patterns substantially penetrating the target sub-layers111,112, and113. The conductive lands202may be disposed at interfaces between the target sub-layers111,112, and113. The conductive vias201may be connected to the conductive lands202. FIG.14is a schematic cross-sectional view illustrating a semiconductor device10F according to another embodiment of the present disclosure. InFIG.14, elements indicated by the same reference numerals as inFIGS.3and13may indicate substantially the same elements. Referring toFIG.14, the semiconductor device10F may include a crack sensor200-3and a target layer110-1that is a crack detection target. The target layer110-1may include a plurality of target sub-layers111,112, and113. The crack sensor200-3may include a first conductive pattern270, a second conductive pattern260, and a plurality of resistors210-3,220-3,230-3, and240-3. The first, second, third, and fourth resistors210-3,220-3,230-3, and240-3may include structures in which conductive vias201and conductive lands are combined. Some of the conductive lands202-1may be extended to be connected to adjacent resistors. FIG.15is a schematic cross-sectional view illustrating a semiconductor device10G according to another embodiment of the present disclosure. InFIG.15, elements indicated by the same reference numerals as inFIG.3may indicate substantially the same elements. Referring toFIG.15, the semiconductor device10G may include a crack sensor200-4. The crack sensor200-4may include a first conductive pattern270, a second conductive pattern260, and a plurality of resistors210,220,230, and240. The crack sensor200-4may further include a first contact pad271-1that is connected to a first node N1 and a second contact pad261-1that is connected to a second node N2. The first contact pad271-1and the second contact pad261-1may be disposed over a chip region of a semiconductor substrate100. As illustrated inFIG.3, the first contact pad271and the second contact pad261may be disposed over a scribe lane region102of the semiconductor substrate100. The first contact pad271-1and the second contact pad261-1may be disposed over the chip region101of the semiconductor substrate100so that it is possible to overcome the size and location limitations of the first contact pad271-1and the second contact pad261-1, compared to the case in which the first contact pad271and the second contact pad261are disposed over the scribe lane region102of the semiconductor substrate100. The resistors210,220,230, and240and the first and second conductive patterns270and260may be disposed over the scribe lane region102of the semiconductor substrate100, while the first contact pad271-1and the second contact pad261-1are disposed over the chip region101. Accordingly, a first extension portion271-2and a second extension portion261-2that connect the first and conductive pattern270and the second conductive pattern260to the first contact pad271-1and the second contact pad261-1, respectively, may extend from the scribe lane region102to the chip region101. FIG.16is a schematic plan view illustrating a semiconductor device10H according to another embodiment of the present disclosure. InFIG.16, elements indicated by the same reference numerals as inFIG.3may indicate substantially the same elements. Referring toFIG.16, the semiconductor device10H may include a crack sensor200-5. The crack sensor200-5may include a first conductive pattern270-4, a second conductive pattern260-4, and a plurality of resistors210-4,220-4,230-4, and240-4. The crack sensor200-5may further include a first contact pad271-4that is connected to a first node N1 and a second contact pad261-4that is connected to a second node N2. The first resistor210-4that is introduced as an additional resistor may include a conductive via disposed while being spaced apart from the third resistor230-4, which is one of the second, third, and fourth resistors220-4,230-4, and240-4. The third resistor230-4and the first resistor210-4may be disposed side by side along a boundary101S between the chip region101and the scribe lane region102. FIG.17is a block diagram illustrating an electronic system including a memory card7800that employs at least one of the semiconductor packages according to the embodiments. The memory card7800may include a memory7810, such as a nonvolatile memory device, and a memory controller7820. The memory7810and the memory controller7820may store data or read out the stored data. At least one of the memory7810and the memory controller7820may include at least one of the semiconductor packages according to the embodiments. The memory7810may include a nonvolatile memory device to which the technology of the embodiments of the present disclosure is applied. The memory controller7820may control the memory7810such that stored data is read out or data is stored in response to a read/write request from a host7830. FIG.18is a block diagram illustrating an electronic system8710including at least one of the semiconductor packages according to the embodiments. The electronic system8710may include a controller8711, an input/output device8712, and a memory8713. The controller8711, the input/output device8712, and the memory8713may be coupled with one another through a bus8715providing a path through which data move. In an embodiment, the controller8711may include one or more microprocessor, digital signal processor, microcontroller, and/or logic device capable of performing the same functions as these components. The controller8711or the memory8713may include at least one of the semiconductor packages according to the embodiments of the present disclosure. The input/output device8712may include at least one selected among a keypad, a keyboard, a display device, a touchscreen and so forth. The memory8713may be a device for storing data. The memory8713may store data and/or commands to be executed by the controller8711, and the like. The memory8713may include a volatile memory device such as a DRAM and/or a nonvolatile memory device, such as a flash memory. For example, a flash memory may be mounted to an information processing system, such as a mobile terminal or a desktop computer. The flash memory may constitute a solid state disk (SSD). In this case, the electronic system8710may stably store a large amount of data in a flash memory system. The electronic system8710may further include an interface8714configured to transmit and receive data to and from a communication network. The interface8714may be a wired or wireless type. For example, the interface8714may include an antenna or a wired or wireless transceiver. The electronic system8710may be realized as a mobile system, a personal computer, an industrial computer or a logic system performing various functions. For example, the mobile system may be any one of a personal digital assistant (PDA), a portable computer, a tablet computer, a mobile phone, a smart phone, a wireless phone, a laptop computer, a memory card, a digital music system and an information transmission/reception system. If the electronic system8710is an equipment capable of performing wireless communication, the electronic system8710may be used in a communication system by using a technique of CDMA (code division multiple access), GSM (global system for mobile communications), NADC (north American digital cellular), E-TDMA (enhanced-time division multiple access), WCDMA (wideband code division multiple access), CDMA2000, LTE (long term evolution), or Wibro (wireless broadband Internet). The inventive concept has been disclosed in conjunction with some embodiments as described above. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the present disclosure. Accordingly, the embodiments disclosed in the present specification should be considered from not a restrictive standpoint but an illustrative standpoint. The scope of the inventive concept is not limited to the above descriptions but defined by the accompanying claims, and all of distinctive features in the equivalent scope should be construed as being included in the inventive concept. | 38,796 |
11860117 | DETAILED DESCRIPTION Before the present disclosure is described in greater detail with reference to the accompanying drawings and embodiments, it should be noted herein that like elements are denoted by the same reference numerals throughout the disclosure. Referring toFIG.1, a pH sensor according to the first embodiment of the present disclosure is configured to detect a pH of a sample (not shown), and includes a reference electrode1configured to contact the sample, and a glass electrode2inserted into the reference electrode1and also configured to contact the sample. In this embodiment, the sample is a liquid sample. The reference electrode1includes a container unit11, two water absorbing units (12a,12b), and a sensing element13. The container unit11includes a hollow tubular housing111extending in a top-bottom direction, an inner tube112disposed in the housing111and having two opposite open ends, and a cover113covering a top end of the housing111. The inner tube112has a support portion114extending in a same direction as the housing111and having a bottom side configured to face the sample, and an abutment portion115extending radially and outwardly from the bottom side of the support portion114and tightly abutting against an inner surface of the housing111. The water absorbing units (12a,12b) are disposed in the housing111and are connected to each other. The water absorbing unit (12a) has a junction member (121a) abutting against the inner surface of the housing111in proximity to a bottom end thereof, and a water absorbing member (122a) disposed in the inner tube112. The junction member (121a) and the water absorbing member (122a) are in contact with each other at an interface (120a). The abutment portion115of the inner tube112further abuts against a top end of the junction member (121a). The water absorbing unit (12b) has a junction member (121b) disposed on a top end of the water absorbing member (122a), and a water absorbing member (122b) inserted into the housing111. The junction member (121b) and the water absorbing member (122b) are in contact with each other at an interface (120b). The junction member (121b) is further in contact with the top end of the water absorbing member (122a) at an interface (120c). The water absorbing member (122b) has a lower portion located between the inner surface of the housing111and an outer periphery of the inner tube112, and an upper portion located between the inner surface of the housing111and an outer periphery of the junction member (121b). The junction member (121a,121b) of each water absorbing unit (12a,12b) is configured to contact the sample. The water absorbing members (122a,122b) of the water absorbing units (12a,12b) are not in contact with each other. In this embodiment, the junction member (121a,121b) of each water absorbing unit (12a,12b) is a porous structure formed with a plurality of channels for ions to pass through, is usually made of sintered Teflon and potassium chloride, has sufficient acid and alkali resistance, and can be applied to samples having different pH levels. However, in other implementations of this embodiment, as long as a porous structure can be formed and the sample can be introduced as liquid, any type of the junction member (121a,121b) is acceptable. On the other hand, the water absorbing member (122a,122b) of each water absorbing unit (12a,12b) is made by soaking superabsorbent polymer (SAP) in potassium chloride solution. The superabsorbent polymer is polyacrylate or polyacrylamide. Preferably, the powdered superabsorbent polymer is placed in a saturated potassium chloride solution, so that it will absorb the potassium chloride solution and expand into a jelly-like elastomer. After the superabsorbent polymer is saturated and no longer expands in volume, the making of each water absorbing unit (12a,12b) is completed. The sensing element13extends outwardly from the water absorbing unit (12b), and has one end inserted into the water absorbing member (122b). In this embodiment, the sensing element13is a silver/silver chloride (Ag/AgCl) wire that can output an electric potential. However, in other implementations of this embodiment, as long as electrical conductivity can be ensured and potential information can be output, any type of sensing element13is acceptable. The glass electrode2is disposed in the housing111of the reference electrode1, and is inserted into the junction member (121b) and the water absorbing member (122a), and extends out of the junction member (121a) for contact with the sample. In this embodiment, the glass electrode2further has a temperature compensation function. However, since the function of the glass electrode2is well known to those skilled in the art, a detailed description thereof is omitted herein for the sake of brevity. In this embodiment, the inner tube112can be supported inside the housing111through tight abutment of the abutment portion115against the inner surface of the housing111, and the water absorbing members (122a,122b) can be supported through cooperation of the support portion114of the inner tube112with the housing111and the glass electrode2, so that the overall structural strength of the first embodiment can be improved, thereby preventing the water absorbing members (122a,122b) from being squeezed out or deformed due to an external force. The cover113can cover the top end of the housing111to delay evaporation of water contained in the water absorbing members (122a,122b) so as to prolong the service life of the first embodiment. Moreover, because the water absorbing members (122a,122b) of the water absorbing units (12a,12b) are made of elastic and soft superabsorbent polymers, there is no need to make each water absorbing member (122a,122b) into a predetermined size in advance when making the first embodiment, it is only necessary to directly fill a sufficient amount of the superabsorbent polymers into the housing111of the container unit11. The water absorbing members (122a,122b) can close gaps among the housing111, the inner tube112and the glass electrode2by their own elasticity, thereby ensuring tightness between the water absorbing members (122b) and the housing111, between the water absorbing members (122a,122b) and the inner tube112, and between the water absorbing member (122a) and the glass electrode2. As such, the tolerance problem caused by the manufacturing and assembling of the water absorbing members (122a,122b) can be reduced, so that the assembly yield of the first embodiment can be effectively improved, the material cost can be minimized, and the labor cost can be reduced due to convenience of the assembly. In use, the junction member (121a) and the lower portion of the glass electrode2are soaked in the liquid sample, so that anions and water molecules in the liquid sample can pass through the porous junction member (121a) and reach the interface (120a). Next, since there is no gap between the water absorbing members (122b) and the housing111, between the water absorbing members (122a,122b) and the inner tube112, and between the water absorbing member (122a) and the glass electrode2for the anions to pass through, and since the water absorbing members (122a,122b) are in a saturated state that neither absorbs nor allows anions to pass through, the anions of the liquid sample are blocked by the water absorbing member (122a) and stay in the interface (120a). Even if there is a gap between the water absorbing members (122b) and the housing111, between the water absorbing members (122a,122b) and the inner tube112, and between the water absorbing member (122a) and the glass electrode2due to assembly errors, the water absorbing member (122b) can prevent the anions of the liquid sample from contacting the sensing element13, so that the anions stay in the interface (120b). With the interfaces (120a,120b) and the water absorbing members (122a,122b) serving as two lines of defense, the sensing element13is prevented from being contaminated and from failure, so that the service life of the first embodiment can indeed be prolonged. Subsequently, the water molecules of the sample will continue to pass through the interface (120a), the water absorbing member (122a), the interface (120c), the junction member (121b), the interface (120b) and the water absorbing member (122b) in sequence, so that the reference electrode1can provide a stable reference potential. Furthermore, since the superabsorbent polymer has high hydrophilicity and high water retention, and is insoluble in water, the water absorbing members (122a,122b) can retain sufficient water for a long time and not easy to dry up, and can be maintained in an elastic solid state, thereby prolonging the service life and the storage period of the first embodiment. Moreover, since the superabsorbent polymer is electrically neutral and has high absorption, and since the water absorbing members (122a,122b) will not absorb other ions, compared with the prior art, in which the solid potassium chloride has lower potassium chloride content due to poor absorption of the wood blocks and the liquid potassium chloride is easily penetrated by other ions, the water absorbing members (122a,122b) of this disclosure contain large amount of high purity potassium chloride, so that the water absorbing members (122a,122b) have low impedance and fast conveying speed. Therefore, the first embodiment will not be easily affected by other ions, the error in measurement is low, and the reaction speed is fast. It should be noted that the measurement principle of the glass electrode2is the same as that of a conventional pH sensor, and since the measurement principle of the glass electrode2is well known to those skilled in the art, a detailed description thereof is omitted herein for the sake of brevity. Further, the number of the water absorbing unit (12a,12b) is not limited to two, and may be one. Even if the number of the water absorbing unit of the reference electrode1is reduced, the effect of blocking the anions in the sample and outputting a stable reference potential can still be achieved. Referring toFIG.2, the second embodiment of the pH sensor of this disclosure is identical to the first embodiment, and differs in the arrangement of the water absorbing units (12a,12b) of the reference electrode1. To clearly illustrate the arrangement of the water absorbing units (12a,12b) of this embodiment, the glass electrode2is omitted inFIG.2. In this embodiment, the junction members (121a,121b) and the water absorbing members (122a,122b) are stacked in an alternate manner. That is, the water absorbing member (122a) of the water absorbing unit (12a) is disposed on top of the junction member (121a), the junction member (121b) is disposed on top of the water absorbing member (122a), and the water absorbing member (122b) is disposed on top of the junction member (121b). Similarly, the junction member (121a) and the water absorbing member (122a) are in contact with each other at the interface (120a), the junction member (121b) and the water absorbing member (122b) are in contact with each other at the interface (120b), and the junction member (121b) is in contact with the top end of the water absorbing member (122a) at the interface (120c). With the water absorbing members (122a,122b) cooperatively blocking the anions of the sample, the effect of prolonging the service life of the sensing element13can be achieved. In summary, the water absorbing member (122a,122b) of each water absorbing unit (12a,12b) is made by soaking superabsorbent polymer (SAP) in potassium chloride solution. The superabsorbent polymer is polyacrylate or polyacrylamide. Thus, by using the polyacrylate or polyacrylamide superabsorbent polymers to absorb potassium chloride solution and expand to form a jelly-like elastomer, the water absorbing members (122a,122b) of the water absorbing units (12a,12b) are elastic and soft, and can improve the tightness by its own elasticity, so that the water absorbing members (122a,122b) can seal the gaps with the housing111, the inner tube112and the glass electrode2to ensure the tightness with the same and reduce the tolerance problem during making and assembling of this disclosure. Thus, the assembly yield of this disclosure can be effectively improved, and the labor and material costs can be reduced. Simultaneously, because the superabsorbent polymers has the characteristics of high hydrophilicity and high water retention and insoluble in water, the water absorbing members (122a,122b) can maintain an elastic solid state for a long time, thereby prolonging the service life of this disclosure. Finally, because the water absorbing members (122a,122b) no longer absorb other ions, the impedance is low, the conveying speed is fast, the error in measurement is low, and the reaction speed is fast. Therefore, the drawbacks of the reference electrode described in the background can be effectively improved. Furthermore, because the inner tube112can be supported inside the housing111through the tight abutment of the abutment portion115against the inner surface of the housing111, and because the water absorbing members (122a,122b) can be supported through cooperation of the support portion114of the inner tube112with the housing111and the glass electrode2, the water absorbing members (122a,122b) are prevented from being squeezed out or deformed due to an external force, and the sensing element13is prevented from being contaminated and from failure, so that the reference electrode1can provide a stable reference potential, and the water absorbing units (12a,12b) of this disclosure can be stably assembled to the container unit (11). Additionally, by virtue of high water retention of the water absorbing members (122a,122b), and in cooperation with the inner tube112to strengthen the overall structure, the service life of this disclosure can be prolonged. Therefore, the object of this disclosure can indeed be achieved. In the description above, for the purposes of explanation, numerous specific details have been set forth in order to provide a thorough understanding of the embodiments. It will be apparent, however, to one skilled in the art, that one or more other embodiments may be practiced without some of these specific details. It should also be appreciated that reference throughout this specification to “one embodiment,” “an embodiment,” an embodiment with an indication of an ordinal number and so forth means that a particular feature, structure, or characteristic may be included in the practice of the disclosure. It should be further appreciated that in the description, various features are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of various inventive aspects, and that one or more features or specific details from one embodiment may be practiced together with one or more features or specific details from another embodiment, where appropriate, in the practice of the disclosure. While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements. | 15,502 |
11860118 | DETAILED DESCRIPTION The illustrative examples recognize and take into account one or more different considerations. The illustrative examples recognize and take into account that Tuberculosis (TB) caused by thebacillus Mycobacterium tuberculosis(MTB) has been one of the deadliest “big 3” infectious diseases resulting in around 1.5 million deaths annually, particularly in low-income countries. The illustrative examples recognize and take into account that various detection methods targeting MTB DNA have been developed including colorimetry, electrochemistry, fluorescence, and chemiluminescence. However, most of these methods require complicated DNA amplification procedures, sophisticated infrastructure, and well-trained personnel, which significantly lead to the high cost of TB diagnosis and limit their accessibility in low-resource settings. In the illustrative examples, MTB DNA is one of the target DNAs. MTB DNA has been to demonstrate the application of the novel gold nanoparticle aggregation-based photothermal biosensing in genetic analysis using a common thermometer, with no assistance from any DNA amplification process or expensive instruments. Under optimal conditions, quantitative photothermal biosensing of target DNA could be achieved by simply monitoring the temperature changes of gold nanoparticle suspensions using a thermometer. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The terms “about” or “approximately” are defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%. The term “substantially” and its variations are defined to include ranges within 10%, within 5%, within 1%, or within 0.5%. The terms “wt. %,” “vol. %,” or “mol. %” refer to a weight, volume, or molar percentage of a component, respectively, based on the total weight, the total volume, or the total moles of material that includes the component. In a non-limiting example, 10 moles of component in 100 moles of the material is 10 mol. % of component. As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Turning now toFIG.1, an illustration of a block diagram of a biosensing environment is depicted in which an illustrative embodiment may be implemented. Biosensing environment100includes biosensing system102. Biosensing system102is configured to detect and quantify target DNA118. Biosensing system102comprises biosensor104comprising suspension106of hybridization buffer108containing dispersed109gold nanoparticles110, sodium chloride112, and single-stranded DNA probes114configured to undergo DNA hybridization116with target DNA118. Target DNA118takes any desirable form. In some illustrative examples, target DNA118is the DNA of a bacteria. In some illustrative examples, target DNA118isM. tuberculosis. In some illustrative examples, target DNA118isB. pertussis. In some illustrative examples, target DNA118isE. coli. In some illustrative examples, target DNA118is MCF-7 breast cancer cells. In some illustrative examples, target DNA118is a genomic DNA sequence extracted from one ofM. tuberculosisgenomic DNA,B. pertussis, E. coli, breast cancer cells (MCF-7), cancer associated miRNA (miRNA-141), or parasitic infection relatedG. lamblia. Gold nanoparticles110are not functionalized. Gold nanoparticles110may be referred to as “bare” or “unmodified”. Gold nanoparticles110have exposed surfaces of gold. Gold nanoparticles110have any desirable diameter. Gold nanoparticles110have a diameter selected such that gold nanoparticles110will be in suspension. In some illustrative examples, gold nanoparticles110have a diameter selected such that gold nanoparticles110do not precipitate out of solution. In some illustrative examples, it is desirable for gold nanoparticles110to have as large a diameter as possible without gold nanoparticles110precipitating out of suspension106. In some illustrative examples, gold nanoparticles110have a diameter in the range of 5 nm to 300 nm. In some illustrative examples, gold nanoparticles110have a diameter of 20 nm. In some illustrative examples, the concentration of gold nanoparticles110can be from 10 pM to 1 μM. Single-stranded DNA probes114are adsorbed onto dispersed109gold nanoparticles110via van der Waals attraction. The van der Waals attraction is between the exposed bases of single-stranded DNA probes114and the gold surfaces of dispersed109gold nanoparticles110. Single-stranded DNA probes114protect dispersed109gold nanoparticles110from aggregation induced by sodium chloride112. Without single-stranded DNA probes114in biosensor104, sodium chloride112induces aggregation of dispersed109gold nanoparticles110into aggregated gold nanoparticles132. Dispersed109gold nanoparticles110maintain their dispersed109status due to protection by single-stranded DNA probes114. Hybridization buffer108is selected to provide an environment for DNA hybridization116of single-stranded DNA probes114with target DNA118upon introduction of target DNA118. When target DNA118is introduced into biosensor104, target DNA118and single—stranded DNA probes114undergo DNA hybridization116to form double-stranded DNA119. When single-stranded DNA probes114undergo DNA hybridization116, single-stranded DNA probes114no longer protect gold nanoparticles110from aggregation. There is a disparity in adsorption onto gold nanoparticles110between single-stranded DNA probes114and double-stranded DNA, such as double-stranded DNA119. Specifically, single-stranded DNA probes114are able to be adsorbed onto gold nanoparticles110via van der Waals attraction between the exposed bases and the gold surface. Double-stranded DNAs, such as double-stranded DNA119, have little affinity to gold nanoparticles110. Double-stranded DNA119has a stable double-helix geometry that inhibits the exposure of nucleobases and leads to the electrostatic repulsive interaction between gold nanoparticles110and the negatively charged phosphate backbone of double-stranded DNA119. Concentration120of single-stranded DNA probes114in biosensor104is sufficient to protect dispersed109gold nanoparticles110from aggregation induced by sodium chloride112. Concentration120of single-stranded DNA probes114is selected such that suspension106has absorbance and temperature changes substantially similar to a suspension of only gold nanoparticles110in hybridization buffer108. In some illustrative examples, concentration120of single-stranded DNA probes114is selected such that substantially all single-stranded DNA probes114are adsorbed to gold nanoparticles110. In some illustrative examples, concentration120of single-stranded DNA probes114is selected such that few, if any, of single-stranded DNA probes114are free in suspension106. In some illustrative examples, concentration120of single-stranded DNA probes114is 160 nanoMolar. In some illustrative examples, biosensor104comprises concentration120of single-stranded DNA probes114configured to interact with dispersed109gold nanoparticles110. In some illustrative examples, biosensor104comprises concentration120of single-stranded DNA probes114configured to provide an absorbance at 520 nm equivalent to an absorbance at 520 nm of a suspension of dispersed109gold nanoparticles110. In some illustrative examples, biosensor104comprises concentration120of single-stranded DNA probes114configured to provide biosensor104with temperature124increase122in response to beam of near-infrared radiation128equivalent to a temperature increase in response to a beam of near-infrared radiation of a suspension of dispersed109gold nanoparticles110.FIGS.12and13provide a description of selecting concentration120of single-stranded DNA probes114for biosensor104. Analysis of DNA sample121using biosensing system102is based on increase122in temperature124of biosensor104when subjected to irradiation by laser126. After introduction of DNA sample121to biosensor104, laser126sends beam of near-infrared radiation128into biosensor104. Photothermic effect134converts the light energy of beam of near-infrared radiation128into heat energy. A difference in photothermic effect134exists between gold nanoparticles110in different statuses. Dispersed109gold nanoparticles110have a weak photothermic effect134under the irradiation of a near-infrared (NIR) laser, laser126. Increase122in temperature124is minimal when suspension106with dispersed109gold nanoparticles110is under the irradiation of a near-infrared (NIR) laser, laser126. Aggregated gold nanoparticles132have a much stronger photothermic effect134. The aggregation of gold nanoparticles110is mainly due to the adsorption disparity onto gold nanoparticles110between single-stranded DNA and double-stranded DNA (dsDNA) under the presence of sodium chloride112. When target DNA118is introduced to biosensor104, target DNA118undergoes DNA hybridization116with single-stranded DNA probes114. When single-stranded DNA probes114undergo DNA hybridization116with target DNA118, protection of the single-stranded DNA probes114is removed. When single-stranded DNA probes114undergo DNA hybridization116with target DNA118, sodium chloride112is able to access those of gold nanoparticles110that are no longer protected. Following DNA hybridization116, at least some of gold nanoparticles110aggregate upon exposure to sodium chloride112to form aggregated gold nanoparticles132. The quantity of gold nanoparticles110that aggregate to form aggregated gold nanoparticles132is related to the quantity of single-stranded DNA probes114that undergo DNA hybridization116. The quantity of single-stranded DNA probes114that undergo DNA hybridization116is related to the concentration154of target DNA118that is introduced to biosensor104. Increase122in temperature124is related to the quantity of gold nanoparticles110that undergo aggregation to form aggregated gold nanoparticles132. Increase122in temperature124is thus related to the concentration154of target DNA118introduced to biosensor104. Using biosensor104, the presence and the concentration of target DNA118introduced to biosensor104is detected using increase122in temperature124. There is a direct relationship between increase122in temperature124and concentration154of target DNA118. In some illustrative examples, there is a logarithmic relationship between increase122of temperature124with the logarithm of concentration154of target DNA118. Sodium chloride112has concentration130in biosensor104. Concentration130is selected to trigger aggregation of gold nanoparticles110without causing gold nanoparticles110to precipitate out of solution. In some illustrative examples, biosensor104comprises concentration130of sodium chloride112configured to produce a maximum temperature increase due to gold nanoparticle aggregation. In some illustrative examples, biosensor104comprises concentration130of sodium chloride112configured to produce a large temperature increase due to gold nanoparticle aggregation without triggering precipitation of aggregated gold nanoparticles132.FIGS.8and9provide a description of selecting concentration130of sodium chloride112for biosensor104. In some illustrative examples, sodium chloride112has concentration130of 40 milliMolar. Concentration130of sodium chloride112is selected prior to selecting concentration120of single-stranded DNA probes114. Laser126is configured to direct beam of near-infrared radiation128into biosensor104. Beam of near-infrared radiation128is configured to induce photothermal effect134in any aggregated gold nanoparticles132present in biosensor104. Laser126has power136, power density138, and wavelength140. Laser126can have power136in any desirable range to initiate photothermic effect134in gold nanoparticles110. Laser126has power density138sufficient to initiate photothermic effect134in gold nanoparticles110. In some illustrative examples, laser126has power density138of 5.2 W/cm2. Power density138is desirably substantially the same throughout biosensor104. Laser126is configured to provide beam of near-infrared radiation128having wavelength140configured to excite gold nanoparticles110and initiate photothermic effect134. In some illustrative examples, wavelength140is in the near-infrared range. The near-infrared range includes wavelengths in the range of 700 nm-1400 nm. In some illustrative examples, wavelength140is 808 nm. Other aspects of laser126that may be taken into consideration when choosing laser126include portability, commercial availability, cost, use without specialized training, and laser regulations. Taking into account any of these aspects of laser126may provide this biosensing method as part of point-of-care testing (POCT). Aggregation of gold nanoparticles110to form aggregated gold nanoparticles132results in change144in color142of suspension106of biosensor104. Spectrometer146can be used to perform colorimetric detection of change144of color142. However, limit of detection148of spectrometer146is undesirable. It is desirable to have a higher sensitivity than provided by spectrometer146. It is desirable to have a lower limit of detection than limit of detection148provided by spectrometer146. In one illustrative example, limit of detection148was calculated based on three folds standard deviation above the blank (hybridization buffer108) and determined to be 2.0 nM. Additionally, spectrometer146may be at least one of undesirably costly, undesirably bulky, and using trained personnel. Change144in color142is due to aggregation of gold nanoparticles110to form aggregated gold nanoparticles132. Increase122in temperature124is also due to presence of aggregated gold nanoparticles132. Thus, biosensor104can be used to detect target DNA118using DNA hybridization116to form aggregated gold nanoparticles132. Increase122in temperature124is measured after biosensor is irradiated by laser126for a selected time. In some illustrative examples, irradiation using a near-infrared (NIR) laser, laser126, is performed until a heat balance is achieved. A heat balance is achieved at a plateau in increase122in temperature124. The heat balance is achieved between heat generation from photothermal effect134and heat dissipation to the environment. In some illustrative examples, the irradiation time is selected to acquire a stable and sensitive temperature measurement. In some illustrative examples, a laser irradiation time of between 5 minutes and 10 minutes is selected as the irradiation time for biosensing using biosensor104. In some illustrative examples, a laser irradiation time of 8 minutes is selected as the irradiation time for biosensing using biosensor104. Thermometer150is configured to detect temperature changes of suspension106. Thermometer150is used to detect increase122in temperature124. Although thermometer150can be a low-cost thermometer, high sensitivity can be achieved with the low-cost thermometer. Thermometer150can have a greater sensitivity than spectrometer146. For example, thermometer150can have limit of detection152as low as 0.28 nM, about 10-fold lower than limit of detection148in the colorimetric detection method using spectrometer146. Photothermal biosensing using thermometer150in biosensing system102provides improvements over colorimetric detection utilizing spectrometer146. Compared to change144in color142observed by the naked eye, photothermal biosensing based on target DNA118induced aggregation of gold nanoparticles110provides a simple yet reliable platform for the quantitative detection of target DNA118. Most colorimetric methods pose the issue of low detection sensitivity and are not suitable for quantitative detection unless using specialized instruments, such as spectrometer146. Photothermal biosensing using biosensor104and thermometer150in biosensing system102is a low-cost and universal biosensing process. Biosensor104is referred to as universal due to the use of bare gold nanoparticles110without functionalization. The use of bare gold nanoparticles110allows for biosensor104to be easily modified for detecting different types of target DNA118. To change target DNA118detected by biosensor104, gold nanoparticles110can be used with a different set of single-stranded DNA probes configured to hybridize with the new target DNA. Single-stranded DNA probes114are configured to undergo DNA hybridization116with target DNA118. Single-stranded DNA probes114will not undergo DNA hybridization without exposure to target DNA118. The specificity of biosensor104has been confirmed. In one illustrative example, target DNA118isM. tuberculosisgenomic DNA. In one illustrative example, various nucleic acids including genomic DNA sequences extracted fromB. pertussis, E. coli, and breast cancer cells (MCF-7), cancer associated miRNA (miRNA-141), and parasitic infections relatedG. lambliaDNA, were presented in different DNA samples at 3-fold higher concentrations as interfering substances than target DNA118in DNA sample121introduced to biosensor104. UV-vis characterization using absorbances at 650 nm and photothermal biosensing using temperature as readouts were performed on each sample introduced to a biosensor. Only upon the addition of target DNA118does an obvious absorbance at 650 nm appear with a dramatic temperature increase of ˜9° C. Other samples with the addition of ssDNA,B. pertussis, E. coli, and MCF-7 cells extracted genomic DNA, miRNA-141, andG. lambliaDNA had no apparent temperature change as compared to hybridization buffer108as blank. The temperature increase for each of ssDNA,B. pertussis, E. coli, and MCF-7 cells extracted genomic DNA, miRNA-141, andG. lambliaDNA samples were less than 50% of the temperature increase of DNA sample121with target DNA118. More than 200% higher temperature increases were still obtained when detecting target DNA118at a 3-fold lower concentration compared to other nucleic acids. The results demonstrated high specificity of biosensor104in biosensing system102for quantitative genetic analysis even in the presence of higher concentrations of interfering substances. Although specificity is described as being confirmed usingM. tuberculosisgenomic DNA as target DNA118, any desirable DNA can be detected. In some illustrative examples, target DNA118isB. pertussis. In some illustrative examples, target DNA118isE. coli. Target DNA118can be any desirable DNA for detection. Biosensing system102was tested using three pairs of respective single-stranded DNA probes and their complementary targets. For each biosensor, the optimal procedures for determining irradiation time, sodium chloride112concentration130, and single-stranded DNA probes114concentration120. Biosensing system102has been tested usingN. meningitidis, miRNA-141, andG. lambliaas target DNA118. A distinctive increase122(>10° C.) in temperature124was obtained in all three targets, compared with the blank of hybridization buffer108, indicating wide application of biosensing system102as a universal photothermal biosensing platform. Biosensor104is referred to as universal due to the ability to modify biosensor104to detect any desirable target. The use of bare gold nanoparticles110without functionalization allows for biosensor104to be easily modified for detecting different types of target DNA118. To change target detected by biosensor104, gold nanoparticles110can be used with a different set of single-stranded DNA probes configured to hybridize with the new target. The illustration of biosensing system102inFIG.1is not meant to imply physical or architectural limitations to the manner in which an illustrative embodiment may be implemented. Other components in addition to or in place of the ones illustrated may be used. Some components may be unnecessary. Also, the blocks are presented to illustrate some functional components. One or more of these blocks may be combined, divided, or combined and divided into different blocks when implemented in an illustrative embodiment. For example, although spectrometer146is displayed, spectrometer146may not be present in biosensing system102. In some illustrative examples, spectrometer146is only utilized when testing DNA hybridization116of single-stranded DNA probes114. In some illustrative examples, spectrometer146is not present in biosensing system102in point-of-care biosensing applications. As another example, although biosensor104is described as configured to detect target DNA118, biosensor104can be utilized to quantitatively detect a wide range of biochemicals and biological organisms, not solely nucleic acids. In some illustrative examples, biosensor104can use DNA-based aptamers to detect a variety of different chemicals. In some illustrative examples, biosensor104can use DNA-based aptamers to detect at least one of protein biomarkers, microorganisms, cancer cells, or metal ions. Turning now toFIG.2, an illustration of a biosensor is depicted in accordance with an illustrative embodiment. View200depicts biosensor201. Biosensor201is a physical implementation of biosensor104ofFIG.1. In view200of biosensor201, a DNA sample to be tested has not been added to biosensor201. Biosensor201comprises suspension202of hybridization buffer204containing dispersed gold nanoparticles206, sodium chloride208, and single-stranded DNA probes210configured to undergo DNA hybridization with the target DNA. Hybridization buffer204is selected to provide an environment for hybridization of single-stranded DNA probes210with a target DNA upon introduction of the target DNA. Single-stranded DNA probes210are adsorbed onto dispersed gold nanoparticles206via van der Waals attraction. The van der Waals attraction is between the exposed bases of single-stranded DNA probes210and the gold surfaces of dispersed gold nanoparticles206. Single-stranded DNA probes210protect dispersed gold nanoparticles206from aggregation induced by sodium chloride208. Dispersed gold nanoparticles206maintain their dispersed status due to protection by single-stranded DNA probes210. Dispersed gold nanoparticles206have a weak photothermal effect under the irradiation of a near-infrared (NIR) laser. Turning now toFIG.3, an illustration of a biosensor with a DNA sample added that includes a target DNA is depicted in accordance with an illustrative embodiment. In view300of biosensor201, a DNA sample has been added to biosensor201. The DNA sample included target DNA302. Target DNA302and Single-stranded DNA probes210underwent DNA hybridization to form double-stranded DNA304. In view300of biosensor201, gold nanoparticles206are no longer dispersed. Dispersed gold nanoparticles206ofFIG.2have aggregated to form aggregated gold nanoparticles306in view300. The aggregation of gold nanoparticles206is mainly due to the adsorption disparity between single-stranded DNA and double-stranded DNA (dsDNA). An adsorption disparity exists between single-stranded DNA probes210ofFIG.2and double-stranded DNA304. Double-stranded DNA, such as double-stranded DNA304, has little affinity to gold nanoparticles206because of the stable double-helix geometry of double-stranded DNA304. The double-helix geometry inhibits the exposure of nucleobases and leads to the electrostatic repulsive interaction between the negatively charged phosphate backbone and gold nanoparticles206. In the presence of target DNA302, the protection of gold nanoparticles206is damaged due to DNA hybridization. Damaging the protection of gold nanoparticles206causes the aggregation of gold nanoparticles206upon the exposure to sodium chloride208. Aggregated gold nanoparticles306is formed and used as a photothermal agent for photothermal biosensing. As such, an obvious temperature increase can be obtained from biosensor201with aggregated gold nanoparticles306while being irradiated by the NIR laser. Turning now toFIG.4, an illustration of a biosensor with a DNA sample added that does not include a target DNA is depicted in accordance with an illustrative embodiment. In view400of biosensor201, DNA sample402has been added to biosensor201. DNA sample402does not include target DNA302ofFIG.3. Single-stranded DNA probes210are configured to undergo DNA hybridization with target DNA302. Single-stranded DNA probes210will not undergo DNA hybridization without exposure to target DNA302. Single-stranded DNA probes210will not undergo DNA hybridization with DNA sample402. Gold nanoparticles206remain in dispersed state while adding DNA sample402that does not include target DNA302. Gold nanoparticles206in dispersed state cause a negligible temperature increase due to a weak PT effect. As such, a temperature change of biosensor201with gold nanoparticles206in dispersed state would be minimal while being irradiated by near infrared radiation from a laser. Detection of target DNA302can be achieved by recording the temperature change of biosensor201while being irradiated by near infrared radiation from a laser. By recording the temperature change of biosensor201while being irradiated by near infrared radiation, target DNA302can be detected by using a thermometer. For example, using a thermometer, the temperature change of biosensor201depicted inFIG.3would be sufficient to indicate the presence of target DNA302. As another example, using a thermometer, the temperature change of biosensor201depicted inFIG.4is insufficient to indicate the presence of target DNA302. Turning now toFIG.5, an illustration of scanning electron microscopy (SEM) images of different suspensions comprising gold nanoparticles is depicted in accordance with an illustrative embodiment. View500includes scanning electron microscope (SEM) images of four suspensions comprising gold nanoparticles. SEM image502is of gold nanoparticle suspension503. SEM image502is of gold nanoparticles504suspended in hybridization buffer506. Gold nanoparticles504are in a dispersed state in hybridization buffer506. Under laser irradiation, no obvious temperature increase is obtained for gold nanoparticle suspension503. SEM image508is of suspension509comprising gold nanoparticles510, sodium chloride (not visible), and hybridization buffer512. The sodium chloride induces aggregation of gold nanoparticles510to form aggregated gold nanoparticles514. The addition of sodium chloride results in a change of the color of gold nanoparticles510. The color of gold nanoparticles510is changed to blue due to gold nanoparticle aggregation. In addition, an obvious temperature increase is obtained under laser irradiation of suspension509. In this illustrative example, a temperature increase of about 13 degrees Celsius was obtained under laser irradiation. SEM image516is of suspension517comprising gold nanoparticles518, sodium chloride (not visible), single-stranded DNA (not visible), and hybridization buffer520. In suspension517, the presence of single-stranded DNA probes maintains gold nanoparticles518in a dispersed state. The adsorption of single-stranded DNA probes on the surface of gold nanoparticles518protects gold nanoparticles518from aggregation due to the presence of sodium chloride. As gold nanoparticles518are protected from aggregation by the single-stranded DNA probes, suspension517has no obvious changes are observed in UV-vis spectra, SEM image516, and temperature measurement when compared to suspension503. SEM image522is of suspension523comprising gold nanoparticles524, sodium chloride (not visible), single-stranded DNA (not visible), hybridization buffer526, and a DNA sample (not visible). Suspension523is the same as suspension517with a DNA sample introduced. In suspension523, the single-stranded DNA probes are no longer protecting gold nanoparticles518from aggregation. In suspension523, the sodium chloride has induced aggregation of gold nanoparticles524to form aggregated gold nanoparticles528. In the presence of the DNA sample, aggregation of gold nanoparticles524has occurred to form aggregated gold nanoparticles528. Thus, the DNA sample includes the target DNA. Upon introduction of the DNA sample, the hybridization of the single-stranded DNA probes and the target DNA left gold nanoparticles524unprotected from the sodium chloride. After DNA hybridization of the DNA sample and the single-stranded DNA probes, the gold nanoparticles524are aggregated by the sodium chloride. The color of suspension523changed from red to purple/blue and a shoulder peak centered at around 650 nm appeared. The color change of suspension523was primarily due to DNA hybridization and gold nanoparticle aggregation. An obvious temperature increase was recorded in suspension523under laser irradiation. In this illustrative example, the temperature change was ˜11° C. As evidenced by the substantially similar UV-vis spectra, SEM image, and temperature measurement for suspension509and suspension523, biosensing of the DNA sample can be performed using a suspension including single-stranded DNA probes configured to undergo DNA hybridization with the target DNA. A temperature change occurs upon radiating suspension517in the presence of the target DNA. Temperature signals of biosensors including gold nanoparticles, sodium chloride, and single-stranded DNA probes be used as readouts for biosensing. Target DNA-induced gold nanoparticle aggregation can be used for the photothermal biosensing using a thermometer. Turning now toFIG.6, an illustration of a line graph of UV-vis spectra of different suspensions comprising gold nanoparticles is depicted in accordance with an illustrative embodiment. Line graph600displays the UV-vis spectra of the suspensions ofFIG.5. Line graph600includes x-axis602of wavelength in nanometers and y-axis604of absorbance in absorbance units. Line606is of a suspension of only the hybridization buffer. Line608is of a suspension of the hybridization buffer and gold nanoparticles. Line608displays absorbance data of suspension503ofFIG.5. Line608includes absorption peak609at 520 nm. Line610is of a suspension of the hybridization buffer, gold nanoparticles, and sodium chloride. In this illustrative example, the sodium chloride provided was 40 mM (milliMolar). Line610displays absorbance data of suspension509ofFIG.5. Line610displays peak611at 750 nm due to red shift of the SPR peak, which resulted from aggregation of the gold nanoparticles. Line612is of a suspension of the hybridization buffer, gold nanoparticles, sodium chloride, and single-stranded DNA probes. Line612displays absorbance data of suspension517ofFIG.5. Line614is of a suspension of the hybridization buffer, gold nanoparticles, sodium chloride, single-stranded DNA probes, and DNA sample including the target DNA. In this illustrative example, the sodium chloride provided was 40 mM and the target DNA had a concentration of 1600 nM. Line614displays absorbance data of suspension523ofFIG.5. The color of suspension523changed from red to purple/blue. As can be seen in line graph600, shoulder peak615centered at around 650 nm appeared in line614, which was mainly due to DNA hybridization and gold nanoparticle aggregation. As evidenced inFIGS.5and6, the biosensing system can be used to detect a target DNA using temperature detection. In this photothermal biosensing system, different factors can significantly affect the detection performance, such as concentrations of sodium chloride, concentrations of single-stranded DNA probes, and laser irradiation time. Therefore, the factors should be selected to provide desirable levels of detection of the target DNA. FIGS.7-9depict data collected in determining a sodium chloride concentration for a biosensor such as biosensor104ofFIG.1.FIG.10depicts data collected in determining an irradiation time for the photothermal biosensing method.FIGS.11-13depict data collected in determining a concentration of single-stranded DNA probes for a biosensor such as biosensor104ofFIG.1. Turning now toFIG.7, an illustration of a line graph of the UV-vis spectra of suspensions having different sodium chloride concentrations in the gold nanoparticle aggregation-induced photothermal biosensing method is depicted in accordance with an illustrative embodiment. Line graph700has x-axis702of wavelength in nanometers and y-axis704of absorbance in absorbance units. In line graph700different concentrations of sodium chloride were added to suspensions of gold nanoparticles. The concentration of sodium chloride was first optimized because of its key role in causing gold nanoparticle aggregation, resulting in a strong photothermic effect. The gold nanoparticle suspensions were characterized by UV-vis spectroscopy. Line706is the absorbance data of a suspension of only the hybridization buffer. Line706does not have any peaks present. Line708is the absorbance data of a suspension of gold nanoparticles in the hybridization buffer without sodium chloride. Line708has a peak at 520 nm. The peak at 520 nm is indicative of dispersed gold nanoparticles. Line710is the absorbance data of a suspension of gold nanoparticles in the hybridization buffer with 20 mM sodium chloride. Line710has a peak at 520 nm and a shoulder peak at around 650 nm. Line712is the absorbance data of a suspension of gold nanoparticles in the hybridization buffer with 40 mM sodium chloride. Line712has a small peak at 520 nm and a larger peak at around 750 nm. The peak at around 750 nm has appeared due to the formation of aggregates of gold nanoparticles. Line714is the absorbance data of a suspension of gold nanoparticles in the hybridization buffer with 50 mM sodium chloride. Line716is the absorbance data of a suspension of gold nanoparticles in the hybridization buffer with 100 mM sodium chloride. Both line714and line716have peaks at around 750 nm. As can be seen in line graph700, with the increase of sodium chloride concentrations, along with the peak at 520 nm, a second peak at a longer wavelength around 750 nm appeared due to the formation of aggregates. The appearance of the second peak at 750 nm indicates stronger photothermal effects in the NIR range. Viewing line graph700, absorbance at 520 nm can be used to study the gold nanoparticles dispersed status. Viewing line graph700, absorbance at 800 nm can be used to study the photothermal effect of aggregated gold nanoparticles in the near-infrared range. These wavelengths are viewed in greater detail inFIG.8. Turning now toFIG.8, an illustration of a bar graph of absorbances at 520 and 800 nm for suspensions having different sodium chloride concentrations in the gold nanoparticle aggregation-induced photothermal biosensing method is depicted in accordance with an illustrative embodiment. Bar graph800has x-axis802of sodium chloride concentration in millimolar and y-axis804of absorbance in absorbance units. Absorbances806at 520 nm decreased sharply moving from sodium concentration of 0 mM to sodium concentration of 40 mM owing to the depletion of dispersed gold nanoparticles. Absorbances806at 520 nm then reached a plateau when the sodium chloride concentration was higher than 40 mM because of the complete formation of aggregated nanoparticles. For example, absorbance measurement810at sodium concentration of 0 mM is approximately 0.4 absorbance units. Absorbance measurement812at sodium concentration of 20 mM is at approximately 0.34 absorbance units. Absorbance measurement814is significantly lower than absorbance measurement812due to depletion of dispersed gold nanoparticles. Absorbance measurement814is approximately 0.17 absorbance units. Each absorbance measurement for sodium concentrations of mM to 100 nM is between approximately 0.12 and 0.15 absorbance units. Absorbances808at 800 nm increased moving from sodium concentration of 0 mM to sodium concentration of mM. Absorbances808at 800 nm reached a plateau at sodium chloride concentrations higher than 40 mM and up to a sodium chloride concentration of 100 mM. For example, absorbance measurement816at sodium concentration of 0 mM is approximately 0 absorbance units. Absorbance measurement818at sodium concentration of 20 mM is at approximately 0.05 absorbance units. Absorbance measurement820is significantly higher than absorbance measurement818due to depletion of dispersed gold nanoparticles. Absorbance measurement820is approximately 0.2 absorbance units. Each absorbance measurement for sodium concentrations of mM to 75 nM is between approximately 0.18 and 0.19 absorbance units. The optical absorption changes at 800 nm displayed in absorbances808further proved the transformation of dispersed nanoparticles to aggregated nanoparticles when increasing the concentration of sodium chloride. The optical absorption changes at 800 nm displayed in absorbances808also suggested the changes from weak Photothermic effects to strong Photothermic effects. Turning now toFIG.9, an illustration of a bar graph of temperature increases of suspensions having different sodium chloride concentrations in the gold nanoparticle aggregation-induced photothermal biosensing method under 808 nm laser irradiation is depicted in accordance with an illustrative embodiment. Bar graph900has x-axis902of sodium chloride concentration in millimolar and y-axis904of temperature increase in degrees Celsius. The changes from weak photothermic effects to strong photothermic effects due to transformation of dispersed gold nanoparticles to aggregated gold nanoparticles when increasing the concentration of sodium chloride is suggested by the results inFIGS.7and8. the changes from weak photothermic effects to strong photothermic effects due to transformation of dispersed gold nanoparticles to aggregated gold nanoparticles when increasing the concentration of sodium chloride is confirmed by the data inFIG.9. Under the irradiation of a near-infrared laser, the temperatures of suspensions having different sodium concentrations are measured. The temperature first increased rapidly upon adding sodium chloride in the concentration range from 0 to 40 mM. Temperature change906for hybridization buffer is small, approximately 2.5 degrees Celsius. Temperature change908for gold nanoparticles in a hybridization buffer is small, approximately 5 degrees Celsius. Temperature change910for a suspension with a sodium chloride concentration of mM had a marked increase from both temperature change906and temperature change908. Temperature change910for a suspension with sodium chloride concentration of 20 mM is approximately 14 degrees Celsius. The temperature increase then reached a plateau at approximately 18 degrees Celsius in the sodium chloride concentration range of 40-65 mM, and slightly decreased afterward. The maximum temperature increase was mainly caused by increasing the aggregated gold nanoparticles formed when adding sodium chloride to bare gold nanoparticle suspensions. Upon the addition of excess sodium chloride, the aggregated gold nanoparticles further accumulated and started to precipitate, which caused lower signals in both absorbances at 800 nm inFIG.8and temperature measurement inFIG.9. For example, temperature change912at 100 mM sodium chloride concentration is lower than temperature change914at 40 mM sodium chloride concentration. Precipitation of the gold nanoparticles from the suspension at greater sodium chloride concentrations causes lower temperature changes at 65 mM, 75 mM, and 100 mM concentrations of sodium chloride. Maximum temperature change inFIG.9and maximum absorbance in 800 nm inFIG.8both occur at 40 mM sodium chloride concentration. From the data inFIGS.8and9, it can be asserted that the aggregated gold nanoparticles further accumulated and started to precipitate at concentrations over 40 mM sodium chloride. The further aggregation and precipitation of the gold nanoparticles caused lower signals in both absorbances at 800 nm inFIG.8and temperature measurement inFIG.9. Therefore, 40 mM of sodium chloride was selected as the concentration of sodium chloride to be used in one example of the biosensor. FIGS.8and9are formed from data for a suspension including a saline-sodium citrate hybridization buffer and 20 nm diameter gold nanoparticles. A concentration of 40 mM of sodium chloride is selected for the biosensor having these biosensor characteristics. However, the selected sodium chloride concentration may be different for a biosensor with at least one of a different hybridization buffer, different sized gold nanoparticles, or a different concentration of gold nanoparticles. The concentration of sodium chloride in a biosensor is selected using the methods set forth above based on the absorbance and temperature increase data of different sodium chloride concentrations in the set suspension. A sodium chloride concentration is selected from a group of possible sodium chloride concentrations. The selected sodium chloride concentration is the concentration that provides a highest absorbance at 800 nm and a greatest temperature increase under irradiation without undesirably causing precipitation of the gold nanoparticles. In some illustrative examples, a concentration of the sodium chloride is selected such that the concentration of sodium chloride is configured to produce a maximum temperature increase due to gold nanoparticle aggregation. In some illustrative examples, a concentration of the sodium chloride is selected such that the concentration of sodium chloride is configured to produce a large temperature increase due to gold nanoparticle aggregation without triggering precipitation of aggregated gold nanoparticles. Turning now toFIG.10, an illustration of a line graph of temperature increase versus irradiation time under the 808 nm laser irradiation from different suspensions is depicted in accordance with an illustrative embodiment. The irradiation time in the gold nanoparticle aggregation-based photothermal biosensing was also selected prior to the detection of target DNA. The irradiation time could significantly affect the measurement of readout signals. The sodium chloride concentration for suspensions utilized in the irradiation time tests is desirably the sodium chloride concentration selected based on absorbance and temperature change data, such as the data presented inFIGS.4and5. The single-stranded DNA probe concentration for a suspension utilized in the irradiation time tests is desirably the single-stranded DNA probe concentration selected based on absorbance and temperature change data, such as the data presented inFIGS.12and13. Line graph1000is a line graph of temperature increase for some of the suspensions depicted inFIGS.6and7. Line graph1000has x-axis1002of irradiation time in seconds and y-axis1004of temperature increase in degrees Celsius. Line1006is of a suspension of only the hybridization buffer. Line1008is of a suspension of the hybridization buffer and gold nanoparticles. Line1010is of a suspension of the hybridization buffer, gold nanoparticles, and 40 mM sodium chloride. Line1012is of a suspension of the hybridization buffer, gold nanoparticles, 40 mM sodium chloride, and 160 nM single-stranded DNA probes. The laser power density was 5.2 W/cm2. The temperature was monitored for irradiation time in the range of 0-600 s under continuous irradiation of the 808 nm laser irradiation. Under the continuous irradiation of a near-infrared laser, the temperature of the hybridization buffer had only a slight temperature increase of approximately 2 degrees Celsius as can be seen in line1006. Under the continuous irradiation of a near-infrared laser, the temperature of the gold nanoparticle suspension had a temperature increase of about 4 degrees Celsius, as can be seen in line1008. Under the continuous irradiation of a near-infrared laser, the temperature of a gold nanoparticle suspension with sodium chloride and single-stranded DNA probes had a temperature increase of about 4 degrees Celsius, as can be seen in line1012. As can be seen by comparing the gold nanoparticle suspension and a suspension of gold nanoparticles, sodium chloride, and single-stranded DNA probes. The gold nanoparticle and sodium chloride suspension results in aggregated gold nanoparticles. The gold nanoparticle and sodium chloride suspension had a dramatic temperature increase in the first 2.5 minutes, then exhibited a stagnant increase, and finally reached the plateau of 14 degrees Celsius at approximately 8 minutes, as can be seen in line1010. The heat balance seen in line1010was achieved between heat generation from the photothermal effect and heat dissipation to the environment. Therefore, to acquire a stable and sensitive temperature measurement, the laser irradiation time of 8 minutes was selected as the irradiation time for biosensing using the biosensor of the illustrative examples. The irradiation time is selected based on the concentration of sodium chloride, concentration of single-stranded DNA probes, the laser power settings, and other biosensor characteristics. In this illustrative example, 8 minutes was selected as the set irradiation time. In other illustrative examples, a different irradiation time is selected based on when the temperature change remains substantially stable. Turning now toFIG.11, an illustration of a line graph of UV-vis spectra of suspensions having different single-stranded DNA concentrations in the gold nanoparticle aggregation-based photothermal biosensing method is depicted in accordance with an illustrative embodiment. Line graph1100is a graph of suspensions with varying concentrations of single-stranded DNA probes. Each of the suspensions in line graph1100includes the selected amount of sodium chloride. In this illustrative example, the selected amount of sodium chloride is 40 mM. To obtain the best protection performance of gold nanoparticles by oligonucleotides, the concentration of single-stranded DNA probes is selectively determined. To form line graph1100, different concentrations of single-stranded DNA probes in the range of 0-320 nM are added to gold nanoparticle suspensions, followed by the addition of 40 mM NaCl. Line1106is of a suspension of the hybridization buffer, gold nanoparticles, and sodium chloride. Line1106displays absorbance data of suspension509ofFIG.5. Line1106includes a small peak at 520 nm and a larger peak at around 750 nm. The peak at around 750 nm has appeared due to the formation of aggregates of gold nanoparticles. Line1108is of a suspension of the hybridization buffer, gold nanoparticles, sodium chloride, and 16 nM of single-stranded DNA probes. Line1108includes small peaks at both 520 nm and 750 nm. The peak at around 750 nm is smaller due to the protection against aggregation of the gold nanoparticles by the single-stranded DNA probes. Line1108has a greater peak at 520 nm than line1106. Line1110is of a suspension of the hybridization buffer, gold nanoparticles, sodium chloride, and 32 nM of single-stranded DNA probes. Line1110includes a peak at 520 nm and a shoulder peak at around 650 nm. Line1110has a greater peak at 520 nm than both line1106and line1108. Line1112is of a suspension of the hybridization buffer, gold nanoparticles, sodium chloride, and 80 nM of single-stranded DNA probes. Line1112includes a peak at 520 nm and a shoulder peak at around 650 nm. Line1112has a greater peak at 520 nm than each of line1106, line1108, and line1110. Line1114is of a suspension of the hybridization buffer, gold nanoparticles, sodium chloride, and 160 nM of single-stranded DNA probes. Line1114has a peak at 520 nm indicative of dispersed gold nanoparticles. Line1114has a greater peak at 520 nm than any of line1106, line1108, line1110, and line1112. Line1114does not have any other peaks. Line1116is of a suspension of the hybridization buffer and gold nanoparticles. There is no sodium chloride in the suspension represented by line1116. Line1114is substantially the same as line1116, indicating that 160 nM concentration of the single-stranded DNA probes protected all of the dispersed gold nanoparticles from the sodium chloride. In some illustrative examples, the concentration of single-stranded DNA probes in the biosensor is configured to interact with dispersed gold nanoparticles in the biosensor. In some illustrative examples, the concentration of single-stranded DNA probes in the biosensor is configured to provide an absorbance at 520 nm equivalent to an absorbance at 520 nm of a suspension of dispersed gold nanoparticles. It can be seen fromFIG.11that as the concentration of single-stranded DNA probes is increased in a suspension, the peak at a longer wavelength around 750 nm became weaker until it disappeared. Disappearance of the peak at 750 nm indicates reduced aggregated gold nanoparticles and better protection of the dispersed gold nanoparticles by the single-stranded DNA probes. Turning now toFIG.12, an illustration of a bar graph of absorbances at 520 nm and 800 nm for suspensions having different concentrations of single-stranded DNA concentrations in the gold nanoparticle aggregation-based photothermal biosensing method is depicted in accordance with an illustrative embodiment. The suspensions depicted inFIG.12include each of the suspensions shown inFIG.11as well as suspensions with greater concentrations of single-stranded DNA probes than those depicted inFIG.11. Absorbances1206at 520 nm increased gradually in the concentration range from 0 to 160 nM, implying greater quantity of dispersed gold nanoparticles due to increased single-stranded DNA probe protection. When the single-stranded DNA probe concentration was higher than 160 nM, no obvious change in UV-vis spectra was observed as compared to bare gold nanoparticles. No obvious change in absorbances1206when DNA probe concentration is higher than 160 nM indicates that the maximum protection of gold nanoparticles can be achieved at the single-stranded DNA probe concentration of 160 nM. Moreover, absorbance1206at 520 nm reached the highest value at the single-stranded DNA probe concentration of 160 nM. Absorbance value1210at 160 nm of single-stranded DNA probes is almost the same as absorbance value1212of bare gold particles. Absorbance value1210being almost the same as absorbance value1212suggests that the dispersed status of the gold nanoparticles is protected from salt-induced aggregation by the protection of ssDNA probes. Absorbances1208at 800 nm decreased as the single-stranded DNA probe concentrations increased from 0 to 160 nM and reached the lowest value at 160 nM. The decrease of absorbances1208indicates decreased quantities of aggregated gold nanoparticles as well as weaker photothermic effects. Absorbance value1214at 160 nM of single-stranded DNA probes is substantially the same as absorbance value1216of bare gold nanoparticles. Turning now toFIG.13, an illustration of a bar graph of temperature increases of suspensions having different single-stranded DNA concentrations in the gold nanoparticle aggregation-based photothermal biosensing method under the 808 nm laser irradiation is depicted in accordance with an illustrative embodiment. Bar graph1300is a graph of temperature increases for the suspensions depicted inFIGS.11and12. Bar graph1300has x-axis1302of single-stranded DNA probe concentration in nanomolar and y-axis1304of temperature increase in degrees Celsius. As can be seen in bar graph1300, under the irradiation of a near infrared laser, temperature increase1306of a suspension without single-stranded DNA probes is approximately 18° C. owing to the strong PT effect of gold nanoparticle aggregation. In bar graph1300, temperature signals decrease with the increase of single-stranded DNA probe concentration. For example, temperature increase1308of a suspension with 16 nM of single-stranded DNA probes is lower than temperature increase1306. Temperature signals decrease with the increase of single-stranded DNA probe concentration until reaching a plateau of approximately 6° C. temperature increase. At concentration of 160 nM single-stranded DNA probes, temperature increase1310is similar to temperature increase1312for a suspension of bare gold nanoparticles. The weaker photothermic effects of the suspensions with greater single-stranded DNA probe concentrations indicates that gold nanoparticles are protected from sodium chloride-induced aggregation by the addition of single-stranded DNA probes. Minimum temperature change inFIG.13and maximum absorbance in 520 nm inFIG.12both occur at 160 nM single-stranded DNA probe concentration. From the data inFIGS.12and13, it can be asserted that the single-stranded DNA probes do not provide any additional protection to dispersed gold nanoparticles at concentrations over 160 nM single-stranded DNA probe. From the data inFIGS.12and13, it can be asserted that concentrations over 160 nM of single-stranded DNA probes would not be beneficial in the biosensor. From the data inFIGS.12and13, it can be asserted that at 160 nM concentration of single-stranded DNA probes, the dispersed gold nanoparticles have substantially the same absorbances and temperature increase as bare gold nanoparticles. Therefore, 160 nM of single-stranded DNA probes was selected as the concentration of single-stranded DNA probes to be used in one example of the biosensor. FIGS.12and13are formed from data for a suspension including a saline-sodium citrate hybridization buffer, 20 nm diameter gold nanoparticles, and 40 mM sodium chloride. A concentration of 160 nM of single-stranded DNA probes is selected for the biosensor having these biosensor characteristics. However, the selected single-stranded DNA probe concentration may be different for a biosensor with at least one of a different hybridization buffer, different sized gold nanoparticles, a different concentration of sodium chloride, or a different concentration of gold nanoparticles. The concentration of single-stranded DNA probe in a biosensor is selected using the methods set forth above based on the absorbance and temperature increase data of different single-stranded DNA probe concentrations in the set suspension. A single-stranded DNA probe is selected from a group of possible single-stranded DNA probe concentrations. The selected single-stranded DNA probe concentration is the concentration that provides a highest absorbance at 520 nm and a lowest temperature increase under irradiation without undesirably causing precipitation of the gold nanoparticles. Turning now toFIG.14, an illustration of a line graph of temperature increases vs target DNA logarithmic concentrations forbacillus Mycobacterium tuberculosisis depicted in accordance with an illustrative embodiment. Line graph1400is a graph of temperature increases for biosensors having varying concentrations of introduced target DNA. Line graph1400is formed using data from biosensors having 40 mM of sodium chloride and 160 nM of single-stranded DNA probes. Line graph1400has x-axis1402of target DNA concentration in nanomolar and y-axis1404of temperature increase in degrees Celsius. Line graph1400is a study of target DNA-induced gold nanoparticle aggregation based photothermal biosensing platform for quantitative detection ofMycobacterium tuberculosis(MTB) DNA using a thermometer. Biosensors, such as biosensor104ofFIG.1, receive different concentrations of the target DNA. In this illustrative example, the target DNA is MTB DNA. Each biosensor with the target DNA was irradiated by a near infrared laser (808 nm) for 8 minutes at the power density of 5.2 W/cm2, and the temperature was recorded using a portable digital thermometer after the irradiation. In line graph1400, the temperature increased with the increase of target DNA concentration. Linear relationship1406is obtained between the temperature increase with the logarithm of target MTB DNA concentration in the range from 2 to 1200 nM, with a squared correlation coefficient of 0.996. The limit of detection (LOD) was calculated to be 0.28 nM based on three folds standard deviation above the blank, which was about 10-fold lower than that obtained from our colorimetric detection method, indicating high detection sensitivity of this photothermal biosensing method. Moreover, as compared to conventional colorimetric methods, the proposed photothermal biosensing platform is simple and convenient for quantitative analysis simply using an inexpensive thermometer as a signal reader, whereas conventional colorimetric methods require the use of expensive spectrometers for quantitative analysis. Furthermore, no DNA amplification was needed, and the detection could be completed within 40 minutes with no assistance from analytical instrumentation, which greatly reduced the complexity, cost, and detection time of the entire assay. Although line graph1400displays data forbacillus Mycobacterium tuberculosis, temperature data can be collected for any biological material detected using quantitative photothermal biosensing. For example, biosensors of the illustrative examples could be used to detect any desirable nucleic acid. The analytical performance of this photothermal biosensing platform was investigated by testing recovery in the quantitation ofM. tuberculosisgenomic DNA. The analytical recovery was evaluated by spiking different concentrations of targetM. tuberculosisgenomic DNA. Color images of different samples were captured, and temperature increases were recorded immediately after the laser irradiation. Turning now toFIG.15, an illustration of a flowchart of a method of detecting a target DNA is depicted in accordance with an illustrative embodiment. Method1500is a method of detecting target DNA118ofFIG.1using biosensor104ofFIG.1. Method1500can be performed using biosensor201ofFIGS.2-4. Characteristics for a biosensor to be used in method1500can be determined using analysis described inFIGS.7-13. For example, a sodium chloride concentration in a biosensor utilized in method1500can be selected based on an analysis described inFIGS.8and9. The irradiation time utilized in method1500can be determined according to the analysis described in FIG.10. As another example, a single-stranded DNA probe concentration in a biosensor utilized in method1500can be selected based on an analysis described inFIGS.12and13. Method1500adds a DNA sample to a biosensor comprising a suspension of a hybridization buffer containing dispersed gold nanoparticles, sodium chloride, and single-stranded DNA probes configured to undergo DNA hybridization with the target DNA (operation1502). Method1500sends, by a laser, a beam of near-infrared radiation into the biosensor, wherein the beam of near-infrared radiation is configured to induce a photothermal effect in any aggregated gold nanoparticles present in the biosensor (operation1504). Method1500detects a temperature of the biosensor after sending the beam of near-infrared radiation into the biosensor (operation1506). Method1500determines whether the target DNA is present in the DNA sample based on the temperature of the biosensor (operation1508). Afterwards, method1500terminates. In some illustrative examples, method1500determines, in response to a determination that the target DNA is present in the DNA sample, a concentration of the target DNA in the DNA sample (operation1520). In some illustrative examples, determining the concentration of the target DNA comprises determining a temperature change using the temperature of the biosensor and an initial temperature of the biosensor (operation1518) and determining the concentration of the target DNA using the temperature change (operation1522). In these illustrative examples, the concentration of the target DNA is determined by comparing the temperature change to a known relationship between target DNA concentration and temperature increase. In some illustrative examples forming the biosensor comprises adding a first solution of the single-stranded DNA probes in the hybridization buffer to a suspension of the dispersed gold nanoparticles to form a protected gold nanoparticle suspension (operation1510). In some illustrative examples forming the biosensor further comprises adding a second solution of sodium chloride in the hybridization buffer to the protected gold nanoparticle suspension to form the biosensor (operation1512). In some illustrative examples, the first solution of the single-stranded DNA probes comprises a minimum concentration of single-stranded DNA probes configured to protect the dispersed gold nanoparticles from the sodium chloride, and wherein the second solution of the sodium chloride comprises a concentration of sodium chloride configured to produce a maximum temperature increase due to gold nanoparticle aggregation (operation1514). In some illustrative examples, the second solution of the sodium chloride comprises a concentration of sodium chloride configured to produce a large temperature increase due to gold nanoparticle aggregation without triggering precipitation of aggregated gold nanoparticles. Method1500is a simpler method of quantitative biosensing method. To perform method1500, labeling of DNA probes or DNA amplification processes are not required. Method1500is both DNA label-free and gold nanoparticle label free. In some illustrative examples, method1500is described as label-free and amplification-free. In some illustrative examples, method1500forms the DNA sample without DNA amplification (operation1516). Turning now toFIG.16, an illustration of a flowchart of a method of detecting a target DNA is depicted in accordance with an illustrative embodiment. Method1600is a method of detecting target DNA118ofFIG.1using biosensor104ofFIG.1. Method1600can be performed using biosensor201ofFIGS.2-4. Characteristics for a biosensor to be used in method1600can be determined using analysis described inFIGS.7-13. For example, a sodium chloride concentration in a biosensor utilized in method1600can be selected based on an analysis described inFIGS.8and9. The irradiation time utilized in method1600can be determined according to the analysis described inFIG.10. As another example, a single-stranded DNA probe concentration in a biosensor utilized in method1600can be selected based on an analysis described inFIGS.12and13. Method1600irradiates a biosensor containing a sample with near-infrared radiation for a selected time, wherein the biosensor comprises a suspension of a hybridization buffer containing gold nanoparticles, sodium chloride, and single-stranded DNA probes configured to undergo DNA hybridization with a target, and wherein the near-infrared radiation is configured to induce a photothermal effect in any aggregated gold nanoparticles present in the biosensor (operation1602). Method1600determines a temperature change of the biosensor after irradiating the biosensor with the near-infrared radiation for the selected time (operation1604). Method1600determines a concentration of the target present in the sample using the temperature change of the biosensor (operation1606). Afterwards, method1600terminates. In some illustrative examples, irradiating the biosensor further comprises irradiating the suspension at a power density of 5.2 W/cm2for an irradiation time, wherein the irradiation time is from about 5 minutes to about 10 minutes (operation1608). In some illustrative examples, the irradiation time is selected such that the irradiation time is a first time at which heat generation from the photothermal effect and heat dissipation to the environment are substantially the same. As used herein, the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items may be used and only one of each item in the list may be needed. For example, “at least one of item A, item B, or item C” may include, without limitation, item A, item A and item B, or item B. This example also may include item A, item B, and item C or item B and item C. Of course, any combinations of these items may be present. In other examples, “at least one of” may be, for example, without limitation, two of item A; one of item B; and ten of item C; four of item B and seven of item C; or other suitable combinations. The item may be a particular object, thing, or a category. In other words, at least one of means any combination items and number of items may be used from the list but not all of the items in the list are required. As used herein, “a number of,” when used with reference to items means one or more items. The flowcharts and block diagrams in the different depicted embodiments illustrate the architecture, functionality, and operation of some possible implementations of apparatuses and methods in an illustrative embodiment. In this regard, each block in the flowcharts or block diagrams may represent at least one of a module, a segment, a function, or a portion of an operation or step. In some alternative implementations of an illustrative embodiment, the function or functions noted in the blocks may occur out of the order noted in the figures. For example, in some cases, two blocks shown in succession may be executed substantially concurrently, or the blocks may sometimes be performed in the reverse order, depending upon the functionality involved. Also, other blocks may be added in addition to the illustrated blocks in a flowchart or block diagram. Some blocks may be optional. For example, any of operation1510through operation1522may be optional. As another example, operation1608may be optional. The illustrative examples provide biosensors and biosensing methods that leverage the photothermic effect of gold nanoparticles to provide quantitative genetic detection. The illustrative examples present improvements to quantitative genetic detection by providing increased sensitivity over other optical methods. The illustrative examples present improvements to quantitative genetic detection by reducing the cost of testing. The illustrative examples present improvements to quantitative genetic detection by reducing the time between collection of a DNA sample and results of testing the DNA sample. The illustrative examples present improvements to quantitative genetic detection by providing a lower cost tool for signal detection of the biosensor. Use of a thermometer, unlike conventional spectroscopy, is inexpensive and does not require sophisticated training to operate. In the illustrative examples, unmodified gold nanoparticles are first protected by ssDNA probes from sodium chloride-induced aggregation. The unmodified gold nanoparticles are in the dispersed status and have weak PT effect under the irradiation of a near-infrared (NIR) laser. In the presence of target DNA, the protection is damaged due to DNA hybridization, which causes the aggregation of gold nanoparticles upon the addition of sodium chloride. In the illustrative examples, the gold nanoparticle aggregation is formed and used as a photothermal agent for photothermal biosensing. An obvious temperature increase can be obtained from the aggregated gold nanoparticle suspension while being irradiated by the NIR laser. Gold nanoparticles remain in dispersed status while adding nontarget DNA, which causes a negligible temperature increase due to a weak PT effect. DNA quantification can be achieved by simply recording the temperature change by using a thermometer. The photothermic effect of different components was studied by measuring temperature changes (ATs) of different solutions after the irradiation of NIR laser for 5 minutes at a power density of 5.2 W/cm2. The biosensor only had significant increase in temperature upon introduction of the target DNA. Herein, we developed a new simple and universal method for quantitative genetic analysis using a thermometer on the basis of gold nanoparticle aggregation-induced photothermal effects. In this method, two competing processes were present between the protection and the destabilization of dispersed gold nanoparticles by single-stranded DNA (ssDNA) probes and salt (sodium chloride), respectively. When target DNA is added to the biosensor, ssDNA probes are deprived of the surface protection of gold nanoparticles because of the hybridization, promoting gold nanoparticles to change from the dispersed status to the aggregated status. The obtained gold nanoparticle aggregation was used as a novel photothermal biosensor, correlating quantitative analysis of nucleic acids with temperature readouts, which could be simply recorded by using a common thermometer. In the illustrative examples there is no need for DNA amplification, surface modification of gold nanoparticles, and single-stranded DNA probe modification, which greatly reduces the complexity and cost of genetic assays. The entire assay can be accomplished within 40 minutes using only a thermometer as a signal reader, without the assistance from any bulky and costly instrumentation. In addition, the unmodified gold nanoparticles can be used to adsorb various DNA probes, making it a universal platform for a broad range of genetic targets. By usingMycobacterium tuberculosis(MTB) DNA as a model target, high sensitivity and specificity were achieved with the limit of detection (LOD) as low as 0.28 nM, which was nearly 10-fold lower than that in the colorimetric method using a microplate reader. The illustrative examples present a simple, low-cost, and universal gold nanoparticle aggregation-induced photothermal biosensing platform. The illustrative examples apply the gold nanoparticle aggregation-induced photothermal biosensing platform for visual quantitative genetic detection using a common thermometer. The illustrative examples exploit the photothermal effect of target-induced gold nanoparticle aggregation. By exploiting the photothermal effect of target-induced gold nanoparticle aggregation, visual quantitative biochemical analysis can be achieved by simply recording temperature signals using a common thermometer. Compared to conventional genetic testing methods, the biosensor and biosensing methods of the illustrative examples are label- and amplification-free and can be completed in 40 minutes without the aid of any advanced analytical instruments.Mycobacterium tuberculosis(MTB) DNA was used as a model target to demonstrate the application of this photothermal biosensing platform. Although no costly instrument was used, high sensitivity and specificity were achieved with the limit of detection (LOD) of 0.28 nM, which was nearly lower than that of the colorimetric method using a spectrometer. This gold nanoparticle aggregation-induced photothermal biosensing strategy provides a simple, low-cost, and universal platform for broad application of visual quantitative detection of nucleic acids and many other biomolecules, particularly in point-of-care (POC) biosensing applications. The analytical performance of this photothermal biosensing platform was investigated by testing recovery in the quantitation ofM. tuberculosisgenomic DNA. The analytical recovery was evaluated by spiking different concentrations of targetM. tuberculosisgenomic DNA. Color images of different samples were captured, and temperature increases were recorded immediately after the laser irradiation. The analytical recoveries were obtained from 94.5 to 110.2% when testing varying concentrations of the target genomic DNA spiked in SSC buffer from 13 to 39 nM (i.e., 2.5-7.5 μg/mL), which were within the acceptable recovery range for the validation of bioanalytical methods. Although genetic assays usually require cell lysis and DNA extraction before DNA hybridization under optimal hybridization conditions, this biosensing system was also challenged by directly spiking target DNA in a 50% normal human serum sample. The acceptable analytical recovery of 91.5% was acquired, even with 50% normal human serum sample, which further demonstrated the excellent performance of this method even in a complex matrix. Furthermore, as compared to color changes observed by the naked eye, the photothermal biosensing based on target-induced gold nanoparticle aggregation provided a simple yet reliable platform for the quantitative detection of nucleic acids. We, for the first time, developed a simple yet versatile gold nanoparticle aggregation-induced photothermal biosensing platform for sensitive and quantitative detection of nucleic acids using a thermometer. The quantitation detection can be achieved by simply using a thermometer as the signal reader with no assistance from any specialized and costly analytical instrumentation. Although a low-cost thermometer was used as the signal reader, high sensitivity was achieved with the LOD as low as 0.28 nM, about 10-fold lower than the LOD in the colorimetric detection method using a spectrometer. Moreover, it is a universal platform for DNA detection, as labeling of DNA probes, gold nanoparticles, or DNA amplification processes are not required, which greatly reduces the complexity and cost of detection assays. Furthermore, this photothermal biosensing platform also provides unprecedented potential for the quantitative detection of a wide range of biochemicals and biological organisms, not solely nucleic acids. For instance, by using DNA-based aptamers, this platform can be applied to detect a variety of chemicals ranging from protein biomarkers, microorganisms, cancer cells, to metal ions (e.g., Hg2+). Considering more and more inexpensive portable yet powerful NIR laser pointers become commercially available, this novel biosensing method will bring a new horizon to conventional detection methods particularly for point-of-care testing (POCT). The description of the different illustrative embodiments has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. Further, different illustrative embodiments may provide different features as compared to other illustrative embodiments. The embodiment or embodiments selected are chosen and described in order to best explain the principles of the embodiments, the practical application, and to enable others of ordinary skill in the art to understand the disclosure for various embodiments with various modifications as are suited to the particular use contemplated. | 74,995 |
11860119 | Throughout the description and the drawings, like reference numerals refer to like parts. DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION FIG.1is a diagram of a sensor100in accordance with an embodiment of the invention. A sensor100comprises a first terminal110and a second terminal120coupled via a conductive connection130. In the embodiment shown inFIG.1, the first terminal110and the second terminal120are provided on the same body/electrode150, and in this embodiment the electrode150comprises the first terminal110, the second terminal120and the conductive connection130. However, it is to be understood that the first terminal110and the second terminal120may be provided on separate bodies/electrodes such that they are physically distinguishable from one another. A mesoscopic probe element140is capacitively coupled to the conductive connection130, as will be explained in further detail below. The sensor100may be considered as a Density-of-State sensitive field effect transistor (DO2S-FET). A field effect transistor (FET) is a transistor in which most current is carried along a channel between source and drain terminals of the FET, the channel having an effective resistance controllable by a transverse electric field. In particular, the source terminal is a terminal through which charge carriers (electrons) enter the channel, the drain terminal is a terminal through which the charge carriers leave the channel, and a gate terminal modulates the channel conductivity. By applying a voltage to the gate terminal, one can control the current at the drain terminal. A classical FET device operates based on a direct current-voltage (DC) response; by applying a potential difference between the source and drain terminals an electric current is permitted to flow in the channel, subject to moderation by the gate potential, otherwise the FET is said to be in an equilibrium condition. For the sensor100(which may also be referred to as a DO2S-FET in what follows), the first terminal110and the second terminal120may be considered as source and drain terminals of a transistor. However, as opposed to having a direct current (DC) biasing potential applied across the first and second terminals, the first and second terminals (110,120) may be considered as at equilibrium condition (when no current is applied) such that the potential difference between the first and second terminals is substantially zero. In such a scenario, the resistive and capacitive properties of the channel/conductive connection may be probed using a time-dependent electrical signal, such as an alternating current or an electrical pulse. The relaxation time of the sensor can be tailored during fabrication by doping or adjusting the thickness of the body. The conductive connection130is depicted by a solid line inFIG.1, although the skilled person would appreciate that the conductive connection may be any suitable conductor and of any suitable thickness. The conductive connection between the first terminal110and the second terminal120is capacitively coupled to an environment via the mesoscopic probe element140. As the mesoscopic probe element140has at least one mesoscopic dimension, the quantum capacitance Cqvaries as a function of the density-of-states occupancy of the probe element140. In this way, and as will be explained in further detail below, any interaction between the mesoscopic probe element140and a measurand which results in a change in the electron density of states of the mesoscopic probe element140, will have an influence on the capacitive and resistive properties of the sensor100(and therefore the relaxation time of the sensor100), as probed by a time-dependent electrical signal. Accordingly, an electroactive surface160of the mesoscopic probe element140may be considered to function as a gate terminal of the DO2S-FET architecture, as emphasised by the solid line connecting the conductive connection130to the mesoscopic probe element140inFIG.1. FIG.2is a flowchart of a sensing method which may be carried out using, for example, the sensor100ofFIG.1. At step210, a time-dependent electrical signal is provided across the conductive connection130between the first terminal110and the second terminal120. For example, the first terminal110and the second terminal120may be connected to an alternating current/voltage or connected to a pulsed electrical signal. As has been explained above, the conductive connection130of the sensor100is capacitively coupled to an environment via a mesoscopic probe element140of the sensor, the mesoscopic probe element140having an electroactive surface160for exposure to the environment. The conductive connection also has an associated relaxation time/RC response. The electrical signal may be any suitable electrical signal having a time-varying profile. For example, the electrical signal may be an alternating current/voltage, or may be an electrical pulse. At step220, a time-dependent response signal is received from the sensor100. The time-dependent response signal may comprise an alternating current/voltage or an electrical pulse. The time-dependent response signal depends on the time-dependent electrical signal provided to the sensor100and the response characteristics or relaxation time of the sensor100. At step230, the time-dependent response signal is analysed with respect to the time-dependent electrical signal. For example, the analysis may comprise determining a phase difference between the time-dependent response signal and the time-dependent electrical signal. Analysing the time-dependent response signal with respect to the time-dependent electrical signal may comprise determining an immittance function of the sensor from the time-dependent electrical signal and the time-dependent response signal. Analysing may comprise determining the impedance of the sensor100from the ratio of the time-dependent electrical signal to the time-dependent response signal. Analysing may comprise determining the capacitance of the sensor100using any suitable method such as impedance-derived capacitance spectroscopy. At step240, it is determined, from the analysis at step230, whether a change in the relaxation time of the sensor has been detected, a change in the relaxation time being correlated with a change in the density of states of the surface160of the mesoscopic probe element140. A change in density of states can occur from an interaction between the electroactive surface160of the mesoscopic probe element140and a measurand of the environment. For example, when the analysing at step240comprises determining a phase difference between the time-dependent response signal and the time-dependent electrical signal, determining whether a change in the relaxation time of the sensor has been detected may comprise determining that a change in the relaxation time has occurred based on the phase difference. If a determination is made, at step240, that a change in relaxation time has not occurred, then a determination is made that a measurand has not been detected (250). If a determination is made, at step240, that a change in relaxation time has occurred, then a determination is made that a measurand has been detected (260). FIG.3is a block diagram of a sensing system300in accordance with an embodiment. The sensing system comprises a sensor100, which may correspond to the sensor100ofFIG.1. The sensing system further comprises a signal generating means310and a signal analysing means320. Other architectures to that shown inFIG.3may be used as will be appreciated by the skilled person. For example, the signal generating means310and the signal analysing means320may be provided in the same device or controller. As described above in relation toFIG.1, the sensor100comprises a first terminal110and a second terminal120coupled via a conductive connection130, which in turn is capacitively coupled to an environment via a mesoscopic probe element140having an electroactive surface in contact with the environment. The properties of the conductive connection130including the capacitance, impedance and the relaxation time of the conductive connection130, can be explored with the use of a time-dependent electrical signal. In this way it may be determined whether or not an interaction has occurred between the electroactive surface and a measurand of the environment. Signal generating means310, which in the present embodiment comprises a signal generator, is configured to send a time-dependent electrical signal such as an alternating current or an electrical pulse, to the sensor100. The signal generating means310is also configured to communicate the generated signal to the signal analysing means320for analysis. The signal analysing means320, which in the present embodiment comprises a signal analyser, is configured to receive a time-dependent response signal from the sensor100and to receive an indication of the sent time-dependent electrical signal from the signal generator310. Referring to the figure, the signal analysing means320comprises a communications module370for receiving information from the sensor100and the signal generating means310. The communications module370may be in any suitable form. For example, the communications module270may comprise one or more measurement apparatuses for monitoring or measuring the time-dependent electrical signal sent from the signal generating means310and the time-dependent response signal from the sensor100. For example, the communications module370may comprise one or more ammeters and/or voltmeters for directly measuring the response signal from the sensor100. In the present embodiment, the signal analysing means further comprises processing means in the form of a processor330. A storage means in the form of a memory340, and a powering means in the form of a power system350. The processing means330is configured to receive data, access the memory340, and to act upon instructions received either from said memory340, from communications module370, or from one or more virtual or dedicated user input devices380. The processor330is arranged to receive a time-dependent response signal from the sensor100. The processor330is arranged to analyse the time-dependent response signal with respect to the time-dependent electrical signal. The processor330is arranged to determine, from the analysis, whether a change in the relaxation time of the conductive connection of the sensor100has occurred. This may be by, for example, determining that a change in phase difference between the time-dependent electrical signal and the time-dependent response signal has occurred. The processor330may be arranged to determine, from the analysis, a concentration or amount of the measurand in the environment about the sensor100. The processor330is arranged to indicate to visual display360whether or not a change in the relaxation time has occurred. The signal generating means310may comprise similar components to the signal analysing means320. In some embodiments, the signal generating means310and the signal analysing means320may be integrated into a single controller which controls both the signal generation and the signal analysis. How the sensor100functions will become more apparent to the reader from the following, in which the principles of DOS-based sensing are explained and demonstrated, with reference toFIGS.4to8. A molecular junction immersed into an electrolyte generates a capacitance that is largely modelled by classical means using double layer interfacial capacitance models. The simplest model that works adequately at high (>0.1 M) ionic strength is that presented by Gouy-Chapman where the double layer capacitance (per unit of area) is given by Ci=εrεOκ, wherein εris the relative static permittivity (generally referred to as the dielectric constant) of the material, εO(˜8.85×10−12 F m−1) is the dielectric constant or dielectric permittivity of vacuum and κ is the inverse of Debye length (LD=1/κ), classically given by κ=[(2e2N)/(εrεOkBT)]1/2. N denotes the density (molar concentration) of ionic charge (positive or negative) in the bulk of the electrolyte, kBis the Boltzmann constant, and T absolute temperature. Nonetheless this, and any other presented double layer model, fails when electronic states of an adsorbate mix with electrode states. This is the case, for instance, with electro-active molecular films wherein the innate presence of quantized and redox accessible electronic sites generates additional capacitive effects (usually engrained within what is described as a “pseudo capacitance”). This additional capacitive effect is associated with the charging of localized chemical states in a way somewhat detached from pure electrostatics. Under such circumstances, both ionic and electronic contributions must be considered in a combined electrochemical capacitance Cμwhich includes a (density-of-states based) consideration of Thomas-Fermi screening [(1/Cμ=1/Ce+1/Cq), where electrostatic (Ce) and quantic (Cq) capacitance terms contribute] and κ=(Cμ/εrεO)1/2. Capacitive contributions from both ionic (distinguishable particles) and electronic (indistinguishable particles) sources are engrained in experimentally resolvable Cμ. In the former case, a Boltzmann approximation can be assumed for the occupancy such that N∝exp[μ/κBT]. For indistinguishable (electronic) particles N∝(1+exp[μ/κBT])−1. If the former dominates then κ=[(2e2N)/(εrεOκBT)]1/2is recovered from κ=(Cμ/εrεO)1/2Accordingly, the classic double layer phenomenology is thus only a particular form of Cμarising specifically when charging at the interface is exclusively ionic in origin. In situations where there is a significant accessible nanoscale electronic density of states (DOS), this dominates in measured Cμand the electrical field screening is primarily governed by electrons. Under such circumstances, one can directly relate the 1/Cμand its relationship to electronic occupation of states, to energy storage (E=qV and where q=Ne is the charge; e is the elementary electron charge) as expressed by E=q22Cμ_=∑ɛi+q22Ce(1) wherein Σεiis the sum of individual occupied state energies, εi. If the occupation of states is environmentally responsive then the associated experimentally resolved Cμ, which directly reports on DOS occupation, responds likewise. One can experimentally measure Cμusing a sensor100such as that shown inFIG.1and relate the capacitance directly to quantised occupancy according to N∝(1/e2)∫CμdV, wherein ΔV=∫vivfdV accounts for the potential window being scanned. Cμis recorded at a fixed frequency low enough for there to be no kinetic limitations. FIG.4ais a schematic illustration of the energy levels for an electrochemical junction composed of an electrode (yellow) and a generic electroactive system immersed in electrolyte (blue). The electrode may correspond to the conductive connection and/or mesoscopic probe element described above in relation toFIGS.1to3, and the electrolyte may correspond to the environment. The redox reaction free energy is defined as ΔGr=e(V−Vr)=E−EF, where e is the electron charge, and V is the electrode over potential. The expression Vred−Voxcorresponds to the difference between fully reduced and oxidised states of the junction whereas ERis the reference energy level.FIG.4bis equivalent toFIG.4awith integrated biological receptors within a mixed junction also containing electro-active states. When a target biomarker is recruited the occupation of the electronic states changes and the experimentally measured change in interfacial energy transduces this through its effect on the film localized DOS.FIG.4cdenotes the equivalent operation of a field effect transistor (FET) operating under equilibrium conditions, wherein the electrochemical potential of the left- and right-hand electrodes are equal. In a classical FET, the electron density in the channel/conductive connection between the source and drain terminals (first and second terminals) changes as the gate voltage changes. In much the same way, the conductive properties of the conductive connection130of the sensor100ofFIG.1are altered when an interaction occurs between the mesoscopic probe element140and a measurand of the environment.FIG.4ddemonstrates the FET associated equivalent circuit that governs behaviour when only gate voltage (VG) is varied under equilibrium conditions, wherein the first and second terminals (source and drain terminals) are poised at the same potential. In such a configuration, the equivalent capacitance is the electrochemical capacitance, measured as Cμ=CeCq/(Ce+Cq). FIG.5ais a graph showing the effect of environment dielectric on resolved DOS distribution of an electroactive molecular layer; solvent polarity tunes both the DOS energetic dispersion and the associated Fermi energy (dotted lines). Notably, the integrated accessible electron density is not affected (being here a substantially constant ˜1019states per cm−3).FIG.5bis a graph showing the responsiveness of redox DOS to molecular recognition within a mixed redox switchable and antibody constrained film (seeFIG.4b). The inlay inFIG.5bshows the invariance in both Fermi energy of the junction (˜0.49V versus Ag|AgCl chemical reference) and state dispersion with C-Reactive Protein target concentration.FIG.5cshows the cumulative distribution function [ΔN[target]∝(1/e2)∫CμdV] of electronic states as a function of electrode potential, where the DOS occupancy changes as directly reported. N varies as a function of target protein concentration such as ΔN∝Δ(dN/dE)[target].FIG.5dis a graph showing the density of state function data ofFIG.5bnormalized for the total state occupancy at each target concentration (ΔNt[target]; obtained by integration of the DOS across all relevant energies). The superposition of the resulting plots confirms that the energetic change associated with occupancy change, ΔE/ΔNt[target]=(e2/Cμ), is a constant. All values/curves obtained represent means across triplicate measurements. As shown inFIG.5a, a change in local dielectric constant translates into a resolved DOS energetic redistribution without a change in total state occupancy [ΔNt=(1/e2)∫−∞∞Cμ(V)dV, as expressed by the total area of the normal DOS distribution function]. A molecular binding event, however (such as that occurring at a neighbouring receptive site) triggers a resolved change in DOS occupation (FIG.5b). One can seek to distinguish between these two environmental triggers by looking at the DOS shape (that follows a normal distribution) and its associated energetic spread as expressed through S[dN/dE]=lnσg√{square root over (2πen)} (the entropy function of the normal DOS distribution), where enis the base of natural logarithms and σgthe standard deviation. In the case of dielectric change, there is a clearly resolved change in dispersion (FIG.5a). The effects of a local binding event are different in that there is an associated change in chemical potential resolvable through DOS occupation or measured Cμ) without change in electronic dispersion (insetFIG.5b) or in Fermi energy. From Equation (1) the variation in energy per number of particles (the energy/chemical potential change associated with DOS occupancy change) is dE/dN=N(e2/Cμ), from which the associated change in energy and capacitance are quantified as ΔE=(e2Cμ) and Cμ/N=e2(DN/dE), respectively. If one normalizes the experimentally measured electrochemical capacitance change (which is proportional to the DOS) across target binding concentrations for the total number of states ΔNt[target], that is ΔCμ/ΔNt[target]=[e2(dN/dE)] the responses collapse to constant e2(dN/dE) value (FIG.5d). Ultimately this confirms that a neighbouring molecular recognition and associated chemical potential change perturbs only occupancy of the DOS (without a change in the entropy associated with electronic states occupancy, in the present example). This is especially notable close to the electrode Fermi level, wherein the relationship is linear (as expected for an idealised two-dimensional electron gas, where e2/Cμis constant). In summary, the electronic DOS, presented by a redox molecular film, responds to a neighbouring molecular recognition such that the resolved electronic charge ΔN, is proportional to the target/analytic concentration. This is illustrated inFIG.6, in which the target is C-reactive protein (CRP). FIG.6ais a graph showing the linear relationship between the variation of total electron density obtained from DOS integration (1/e2)∫−∞∞Cμ(V)dV in the entire interval of potential shown inFIG.5das a function of target CRP concentration at a redox active SAM-anti-CRP modified gold electrode. The redox unit acts as a mesoscopic probe element140. The resolved redox DOS is unresponsive both to an equivalent concentration of a negative control (here Fetuin-A) and to CRP in the absence of a specific antibody receptor (yellow triangles). From the specific response, binding affinities are readily resolved from subsequent fitting to a Langmuir isothermal model; here 3.2±4×108mol−1L, comparable to that obtained by other methods for the same interfacial immuno-complexation. It is possible to generate analytical curves (FIG.6b) using the local recognition induced energy change (ΔE), as resolved at a single potential, 0.49 V versus Ag|AgCl, the Fermi energy (EF) of the junction. Energy and occupancy are anti-correlated. The error bars represent standard deviation across three independent receptive junctions. This DOS based sensing is, of course, a general principal that the skilled person would appreciate can be readily extended to the selective detection of other markers alternatively using specific interfacial chemistries (seeFIG.7). FIGS.7aand7bshow analytical curves obtained by plotting variation of the energy of the accessible interfacial states (ΔE) at their fixed Fermi energy (EF) for a range of clinically relevant markers. In particular,FIG.7ashows analytical curves for C-reactive protein and α-synuclein (respectively markers for cardiac and general inflammation, and Parkinson's disease);FIG.7bshows analytical curves for C-reactive protein and prostatic acid phosphatase (respectively markers for cardiac and general inflammation, and prostate cancer). The transduction principles active here are independent of specific film composition; the receptive films utilized are mixed thiolated ferrocene (providing the accessible responsive DOS) and thiolated alkyl or pegylated carboxylate. Assay sensitivity shows some predictable dependence on film. Error bars represent standard deviations derived from measurements conducted at three independent electrodes. In all cases ΔN and/or ΔE remains a linear function of the logarithm of the target concentration (as noted previously that ΔE[target]∝e2/Cμ[target] (variations rationalized in terms of the effective variation on the chemical energy state of the surface). Note that occupancy (ΔN) or chemical potential/energy change (ΔE) is most sensitive to target binding at potentials close to the Fermi level such that analytical curves (ΔE versus logarithm of [target]) can be derived by consideration of changes at this specific energy (FIG.6bandFIG.7). The general principles of utilizing the resolved sensitivity of a surface confined electrode-chargeable DOS as a transducer signal of captured target can be extrapolated readily to antibody-modified non-redox active graphene derived interfaces assembled on metallic electrodes. In other words, the mesoscopic principles active at a redox-confined surface extend readily to non-redox active interfaces where an accessible DOS is confined to nanometre scale entities and accordingly highly responsive to environment. This is exemplified inFIG.8athrough the use of electrode immobilized and antibody modified reduced graphene oxide films. FIG.8ais a graph showing capacitance resolved variations in ΔE∝Δ1/Cμfor electrochemically reduced graphene oxide multilayers functionalized with anti-CRP antibodies.FIG.8bshows demonstrative selectivity obtained at micro-fabricated arrays modified with anti-CRP-modified redox-modified graphene oxide; here the response to 105pM CRP spiked into serum is shown relative to a that at native serum prior to spiking.FIG.8c, equivalent to8a, is the analytical curve comprising the linear relationship between and the logarithm of CRP concentration in serum as resolved at redox and graphene oxide modified micro-fabricated gold electrode arrays (detection limit 55 pM, linear analytical range is >103pM and <105pM, relevant to clinical need). InFIG.8cthe errors bars are standard deviations calculated across three measurements performed at the same disposable array. It is now demonstrated that the operational principles associated with these capacitive spectroscopy resolved DOS assays are analogous to those operating in FET-based devices under equilibrium conditions (seeFIG.4c), wherein the quantum capacitance behaves in a manner equivalent to the channel states of FET devices. One first observes that the difference between the potential in the channel/bridge (VC) and in the gate (VG) is dependent on N as VC−VG=−(eN/Ce) (in electro-active molecular layers this corresponds to the potential difference between the local potential of the accessible states and the external potential in the electrolyte). The gate capacitance is thus dq/dVG=(dq/dVC)(dVC/dVG). By noting that dq/dVGis Cμand dq/dVCis the quantum capacitance (Cq), dq/dVGrearranges to give q2Cμ_=q2(dVGdq)=q2(Cq+CeCeCq)=q2(1Ce+1Cq)(2) an expression explicitly equivalent to Equation (1). Equation (2) implicitly demonstrates that, in experimentally accessing Cμ, the thermodynamic properties of the junctions are monitorable, potentially as a function of a neighbouring/integrated target recognition. This is accessible through ΔG=e2/Cμ=−κBT ln Kawhich additionally implies the observed semi-logarithmic linear relationship (shown inFIGS.6b,7and8) resolved under equilibrium binding conditions. It is worth noting that these binding induced changes in electronic free energy are accompanied by associated change in state occupation (discharging/charging Cμand de-populating/populating states) such that the Fermi energy does not change (FIG.5b). The capacitive DOS sensing introduced here is therefore similar to those operating in micro-fabricated FET devices but, significantly, is both highly chemically tailorable and requires just one contact;FIGS.8aand8cspecifically show how the resolved electrochemical energy (electronically dominated) of an anti-CRP antibody functionalized reduced graphene oxide and composite interfaces responds to clinically relevant levels of CRP at macro-disk and micro-fabricated disposable electrode arrays. It has therefore been shown by the disclosures herein that a surface confined and electronically addressable DOS (comprising either redox switchable centres or appropriately immobilized mesoscopic units) contributes to a readily resolved quantum capacitance. This charging reports on the occupancy of quantized states and responds sensitively to changes in local chemical potential. By appropriately introducing receptors this entirely reagentless sensing becomes highly specific and very sensitive. This transduction mechanism operates in a manner analogous to FET devices but in a markedly more experimentally accessible and chemically flexible manner. With reference toFIGS.4to8above, it has therefore been shown that a change in the relaxation time (corresponding to a change in the capacitance of the sensor) is correlated with a change in the density of states. This concept is readily applicable to the sensor100ofFIG.1in which the conductive connection is capacitively coupled to the mesoscopic probe element, and wherein an interaction between the mesoscopic probe element and a measurand of the environment affects the capacitance (and relaxation time) of the conductive connection. Although the effect on capacitance has been described herein, it should be appreciated that not only the capacitance will be affected by a detection event. Notably, the impedance and several other measurable quantities will likewise be affected, all of which are indicative of a change in the relaxation time. Variations of the described embodiments are envisaged, for example, the features of all of the disclosed embodiments may be combined in any way and/or combination, unless such features are incompatible. The mesoscopic probe element may be of any suitable form. For example, the mesoscopic probe element may be formed of any zero- (less than 10 nm), one- or two-dimensional compounds (providing that the others dimensions in the case of one- and two-dimensional compounds are below 10 nm). The mesoscopic probe element may comprise a metal oxide semiconductor, a mixed-valence semiconductor, an organic semiconductor, graphene, reduced graphene, fluorene, redox active polymers, or some other suitable material. A conductive connection may be formed of any suitable electrically conductive material. For example, the conductive connection may comprise indium tin oxide (ITO), fluorine doped tin oxide (FTO), or a conductive carbonaceous compound such as graphene or glassy carbon. The conductive connection may comprise for example a metal such as gold, platinum, titanium, copper or a copper alloy. The conductive connection or electrode or contact or body may comprise gold. They may comprise platinum. They may comprise Indium Tin Oxide (ITO). They may comprise Fluorine Doped Tin Oxide (FTO). They may comprise conductive polymers. They may comprise any other suitable conductor. The mesoscopic probe element may comprise any suitable material, such as any mixed valence oxide, graphene oxide, or titanium dioxide. The mesoscopic probe element may comprise titanium nanotubes, for example. Although only a handful of possible measurands have been described above, any measurand may be detected provided that there is a corresponding change in the density of states. For example, the sensor(s), sensing system(s) and sensing method(s) as described herein may be used to detect biological markers such as metabolytes or other small molecules and may be used to detect electromagnetic radiation. For example, the sensors, sensing systems and sensing methods disclosed herein may be used for the detection of disease marker proteins such as CRP, PAP, PSA, Insulin, HER2, Alpha-synuclein, CA19-9, or NS1. The sensors, sensing systems and sensing methods disclosed herein may be used for the detection of sugar molecules for glycoarrays, D-dimers, hormones, amino acids, alcohols, vitamins (such as B2 and B12), polyols, organic adds, nucleotides (for example inosine-5′-monophosphate and guanosine-5′-monophosphate), and so on. The skilled person would appreciate that the sensors, sensing systems and sensing methods disclosed herein may be used for the detection of a variety of measurands in a variety of settings. It will be appreciated that embodiments of the present invention can be realised in the form of hardware, software or a combination of hardware and software. Any such software may be stored in the form of volatile or non-volatile storage such as, for example, a storage device like a ROM, whether erasable or rewritable or not, or in the form of memory such as, for example, RAM, memory chips, device or integrated circuits or on an optically or magnetically readable medium such as, for example, a CD, DVD, magnetic disk or magnetic tape. It will be appreciated that the storage devices and storage media are embodiments of machine-readable storage that are suitable for storing a program or programs that, when executed, implement embodiments of the present invention. Accordingly, embodiments provide a program comprising code for implementing a system or method as claimed in any preceding claim and a machine readable storage storing such a program. Still further, embodiments of the present invention may be conveyed electronically via any medium such as a communication signal carried over a wired or wireless connection and embodiments suitably encompass the same. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The claims should not be construed to cover merely the foregoing embodiments, but also any embodiments which fall within the scope of the claims. | 33,408 |
11860120 | DETAILED DESCRIPTION The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. Further, spatially relative terms, such as “beneath,” “below,” “lower,” “above,” “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. The spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. The apparatus may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein may likewise be interpreted accordingly. One type of biosensor includes a semiconductor substrate that is covered by an isolation dielectric layer and that accommodates a biologically sensitive field-effect transistor (BioFET). One advantage of BioFETs is the prospect of label-free operation. Specifically, BioFETs enable the avoidance of costly and time-consuming labeling operations such as the labeling of analytes (e.g., cardiac cells) with, for instance, fluorescent or radioactive probes. The BioFET includes a source region and a drain region that are arranged within the semiconductor substrate and that define a channel region therebetween. Further, the BioFET includes a gate arranged under the semiconductor substrate, laterally between the source region and the drain region. The isolation dielectric layer includes a sensing well that exposes the semiconductor substrate, laterally between the source region and the drain region, and that is lined by a biosensing film. The biosensing film is configured to detect an impedance change, molecule charge and/or ion release resulting from bio-entities (e.g., cardiac cells), such that 2D electrical image profile of the bio-entities (e.g., cardiac cells) can be obtained and/or the bio-entities may be monitored. FIG.1illustrates a cross-sectional view of an example integrated circuit10including an array of BioFETs100in accordance with some embodiments of the present disclosure. The BioFETs100each include a pair of source/drain regions104and, in some embodiments, a gate electrode106. The source/drains regions104have a first conductivity type (i.e., doping type) and are arranged within an active semiconductor layer102, respectively on opposite sides of a channel region108of the BioFET100. The channel region108has a second conductivity type opposite the first conductivity type and is arranged in the active semiconductor layer102, laterally between the source/drain regions104. The first and second doping types may, for example, respectively be n-type and p-type, or vice versa. In some embodiments, the source/drain regions104and the channel region108are arranged within a doped well region of the active semiconductor layer102that has the second conductivity type, and/or are electrically coupled to a back-end-of-line (BEOL) interconnect structure110that is arranged over a carrier substrate112. Further, in some embodiments, the source/drain regions104and the channel region108extend from a front surface102fof the active semiconductor layer102to a back surface102bof the active semiconductor layer102. Stated differently, the source/drain regions104and the channel region108can extend through the full thickness of the active semiconductor layer102to facilitate functioning of biosensening. The gate electrode106is arranged on the front surface102fof the active semiconductor layer102, laterally between the source/drain regions104, and is spaced from the front surface102fof the active semiconductor layer102by a gate dielectric layer114. In some embodiments, the gate electrode106is electrically coupled to the BEOL interconnect structure110is metal, doped polysilicon, or a combination of the foregoing. In some embodiments, the BioFETs100may be separated from each other using shallow trench isolation regions (not shown) laterally surrounding each of the BioFETs100. Gate dielectric layer114includes, for example, silicon dioxide and/or a high-k gate dielectric material with a dielectric constant higher than that of silicon dioxide. Exemplary high-k gate dielectric materials include, but are not limited to, silicon nitride, silicon oxynitride, hafnium oxide (HfO2), hafnium silicon oxide (HfSiO), hafnium silicon oxynitride (HfSiON), hafnium tantalum oxide (HfTaO), hafnium titanium oxide (HfTiO), hafnium zirconium oxide (HfZrO), metal oxides, metal nitrides, metal silicates, transition metal-oxides, transition metal-nitrides, transition metal-silicates, oxynitrides of metals, metal aluminates, zirconium silicate, zirconium aluminate, zirconium oxide, titanium oxide, aluminum oxide, hafnium dioxide-alumina (HfO2—Al2O3) alloy, other suitable high-k dielectric materials, and/or combinations thereof. In some embodiments, the gate dielectric includes a stack of an interfacial dielectric material and a high-k dielectric material. In some embodiments, the active semiconductor layer102may be a silicon substrate or wafer. Alternatively, the semiconductor layer102may include another elementary semiconductor, such as germanium (Ge); a compound semiconductor including silicon carbide (SiC), gallium arsenic (GaAs), gallium phosphide (GaP), indium phosphide (InP), indium arsenide (InAs), and/or indium antimonide (InSb); an alloy semiconductor including SiGe, GaAsP, AlInAs, AlGaAs, GaInAs, GaInP, and/or GaInAsP; or combinations thereof. In the depicted embodiments, the active semiconductor layer102is a semiconductor layer of a semiconductor-on-insulator (SOI) substrate (e.g., silicon layer). In some embodiments, the carrier substrate (interchangeably referred to as handle substrate as well)112may be, for example, a bulk semiconductor substrate, such as a bulk substrate of monocrystalline silicon. An isolation dielectric layer116is arranged on the back surface102bof the active semiconductor layer102, and includes a plurality of sensing wells118over corresponding channel regions108of BioFETs100. The sensing wells118extend into the isolation dielectric layer116to proximate the channel regions108of BioFETs100and are at least partially lined by a biosensing film120. Further, in some embodiments, the sensing wells118extend through the isolation dielectric layer116to expose the respective channel regions108of BioFETs100. The isolation dielectric layer116may be, for example, silicon dioxide, a buried oxide (BOX) layer of a SOI substrate, some other dielectric, or a combination of the foregoing. In some specific embodiments, the active semiconductor layer102is a silicon layer of a SOI substrate, and the isolation dielectric layer116is a BOX layer of the SOI substrate. The biosensing film120lines the sensing wells118and, in some embodiments, covers the entire isolation dielectric layer116. The biosensing film120is operative to modulate the source to drain conductivity of each bioFET100when contacted by a fluid190having a suitable composition or carrying specific analytes. For example, the fluid190is an aqueous solution containing cardiac cells192. Examples of materials for biosensing film120that provide the functionality of biosensing include HfO2, SiO2, Si3N4, Al2O3, and Ta2O5. An upper surface of the biosensing film120includes a coating of a selective binding agent122. The selective binding agent122includes one or more biological materials having the property of selectively binding with the cardiac cells192. If the cardiac cell192is stably bound on the upper surface of the biosensing film120, the overall charge concentration at the biosensing film120can become sufficient to modulate the source to drain conductivity of BioFETs100, thus improving the biosensing performance. Because the selective binding agent122has a greater binding ability (i.e., greater adhesion) to the cardiac cell192than that of the biosensing film120, the biosensing performance can be improved as long as the selective binding agent122is coated on the biosensing film120. In some embodiments, the selective binding agent122for selectively binding with the cardiac cell192includes, for example, collagen, laminin, fibronectin, and mucopolysaccharides, heparin sulfate, hyaluronidate, chondroitin sulfate, the like, or combinations thereof. The coating of the selective binding agent122is illustrated as a blanket layer covering the entire biosensing film120merely for the sake of clarity, but in practice the coating of the selective binding agent122is porous and thus does not cover the entire biosensing film120, which allows the biosensing film120to be in contact with the cardiac-cell-containing fluid190. The sensor array of BioFETs100can use the biosensing film120to monitor beating of the cardiac cells192and/or generate 2D image profiles of the cardiac cells192by detecting impedance change, module charge and/or ion release. Take ion release detection for example, in operation a reference electrode194gives the cardiac-cell-containing solution190a voltage potential, then the biosensing film120becomes charged when brought in contact with the cardiac cell containing fluid190having a suitable ion concentration. Moreover, they can become sufficiently charged to switch the source/drain conductivity of bioFETs100. In this way, the sensor array of bioFETs100has the biosensing film120functional to detect ion released from or captured on the cardiac cell192and/or ion released from or captured on the selective binding agent122. In a similar manner, the sensor array of bioFETs100can detect molecule charge released from or captured on the cardiac cell192and/or charge released from or captured on the selective binding agent122. In examples, the current between the source/drain regions104is measured, and the measured current (or a change in the measured current caused by the fluid190and/or the cardiac cell192) is indicative of impedance change, molecule charge and/or ion release caused by the fluid190and/or the cardiac cell192. Thus, in such examples, the detection mechanism is a conduction modulation of the transducer due to the binding of the cardiac cell192over the biosensing film120. In other examples, one or more components (e.g., a trans-impedance amplifier) are used to translate the current or change of current induced by the fluid190and/or the cardiac cell192into another electrical signal, such as a measurable voltage. To illustrate this, reference is made toFIG.2, which depicts a trans-impedance amplifier202used in generating an electrical signal204, in accordance with some embodiments of the present disclosure.FIG.2illustrates an equivalent circuit that corresponds to a BioFET (e.g., BioFET100as illustrated inFIG.1) and shows that a drain current induced by the fluid190and/or cardiac cell192on the upper surface of the biosensing film120is translated into the electrical signal204by the trans-impedance amplifier202. The electrical signal204may include, for example, voltages (e.g., voltage signals) that can be measured. In some embodiments, each of the BioFETs100may be controlled (i.e., turned on and turned off) by an access transistor. For example, as illustrated in toFIG.3, a cross section of a BioFET100and an access transistor300is provided, according to some embodiments. The BioFET100includes source/drain regions104, a channel region108laterally between the source/drain regions104, a gate dielectric layer114over the channel region108and a gate electrode106over the gate dielectric layer114, all of which are discussed previously with respect toFIG.1and thus are not repeated for the sake of brevity. The access transistor300is coupled to the BioFET100, as illustrated in the circuit diagram ofFIG.4. The access transistor300similarly includes source/drain regions304formed in the semiconductor substrate102, a channel region308laterally between the source/drain regions304, a gate dielectric layer314over the channel region308and a gate electrode306over the gate dielectric layer314. The source/drain regions304are doped regions having a conductivity type opposite the conductivity type of the channel region308. For example, the source/drain regions304are of an n-type conductivity and the channel region308is of a p-type conductivity, or vice versa. In some embodiments, the gate electrode306of the access transistor300has a same material composition as the gate electrode106of the BioFET100and is formed simultaneously with the gate electrode106. For example, the gate electrode306may be metal, doped polysilicon, or a combination of the foregoing. In some embodiments, the gate dielectric layer314has a same material composition as the gate dielectric layer114and is formed simultaneously with the gate dielectric layer114, and thus example materials of the gate dielectric layer314are not repeated for the sake of brevity. The access transistor300is similar with the BioFET100, except that the channel region308and/or the source/drain regions304of the access transistor300are separated from the biosensing film120by the isolation dielectric layer116. In this way, operation of the access transistor300is not affected by the fluid190and/or the cardiac cell192. FIG.4illustrates an equivalent circuit that corresponds to the relationship between the BioFET100and the access transistor300as shown inFIG.3. As illustrated inFIG.4, the access transistor300serves as a switching device coupled to source/drain terminal of the BioFET100, and thus the access transistor300can turn on the BioFET100to initiate detecting and/or monitoring the cardiac cell, and can also turn off the BioFET100to stop detecting and/or monitoring the cardiac cell. FIG.5is a functional block diagram of an integrated circuit50in accordance some embodiments of the present disclosure. The integrated circuit50includes a sensing pixel array SA that includes sensing pixels502arranged into M columns and N rows. M and N are positive integers. In some embodiments, M ranges from 1 to 256. In some embodiments, N ranges from 1 to 256. The number of M and N are selected based on a normal size of a cardiac cell192, which in turn allows for detecting/monitoring a single cardiac cell192using a single sensing pixel array SA. Each sensing pixel502of the array SA at least includes a BioFET (e.g., BioFET100as illustrated inFIGS.3and4) and an access transistor (e.g., access transistor300as illustrated inFIGS.3and4) coupled to the BioFET. The integrated circuit50also includes a column decoder504coupled to the sensing pixel array SA via column lines CL1-CLM. The column decoder504decodes a column address of sensing pixels502selected to be accessed in a cardiac cell detection/monitor operation. The column decoder504then enables, via the column pixel selector506, the column line corresponding to the decoded column address to permit access to the selected sensing pixels502. The integrated circuit50also includes a row decoder508coupled to the sensing pixel array SA via row lines RL1-RLN. The row decoder decodes a row address of sensing pixels502selected to be accessed in a cardiac cell detection/monitor operation. The row decoder508then enables, via the row pixel selector510, the row line corresponding to the decoded row address to permit reading out biosensing measurements from the selected sensing pixels502. In some embodiments, the integrated circuit50further includes one or more trans-impedance amplifiers512configured to receive readout biosensing measurements from an output of the row decoder508and generate an electrical signal based on the readout biosensing measurements of the selected sensing pixels502. By way of example and not limitation, the readout biosensing measurements of selected sensing pixels502include drain currents of BioFETs of the selected sensing pixels502and can be translated into electric signals514by the trans-impedance amplifier512. The electric signals514may include, for example, voltages (e.g., voltage signals). The integrated circuit50allows for detecting/monitoring a single cardiac cell. However, in some embodiments, multiple cardiac cells can be detected/monitored using an integrated circuit.FIG.6is a functional block diagram of an example integrated circuit60that is designed to detect/monitor multiple cardiac cells192in accordance some embodiments of the present disclosure. The integrated circuit60includes multiple sensing pixel arrays SA. Each sensing pixel arrays SA includes sensing pixels602each including a BioFET (e.g., BioFET100as illustrated inFIGS.3and4) and an access transistor (e.g., access transistor300as illustrated inFIGS.3and4) coupled to the BioFET. The sensing pixels602in each sensing pixel array SA are arranged into M columns and N rows. M and N are positive integers. In some embodiments, both M and N range from 1 to 256. The number of M and N are selected based on a normal size of a cardiac cell192, which in turn allows for detecting/monitoring a single cardiac cell192using a single sensing pixel array SA. Therefore, the integrated circuit60having multiple sensing pixel arrays SA can detect and/or monitor multiple cardiac cells192. The sensing pixel arrays SA are arranged into P columns and Q rows. P and Q are positive integers. In some embodiments, P is less than M, and Q is less than N. In some other embodiments, P is greater than M, and Q is greater than N. The number of P and Q are selected depending on a desired number of cardiac cells to be detected and/or monitored. The integrated circuit60includes a column decoder604coupled to the sensing pixel arrays SA via column cell spot lines CCL1-CCLPand to the sensing pixels602via column pixel lines CPL1-CPLM. The column decoder604decodes a column cell spot address of sensing pixel arrays SA and a column pixel address of sensing pixels602of the selected sensing pixel array SA. The column decoder604then enables, via the column cell spot selector606, the column cell spot line corresponding to the decoded column cell spot address, to permit access to the selected sensing pixel arrays SA. The column decoder604then enables, via the column pixel selector608, the column pixel line corresponding to the decoded column pixel address, so as to permit access to the selected sensing pixels602of the selected sensing pixel arrays SA. The integrated circuit60includes a row decoder610coupled to the sensing pixel arrays SA via row cell spot lines RCL1-RCLqand to the sensing pixels602via row pixel lines RPL1-RPLN. The row decoder610decodes a row cell spot address of sensing pixel arrays SA and a row pixel address of sensing pixels602of the selected sensing pixel array SA. The row decoder610then enables, via the row cell spot selector612, the row cell spot line corresponding to the decoded row cell spot address. The row decoder610then enables, via the row pixel selector614, the row pixel line corresponding to the decoded row pixel address, so as to permit reading out biosensing measurements from the selected sensing pixels602of the selected sensing pixel arrays SA. FIG.7illustrates an example cross-sectional view of a partial region of the integrated circuit60ofFIG.6.FIG.7illustrates two neighboring sensing pixel array SA spaced apart by one or more fluid channel walls702. The fluid channel walls702laterally define fluid containment regions704over the respective sensing pixel arrays SA. Each sensing pixel array SA includes BioFETs100of sensing pixels. The fluid containment region704can be a well or a length of channel bound by fluid channel walls702. The fluid channel walls702can be formed of waterproof material(s). In some embodiments, the fluid channel walls702are an elastomer. In some of these embodiments, the elastomer of polydimethylsiloxane (PDMS). In some embodiments, fluid containment regions704are capped to provide closed channels or reservoirs. Spacing between the fluid channel walls702are selected such that a single fluid containment region704's size matches with a normal size of a cardiac cell192. By way of example, the fluid containment region704has a width in a range from about 10 um to about 300 um. If the width of the fluid containment region704is less than about 10 um, the fluid containment region704may be too narrow to accommodate a single cardiac cell192. If the width of the fluid containment region704is greater than about 300 um, the fluid containment region704may accommodate multiple cardiac cells193. The BioFETs100are discussed previously with respect toFIG.1and thus are not repeated for the sake of brevity. FIG.8illustrates a cross-sectional view of an example integrated circuit80including an array of BioFETs100in accordance with some embodiments of the present disclosure. The integrated circuit80is similar to the integrated circuit10ofFIG.1, except that the integrated circuit80includes additional heaters802. In some embodiments, the heaters802are doped regions in the active semiconductor layer102. In some embodiments, the heaters802are formed together with (i.e., simultaneously with) the source/drain regions104, and thus have the same dopant type and dopant concentration profile as the source/drain regions104. Different from the source/drain regions104, the heaters802in the active semiconductor layer102are separated from the channel regions104by, for example, shallow trench isolation (STI), and thus the voltage applied to the heaters802does not affect functionality of the BioFETs100. With the heaters802, the fluid190and/or the cardiac cell192can be heated to enhance the detection and/or monitoring of the cardiac cell192. In some embodiments, the heaters802are operated in response to a temperature measurement from a temperature-sensing device (not shown inFIG.8), as discussed below. FIG.9is a circuit diagram of a BioFET100and its surrounding heaters802of the integrated circuit80in accordance with some embodiments. The integrated circuit80includes a sensing pixel902having a BioFET100, a first switching device904, a temperature-sensing device906, and a second switching device908. The first switching device904is coupled between a first end of the BioFET100and a corresponding row line (e.g., one of the row lines RL1-RLNas illustrated inFIG.5). The second switching device908is coupled between a first end of the temperature-sensing device906and a corresponding signal path for reading out the temperature measurement of the temperature-sensing device906. The first switching device904and the second switching device908are N-type transistors having gates coupled with a corresponding column line (e.g., one of the column lines CL1-CLMas illustrated inFIG.5). A second end of the BioFET100and a second end of temperature-sensing device906are coupled together and configured to receive a reference voltage. In some embodiments, temperature-sensing device906includes a p-n diode formed in the active semiconductor layer102(as shown inFIG.8). In some embodiments, the first switching device904or second switching device908is implemented by other types of switching devices, such as a transmission gate or a P-type transistor. Temperature-sensing device906is configured to measure a temperature of the biosensing film of the BioFET100and then generate a temperature-sensing signal responsive to the measured temperature of the biosensing film. The heaters802are configured to adjust the temperature of the biosensing film of the BioFET100, which in turn adjusts the temperature of the cardiac-cell-containing fluid and the cardiac cell over the BioFET100. The temperature-sensing signal generated from the temperature-sensing device906can serve as feedback to control the heaters802, which in turn promotes good temperature control and uniformity. By way of example and not limitation, when the measured temperature from the temperature-sensing device906is higher than an expected temperature range suitable for detecting and/or monitoring the cardiac cell192, the heaters802are turned off; when the measured temperature from the temperature-sensing device906is lower than the expected temperature range, the heaters802are turned off. In some embodiments, as illustrated inFIG.9, separate heaters802together surround four sides of the sensing pixel902. However, in some other embodiments an integrated circuit has a different heater configuration, as illustrated in a layout diagram ofFIG.10. As shown inFIG.10, the layout1000includes a plurality of first elongated heaters1002extending in a first direction, a plurality of second elongated heaters1004extending across the first elongated heaters1002in a second direction perpendicular to the first direction, and a plurality of sensing pixels902bound by the first elongated heaters1002and the second elongated heaters1004. The sensing pixels902each include a BioFET100, a temperature-sensing device906, and first and second switching devices904and908, as discussed previously with respect toFIG.9. FIGS.11and12illustrate cross-sectional views of an example integrated circuit1100having the top view layout1000ofFIG.10in accordance with some embodiments, whereinFIG.11is a cross-sectional view taken along line A-A′ ofFIG.10, andFIG.12is a cross-sectional view taken along line B-B′ ofFIG.10. As illustrated inFIGS.11and12, the first elongated heaters1002are doped regions formed in the active semiconductor layer102, and the second elongated heaters1004are doped polysilicon structure formed on the front surface102fof the active semiconductor layer102. In some embodiments where the gate electrodes106are polysilicon, the second elongated heaters1004are formed together with the polysilicon gates106, and thus have the same thickness and material composition as the polysilicon gates106. The first elongated heaters1002are thus interchangeably referred to as doped silicon heaters, and the second elongated heaters1004are thus interchangeably referred to as polysilicon heaters. The polysilicon heaters1004are vertically spaced apart from the doped silicon heaters1002by a dielectric layer1006, as illustrated inFIG.12. In some embodiments, the dielectric layer1006is formed together with the gate dielectric layers114of the BioFETs100, and thus the dielectric layer1006has the same thickness and material composition as the gate electric layers114. By way of example and not limitation, the dielectric layer1006includes silicon dioxide, a high-k dielectric material with a dielectric constant higher than a dielectric constant of silicon dioxide or combinations thereof. In some embodiments, the doped silicon heaters1002and the polysilicon heaters1004non-overlap with BioFETs100of all sensing pixels902from a top view as illustrated inFIG.10, and thus the doped silicon heaters1002and the polysilicon heaters1004do not affect the functionality of BioFETs100. FIG.13illustrates a cross-sectional view of another example integrated circuit1300having the layout1000ofFIG.10in accordance with some embodiments, whereinFIG.13is a cross-sectional view taken along line B-B′ ofFIG.10. The second elongated heaters1004in the integrated circuit1300is formed in the BEOL interconnect structure110, not in the active semiconductor layer102. In some embodiments, the second elongated heaters1004include titanium aluminum nitride, platinum, indium tin oxide, titanium nitride, or a combination of the foregoing. In some embodiments, the second elongated heaters1004have a thickness in a range from about 5600 angstroms to about 6600 angstroms and a sheet resistance in a range from about 4 ohm/sq to about 6 ohm/sq. In some embodiments, the second elongated heaters1004are formed in a metallization layer of the interconnect structure110and laterally surrounded by an inter-metal dielectric (IMD) layer of the multi-layer dielectric structure128. Moreover, other metallization layers including the metal lines124and metal vias126are formed of a different metal composition (e.g., copper) than that of the second elongated heaters1004, because these metallization layers including the metal lines124and metal vias126are not designed for heating. FIG.14illustrates a cross-sectional view of an example integrated circuit1400in accordance with some embodiments of the present disclosure. The integrated circuit1400includes a BioFET100formed on the active semiconductor layer102. The BioFET100is similar to that described previously with respect toFIG.1, except that the source/drain regions104are formed in a well region1042of the active semiconductor layer102. The well region1042has a conductivity type opposite the source/drain regions104. Moreover, the integrated circuit1400includes one or more first heaters1404and one or more temperature-sensing devices1406formed in the active semiconductor layer102. The first heater1404is a doped region in the active semiconductor layer102. In some embodiments, the first heater1404is formed together with (i.e., simultaneously with) the source/drain regions104, and thus has the same dopant type and dopant concentration profile as the source/drain regions104. In some other embodiments, the first heater1404is formed together with (i.e., simultaneously with) the well region1402, and thus has the same dopant type and dopant concentration profile as the well region1402. The temperature-sensing device1406is a diode formed in the active semiconductor layer102, and as such the temperature-sensing device1046includes at least one P-N junction (as indicated by the dash line DL) forming the diode within the active semiconductor layer102. In some embodiments, the temperature-sensing device1046has a p-type doped region and an n-type doped region to form the P-N junction. In some embodiments where the BioFET100is a PFET, the p-type doped region of the temperature-sensing device1406is formed together with the source/drain regions104of the BioFET100, and the n-type doped region of the temperature-sensing device1406is formed together with the well region1402. In some embodiments where the BioFET100is an NFET, the n-type doped region of the temperature-sensing device1406is formed together with the source/drain regions104of the BioFET100, and the p-type doped region of the temperature-sensing device1406is formed together with the well region1402. The integrated circuit1400further includes one or more second heaters1408formed in the interconnect structure110below the active semiconductor layer102. In some embodiments, the second heaters1408include titanium aluminum nitride, platinum, indium tin oxide, titanium nitride, or a combination of the foregoing. In some embodiments, the second heaters1408have a thickness in a range from about 5600 angstroms to about 6600 angstroms and a sheet resistance in a range from about 4 ohm/sq to about 6 ohm/sq. In some embodiments, the second heaters1408are formed in a metallization layer of the interconnect structure110and laterally surrounded by an inter-metal dielectric (IMD) layer of the multi-layer dielectric structure128. Moreover, other metallization layers including the metal lines124and metal vias126are formed of a different metal composition (e.g., copper) than that of the second elongated heaters1004, because these metallization layers including the metal lines124and metal vias126are not designed for heating. The integrated circuit1400has a pad opening1410extending through the coating of selective binding agent122, the biosensing film120, the isolation dielectric layer160and the active semiconductor layer102to expose a pad structure1412formed within the interconnect structure110. In some embodiments, the pad structure1412is formed in a metallization layer of the interconnect structure110and laterally surrounded by an inter-metal dielectric (IMD) layer of the multi-layer dielectric structure128. In some embodiments, a vertical distance from the pad structure1412to the active semiconductor layer102is less than a vertical distance from the second heater1408to the active semiconductor layer102. Moreover the integrated circuit1400further includes fluid channel walls1414. The fluid channel walls1414laterally define a fluid containment region1416over the BioFET100. The fluid containment region1416may be a well or a length of channel bound by fluid channel walls1414. The fluid channel walls1414can be formed of waterproof material(s). In some embodiments, the fluid channel walls1414are an elastomer. In some of these embodiments, the elastomer of polydimethylsiloxane (PDMS). FIGS.15-22illustrate cross-sectional views of various intermediate stage of a method for forming the integrated circuit1400as illustrated inFIG.14. Throughout the various views and illustrative embodiments, like reference numbers are used to designate like elements. It is understood that additional operations can be provided before, during, and after the processes shown byFIGS.15-22, and some of the operations described below can be replaced or eliminated, for additional embodiments of the method. The order of the operations/processes may be interchangeable. As illustrated inFIG.15, an SOI substrate1500is formed. The SOI substrate1500comprises a bulk semiconductor substrate1502over which an isolation dielectric layer116and an active semiconductor layer102are stacked. As seen hereafter, the bulk semiconductor substrate1502is sacrificial. The bulk semiconductor substrate1502and the active semiconductor layer102may be, for example, monocrystalline silicon, and/or the isolation dielectric layer116may be, for example, silicon dioxide. The SOI substrate1500can be formed by any suitable process. In some embodiments, SOI substrate1500is formed through separation by implanted oxygen (SIMOX). After the SOI substrate1500is prepared, one or more isolation regions1504are optionally formed in the active semiconductor layer102of the SOI substrate1500. In the depicted embodiments, the isolation regions1504are formed through the full thickness of the active semiconductor layer102. In some other embodiments, the isolation regions1504are STI regions that do not extend through the full thickness of the active semiconductor layer102. The isolation regions1504extend laterally to enclose subsequently formed BioFETs100, first heaters1404and temperature-sensing devices1406, so as to provide electrical isolation to these devices. The process for forming the isolation regions1504may comprise, for example, patterning the active semiconductor layer102to define one or more trenches corresponding to the isolation regions1504, subsequently depositing or growing one or more dielectric materials filling the one or more trenches, followed by performing a planarization process (e.g., chemical mechanical polish (CMP)) on the dielectric materials until the active semiconductor layer102is exposed. The active semiconductor layer102is patterned using suitable photolithography and etching techniques. For example, a photoresist (not shown) may be formed over the active semiconductor layer102using a spin-on coating process, followed by patterning the photoresist to forming a plurality of trenches using suitable photolithography techniques, and then the active semiconductor layer102is etched using the patterned photoresist as an etch mask until the isolation dielectric layer116is exposed. The active semiconductor layer can be etched using, for example, a reactive ion etching (RIE) process or other suitable etching processes. The one or more dielectric materials (e.g., silicon dioxide) may be deposited in the trenches using a high density plasma chemical vapor deposition (HDP-CVD), a low-pressure CVD (LPCVD), sub-atmospheric CVD (SACVD), a flowable CVD (FCVD), spin-on, and/or the like, or a combination thereof. FIG.15also illustrates various doped regions formed in the active semiconductor layer102. The active semiconductor layer102can be doped before or after the isolation regions1504are formed. Multiple ion implantation processes are carried out to form the doped regions. In greater detail, a well region1402is formed in the active semiconductor layer102by a first ion implantation process, and then source/drain regions104are formed in the well region1402by a second ion implantation process. The source/drain regions104are formed with a first doping type (e.g., n-type) and the well region1402is formed with a second doping type (e.g., p-type) opposite the first doping type, such that a channel region108is formed with a second doping type in the active semiconductor layer102between the source/drain regions104. In some embodiments where the first heaters1404has the same doping type as the source/drain regions104, the first heaters1404can be formed in the active semiconductor layer102simultaneously with the source/drain regions104using the same ion implantation process. In some embodiments where the first heaters1404has the same doping type as the well region1402, the first heaters1404can be formed in the active semiconductor layer102simultaneously with the well region1402using the same ion implantation process. In some embodiments where the temperature-sensing device1406is a diode, a first doped region of the diode1406with the same doping type as the source/drain regions104can be formed in the active semiconductor layer102simultaneously with the source/drain regions104using the same ion implantation process, and a second doped region of the diode1406with the same doping type as the well region1402can be formed in the active semiconductor layer102simultaneously with the well region1402using the same ion implantation process. In some embodiments, in each ion implantation process photoresist is coated on the active semiconductor layer102and patterned on to serve as an implantation mask, which is removed by ashing once the corresponding ion implantation process is complete. A gate dielectric layer114and a gate electrode106are formed stacked over the channel region108, laterally between the source/drain regions104. In some embodiments, the process for forming the gate dielectric layer114and gate electrode106comprises sequentially depositing or growing a dielectric layer and a conductive layer stacked over the active semiconductor layer102. For example, the dielectric and conductive layers may be deposited or grown by, for example, thermal oxidation, electro chemical plating (ECP), vapor deposition, sputtering, or a combination of the foregoing. Further, in some embodiments, the process comprises patterning the dielectric and conductive layers using, for example, photolithography to selectively etch the dielectric and conductive layers respectively into the gate dielectric layer114and the gate electrode106. In some embodiments, the gate dielectric layer114includes silicon dioxide, and the gate electrode106includes doped polysilicon. In some embodiments, the ion implantation for forming the source/drain regions104are performed after forming the gate dielectric layer114and the gate electrode106, so that a gate stack of gate dielectric layer114and the gate electrode106can serve as a implantation mask, which allows for the source/drain regions104self-aligned to the gate stack. As illustrated inFIG.16, a BEOL interconnect structure110is partially formed over the SOI substrate1500. The BEOL interconnect structure110is formed with metal lines124and metal vias126alternatingly stacked within a multi-layer dielectric structure128. The BEOL interconnect structure110further include a second heater1408and a pad structure1412within the multi-layer dielectric structure128. In some embodiments, the second heater1408is formed of a material different from the metal lines124, metal vias126and the pad structure1412. By way of example and not limitation, the metal lines124, metal vias126and the pad structure1412include copper, but the second heater1408is free of copper. Instead, the second heater1408includes a metal composition with a thermal conductivity lower than copper. For example, the second heater1408may include titanium aluminum nitride, platinum, indium tin oxide, titanium nitride, or a combination of the foregoing. The reduced thermal conductivity of the second heater1408is helpful in temperature increase in a shorter time. In some embodiments, the pad structure1412has a larger plan view area (or larger top view area) than the metal lines124and vias126, which in turn helps for wire bonding on the pad structure1412. The second heater1408, metal lines124, metal vias126and pad structure1412may be, for example, formed by a single-damascene-like process or a dual-damascene-like process. A single-damascene-like or dual-damascene-like process is a single-damascene or dual-damascene process that is not restricted to copper. By way of example, a first inter-metal dielectric (IMD) layer is formed over the gate electrode106and then patterned to form an opening in the first IMD layer, one or more metals (e.g., copper) is then deposited to overfill the opening in the first IMD layer, followed by performing a CMP process on the one or more metals until the first IMD layer is exposed, resulting in the pad structure1412inlaid in the first IMD layer. Thereafter, a second IMD layer is formed over the first IMD layer and patterned to form via openings in the second IMD layer, one or more metals (e.g., copper) is then deposited to overfill the via openings in the second IMD layer, followed by performing a CMP process on the one or more metals until the second IMD layer is exposed, resulting in the metal vias126inlaid in the second IMD layer. Afterwards, a third IMD layer is formed over the second IMD layer and patterned to form trenches laterally extending in the third IMD layer, one or more metals (e.g., copper) is then deposited to overfill the trenches in the third IMD layer, followed by performing a CMP process on the one or more metals until the third IMD layer is exposed, resulting in the metal lines124inlaid in the third IMD layer. The second heater1408is formed in a manner similar to that of the metal lines124. By way of example and not limitation, an upper IMD layer is formed over a lower IMD layer having metal vias126(both the upper and lower IMD layers are higher than the third IMD layer as described above), the upper IMD layer is then patterned to form trenches laterally extending in the upper IMD layer, one or more non-copper metals (e.g., titanium aluminum nitride) is then deposited to overfill the trenches in the upper IMD layer, followed by performing a CMP process on the one or more metals until the upper IMD layer is exposed, resulting in the second heater1408inlaid in the upper IMD layer. Another IMD layer is then formed over the second heater1408, and these IMD layers are in combination referred to as a multi-layer dielectric structure128. In some embodiments, the second heater1408includes metal lines laterally extending within the multi-layer dielectric structure128, and the metal lines of the second heater1408may have a line width less than line widths of the metal lines124, which in turn results in an increased thermal resistance for the second heater1408, thus facilitating temperature increase in a shorter time. As illustrated in a cross-sectional view ofFIG.17, a carrier substrate112is bonded to the SOI substrate1500through the BEOL interconnect structure110. For example, the carrier substrate112may be bonded to the BEOL interconnect structure110by a fusion bonding process, such as a hydrophilic fusion bonding process. As illustrated in a cross-sectional view ofFIG.18, the structure ofFIG.17is flipped vertically and the SOI substrate1500is thinned to remove the bulk semiconductor substrate1502(see, e.g.,FIG.17). In some embodiments, the bulk semiconductor substrate1502is removed by grinding, CMP, etching back, or a combination of the foregoing. The isolation dielectric layer (e.g., BOX layer)116remains after removing the bulk semiconductor substrate1502. As illustrated in a cross-sectional view ofFIG.19, the isolation dielectric layer116is patterned to form a sensing well O over the channel region108and laterally between the source/drain regions104. The isolation dielectric layer116is patterned using suitable photolithography and etching techniques. For example, a photoresist (not shown) may be formed over the isolation dielectric layer116using a spin-on coating process, followed by patterning the photoresist to forming an opening using suitable photolithography techniques, and then the isolation dielectric layer116is etched using the patterned photoresist as an etch mask until the channel region108is exposed. Example etchant for etching the isolation dielectric layer116includes hydrofluoric acid, if the isolation dielectric layer116is silicon dioxide. Once the sensing well O is formed, a biosensing film120is formed lining the sensing well O. In some embodiments, the biosensing film120is also formed covering the isolation dielectric layer116. The biosensing film120may be deposited using, for example, vapor deposition, sputtering, atomic layer deposition (ALD), or a combination of the foregoing. Moreover, the biosensing film120includes, for example, include HfO2, SiO2, Si3N4, Al2O3, Ta2O5or combinations thereof. As illustrated in a cross-sectional view ofFIG.20, the biosensing film120is coated with a selective binding agent122. Coating the biosensing film120with the selective binding agent122includes, but is not limited to, immersing the wafer having the structure ofFIG.19in a selective binding agent bath at a suitable temperature (e.g., from about 25 degrees Celsius to about 300 degrees Celsius) for a suitable time duration (e.g., from about 5 mins to about 10 hrs) that is sufficient to allow the selective binding agent122to be attached to the biosensing film120, thus resulting in a thin film coating of the selective binding agent122in contact with the selective binding agent122. In some embodiments, the thin film coating of selective binding agent122is porous, which allows for the biosensing film120to be in contact with the cardiac-cell-containing fluid. In some embodiments, the binding agent122includes silane coupling agents that are compounds whose molecules contain functional groups that bond with both organic and inorganic materials. A silane agent acts as a sort of intermediary which bonds organic materials to inorganic materials. The silane coupling agent may include, by way of example and not limitation, silane having vinyl functional group (e.g., Vinyltrimethoxysilane ((CH3O)3SiCH═CH2), Vinyltriethoxysilane ((C2H5O)3SiCH═CH2) or the like), silane having epoxy functional group (e.g., 2-(3, 4 epoxycyclohexyl) ethyltrimethoxysilane, 3-Glycidoxypropyl methyldimethoxysilane, 3-Glycidoxypropyl trimethoxysilane, 3-Glycidoxypropyl methyldiethoxysilane, 3-Glycidoxypropyl triethoxysilane or the like), silane having styryl functional group (e.g., p-Styryltrimethoxysilane or the like), silane having methacryloxy functional group (e.g., 3-Methacryloxypropyl methyldimethoxysilane, 3-Methacryloxypropyl trimethoxysilane, 3-Methacryloxypropyl methyldiethoxysilane, 3-Methacryloxypropyl triethoxysilane or the like), silane having acryloxy functional group (e.g., 3-Acryloxypropyl trimethoxysilnae or the like), silane having amino functional group (e.g., N-2-(Aminoethyl)-3-amonopropylmethyldimethoxysilane, N-2-(Aminoethyl)-3-aminopropyltrimethoxysilane, 3-Aminopropyltrimethoxysilane, 3-Aminopropyltriethoxysilane, 3-Triethoxysilyl-N-(1, 3 dimethy-butylidene) propylamine, N-Pheny-3-aminopropyltrimethoxy silane, N-(Vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride or the like), silane having ureide functional group (e.g., 3-Ureidopropyltrialkoxysilane or the like), silane having isocyanate functional group (e.g., 3-Isocyanatepropyltriethoxysilane or the like), silane having isocyanurate functional group (e.g., Tris-(trimethoxysilylpropyl)isocyanurate or the like), silane having mercapto functional group (e.g., 3-Mercaptopropylmethyldimethoxysilane, 3-Mercaptopropyltrimethoxysilane or the like) or silane having other suitable functional groups. In some embodiments, the selective binding agent122for selectively binding with the cardiac cell192includes, for example, collagen, laminin, fibronectin, and mucopolysaccharides, heparin sulfate, hyaluronidate, chondroitin sulfate, the like, or combinations thereof. In some embodiments, an additional surface treatment is performed on the biosensing film120before forming the coating of selective binding agent122. The surface treatment includes, for example, a plasma treatment and/or a liquid-phase chemistry treatment that is capable of improving hydrophilicity of the biosensing film120. For example, the biosensing film120may undergo O2or O3plasma treatment before forming the coating of selective binding agent122, so as to improve hydrophilicity of the biosensing film120. The biosensing film120with improved hydrophilicity will be helpful in attachment to the cardiac cell, thus improving the detection and/or monitoring on the cardiac cell. As illustrated in a cross-sectional view ofFIG.21, in some embodiments, an etching process is performed into the coating of selective binding agent122, the biosensing film120, the isolation dielectric layer116, the active semiconductor layer102, the multi-layer dielectric structure128to form a pad opening1410exposing the pad structure1412of the BEOL interconnect structure110. The process for performing the etching may comprise, for example, coating a photoresist over the coating of selective binding agent122and patterning the photoresist using photolithography, such that the patterned photoresist has an opening corresponding to the pad opening1410. With the patterned photoresist in place, the etching process may comprise, for example, applying one or more etchants to the coating of selective binding agent122, the biosensing film120, the isolation dielectric layer116, the active semiconductor layer102, the multi-layer dielectric structure128, and subsequently stripping the patterned photoresist by ashing. As illustrated by a cross-sectional view ofFIG.22, fluid channel walls1414are formed over the coating of selective binding agent122to define a fluid containment region1416over the sensing well O. In some embodiments, the fluid channel walls1414include an elastomer. In some embodiments, the elastomer is polydimethylsiloxane (PDMS). In some embodiments, a layer of elastomer is patterned and then attached to the structure ofFIG.21to form fluid channel walls1414. In some embodiments, the material of fluid channel walls1414is first deposited and then pattern on the structure ofFIG.21. FIG.23is a chart illustrating an experimental result of a cardiac cell measured using an integrated circuit having BioFETs100as discussed above. The experimental result includes a time domain signal2500measured from a cardiac cell using the BioFETs100. The time domain signal2500indicates that beating pulse2502is greater than about 4 μA. The time domain signal2500detected by the BioFETs100is similar to a normal cardiac cycle, and thus the experimental result shows that the integrated circuit having BioFETs100can serve as a promising candidate for monitoring beating of cardiac cells. FIG.24is a 2D electrical image of a cardiac cell obtained using an integrated circuit having an array of BioFETs100as discussed above. The array of BioFETs100includes sensing pixels2601,2602,2603and2604. In the experiment a cardiac cell is placed on the sensing pixel2601and no cardiac cell is placed on the sensing pixels2602-2604, and the 2D electrical image clearly indicates that a cardiac cell in on the sensing pixel2601and no cardiac cell is placed on the sensing pixels2602-2604. Moreover, the 2D electrical image properly reflects the 2D image profile of the cardiac cell. This experimental result shows that the integrated circuit having BioFETs100can serve as a promising candidate for generating a 2D image of one or more cardiac cells. Based on the above discussions, it can be seen that the present disclosure offers advantages. It is understood, however, that other embodiments may offer additional advantages, and not all advantages are necessarily disclosed herein, and that no particular advantage is required for all embodiments. One advantage is that cardiac cells can be detected, measured and/or monitored using an IC having BioFETs. Another advantage is that the coating of selective binding agent on the biosensing film aids in binding the cardiac cell to the biosensing film, thus improving the accuracy of the measurement result of cardiac cell. In some embodiments, an IC includes a source region and a drain region in a semiconductor layer. A channel region is laterally between the source region and the drain region. A sensing well is on a back surface of the semiconductor layer and over the channel region. An interconnect structure is on a front surface of the semiconductor layer opposite the back surface of the semiconductor layer. A biosensing film lines the sensing well and contacts a bottom surface of the sensing well that is defined by the semiconductor layer. A coating of selective binding agent is over the biosensing film and configured to bind with a cardiac cell. In some embodiments, an IC includes a semiconductor substrate having a source region and a drain region. A sensing well is on a back surface of the semiconductor substrate. A biosening film lines the sensing well and contacts the back surface of the semiconductor substrate. A biological material coating layer is over the biosensing film. A first heater is in the semiconductor substrate and laterally spaced from the source region and the drain region. The first heater vertically overlaps with the biological material coating layer. In some embodiments, a method includes forming a semiconductor-on-insulator (SOI) substrate comprising a semiconductor substrate, a sacrificial substrate and a dielectric layer between the semiconductor substrate and the sacrificial substrate; forming source/drain regions in the semiconductor substrate; forming a back-end-of-line (BEOL) interconnect structure on a first side of the semiconductor substrate; bonding a carrier substrate to the semiconductor substrate through the BEOL interconnect structure; after bonding the carrier substrate to the semiconductor substrate; thinning the SOI substrate to remove the sacrificial substrate and to expose the dielectric layer; etching the dielectric layer until a second side of the semiconductor substrate is exposed, resulting in a sensing well extending through the dielectric layer and laterally between the source/drain regions; forming a biosensing film lining the sensing well; and immersing the biosensing film in a biological material bath until the biosensing film is coated with a biological material layer. The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. | 56,402 |
11860121 | DETAILED DESCRIPTION The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. Further, spatially relative terms, such as “beneath,” “below,” “lower,” “above,” “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. The spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. The apparatus may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein may likewise be interpreted accordingly. One type of biosensor includes a semiconductor substrate that is covered by an isolation dielectric layer and that accommodates a biologically sensitive field-effect transistor (BioFET). One advantage of BioFETs is the prospect of label-free operation. Specifically, BioFETs enable the avoidance of costly and time-consuming labeling operations such as the labeling of analytes (e.g., cardiac cells) with, for instance, fluorescent or radioactive probes. The BioFET includes a source region and a drain region that are arranged within the semiconductor substrate and that define a channel region therebetween. Further, the BioFET includes a gate arranged under the semiconductor substrate, laterally between the source region and the drain region. The isolation dielectric layer includes a sensing well that exposes the semiconductor substrate, laterally between the source region and the drain region, and that is lined by a biosensing film. The biosensing film is configured to detect an impedance change, molecule charge and/or ion release resulting from bio-entities (e.g., cardiac cells), such that 2D electrical image profile of the bio-entities (e.g., cardiac cells) can be obtained and/or the bio-entities may be monitored. FIG.1illustrates a cross-sectional view of an example integrated circuit10including an array of BioFETs100in accordance with some embodiments of the present disclosure. The BioFETs100each include a pair of source/drain regions104and, in some embodiments, a gate electrode106. The source/drains regions104have a first conductivity type (i.e., doping type) and are arranged within an active semiconductor layer102, respectively on opposite sides of a channel region108of the BioFET100. The channel region108has a second conductivity type opposite the first conductivity type and is arranged in the active semiconductor layer102, laterally between the source/drain regions104. The first and second doping types may, for example, respectively be n-type and p-type, or vice versa. In some embodiments, the source/drain regions104and the channel region108are arranged within a doped well region of the active semiconductor layer102that has the second conductivity type, and/or are electrically coupled to a back-end-of-line (BEOL) interconnect structure110that is arranged over a carrier substrate112. Further, in some embodiments, the source/drain regions104and the channel region108extend from a front surface102fof the active semiconductor layer102to a back surface102bof the active semiconductor layer102. Stated differently, the source/drain regions104and the channel region108can extend through the full thickness of the active semiconductor layer102to facilitate functioning of biosensening. The gate electrode106is arranged on the front surface102fof the active semiconductor layer102, laterally between the source/drain regions104, and is spaced from the front surface102fof the active semiconductor layer102by a gate dielectric layer114. In some embodiments, the gate electrode106is electrically coupled to the BEOL interconnect structure110is metal, doped polysilicon, or a combination of the foregoing. In some embodiments, the BioFETs100may be separated from each other using shallow trench isolation regions (not shown) laterally surrounding each of the BioFETs100. Gate dielectric layer114includes, for example, silicon dioxide and/or a high-k gate dielectric material with a dielectric constant higher than that of silicon dioxide. Exemplary high-k gate dielectric materials include, but are not limited to, silicon nitride, silicon oxynitride, hafnium oxide (HfO2), hafnium silicon oxide (HfSiO), hafnium silicon oxynitride (HfSiON), hafnium tantalum oxide (HfTaO), hafnium titanium oxide (HfTiO), hafnium zirconium oxide (HfZrO), metal oxides, metal nitrides, metal silicates, transition metal-oxides, transition metal-nitrides, transition metal-silicates, oxynitrides of metals, metal aluminates, zirconium silicate, zirconium aluminate, zirconium oxide, titanium oxide, aluminum oxide, hafnium dioxide-alumina (HfO2—Al2O3) alloy, other suitable high-k dielectric materials, and/or combinations thereof. In some embodiments, the gate dielectric includes a stack of an interfacial dielectric material and a high-k dielectric material. In some embodiments, the active semiconductor layer102may be a silicon substrate or wafer. Alternatively, the semiconductor layer102may include another elementary semiconductor, such as germanium (Ge); a compound semiconductor including silicon carbide (SiC), gallium arsenic (GaAs), gallium phosphide (GaP), indium phosphide (InP), indium arsenide (InAs), and/or indium antimonide (InSb); an alloy semiconductor including SiGe, GaAsP, AlInAs, AlGaAs, GaInAs, GaInP, and/or GaInAsP; or combinations thereof. In the depicted embodiments, the active semiconductor layer102is a semiconductor layer of a semiconductor-on-insulator (SOI) substrate (e.g., silicon layer). In some embodiments, the carrier substrate (interchangeably referred to as handle substrate as well)112may be, for example, a bulk semiconductor substrate, such as a bulk substrate of monocrystalline silicon. An isolation dielectric layer116is arranged on the back surface102bof the active semiconductor layer102, and includes a plurality of sensing wells118over corresponding channel regions108of BioFETs100. The sensing wells118extend into the isolation dielectric layer116to proximate the channel regions108of BioFETs100and are at least partially lined by a biosensing film120. Further, in some embodiments, the sensing wells118extend through the isolation dielectric layer116to expose the respective channel regions108of BioFETs100. The isolation dielectric layer116may be, for example, silicon dioxide, a buried oxide (BOX) layer of a SOI substrate, some other dielectric, or a combination of the foregoing. In some specific embodiments, the active semiconductor layer102is a silicon layer of a SOI substrate, and the isolation dielectric layer116is a BOX layer of the SOI substrate. The biosensing film120lines the sensing wells118and, in some embodiments, covers the entire isolation dielectric layer116. The biosensing film120is operative to modulate the source to drain conductivity of each bioFET100when contacted by a fluid190having a suitable composition or carrying specific analytes. For example, the fluid190is an aqueous solution containing cardiac cells192. Examples of materials for biosensing film120that provide the functionality of biosensing include HfO2, SiO2, Si3N4, Al2O3, and Ta2O5. An upper surface of the biosensing film120includes a coating of a selective binding agent122. The selective binding agent122includes one or more biological materials having the property of selectively binding with the cardiac cells192. If the cardiac cell192is stably bound on the upper surface of the biosensing film120, the overall charge concentration at the biosensing film120can become sufficient to modulate the source to drain conductivity of BioFETs100, thus improving the biosensing performance. Because the selective binding agent122has a greater binding ability (i.e., greater adhesion) to the cardiac cell192than that of the biosensing film120, the biosensing performance can be improved as long as the selective binding agent122is coated on the biosensing film120. In some embodiments, the selective binding agent122for selectively binding with the cardiac cell192includes, for example, collagen, laminin, fibronectin, and mucopolysaccharides, heparin sulfate, hyaluronidate, chondroitin sulfate, the like, or combinations thereof. The coating of the selective binding agent122is illustrated as a blanket layer covering the entire biosensing film120merely for the sake of clarity, but in practice the coating of the selective binding agent122is porous and thus does not cover the entire biosensing film120, which allows the biosensing film120to be in contact with the cardiac-cell-containing fluid190. The sensor array of BioFETs100can use the biosensing film120to monitor beating of the cardiac cells192and/or generate 2D image profiles of the cardiac cells192by detecting impedance change, module charge and/or ion release. Take ion release detection for example, in operation a reference electrode194gives the cardiac-cell-containing solution190a voltage potential, then the biosensing film120becomes charged when brought in contact with the cardiac cell containing fluid190having a suitable ion concentration. Moreover, they can become sufficiently charged to switch the source/drain conductivity of bioFETs100. In this way, the sensor array of bioFETs100has the biosensing film120functional to detect ion released from or captured on the cardiac cell192and/or ion released from or captured on the selective binding agent122. In a similar manner, the sensor array of bioFETs100can detect molecule charge released from or captured on the cardiac cell192and/or charge released from or captured on the selective binding agent122. In examples, the current between the source/drain regions104is measured, and the measured current (or a change in the measured current caused by the fluid190and/or the cardiac cell192) is indicative of impedance change, molecule charge and/or ion release caused by the fluid190and/or the cardiac cell192. Thus, in such examples, the detection mechanism is a conduction modulation of the transducer due to the binding of the cardiac cell192over the biosensing film120. In other examples, one or more components (e.g., a trans-impedance amplifier) are used to translate the current or change of current induced by the fluid190and/or the cardiac cell192into another electrical signal, such as a measurable voltage. To illustrate this, reference is made toFIG.2, which depicts a trans-impedance amplifier202used in generating an electrical signal204, in accordance with some embodiments of the present disclosure.FIG.2illustrates an equivalent circuit that corresponds to a BioFET (e.g., BioFET100as illustrated inFIG.1) and shows that a drain current induced by the fluid190and/or cardiac cell192on the upper surface of the biosensing film120is translated into the electrical signal204by the trans-impedance amplifier202. The electrical signal204may include, for example, voltages (e.g., voltage signals) that can be measured. In some embodiments, each of the BioFETs100may be controlled (i.e., turned on and turned off) by an access transistor. For example, as illustrated in toFIG.3, a cross section of a BioFET100and an access transistor300is provided, according to some embodiments. The BioFET100includes source/drain regions104, a channel region108laterally between the source/drain regions104, a gate dielectric layer114over the channel region108and a gate electrode106over the gate dielectric layer114, all of which are discussed previously with respect toFIG.1and thus are not repeated for the sake of brevity. The access transistor300is coupled to the BioFET100, as illustrated in the circuit diagram ofFIG.4. The access transistor300similarly includes source/drain regions304formed in the semiconductor substrate102, a channel region308laterally between the source/drain regions304, a gate dielectric layer314over the channel region308and a gate electrode306over the gate dielectric layer314. The source/drain regions304are doped regions having a conductivity type opposite the conductivity type of the channel region308. For example, the source/drain regions304are of an n-type conductivity and the channel region308is of a p-type conductivity, or vice versa. In some embodiments, the gate electrode306of the access transistor300has a same material composition as the gate electrode106of the BioFET100and is formed simultaneously with the gate electrode106. For example, the gate electrode306may be metal, doped polysilicon, or a combination of the foregoing. In some embodiments, the gate dielectric layer314has a same material composition as the gate dielectric layer114and is formed simultaneously with the gate dielectric layer114, and thus example materials of the gate dielectric layer314are not repeated for the sake of brevity. The access transistor300is similar with the BioFET100, except that the channel region308and/or the source/drain regions304of the access transistor300are separated from the biosensing film120by the isolation dielectric layer116. In this way, operation of the access transistor300is not affected by the fluid190and/or the cardiac cell192. FIG.4illustrates an equivalent circuit that corresponds to the relationship between the BioFET100and the access transistor300as shown inFIG.3. As illustrated inFIG.4, the access transistor300serves as a switching device coupled to source/drain terminal of the BioFET100, and thus the access transistor300can turn on the BioFET100to initiate detecting and/or monitoring the cardiac cell, and can also turn off the BioFET100to stop detecting and/or monitoring the cardiac cell. FIG.5is a functional block diagram of an integrated circuit50in accordance some embodiments of the present disclosure. The integrated circuit50includes a sensing pixel array SA that includes sensing pixels502arranged into M columns and N rows. M and N are positive integers. In some embodiments, M ranges from 1 to 256. In some embodiments, N ranges from 1 to 256. The number of M and N are selected based on a normal size of a cardiac cell192, which in turn allows for detecting/monitoring a single cardiac cell192using a single sensing pixel array SA. Each sensing pixel502of the array SA at least includes a BioFET (e.g., BioFET100as illustrated inFIGS.3and4) and an access transistor (e.g., access transistor300as illustrated inFIGS.3and4) coupled to the BioFET. The integrated circuit50also includes a column decoder504coupled to the sensing pixel array SA via column lines CL1-CLM. The column decoder504decodes a column address of sensing pixels502selected to be accessed in a cardiac cell detection/monitor operation. The column decoder504then enables, via the column pixel selector506, the column line corresponding to the decoded column address to permit access to the selected sensing pixels502. The integrated circuit50also includes a row decoder508coupled to the sensing pixel array SA via row lines RL1-RLN. The row decoder decodes a row address of sensing pixels502selected to be accessed in a cardiac cell detection/monitor operation. The row decoder508then enables, via the row pixel selector510, the row line corresponding to the decoded row address to permit reading out biosensing measurements from the selected sensing pixels502. In some embodiments, the integrated circuit50further includes one or more trans-impedance amplifiers512configured to receive readout biosensing measurements from an output of the row decoder508and generate an electrical signal based on the readout biosensing measurements of the selected sensing pixels502. By way of example and not limitation, the readout biosensing measurements of selected sensing pixels502include drain currents of BioFETs of the selected sensing pixels502and can be translated into electric signals514by the trans-impedance amplifier512. The electric signals514may include, for example, voltages (e.g., voltage signals). The integrated circuit50allows for detecting/monitoring a single cardiac cell. However, in some embodiments, multiple cardiac cells can be detected/monitored using an integrated circuit.FIG.6is a functional block diagram of an example integrated circuit60that is designed to detect/monitor multiple cardiac cells192in accordance some embodiments of the present disclosure. The integrated circuit60includes multiple sensing pixel arrays SA. Each sensing pixel arrays SA includes sensing pixels602each including a BioFET (e.g., BioFET100as illustrated inFIGS.3and4) and an access transistor (e.g., access transistor300as illustrated inFIGS.3and4) coupled to the BioFET. The sensing pixels602in each sensing pixel array SA are arranged into M columns and N rows. M and N are positive integers. In some embodiments, both M and N range from 1 to 256. The number of M and N are selected based on a normal size of a cardiac cell192, which in turn allows for detecting/monitoring a single cardiac cell192using a single sensing pixel array SA. Therefore, the integrated circuit60having multiple sensing pixel arrays SA can detect and/or monitor multiple cardiac cells192. The sensing pixel arrays SA are arranged into P columns and Q rows. P and Q are positive integers. In some embodiments, P is less than M, and Q is less than N. In some other embodiments, P is greater than M, and Q is greater than N. The number of P and Q are selected depending on a desired number of cardiac cells to be detected and/or monitored. The integrated circuit60includes a column decoder604coupled to the sensing pixel arrays SA via column cell spot lines CCL1-CCLpand to the sensing pixels602via column pixel lines CPL1-CPLM. The column decoder604decodes a column cell spot address of sensing pixel arrays SA and a column pixel address of sensing pixels602of the selected sensing pixel array SA. The column decoder604then enables, via the column cell spot selector606, the column cell spot line corresponding to the decoded column cell spot address, to permit access to the selected sensing pixel arrays SA. The column decoder604then enables, via the column pixel selector608, the column pixel line corresponding to the decoded column pixel address, so as to permit access to the selected sensing pixels602of the selected sensing pixel arrays SA. The integrated circuit60includes a row decoder610coupled to the sensing pixel arrays SA via row cell spot lines RCL1-RCLqand to the sensing pixels602via row pixel lines RPL1-RPLN. The row decoder610decodes a row cell spot address of sensing pixel arrays SA and a row pixel address of sensing pixels602of the selected sensing pixel array SA. The row decoder610then enables, via the row cell spot selector612, the row cell spot line corresponding to the decoded row cell spot address. The row decoder610then enables, via the row pixel selector614, the row pixel line corresponding to the decoded row pixel address, so as to permit reading out biosensing measurements from the selected sensing pixels602of the selected sensing pixel arrays SA. FIG.7illustrates an example cross-sectional view of a partial region of the integrated circuit60ofFIG.6.FIG.7illustrates two neighboring sensing pixel array SA spaced apart by one or more fluid channel walls702. The fluid channel walls702laterally define fluid containment regions704over the respective sensing pixel arrays SA. Each sensing pixel array SA includes BioFETs100of sensing pixels. The fluid containment region704can be a well or a length of channel bound by fluid channel walls702. The fluid channel walls702can be formed of waterproof material(s). In some embodiments, the fluid channel walls702are an elastomer. In some of these embodiments, the elastomer of polydimethylsiloxane (PDMS). In some embodiments, fluid containment regions704are capped to provide closed channels or reservoirs. Spacing between the fluid channel walls702are selected such that a single fluid containment region704's size matches with a normal size of a cardiac cell192. By way of example, the fluid containment region704has a width in a range from about 10 um to about 300 um. If the width of the fluid containment region704is less than about 10 um, the fluid containment region704may be too narrow to accommodate a single cardiac cell192. If the width of the fluid containment region704is greater than about 300 um, the fluid containment region704may accommodate multiple cardiac cells193. The BioFETs100are discussed previously with respect toFIG.1and thus are not repeated for the sake of brevity. FIG.8illustrates a cross-sectional view of an example integrated circuit80including an array of BioFETs100in accordance with some embodiments of the present disclosure. The integrated circuit80is similar to the integrated circuit10ofFIG.1, except that the integrated circuit80includes additional heaters802. In some embodiments, the heaters802are doped regions in the active semiconductor layer102. In some embodiments, the heaters802are formed together with (i.e., simultaneously with) the source/drain regions104, and thus have the same dopant type and dopant concentration profile as the source/drain regions104. Different from the source/drain regions104, the heaters802in the active semiconductor layer102are separated from the channel regions104by, for example, shallow trench isolation (STI), and thus the voltage applied to the heaters802does not affect functionality of the BioFETs100. With the heaters802, the fluid190and/or the cardiac cell192can be heated to enhance the detection and/or monitoring of the cardiac cell192. In some embodiments, the heaters802are operated in response to a temperature measurement from a temperature-sensing device (not shown inFIG.8), as discussed below. FIG.9is a circuit diagram of a BioFET100and its surrounding heaters802of the integrated circuit80in accordance with some embodiments. The integrated circuit80includes a sensing pixel902having a BioFET100, a first switching device904, a temperature-sensing device906, and a second switching device908. The first switching device904is coupled between a first end of the BioFET100and a corresponding row line (e.g., one of the row lines RL1-RLNas illustrated inFIG.5). The second switching device908is coupled between a first end of the temperature-sensing device906and a corresponding signal path for reading out the temperature measurement of the temperature-sensing device906. The first switching device904and the second switching device908are N-type transistors having gates coupled with a corresponding column line (e.g., one of the column lines CL1-CLMas illustrated inFIG.5). A second end of the BioFET100and a second end of temperature-sensing device906are coupled together and configured to receive a reference voltage. In some embodiments, temperature-sensing device906includes a p-n diode formed in the active semiconductor layer102(as shown inFIG.8). In some embodiments, the first switching device904or second switching device908is implemented by other types of switching devices, such as a transmission gate or a P-type transistor. Temperature-sensing device906is configured to measure a temperature of the biosensing film of the BioFET100and then generate a temperature-sensing signal responsive to the measured temperature of the biosensing film. The heaters802are configured to adjust the temperature of the biosensing film of the BioFET100, which in turn adjusts the temperature of the cardiac-cell-containing fluid and the cardiac cell over the BioFET100. The temperature-sensing signal generated from the temperature-sensing device906can serve as feedback to control the heaters802, which in turn promotes good temperature control and uniformity. By way of example and not limitation, when the measured temperature from the temperature-sensing device906is higher than an expected temperature range suitable for detecting and/or monitoring the cardiac cell192, the heaters802are turned off; when the measured temperature from the temperature-sensing device906is lower than the expected temperature range, the heaters802are turned off. In some embodiments, as illustrated inFIG.9, separate heaters802together surround four sides of the sensing pixel902. However, in some other embodiments an integrated circuit has a different heater configuration, as illustrated in a layout diagram ofFIG.10. As shown inFIG.10, the layout1000includes a plurality of first elongated heaters1002extending in a first direction, a plurality of second elongated heaters1004extending across the first elongated heaters1002in a second direction perpendicular to the first direction, and a plurality of sensing pixels902bound by the first elongated heaters1002and the second elongated heaters1004. The sensing pixels902each include a BioFET100, a temperature-sensing device906, and first and second switching devices904and908, as discussed previously with respect toFIG.9. FIGS.11and12illustrate cross-sectional views of an example integrated circuit1100having the top view layout1000ofFIG.10in accordance with some embodiments, whereinFIG.11is a cross-sectional view taken along line A-A′ ofFIG.10, andFIG.12is a cross-sectional view taken along line B-B′ ofFIG.10. As illustrated inFIGS.11and12, the first elongated heaters1002are doped regions formed in the active semiconductor layer102, and the second elongated heaters1004are doped polysilicon structure formed on the front surface102fof the active semiconductor layer102. In some embodiments where the gate electrodes106are polysilicon, the second elongated heaters1004are formed together with the polysilicon gates106, and thus have the same thickness and material composition as the polysilicon gates106. The first elongated heaters1002are thus interchangeably referred to as doped silicon heaters, and the second elongated heaters1004are thus interchangeably referred to as polysilicon heaters. The polysilicon heaters1004are vertically spaced apart from the doped silicon heaters1002by a dielectric layer1006, as illustrated inFIG.12. In some embodiments, the dielectric layer1006is formed together with the gate dielectric layers114of the BioFETs100, and thus the dielectric layer1006has the same thickness and material composition as the gate electric layers114. By way of example and not limitation, the dielectric layer1006includes silicon dioxide, a high-k dielectric material with a dielectric constant higher than a dielectric constant of silicon dioxide or combinations thereof. In some embodiments, the doped silicon heaters1002and the polysilicon heaters1004non-overlap with BioFETs100of all sensing pixels902from a top view as illustrated inFIG.10, and thus the doped silicon heaters1002and the polysilicon heaters1004do not affect the functionality of BioFETs100. FIG.13illustrates a cross-sectional view of another example integrated circuit1300having the layout1000ofFIG.10in accordance with some embodiments, whereinFIG.13is a cross-sectional view taken along line B-B′ ofFIG.10. The second elongated heaters1004in the integrated circuit1300is formed in the BEOL interconnect structure110, not in the active semiconductor layer102. In some embodiments, the second elongated heaters1004include titanium aluminum nitride, platinum, indium tin oxide, titanium nitride, or a combination of the foregoing. In some embodiments, the second elongated heaters1004have a thickness in a range from about 5600 angstroms to about 6600 angstroms and a sheet resistance in a range from about 4 ohm/sq to about 6 ohm/sq. In some embodiments, the second elongated heaters1004are formed in a metallization layer of the interconnect structure110and laterally surrounded by an inter-metal dielectric (IMD) layer of the multi-layer dielectric structure128. Moreover, other metallization layers including the metal lines124and metal vias126are formed of a different metal composition (e.g., copper) than that of the second elongated heaters1004, because these metallization layers including the metal lines124and metal vias126are not designed for heating. FIG.14illustrates a cross-sectional view of an example integrated circuit1400in accordance with some embodiments of the present disclosure. The integrated circuit1400includes a BioFET100formed on the active semiconductor layer102. The BioFET100is similar to that described previously with respect toFIG.1, except that the source/drain regions104are formed in a well region1042of the active semiconductor layer102. The well region1042has a conductivity type opposite the source/drain regions104. Moreover, the integrated circuit1400includes one or more first heaters1404and one or more temperature-sensing devices1406formed in the active semiconductor layer102. The first heater1404is a doped region in the active semiconductor layer102. In some embodiments, the first heater1404is formed together with (i.e., simultaneously with) the source/drain regions104, and thus has the same dopant type and dopant concentration profile as the source/drain regions104. In some other embodiments, the first heater1404is formed together with (i.e., simultaneously with) the well region1402, and thus has the same dopant type and dopant concentration profile as the well region1402. The temperature-sensing device1406is a diode formed in the active semiconductor layer102, and as such the temperature-sensing device1046includes at least one P-N junction (as indicated by the dash line DL) forming the diode within the active semiconductor layer102. In some embodiments, the temperature-sensing device1046has a p-type doped region and an n-type doped region to form the P-N junction. In some embodiments where the BioFET100is a PFET, the p-type doped region of the temperature-sensing device1406is formed together with the source/drain regions104of the BioFET100, and the n-type doped region of the temperature-sensing device1406is formed together with the well region1402. In some embodiments where the BioFET100is an NFET, the n-type doped region of the temperature-sensing device1406is formed together with the source/drain regions104of the BioFET100, and the p-type doped region of the temperature-sensing device1406is formed together with the well region1402. The integrated circuit1400further includes one or more second heaters1408formed in the interconnect structure110below the active semiconductor layer102. In some embodiments, the second heaters1408include titanium aluminum nitride, platinum, indium tin oxide, titanium nitride, or a combination of the foregoing. In some embodiments, the second heaters1408have a thickness in a range from about 5600 angstroms to about 6600 angstroms and a sheet resistance in a range from about 4 ohm/sq to about 6 ohm/sq. In some embodiments, the second heaters1408are formed in a metallization layer of the interconnect structure110and laterally surrounded by an inter-metal dielectric (IMD) layer of the multi-layer dielectric structure128. Moreover, other metallization layers including the metal lines124and metal vias126are formed of a different metal composition (e.g., copper) than that of the second elongated heaters1004, because these metallization layers including the metal lines124and metal vias126are not designed for heating. The integrated circuit1400has a pad opening1410extending through the coating of selective binding agent122, the biosensing film120, the isolation dielectric layer160and the active semiconductor layer102to expose a pad structure1412formed within the interconnect structure110. In some embodiments, the pad structure1412is formed in a metallization layer of the interconnect structure110and laterally surrounded by an inter-metal dielectric (IMD) layer of the multi-layer dielectric structure128. In some embodiments, a vertical distance from the pad structure1412to the active semiconductor layer102is less than a vertical distance from the second heater1408to the active semiconductor layer102. Moreover the integrated circuit1400further includes fluid channel walls1414. The fluid channel walls1414laterally define a fluid containment region1416over the BioFET100. The fluid containment region1416may be a well or a length of channel bound by fluid channel walls1414. The fluid channel walls1414can be formed of waterproof material(s). In some embodiments, the fluid channel walls1414are an elastomer. In some of these embodiments, the elastomer of polydimethylsiloxane (PDMS). FIGS.15-22illustrate cross-sectional views of various intermediate stage of a method for forming the integrated circuit1400as illustrated inFIG.14. Throughout the various views and illustrative embodiments, like reference numbers are used to designate like elements. It is understood that additional operations can be provided before, during, and after the processes shown byFIGS.15-22, and some of the operations described below can be replaced or eliminated, for additional embodiments of the method. The order of the operations/processes may be interchangeable. As illustrated inFIG.15, an SOI substrate1500is formed. The SOI substrate1500comprises a bulk semiconductor substrate1502over which an isolation dielectric layer116and an active semiconductor layer102are stacked. As seen hereafter, the bulk semiconductor substrate1502is sacrificial. The bulk semiconductor substrate1502and the active semiconductor layer102may be, for example, monocrystalline silicon, and/or the isolation dielectric layer116may be, for example, silicon dioxide. The SOI substrate1500can be formed by any suitable process. In some embodiments, SOI substrate1500is formed through separation by implanted oxygen (SIMOX). After the SOI substrate1500is prepared, one or more isolation regions1504are optionally formed in the active semiconductor layer102of the SOI substrate1500. In the depicted embodiments, the isolation regions1504are formed through the full thickness of the active semiconductor layer102. In some other embodiments, the isolation regions1504are STI regions that do not extend through the full thickness of the active semiconductor layer102. The isolation regions1504extend laterally to enclose subsequently formed BioFETs100, first heaters1404and temperature-sensing devices1406, so as to provide electrical isolation to these devices. The process for forming the isolation regions1504may comprise, for example, patterning the active semiconductor layer102to define one or more trenches corresponding to the isolation regions1504, subsequently depositing or growing one or more dielectric materials filling the one or more trenches, followed by performing a planarization process (e.g., chemical mechanical polish (CMP)) on the dielectric materials until the active semiconductor layer102is exposed. The active semiconductor layer102is patterned using suitable photolithography and etching techniques. For example, a photoresist (not shown) may be formed over the active semiconductor layer102using a spin-on coating process, followed by patterning the photoresist to forming a plurality of trenches using suitable photolithography techniques, and then the active semiconductor layer102is etched using the patterned photoresist as an etch mask until the isolation dielectric layer116is exposed. The active semiconductor layer can be etched using, for example, a reactive ion etching (RIE) process or other suitable etching processes. The one or more dielectric materials (e.g., silicon dioxide) may be deposited in the trenches using a high density plasma chemical vapor deposition (HDP-CVD), a low-pressure CVD (LPCVD), sub-atmospheric CVD (SACVD), a flowable CVD (FCVD), spin-on, and/or the like, or a combination thereof. FIG.15also illustrates various doped regions formed in the active semiconductor layer102. The active semiconductor layer102can be doped before or after the isolation regions1504are formed. Multiple ion implantation processes are carried out to form the doped regions. In greater detail, a well region1402is formed in the active semiconductor layer102by a first ion implantation process, and then source/drain regions104are formed in the well region1402by a second ion implantation process. The source/drain regions104are formed with a first doping type (e.g., n-type) and the well region1402is formed with a second doping type (e.g., p-type) opposite the first doping type, such that a channel region108is formed with a second doping type in the active semiconductor layer102between the source/drain regions104. In some embodiments where the first heaters1404has the same doping type as the source/drain regions104, the first heaters1404can be formed in the active semiconductor layer102simultaneously with the source/drain regions104using the same ion implantation process. In some embodiments where the first heaters1404has the same doping type as the well region1402, the first heaters1404can be formed in the active semiconductor layer102simultaneously with the well region1402using the same ion implantation process. In some embodiments where the temperature-sensing device1406is a diode, a first doped region of the diode1406with the same doping type as the source/drain regions104can be formed in the active semiconductor layer102simultaneously with the source/drain regions104using the same ion implantation process, and a second doped region of the diode1406with the same doping type as the well region1402can be formed in the active semiconductor layer102simultaneously with the well region1402using the same ion implantation process. In some embodiments, in each ion implantation process photoresist is coated on the active semiconductor layer102and patterned on to serve as an implantation mask, which is removed by ashing once the corresponding ion implantation process is complete. A gate dielectric layer114and a gate electrode106are formed stacked over the channel region108, laterally between the source/drain regions104. In some embodiments, the process for forming the gate dielectric layer114and gate electrode106comprises sequentially depositing or growing a dielectric layer and a conductive layer stacked over the active semiconductor layer102. For example, the dielectric and conductive layers may be deposited or grown by, for example, thermal oxidation, electro chemical plating (ECP), vapor deposition, sputtering, or a combination of the foregoing. Further, in some embodiments, the process comprises patterning the dielectric and conductive layers using, for example, photolithography to selectively etch the dielectric and conductive layers respectively into the gate dielectric layer114and the gate electrode106. In some embodiments, the gate dielectric layer114includes silicon dioxide, and the gate electrode106includes doped polysilicon. In some embodiments, the ion implantation for forming the source/drain regions104are performed after forming the gate dielectric layer114and the gate electrode106, so that a gate stack of gate dielectric layer114and the gate electrode106can serve as a implantation mask, which allows for the source/drain regions104self-aligned to the gate stack. As illustrated inFIG.16, a BEOL interconnect structure110is partially formed over the SOI substrate1500. The BEOL interconnect structure110is formed with metal lines124and metal vias126alternatingly stacked within a multi-layer dielectric structure128. The BEOL interconnect structure110further include a second heater1408and a pad structure1412within the multi-layer dielectric structure128. In some embodiments, the second heater1408is formed of a material different from the metal lines124, metal vias126and the pad structure1412. By way of example and not limitation, the metal lines124, metal vias126and the pad structure1412include copper, but the second heater1408is free of copper. Instead, the second heater1408includes a metal composition with a thermal conductivity lower than copper. For example, the second heater1408may include titanium aluminum nitride, platinum, indium tin oxide, titanium nitride, or a combination of the foregoing. The reduced thermal conductivity of the second heater1408is helpful in temperature increase in a shorter time. In some embodiments, the pad structure1412has a larger plan view area (or larger top view area) than the metal lines124and vias126, which in turn helps for wire bonding on the pad structure1412. The second heater1408, metal lines124, metal vias126and pad structure1412may be, for example, formed by a single-damascene-like process or a dual-damascene-like process. A single-damascene-like or dual-damascene-like process is a single-damascene or dual-damascene process that is not restricted to copper. By way of example, a first inter-metal dielectric (IMD) layer is formed over the gate electrode106and then patterned to form an opening in the first IMD layer, one or more metals (e.g., copper) is then deposited to overfill the opening in the first IMD layer, followed by performing a CMP process on the one or more metals until the first IMD layer is exposed, resulting in the pad structure1412inlaid in the first IMD layer. Thereafter, a second IMD layer is formed over the first IMD layer and patterned to form via openings in the second IMD layer, one or more metals (e.g., copper) is then deposited to overfill the via openings in the second IMD layer, followed by performing a CMP process on the one or more metals until the second IMD layer is exposed, resulting in the metal vias126inlaid in the second IMD layer. Afterwards, a third IMD layer is formed over the second IMD layer and patterned to form trenches laterally extending in the third IMD layer, one or more metals (e.g., copper) is then deposited to overfill the trenches in the third IMD layer, followed by performing a CMP process on the one or more metals until the third IMD layer is exposed, resulting in the metal lines124inlaid in the third IMD layer. The second heater1408is formed in a manner similar to that of the metal lines124. By way of example and not limitation, an upper IMD layer is formed over a lower IMD layer having metal vias126(both the upper and lower IMD layers are higher than the third IMD layer as described above), the upper IMD layer is then patterned to form trenches laterally extending in the upper IMD layer, one or more non-copper metals (e.g., titanium aluminum nitride) is then deposited to overfill the trenches in the upper IMD layer, followed by performing a CMP process on the one or more metals until the upper IMD layer is exposed, resulting in the second heater1408inlaid in the upper IMD layer. Another IMD layer is then formed over the second heater1408, and these IMD layers are in combination referred to as a multi-layer dielectric structure128. In some embodiments, the second heater1408includes metal lines laterally extending within the multi-layer dielectric structure128, and the metal lines of the second heater1408may have a line width less than line widths of the metal lines124, which in turn results in an increased thermal resistance for the second heater1408, thus facilitating temperature increase in a shorter time. As illustrated in a cross-sectional view ofFIG.17, a carrier substrate112is bonded to the SOI substrate1500through the BEOL interconnect structure110. For example, the carrier substrate112may be bonded to the BEOL interconnect structure110by a fusion bonding process, such as a hydrophilic fusion bonding process. As illustrated in a cross-sectional view ofFIG.18, the structure ofFIG.17is flipped vertically and the SOI substrate1500is thinned to remove the bulk semiconductor substrate1502(see, e.g.,FIG.17). In some embodiments, the bulk semiconductor substrate1502is removed by grinding, CMP, etching back, or a combination of the foregoing. The isolation dielectric layer (e.g., BOX layer)116remains after removing the bulk semiconductor substrate1502. As illustrated in a cross-sectional view ofFIG.19, the isolation dielectric layer116is patterned to form a sensing well O over the channel region108and laterally between the source/drain regions104. The isolation dielectric layer116is patterned using suitable photolithography and etching techniques. For example, a photoresist (not shown) may be formed over the isolation dielectric layer116using a spin-on coating process, followed by patterning the photoresist to forming an opening using suitable photolithography techniques, and then the isolation dielectric layer116is etched using the patterned photoresist as an etch mask until the channel region108is exposed. Example etchant for etching the isolation dielectric layer116includes hydrofluoric acid, if the isolation dielectric layer116is silicon dioxide. Once the sensing well O is formed, a biosensing film120is formed lining the sensing well O. In some embodiments, the biosensing film120is also formed covering the isolation dielectric layer116. The biosensing film120may be deposited using, for example, vapor deposition, sputtering, atomic layer deposition (ALD), or a combination of the foregoing. Moreover, the biosensing film120includes, for example, include HfO2, SiO2, Si3N4, Al2O3, Ta2O5or combinations thereof. As illustrated in a cross-sectional view ofFIG.20, the biosensing film120is coated with a selective binding agent122. Coating the biosensing film120with the selective binding agent122includes, but is not limited to, immersing the wafer having the structure ofFIG.19in a selective binding agent bath at a suitable temperature (e.g., from about 25 degrees Celsius to about 300 degrees Celsius) for a suitable time duration (e.g., from about 5 mins to about 10 hrs) that is sufficient to allow the selective binding agent122to be attached to the biosensing film120, thus resulting in a thin film coating of the selective binding agent122in contact with the selective binding agent122. In some embodiments, the thin film coating of selective binding agent122is porous, which allows for the biosensing film120to be in contact with the cardiac-cell-containing fluid. In some embodiments, the binding agent122includes silane coupling agents that are compounds whose molecules contain functional groups that bond with both organic and inorganic materials. A silane agent acts as a sort of intermediary which bonds organic materials to inorganic materials. The silane coupling agent may include, by way of example and not limitation, silane having vinyl functional group (e.g., Vinyltrimethoxysilane ((CH3O)3SiCH═CH2), Vinyltriethoxysilane ((C2H5O)3SiCH═CH2) or the like), silane having epoxy functional group (e.g., 2-(3, 4 epoxycyclohexyl) ethyltrimethoxysilane, 3-Glycidoxypropyl methyldimethoxysilane, 3-Glycidoxypropyl trimethoxysilane, 3-Glycidoxypropyl methyldiethoxysilane, 3-Glycidoxypropyl triethoxysilane or the like), silane having styryl functional group (e.g., p-Styryltrimethoxysilane or the like), silane having methacryloxy functional group (e.g., 3-Methacryloxypropyl methyldimethoxysilane, 3-Methacryloxypropyl trimethoxysilane, 3-Methacryloxypropyl methyldiethoxysilane, 3-Methacryloxypropyl triethoxysilane or the like), silane having acryloxy functional group (e.g., 3-Acryloxypropyl trimethoxysilnae or the like), silane having amino functional group (e.g., N-2-(Aminoethyl)-3-amonopropylmethyldimethoxysilane, N-2-(Aminoethyl)-3-aminopropyltrimethoxysilane, 3-Aminopropyltrimethoxysilane, 3-Aminopropyltriethoxysilane, 3-Triethoxysilyl-N-(1, 3 dimethy-butylidene) propylamine, N-Pheny-3-aminopropyltrimethoxysilane, N-(Vinylbenzyl)-2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride or the like), silane having ureide functional group (e.g., 3-Ureidopropyltrialkoxysilane or the like), silane having isocyanate functional group (e.g., 3-Isocyanatepropyltriethoxysilane or the like), silane having isocyanurate functional group (e.g., Tris-(trimethoxysilylpropyl)isocyanurate or the like), silane having mercapto functional group (e.g., 3-Mercaptopropylmethyldimethoxysilane, 3-Mercaptopropyltrimethoxysilane or the like) or silane having other suitable functional groups. In some embodiments, the selective binding agent122for selectively binding with the cardiac cell192includes, for example, collagen, laminin, fibronectin, and mucopolysaccharides, heparin sulfate, hyaluronidate, chondroitin sulfate, the like, or combinations thereof. In some embodiments, an additional surface treatment is performed on the biosensing film120before forming the coating of selective binding agent122. The surface treatment includes, for example, a plasma treatment and/or a liquid-phase chemistry treatment that is capable of improving hydrophilicity of the biosensing film120. For example, the biosensing film120may undergo O2or O3plasma treatment before forming the coating of selective binding agent122, so as to improve hydrophilicity of the biosensing film120. The biosensing film120with improved hydrophilicity will be helpful in attachment to the cardiac cell, thus improving the detection and/or monitoring on the cardiac cell. As illustrated in a cross-sectional view ofFIG.21, in some embodiments, an etching process is performed into the coating of selective binding agent122, the biosensing film120, the isolation dielectric layer116, the active semiconductor layer102, the multi-layer dielectric structure128to form a pad opening1410exposing the pad structure1412of the BEOL interconnect structure110. The process for performing the etching may comprise, for example, coating a photoresist over the coating of selective binding agent122and patterning the photoresist using photolithography, such that the patterned photoresist has an opening corresponding to the pad opening1410. With the patterned photoresist in place, the etching process may comprise, for example, applying one or more etchants to the coating of selective binding agent122, the biosensing film120, the isolation dielectric layer116, the active semiconductor layer102, the multi-layer dielectric structure128, and subsequently stripping the patterned photoresist by ashing. As illustrated by a cross-sectional view ofFIG.22, fluid channel walls1414are formed over the coating of selective binding agent122to define a fluid containment region1416over the sensing well O. In some embodiments, the fluid channel walls1414include an elastomer. In some embodiments, the elastomer is polydimethylsiloxane (PDMS). In some embodiments, a layer of elastomer is patterned and then attached to the structure ofFIG.21to form fluid channel walls1414. In some embodiments, the material of fluid channel walls1414is first deposited and then pattern on the structure ofFIG.21. FIG.23is a chart illustrating an experimental result of a cardiac cell measured using an integrated circuit having BioFETs100as discussed above. The experimental result includes a time domain signal2500measured from a cardiac cell using the BioFETs100. The time domain signal2500indicates that beating pulse2502is greater than about 4 μA. The time domain signal2500detected by the BioFETs100is similar to a normal cardiac cycle, and thus the experimental result shows that the integrated circuit having BioFETs100can serve as a promising candidate for monitoring beating of cardiac cells. FIG.24is a 2D electrical image of a cardiac cell obtained using an integrated circuit having an array of BioFETs100as discussed above. The array of BioFETs100includes sensing pixels2601,2602,2603and2604. In the experiment a cardiac cell is placed on the sensing pixel2601and no cardiac cell is placed on the sensing pixels2602-2604, and the 2D electrical image clearly indicates that a cardiac cell in on the sensing pixel2601and no cardiac cell is placed on the sensing pixels2602-2604. Moreover, the 2D electrical image properly reflects the 2D image profile of the cardiac cell. This experimental result shows that the integrated circuit having BioFETs100can serve as a promising candidate for generating a 2D image of one or more cardiac cells. Based on the above discussions, it can be seen that the present disclosure offers advantages. It is understood, however, that other embodiments may offer additional advantages, and not all advantages are necessarily disclosed herein, and that no particular advantage is required for all embodiments. One advantage is that cardiac cells can be detected, measured and/or monitored using an IC having BioFETs. Another advantage is that the coating of selective binding agent on the biosensing film aids in binding the cardiac cell to the biosensing film, thus improving the accuracy of the measurement result of cardiac cell. In some embodiments, an IC includes a source region and a drain region in a semiconductor layer. A channel region is laterally between the source region and the drain region. A sensing well is on a back surface of the semiconductor layer and over the channel region. An interconnect structure is on a front surface of the semiconductor layer opposite the back surface of the semiconductor layer. A biosensing film lines the sensing well and contacts a bottom surface of the sensing well that is defined by the semiconductor layer. A coating of selective binding agent is over the biosensing film and configured to bind with a cardiac cell. In some embodiments, an IC includes a semiconductor substrate having a source region and a drain region. A sensing well is on a back surface of the semiconductor substrate. A biosening film lines the sensing well and contacts the back surface of the semiconductor substrate. A biological material coating layer is over the biosensing film. A first heater is in the semiconductor substrate and laterally spaced from the source region and the drain region. The first heater vertically overlaps with the biological material coating layer. In some embodiments, a method includes forming a semiconductor-on-insulator (SOI) substrate comprising a semiconductor substrate, a sacrificial substrate and a dielectric layer between the semiconductor substrate and the sacrificial substrate; forming source/drain regions in the semiconductor substrate; forming a back-end-of-line (BEOL) interconnect structure on a first side of the semiconductor substrate; bonding a carrier substrate to the semiconductor substrate through the BEOL interconnect structure; after bonding the carrier substrate to the semiconductor substrate; thinning the SOI substrate to remove the sacrificial substrate and to expose the dielectric layer; etching the dielectric layer until a second side of the semiconductor substrate is exposed, resulting in a sensing well extending through the dielectric layer and laterally between the source/drain regions; forming a biosensing film lining the sensing well; and immersing the biosensing film in a biological material bath until the biosensing film is coated with a biological material layer. The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. | 56,401 |
11860122 | DETAILED DESCRIPTION OF THE DRAWINGS The following description provides embodiments of the present invention, which are generally directed to systems, instruments, devices, and methods for preparing, observing, testing, and/or analyzing biological samples. Such description is not intended to limit the scope of the present invention, but merely to provide a description of embodiments. Exemplary systems and methods related to the various embodiments described in this document include those described in following applications:U.S. patent application Ser. No. 15/124,013, filed on Mar. 7, 2014;U.S. patent application Ser. No. 15/124,129, filed on Mar. 7, 2014;U.S. patent application Ser. No. 15/124,168, filed on Mar. 7, 2014;U.S. design patent application number 29/591,445, filed on Jan. 19, 2017;U.S. design patent application number 29/591,865, filed on Jan. 24, 2017;U.S. design patent application number 29/591,867, filed on Jan. 24, 2017;U.S. provisional patent application No. 62/460,700, filed on Feb. 17, 2017;U.S. provisional patent application No. 62/463,467, filed on Feb. 24, 2017;U.S. provisional patent application No. 62/463,551, filed on Feb. 24, 2017;U.S. provisional patent application No. 62/463,528, filed on Feb. 24, 2017. Embodiments of the present invention may include various sample separation systems and methods including, but not limited to, capillary electrophoresis, chip based electrophoresis, lab-on-a-chip microfluidics, gel electrophoresis, electro-osmosis, chromatography, flow cytometry, and the like. Example embodiments of the present invention will be presented for capillary electrophoresis systems or instruments in order to demonstrate various aspects of the present invention that may be applicable to other separation systems, such as chip based electrophoresis and the like. As used herein the terms “radiation” or “electromagnetic radiation” means radiant energy released by certain electromagnetic processes that may include one or more of visible light (e.g., radiant energy characterized by one or more wavelengths between 400 nanometers and 700 nanometers or between 380 nanometers and 800 nanometers) or invisible electromagnetic radiations (e.g., infrared, near infrared, ultraviolet (UV), X-ray, or gamma ray radiation). As used herein a “radiant source” means a source of electromagnetic radiation that may be directed toward at least one sample mixture or solution in order to produce a detectable signal for determining the presence and/or quantity of one or more target sample molecules or compounds contained within the at least one sample mixture or solution. The radiant source may comprise a single source of light, for example, an incandescent lamp, a gas discharge lamp (e.g., Halogen lamp, Xenon lamp, Argon lamp, Krypton lamp, etc.), a light emitting diode (LED), an organic LED (OLED), a laser (e.g., chemical laser, excimer laser, semiconductor laser, solid state laser, Helium Neon laser, Argon laser, dye laser, diode laser, diode pumped laser, fiber laser, pulsed laser, continuous laser), or the like. Alternatively, the radiant source may comprise a plurality of individual sources (e.g., a plurality of LEDs or lasers). The radiant source may also include one or more excitation filters, such as a high-pass filter, a low-pass filter, or a band-pass filter. For example, the excitation filter comprise a colored filter and/or a dichroic filter. The radiant source may continuous or pulsed, and may comprise either a single beam or a plurality of beams that are spatially and/or temporally separated. The radiant source may be characterized by electromagnetic radiation that is primarily within the visible light range (e.g., a “light source” emitting electromagnetic radiation within a wavelength in the range of 400 nanometers to 700 nanometers or in the range of 380 nanometers and 800 nanometers), near infrared range, infrared range, ultraviolet range, or other ranges within the electromagnetic spectrum. Referring toFIG.1, certain embodiments of the present invention comprise a system or instrument1000for performing a capillary electrophoresis or similar assay, process, test, or experiment. System1000comprises one or more capillaries, tubes, or channels101(four are shown inFIG.1) located on or in a capillary housing, holder, or mount102. Each capillary may comprise a detection portion configured to pass electromagnetic radiation into and/or out of the capillary. In the illustrated embodiment, a capillary array105comprises four capillaries101; however, capillary array105may include more than four capillaries, for example, to provide higher throughput or shorter assay runs. Configurations of instrument1000may include 1, 2, 4, 8, 10, 12, 16, 24, 32, 48, 65, 96, 128, 256, 384, or more than 384 capillaries101. System1000further comprises an optical system110comprising an illumination or excitation optical system111comprising any or all of a radiant source112, a beam shaper or conditioner115, a beam divider118, and/or a beamsplitter or mirror120. Radiant source112is configured to illuminate an optical detection access or optical detection zone121of system1000and/or capillaries101in which electromagnetic radiation (e.g., light, near infrared, or ultraviolet) may pass into and/or out of the detection portion of the one or more capillaries101in order to detect or measure a target, calibration, or other molecules of interest. Optical system110may further comprise a lens122and an emission optical system125. Emission optical system125may comprise lens122, a lens130, an emission filter135, and a detection system136. Radiant source112may comprise one or more of the types of radiant sources discussed above herein. In certain embodiments radiant source112comprises a diode pumped solid state (DPSS) laser having a wavelength of 505 nanometers. Detection system136comprises a detector138configured to receive emissions from the optical detection zone121of capillaries101, for example to receive fluorescent emissions produced by fluorescent dyes, probes, or markers attached to target or other molecules of interest. Detector138may be an optical detector comprising one or more individual photodetectors including, but not limited to, photodiodes, photomultiplier tubes, bolometers, cryogenic detectors, quantum dots, light emitting diodes (LEDs), semiconductor detectors, HgCdTe detectors, or the like. Additionally or alternatively, detector138may be an optical detector comprising an array sensor including an array of sensors or pixels. The array sensor may comprise one or more of a complementary metal-oxide-semiconductor sensor (CMOS), a charge-coupled device (CCD) sensor, a plurality of photodiodes detectors, a plurality of photomultiplier tubes, or the like. In certain embodiments, detector138comprises two or more array sensors. An optical system such as emission optical system125may be used to collect emissions from each capillary101. In the illustrated embodiment inFIG.1, lens122is doublet lens configured to collect emission light from each of the one or more capillaries101and lens130is a doublet lens configured to reimage the emissions from each of the one or more capillaries101to a spot or focus in an image plane of emission optical system125. However, other optical configurations known in the art may be used for these purposes. For applications in which multiple emissions at different wavelengths are produced in each of the one or more capillaries101, detection system136may further comprise one or more spectral dispersion elements139that spread the spectral content contained in different fluorescent signal to different parts (e.g., different groups of pixels) of detector138. In the illustrated embodiment shown inFIG.1, four spectral dispersion elements139are incorporated into a spectrometer140(two spectral dispersion elements139are visible inFIG.1and two more spectral dispersion elements139are located behind the two visible inFIG.1). Spectrometer140may further comprise detector138. Detection system136may be disposed within a housing or enclosure141. Spectrometer140may be optically coupled to capillaries101and/or emission optical system125via one or more fibers or optical fibers145. In the illustrated embodiment, a first pair or bundle of optical fibers145ais configured to receive emission light from first and second capillaries101of capillary array105and a second pair or bundle of optical fibers145bis configured to receive emission light from third and fourth capillaries101of capillary array105. Additionally or alternatively, optical fibers145may be grouped together into a single fiber bundle or each fiber145may be separate from the remaining optical fibers145. Spectrometer140may further comprise the one or more spectral dispersion elements139and the detector138, wherein each spectral dispersion element139is configured to direct emission light from a different one of capillaries101onto a different region of detector138. Spectral dispersion elements139may comprise one or more prisms, diffractive optical elements, holographic optical elements, or the like. Spectral dispersion elements139may comprise reflective or transmissive optical elements. The use of optical fibers145have been discovered to advantageously simplify alignment and calibration of detector138for multi-fluorescent wavelength application, as discuss below herein. In certain embodiments, optical system110, the one or more capillaries101, and capillary mount102are disposed inside of a common housing or enclosure150and spectrometer140is located outside housing150in housing141. Alternatively, spectrometer140and/or housing141may be located within housing150or directly attached to housing150. Housing141may include an opening or port to allow transfer of radiation or light from capillaries101to spectrometer140. Spectrometer140may be contained in a separate housing, as shown inFIG.1, or included inside the same instrument housing as the optical system. In contrast to the embodiment shown inFIG.1, the one or more capillaries101and/or some of associated hardware may be located outside housing150, in which case an interface with system1000may be provided via an opening or port in housing150. In certain embodiments, optical fibers145are part of spectrometer140. Alternatively, optical fibers145may be separate from spectrometer140, wherein the optical fibers145are attached to spectrometer140using an optical coupler (not shown). In the illustrated embodiment, spectral dispersion elements139are advantageously configured to both disperse and focus incident emissions received from optical fiber145onto detector138. During use, capillaries101may contain a polymer or similar solution configured to support an electric field or current. The polymer or similar solution is configured to permit the transfer or migration of one or more samples that may include one or more fluorescent dyes, probes, markers, or the like. The fluorescent dyes, probes, markers, or the like may be selected to produce a fluorescent signal during use that may be correlated to the presence or amount of one or more target molecules or sequences of molecules present at a given time within optical detection zone121. The fluorescent signal(s), light, or radiation produced within any or all of capillaries101may be directed back through lens122and the mirror so as to be received by spectrometer140. Referring again toFIG.1, in certain embodiments, system1000may comprise conditioner115and radiation from radiant source112passes through conditioner115. Conditioner115may comprise a homogenizer configured, for example, to blend different color or wavelength radiant sources and/or to provide a more even illumination cross-section of the output beam. Additionally or alternatively, system1000may comprise divider118. Additionally or alternatively, emitted radiation from radiant source112may pass through beam divider118to provide a plurality of excitation, sample, illumination, or source beams155, each source beam 155 characterized by one or more of, one or more beam diameters, a cross-sectional shape (e.g., square, circular, or elliptical), a predetermined intensity or power profile (e.g., constant, top hat, Gaussian, etc.). As illustrated inFIG.2, beam conditioner115and beam divider118may be configured to produce or provide source beams155, where each source beam155comprising an elliptical cross section or shape. Beam conditioner115may comprise an anamorphic beam shaper, for example, comprising one or more cylindrical lenses configured to produce beams having an elliptical cross section, wherein the beam cross section is wider in one axis than in the other perpendicular axis. Alternatively, beam conditioner115may comprise a Powell lens, for example, configured to provide a line focus and/or an elliptical beam cross section in which an intensity or power over a cross section of the beam uniform, or nearly uniform. In addition, beam conditioner115may be configured so that any diameter of the beam is greater than or less than the diameter of the beam entering beam conditioner115. In the illustrated embodiment, the beam exiting beam conditioner115is collimated. The elliptical cross section of each of source beam155may be oriented so that the long axis or dimension is oriented perpendicular or nearly perpendicular to an axis of the associated capillary101. This orientation of each source beam155and its focus has been found to advantageously reduce the sensitivity of the alignment of the capillary array105to the beams. In the illustrated embodiment shown inFIG.2, the long diameter of the beam focus is less than an inner diameter of an individual capillary101. Alternatively, as illustrated inFIG.3, the long diameter of the focused source beams155may be larger than the inner diameter of the individual capillaries101.FIG.3also illustrates the diameters and pitch of capillaries101within the array for certain embodiments. As seen inFIG.3, the inner diameter of each capillary101is 50 micrometers, while the focused beam has a diameter of about 100 micrometers. Referring again toFIG.1, the excitation beam out of conditioner115enters beam divider118, which may be configured to produce a plurality of identical or similar source beams155from a single input beam into beam divider118. As an example, beam divider118may comprise one or more diffractive optical elements, holographic optical elements, or the like, that is configured to produce or provide four elliptical beams for illuminating each of the four capillaries101, as seen inFIGS.1-3. The four source beams155have the same or a similar cross-section, and each beam diverges at a different angle relative to a system optical axis or general directions of light propagation. Alternatively, beam divider118may be configured to produce a plurality of beams that are parallel to one another or that converge relative to one another. In the illustrated embodiment, the beams out of beam divider118are collimated; however, some or all of the beams may alternatively be converging or diverging as they leave beam divider118. Source beams155originating from beam divider118may each be collimated as they enter lens122, but be divergent from one another. In such embodiments, lens122may be configured focus each of source beam155to a location at or near a respective capillary101, as illustrated in the magnified view ofFIG.1. In addition, lens122and the source beams155out of beam divider118may be configured such that the individual beams155are each collimated relative to one another (e.g., the four beams inFIG.1may all travel parallel to one another after exiting lens122). Source beams155out of beam divider118inFIG.1may be reflected by a mirror120and directed toward capillaries101. Additional mirrors and/or diffractive elements may be included as desired to direct the four beams toward capillaries101, for example, to meet packaging constraints. The beams from beam divider118continue to diverge after reflection off the mirror until they are received by lens122. Mirror120may be a dichroic mirror, or the like, which may be configured to reflect light at a predetermined wavelength or light over a predetermined wavelength range, while transmitting light or other electromagnetic radiation that is outside the predetermined wavelength or wavelength range. In some embodiments, mirror120comprises a dichroic mirror having more than one predetermined wavelength or wavelength range, for example, when the radiant source comprises more than one distinct wavelength or wavelength range. In the illustrated embodiment, the source beams155from beam divider118are reflected by mirror120, while emitted radiation from optical detection zone121is transmitted or largely transmitted by mirror120. Alternatively, the location of capillaries101may be located along the optical axis of beam divider118and mirror120may be configured to transmit, or largely transmit, the excitation beams, while reflecting emissions from the optical detection zone121. Emission filter135may be located between lenses122,130and may be configured block or attenuate light from the radiant source, thereby eliminating or reducing the about of light from the radiant source that is receive by spectrometer140. In certain embodiments, the focal length of lenses122,130are selected to produce a magnification of capillaries101, or of emission radiation from capillaries101, that is different than one (e.g., to produce a magnified or demagnified image). For example, lens122may be selected to have a numerical aperture (NA) that is twice the NA of the lens130, resulting in a system magnification of two. In certain embodiments, lens122,130has an NA of 0.4 and lens130has an NA of 0.2. In some embodiments, the focal length or NA of lenses122,130may be selected to (1) provide a focal spot, or focal point, at or near capillary array105that has a predetermined size or diameter and (2) simultaneously providing an NA that is matched to the NA of spectrometer140and/or the NA of the optical fiber system used to transfer light into spectrometer140. Source beams155are configured to illuminate samples within optical detection zone121of each of the capillaries101to produce respective emissions, for example fluorescent emissions produced by fluorescent dyes, probes, or markers attached to the target molecules or molecules of interest. The emissions may be configured to indicate the presence or amount of target molecules or molecules of interest. The emissions may be focused or re-image onto a plane using lenses122,130or some other suitable emission optical system. Emission filter135may be configured to filter out unwanted radiation, such as excitation light produced by radiant source112. Alternatively, as shown illustrated inFIG.1, emission light from capillaries101may be focused or re-image onto to input or receiving ends of optical fibers145, then propagated by optical fibers145into spectrometer140. Each fiber145may be associated with (e.g., receive radiation from) a corresponding one of capillaries101. Using optical fibers145, radiation from capillaries101is then transferred into spectrometer140, where it is dispersed by wavelength onto a detector138. In the illustrated embodiment, emission radiation from optical fibers145aenter on one side of spectrometer140and radiation from optical fibers145benter on another side of spectrometer140. In this manner, the spectrum from each of fiber140(or capillaries101) is directed onto a different portion of detector138. This configuration has been found to advantageously allow the spectrum from each of multiple capillaries101to be produced and detected simultaneously on a single or reduced number of array detectors138. Detector138may be configured to receive the emissions from the samples contained in capillaries101and to produce emission signal that may be further processed. For example, spectrometer140may be configured to separate the signals created by different fluorescent dyes, probes, or markers, for example, created by dyes, or probes, markers corresponding to different DNA or RNA bases (e.g., adenine, thymine (or uracil), cytosine, and guanine). System1000may further comprise a computer or processing system160including a data processing system, a computer program product161configured to program processing system160, and display or other output device162. Processing system160may be used to control or obtain data from system1000, for example, to monitor and/or control one or more electrical parameters (e.g., radiant source power, detector supply power, cathode/anode voltage, or current through one or more of each capillary101or a group of the capillaries101) or to measure or control various run or process parameters such as temperature or pressure (e.g., system or capillary101temperature, pressure of a pump or syringe for filling capillaries101with a polymer solution or the like). Processing system160may be coupled to detection system136, for example to provide read detected fluorescence signals. In certain embodiments, detection system136passes a signal to processing system160corresponding to the intensity of emissions received at various wavelengths scanned by detection system136. Computer program product161may be used to configure processing system160to process received spectral data from detection system136that may be used during runtime of instrument1000to calibrate instrument1000or to correct for spectral error, for example, as disclosed in U.S. provisional patent application 62/460,700. Display or other output device162is coupled to processing system160and may be used to display or report data related to an assay, process, test, or experiment such as run parameter values, spectral data, run condition data, run quality data, warning flags, and the like, for example, as disclosed in U.S. provisional patent application No. 62/463,551. Referring toFIG.4, computer or processing system160may be configured to execute instruction codes contained in a computer program product161. Computer program product161may comprise executable code in an electronically readable medium that may instruct one or more computers such as computer or processing system160to perform processing that accomplishes the exemplary method steps performed by the embodiments discussed herein. The electronically readable medium may be any non-transitory medium that stores information electronically and may be accessed locally or remotely, for example via a network connection. In alternative embodiments, the medium may be transitory. The medium may include a plurality of geographically dispersed media each configured to store different parts of the executable code at different locations and/or at different times. The executable instruction code in an electronically readable medium directs the illustrated computer or processing system160to carry out various exemplary tasks described herein. The executable code for directing the carrying out of tasks described herein would be typically realized in software or firmware. However, it will be appreciated by those skilled in the art that computers or other electronic devices might utilize code realized in hardware to perform many or all the identified tasks without departing from the present invention. Those skilled in the art will understand that many variations on executable code may be found that implement exemplary methods within the spirit and the scope of the present invention. The code or a copy of the code contained in computer program product161may reside in one or more storage persistent media (not separately shown) communicatively coupled to computer or processing system160for loading and storage in persistent storage device470and/or memory410for execution by a processor420. Computer or processing system160also includes I/O subsystem430and peripheral devices440(e.g., display or output device162). I/O subsystem430, peripheral devices440, processor420, memory410, and persistent storage device470may be coupled via a common bus450. Like persistent storage device470and any other persistent storage that might contain computer program product161, memory410may a non-transitory media (even if implemented as a typical volatile computer memory device). Moreover, those skilled in the art will appreciate that in addition to storing computer program product161for carrying out processing described herein, memory410and/or persistent storage device470may be configured to store various data elements disclosed or referenced and illustrated herein. Those skilled in the art will appreciate computer or processing system160illustrates just one example of a system in which a computer program product in accordance with embodiments of the present invention may be implemented. To cite but one example of an alternative embodiment, execution of instructions contained in a computer program product in accordance with an embodiment of the present invention may be distributed over multiple computers, such as, for example, over the computers of a distributed computing network. Referring toFIG.5, in certain embodiments, a sample separation system or instrument5000, such as a capillary electrophoresis (CE) instrument, is configured for separating biological molecules, for example, for separating sample nucleotide molecules or sample amino acid molecule according to length of the different molecules. Where possible, embodiments of system5000, as well as methods, elements, and/or parameter values associated with system5000, may be incorporated into embodiments of system1000and into methods, elements, and/or parameter values associated with system1000. Conversely, where possible, embodiments of system1000, as well as methods, elements, and/or parameter values associated with system1000, may be incorporated into embodiments of system5000and into methods, elements, and/or parameter values associated with system5000. System5000comprises one or more capillaries101, an electronic or voltage supply502, one or more cathodes503, one or more anodes504, a sample source container505, a sample destination container506, radiant source112, detection system136, and processing system160including a data processing system configured by computer program product161, and display or output device162. Instrument5000may include multiple capillaries101(e.g., four capillaries101, as shown inFIG.1); however, only one capillary101is illustrated inFIG.5for simplicity. Configurations of instrument5000may include 1, 2, 4, 8, 10, 12, 16, 24, 32, 48, 65, 96, 128, 256, 384, or more than 384 capillaries. Sample separation could also be performed by other means including using gel electrophoresis and microfluidics, such as on a lab-on-a-chip. System5000may be used to perform a capillary electrophoresis or other sample separation assay, experiment, or process. A sample mixture or solution515containing various samples or sample molecules515ais first prepared in or delivered into sample source container505. At least a portion of sample mixture515is subsequently loaded into cathode503end of capillary101, for example using a pump or syringe, or by applying a charge or electric field to capillary101. Once loaded into the anode end of capillary101, voltage supply502creates a voltage difference between cathode503and anode504. The voltage difference causes negatively charged, dye-labeled samples515ato move from sample source container505to sample destination container506. During the assay, process, test, or experiment, various samples (e.g., nucleotides or amino acid molecules) pass through an optical detection zone516and are illuminated by radiant source112to produce respective emissions, for example fluorescent emissions produced by fluorescent dyes, probes, or markers attached to the target molecules or molecules of interest. The emissions may be configured to indicate the presence or amount of target molecules or molecules of interest. Longer and/or less charged dye-labeled samples515amove at a slower rate through capillary101than do shorter and/or higher charged dye-labeled samples, thereby creating some separation between samples of varying lengths and charges. As each of samples515apasses through an excitation beam generated by radiant source112, a dye on a leading element (a leading element might, e.g., be a nucleotide) of a sample515aexhibits fluorescence that is detected by detection system136. Detection system136may be coupled to provide signals to processing system160in response to detected fluorescence. In particular, detection system136passes a signal to processing system160corresponding to the intensity of emissions received at various wavelengths scanned by detection system136. Computer program product161configures data processing system160to process the received spectral data and may, for example during runtime of instrument5000, calibrate instrument5000to correct for spectral error, for example, as disclosed in U.S. provisional patent application No. 62/460,700. A display or other output device162is coupled to processing system160and may be used to display or report data related to the assay, process, test, or experiment such as run parameter values, spectral data, run condition data, run quality data, warning flags, and the like, for example, as disclosed in U.S. provisional patent application No. 62/463,551. In certain embodiments, system5000comprises a delivery system520comprising a polymer reservoir522containing a polymer or polymer solution523, a polymer valve525, and a pump528(e.g., a syringe) configured to receive or draw polymer523from polymer reservoir522and to pump or load polymer523into capillary101. Delivery system520further comprises a buffer reservoir530containing a buffer solution532and a buffer valve535. In the illustrated embodiment, buffer reservoir contains the one or more anodes504. In certain embodiments, all or some of components of delivery system520are part of a cassette or cartridge538that may further comprise capillaries101, cartridge538may also comprise the one or more cathodes503(e.g., one cathode503for each of a plurality of capillaries101). Examples of cassette or cartridges suitable for use with embodiments of the present invention are disclosed in U.S. provisional patent application No. 62/463,467. In certain embodiments, the sample separation assay, process, test, or experiment comprises the following activities:Locate cathode503end of capillaries101into wash/waste buffer container540containing a wash/waste buffer solution541.Close buffer valve535, open polymer valve525.Aspirate (draw) polymer solution523from polymer reservoir522into syringe528.Close polymer valve525(buffer valve535remains closed).Dispense (deliver) polymer523to capillaries101using syringe528.Locate cathode503end of capillaries101into sample source container505.Draw at least a portion of sample solution515into cathode503end of capillaries101by inducing a current flow from cathode503to anode504(referred to as electrokinetic injection).Locate cathode503end of capillaries101into run a buffer container545containing a run buffer solution546.Open buffer valve535to provide electrical coupling between anode504and capillaries101(polymer valve525remains closed).Run capillary electrophoresis assay, process, test, or experiment.Locate cathode503end of capillaries101into a wash/waste buffer container540.Close buffer valve535.Optionally open polymer valve525.Optionally aspirate (draw) polymer solution523from polymer reservoir522into syringe528.Optionally close polymer valve525if open.Clean capillaries101by dispensing (delivering) polymer523to capillaries101using syringe528.Repeat above steps for new separation assay, process, test, or experiment. Referring toFIG.6, in certain embodiments a system or instrument6000, such as a capillary electrophoresis (CE) instrument, is configured separating biological molecules, for example, for separating sample nucleotide molecules or sample amino acid molecule according to length of the different molecules. Where possible, embodiments of system6000, as well as methods, elements, and/or parameter values associated with systems1000,5000, may be incorporated into embodiments of systems1000,5000and into methods, elements, and/or parameter values associated with systems1000,5000. Conversely, where possible, embodiments of systems1000,5000, as well as methods, elements, and/or parameter values associated with system6000, may be incorporated into embodiments of system6000and into methods, elements, and/or parameter values associated with system6000. System6000comprises a housing or enclosure600and detection system136shown inFIG.1that may be disposed within housing600. Detection system136comprises a plurality of optical fibers145, the receiving ends of which are coupled, mounted, or attached to an optical fiber mount603. The receiving ends of optical fibers145are configured to receive emissions from optical detection zone121of respective ones of capillaries101. System6000also comprises computer processing system160, computer program product161, and display or other output device162. System6000further comprises a plurality of capillaries101comprising optical detection zone121, which are coupled, mounted, or attached to a capillary mount602. In certain embodiments, capillary mount602may be held or supported by a support structure605that in turn is mounted or attached to a base610. System6000further comprises emission optical system125and an excitation optical system611comprising any or all of a radiant source612. Emission optical system125comprises lenses122,130that are disposed along an optical axis or path613between capillaries101and the entrance end of optical fibers145. Lens122is configured to collect emission light from each of the capillaries101and lens130is configured to reimage the emissions from each of the one or more capillaries101to a spot or focus in image plane of emission optical system125that is at or near the input or receiving ends of optical fibers145; however, other optical configurations known in the art may be used for these purposes. With further reference toFIG.7, capillaries101and capillary mount602may be part of a cartridge or cassette615that may also include support structure605and base610. Base610may be mounted or attached to cartridge615. Cartridge615may be removed from system6000and replaced by another cartridge615′ (not shown) that is configure the same or similar to cartridge615shown inFIGS.6and7. In certain embodiments, cartridge615′ (not shown) may have the same or similar form, but contain modified or different elements than cartridge615. For example, cartridge615′ (not shown) may have more or fewer capillaries101than the four capillaries101of cartridge615, for example, 1, 2, or 8 capillaries101. Capillaries101may be coupled, mounted, or attached to capillary mount602such that portions of capillaries within optical detection zone121are fixedly located relative to one another. In similar fashion to capillaries101, optical fibers145may be coupled, mounted, or attached to optical fiber mount603such that the input or receiving ends of optical fibers145are fixedly located relative to one another. It has been discovered that fixedly mounting capillaries101and the receiving ends of optical fibers145advantageously simplifies alignment between of optical fibers145with respective capillaries101. This arrangement also has been found to improve the accuracy and durability of the alignment between optical fiber145and capillaries101. Referring toFIGS.6and8, in certain embodiments, each capillary101comprises capillary core801made of a core material and an outer coating or layer802surrounding capillary core801. For example, capillary core801may comprise fused silica and outer layer802may comprise a polyimide coating. The central portion of capillary core801comprises a channel803through which sample solution and molecules are contained. In such embodiments, for example, when outer layer802comprises a material that is optically opaque or translucent, optical access to material located in channel803may be provided by removing outer layer802along the portion of capillary101within optical zone121. As illustrated inFIG.8and the magnified view ofFIG.6, in certain embodiments, capillaries101are mounted to capillary mount602so that out layers802of adjacent capillaries101touch or contact one another. In this way, it has been discovered that the spacing between channels can be easily and accurately provided and maintained. Alternatively, spacers of predetermined thickness may be place between at least two adjacent capillaries on each side of optical detection zone121. For example, spacer of differing thickness may be placed between different sets of adjacent capillaries to increase the accuracy of the spacing between adjacent capillaries101and/or to provide a predetermined spacing between adjacent capillaries101. In other embodiments, capillaries101may be place in a fixture, such as a V-block, to provide a predetermined spacing between adjacent capillaries101. The outer diameter of capillaries101may be equal to or about 360 micrometers, for example, 363±10 micrometers. In certain embodiments, the outer diameter of capillaries101is from 100 micrometers to 1000 micrometer, for example, from 200 micrometers to 500 micrometers. In such embodiments, the diameter of channel803may be from 2 micrometers to 700 micrometers, for example, from 25 micrometers to 100 micrometers. In certain embodiments, the thickness of outer layer802is from 12 micrometers to 24 micrometers, for example, from 16 micrometers to 24 micrometers. In certain embodiments, the outer diameter of each capillary101is 363±10 micrometers, the diameter of channel803is 50±3 micrometers, and the thickness of outer layer802is 20 micrometers. In certain embodiments, optical fiber mount603is coupled, mounted, or attached to a motion or translation stage606. In use, capillaries101may be easily aligned using an alignment method comprising:Producing a first alignment signal from detector138by transferring emissions from one or more of capillaries101within optical detection zone through a respective one or more of optical fiber145and to detector138.Using translation stage606, moving the optical fiber mount603one or more times to one or more different locations capillary mount or the fiber mount;At each of the one or more locations, producing a respective alignment signal from detector138by transferring emissions from one or more capillaries101within optical detection zone121through the one or more optical fibers145to detector138;Using translation stage606, aligning capillaries101to the receiving ends of the plurality of capillaries based on the alignment signals. In certain embodiments, the alignment signal comprises a measured signal from detector138based on emissions from a single one of the capillaries101. Additionally or alternatively, the alignment signal comprises a measured signal from detector138based on emissions from a more than one of the capillaries101, for example, based on an average emission from all or some of the capillaries101. It has been discovered that this alignment method advantageously allows all the capillaries to be simultaneously aligned to the respective optical fibers145and, as a consequence, to be simultaneously aligned to the same corresponding areas on detector138each time the alignment method is performed. The reason emissions from each capillary101illuminate the same corresponding areas on detector138each time is because the output (or emitting or distal) ends of each optical fiber145are in a fixed position relative to detector138. Therefore, emitted emissions from the output end of optical fibers145will travel the same path each time to detector138. When capillaries101need to be replaced by a new set of capillaries101and the alignment method rerun, the new capillaries101will have the same or nearly the same spacing between capillaries as the old set of capillaries101. Thus, when the disclosed alignment method is performed again, the only emissions from capillaries101received at detector138are those emission that pass from the same output ends of optical fibers145. In prior art systems that directly reimage capillary emissions (i.e., systems that do not use the optical fiber arrangement disclosed herein), slight changes in a new, replacement set of capillaries will cause emissions from the new set of capillaries to be reimaged onto slightly different portions of the detector. Because of this, the detector itself in non-optical fiber based systems must be recalibrated each time, since different areas or, for example, pixels of a CCD or CMOS array detector, have different sensitivities. Therefore, because of the inventive use of optical fibers145in combination with the fixed mounting configurations of capillaries101and optical fibers145, no recalibration of detector138is necessary when a replacement set of capillaries101is used. In the illustrated embodiment shown inFIG.6, translation stage606is used to translate or move the input ends of optical fibers145in a transverse direction during the above alignment method. Additionally or alternatively, capillary mount602may be attached to a motion or translation stage and move instead of, or in addition to, translation stage606. In other embodiments, relative motion between capillaries101and optical fibers145may be accomplished during the alignment method above by making changes to emission optical system125. For example, a turning mirror or an additional refractive element may be place in the optical path from capillaries101and optical fibers145. Adjusting the turning mirror or additional refractive element can then be used to move the reimaged emissions from capillaries101and so align the reimaged emissions to the receiving ends of optical fibers145. In other embodiments, the alignment method can be implemented using longitudinal motion in place of or in addition to the transverse movement discussed above with translation stage606, for example, in order to move the reimaged emissions toward or away from the input ends of optical fibers145, thereby increasing the amount of emission entering optical fibers145. In yet other embodiments, emission optical system125comprises a zoom lens or other optical elements configured to change the magnification of the reimaged emissions from capillaries101, for example, to accommodate slight changes in spacing between different sets of capillaries101used in system6000. In certain embodiments, the alignment signal used in the above alignment method is produced due to Raman scattering of water molecules within one or more of the channels803of capillaries101, for example, water molecules contained in a polymer solution used to conduct a capillary electrophoresis assay, process, test, or experiment. The use of Raman scattering from water molecules, which is typically a source of noise, has been unexpected discovered to be suitable for the above alignment method because this signal remains constant over time and, for example, between different filling of capillaries101with the polymer solution use in capillary electrophoresis. Because of the stability of this signal source, Raman scattering can also be used to calibrate detector138, as well as provide alignment between capillaries101and optical fibers145. In such embodiments, the signal produced by Raman scatter may be measured during or after the alignment method and the detector may then be calibrated based on the value of the measured signal from detector138. In addition, the use of Raman scatter from water molecules allows the alignment method to be conducted before or after a sample has been introduced into the capillaries101for a capillary electrophoresis run or other sample separation assay, process, test, or experiment using system6000. In other embodiments, the alignment method may be conducted during a sample separation assay, process, test, or experiment. In such embodiments, emissions from one or more of capillaries101may be used to adjust alignment during the assay, process, test, or experiment. Referring toFIGS.6,9, and10, in certain embodiments, system6000further comprises an optical interface, cover, or snout650that is configured to engage, interface, or mate with capillary mount602and/or support structure605. As seen inFIG.9, base610may comprise a spring901, whereby capillary mount602and/or support structure605may be held against, mounted to, or engaged with optical interface650by a contact force that is determined by the amount of compression of spring901as cartridge615is placed or aligned within system6000. Optical interface650may comprise turning mirror652and/or turning mirror654, which are part of excitation optical system611. Mirrors652,654may be configured to a guide a source, source, illumination, or excitation beam655from radiant source612, through capillaries101, and into a beam dump658. Excitation optical system611may further comprise other optical elements not shown inFIG.6, for example, lenses, prisms, polarizers, additional mirrors, and the like. For example, one or more lenses may be place along the optical path between radiant source612and capillaries101to condition source beam655to provide a predetermined illumination characteristic as it passes through the plurality of capillaries101. It has been discovered that mounting turning mirror652with optical interface650advantageously provides a more stable alignment of source beam655to capillaries101, since any expansion or contraction along optical axis613of capillary mount602and/or support structure605due to temperature variations over time is compensated for the same or approximately the same movement of turning mirror652in the direction of optical axis613. Thus, the position of source beam655through capillaries101remains constant or very stable with movement of the of the capillaries due to temperature change. If, for example, source beam655traveled directly from radiant source612to capillaries101(i.e., without first reflecting off turning mirror652), the position of source beam655through capillaries101in the direction parallel to optical axis613would change as the location of capillaries101changed due to temperature variation in capillary mount602and/or support structure605. In certain embodiments, the source beam655comprises a linear polarization, either directly out of radiant source612or through the use of one or more polarization optical elements. It has been discovered that scatter from a polymer solution used in a sample separation assay, process, test, or experiment may be reduced or minimized when (1) the axis of polarization of source beam655perpendicular to the length of capillaries101and (2) the optics axis613of emission optical system125is parallel to the axis of polarization of source beam655. Raman scattering is undesired and adds noise on top of the fluorescent signal from samples during a sample separation assay, process, test, or experiment. The fluorescent signal from samples usually generally less polarization sensitive. Therefore, the polarization criteria discover allows an increase in signal-to-noise ratio during use of system6000. Selected embodiments of the current invention may include, but are not limited to: 1. Embodiment 1 includes a system for separating biological molecules, the system comprising: a plurality of capillaries configured to separate biological molecules in a sample, each capillary comprising a detection portion configured to pass electromagnetic radiation into the capillary;a capillary mount, the plurality of capillaries coupled to the capillary mount such that the detection portions are fixedly located relative to one another;a plurality of optical fibers corresponding to the plurality of capillaries, each optical fiber comprising a receiving end configured to receive emissions from a respective one of the detection portions;a fiber mount, the optical fibers being coupled to the fiber mount such that the receiving ends of the optical fibers are fixedly located relative to one another;an emission optical system configured to direct emissions from the detection portions into the receiving ends of the optical fibers;a optical detector configured to produce an alignment signal when emissions from at least one of the plurality of capillaries is transmitted through a respective at least one of the optical fibers and onto the optical detector; anda motion stage configured to move to a plurality of locations, one or more of the capillary mount, the fiber mount, or at least a portion of the emission optical system;wherein the motion stage is configured to align the receiving ends of the optical fibers to the detection portions based on values of the alignment signal at the plurality of locations. 2. Embodiment 1, wherein the emission optical system comprises one or more lenses disposed along an optical path between the detection portions and the receiving ends. 3. Embodiment 1 or 2, wherein the motion stage comprises a translation stage configured to translate the motion stage within a plane parallel to the receiving ends of the optical fibers and/or parallel to a plane passing through the detection portions. 4. Any of embodiments 1-3, further comprising:a processor; anda memory encoded with instructions for:moving the motion stage to the plurality of locations;for each location, capturing one or more respective values of the alignment signal from the optical detector;determining an alignment position based on the respective values;moving the motion stage to the alignment position so that the detection portions are aligned to the receiving ends of the optical fibers. 5. Embodiment 4, wherein each of the values of the alignment signal comprises one or more of:an average signal from the optical detector for at least two of the capillaries;a mean signal from the optical detector for at least three of the capillaries;a signal from the optical detector corresponding to a highest emission from between at least two of the capillaries. 6. The system of claim4, wherein the memory is further encoded to evaluate whether a signal from the optical detector corresponding to emissions from one or more of the capillaries is a noise signal and/or a signal not produced by Raman scattering from water molecules. 7. Any of embodiments 1-6, further comprising a first electrode and a second electrode, the electrodes configured to produce an electric potential across the capillaries 8. Any of embodiments 1-7, wherein each optical fiber comprises an outer coating surrounding the optical fiber at the receiving end, the outer coating of the optical fiber configured to reflect and/or absorb light from emission signal, wherein the outer coating is not present within the detection portion of each optical fiber. 9. Any of embodiments 1-8, wherein the capillaries are coupled to the capillary mount such that each capillary touches an adjacent capillary of the plurality of capillaries along a portion outside the detection portion. 10. Embodiment 10 includes a system for separating biological molecules, the system comprising:a plurality of capillaries configured to separate biological molecules in a sample, each capillary comprising a detection portion configured to pass electromagnetic radiation into the capillary;a capillary mount, the capillaries coupled to the capillary mount such that the detection portions are fixedly located relative to one another;a radiant source producing a source beam of electromagnetic radiation configured to illuminate the detection portions;a base configured to receive the capillary mount, the base comprising a mirror configured to reflect the source beam and to direct the source beam through the plurality of capillaries. 11. Embodiment 10, further comprising:a optical detector;an emission optical system configured to receive emission signals from the plurality of capillaries and to direct the emission signals to the optical detector. 12. Embodiment 10, further comprising:a spectrometer comprising a dispersive optical element and a optical detector;an emission optical system configured to receive emission signals from the plurality of capillaries and to direct the emission signals to the optical detector. 13. Embodiment 13 includes a system for separating biological molecules, the system comprising:a plurality of capillaries configured to separate biological molecules in a sample, each capillary comprising a detection portion configured to pass electromagnetic radiation into the capillary;a radiant source producing a source beam of electromagnetic radiation configured to illuminate the detection portions;wherein the source beam has a linear polarization disposed along a polarization axis;wherein the polarization axis of the source beam within the detection portions is perpendicular to the capillary plane. 14. Embodiment 14 includes a method of separating biological molecules, comprising:providing a plurality of capillaries, each capillary comprising an detection portion, the capillaries coupled to a capillary mount such that detection portions are fixedly located relative to one another;providing a plurality of optical fibers corresponding to respective ones of the plurality of capillaries, each optical fiber comprising a receiving end configured to receive emissions from a respective one of the detection portions, the optical fibers being coupled to a fiber mount such that the receiving ends are fixedly located relative to one another;producing values of an alignment signal from a optical detector by transferring emissions within the detection portion of at least one of the capillaries through a respective at least one of the optical fiber and to the optical detector;moving, one or more times to one or more different locations, the capillary mount or the fiber mount;at each of the one or more locations, producing a respective value of the alignment signal from the optical detector by transferring emissions within the detection portion of the at least one capillary through the at least one optical fiber to the optical detector;aligning the capillaries to the receiving ends of the plurality of optical fibers based on the values of the alignment signals. 15. Embodiment 14, further comprising:providing an emission optical system configured to direct emissions from each of the detection portions into the receiving end of the respective optical fiber; andmoving, one or more times to one or more locations, at least one of: the capillary mount or the fiber mount, or the emission optical system. 16. Any of embodiments 14-15, wherein the values of the alignment signal are produced by Raman scattering emission from water molecules within a polymer solution contained in the capillaries. 17. Any of embodiments 14-16, wherein the values of the alignment signal comprise emissions from a single one the capillaries. 18. Any of embodiments 14-17, wherein the values of the alignment signal comprise an average of the emissions from more than one of the capillaries. 19. Any of embodiments 14-18, further comprising:loading one or more samples containing a fluorescent molecule into the plurality of capillaries;propagating the one or more samples through the capillaries by producing an electric potential across the capillaries;illuminating each detection portion with a source beam of electromagnetic radiation to produce a plurality of emitted signals from the each of the detection portions;determining a nucleotide sequence of a molecule based on the plurality of emitted signals. 21. Embodiment 21 includes a system for separating biological molecules, the system comprising:a plurality of capillaries configured to separate biological molecules in a sample, the capillaries comprising an optical detection zone;a capillary mount, the capillaries being coupled to the capillary mount such that portions of the capillaries within the optical detection zone are fixedly located relative to one another;a plurality of optical fibers corresponding to the plurality of capillaries, each optical fiber comprising a receiving end configured to receive emissions from a respective capillary within the optical detection zone;a fiber mount, the optical fibers being coupled to the fiber mount such that the receiving ends of the optical fibers are fixedly located relative to one another;optionally, an emission optical system configured to direct emissions from each capillary within the optical detection zone into the receiving end of the respective optical fiber; anda optical detector configured to produce an alignment signal when emissions from at least one of the capillaries is transmitted through a respective at least one of the optical fibers and onto the optical detector;a motion stage coupled to one or more of the capillary mount, the fiber mount, or at least a portion of the optional emission optical system;wherein the motion stage and the optical detector are configured to align receiving ends of the optical fibers to the capillaries based on values of the alignment signal at a plurality of locations of the motion stage. 22. Embodiment 21, wherein the emission optical system comprises one or more lenses disposed along an optical path between the optical detection zone and the receiving ends. 23. Embodiment 21 or 22, wherein the motion stage comprises a translation stage configured to translate the motion stage within a plane parallel to the receiving ends of the optical fibers and/or parallel to a plane passing through each of the capillaries within the optical detection zone. 24. Any of embodiments 21-23, further comprising:a processor; anda memory encoded with instructions for:capturing a first alignment signal from the optical detector for emissions within optical detection zone from the at least one capillary;moving the motion stage to one or more different locations;for each of the one or more different locations, capturing one or more respective alignment signals from the optical detector for emissions within optical detection zone from the at least one capillary;determining an alignment position based on the alignment signals;moving the motion stage to the alignment position so that the capillaries within the optical detection zone are aligned to the receiving ends of the plurality of capillaries. 25. Embodiment 24, wherein the alignment signal comprises one or more of:an average signal from the optical detector for at least two of the capillaries;a mean signal from the optical detector for at least three of the capillaries;a signal from the optical detector corresponding to a highest emission from between at least two of the capillaries. 26. The system of claim24, wherein the memory is further encoded to evaluate whether a signal from the optical detector corresponding to emissions from one or more of the capillaries is a noise signal and/or a signal not produced by Raman scattering from water molecules. 27. Any of embodiments 21-26, further comprising a first electrode and a second electrode, the electrodes configured to produce an electric potential across the capillaries. 28. Any of embodiments 21-27, wherein each optical fiber comprises an outer coating surrounding the optical fiber at the receiving end, the outer coating of the optical fiber configured to reflect and/or absorb light from emission signal. 29. Any of embodiments 21-28, wherein the capillaries are coupled to the capillary mount such that each capillary touches an adjacent capillary of the plurality of capillaries along a portion outside the optical detection zone. 30. Embodiment 30 includes a system for separating biological molecules, the system comprising:a plurality of capillaries configured to separate biological molecules in a sample, the capillaries comprising an optical detection zone;a capillary mount, the plurality of capillaries being fixedly attached to the capillary mount;a light source producing a source beam of electromagnetic radiation configured to illuminate the plurality of capillaries within the optical detection zone;a base configured to receive the capillary mount, the base comprising a mirror configured to reflect the source beam and to direct the source beam through the plurality of capillaries. 31. Embodiment 30, further comprising:a optical detector;an emission optical system configured to receive emission signals from the plurality of capillaries and to direct the emission signals to the optical detector. 32. Embodiment 30, further comprising:a spectrometer comprising a dispersive optical element and a optical detector;an emission optical system configured to receive emission signals from the plurality of capillaries and to direct the emission signals to the optical detector. 33. Embodiment 33 includes a system for separating biological molecules, the system comprising:a plurality of capillaries configured to separate biological molecules in a sample, the capillaries comprising an optical detection zone defining a capillary plane;a light source producing a source beam of electromagnetic radiation configured to illuminate the plurality of capillaries within the optical detection zone;wherein each of the source beam has a linear polarization disposed along a polarization axis;wherein the polarization axis of the source beam within the optical detection zone is perpendicular to the capillary plane. 34. Embodiment 34 includes a method of separating biological molecules, comprising:providing a plurality of capillaries comprising an optical detection zone, the capillaries coupled to a capillary mount such that portions of the capillaries within the optical detection zone are fixedly located relative to one another;providing a plurality of optical fibers corresponding to the plurality of capillaries, each optical fiber comprising a receiving end configured to receive emissions from a respective capillary within the optical detection zone, the optical fibers being coupled to a fiber mount such that the receiving ends are fixedly located relative to one another;producing a first alignment signal from a optical detector by transferring emissions within the optical detection zone from at least one of the capillaries through a respective at least one of the optical fiber and to the optical detector;moving one or more times to one or more different locations at least one of: the capillary mount or the fiber mount;at each of the one or more locations, producing a respective alignment signal from the optical detector by transferring emissions within the optical detection zone from the at least one capillary through the at least one optical fiber to the optical detector;aligning the capillaries to the receiving ends of the plurality of capillaries based on the alignment signals. 35. Embodiment 34 or 35, further comprising:providing an emission optical system configured to direct emissions from each of the capillaries within the optical detection zone into the receiving end of the respective optical fiber; andmoving one or more times to one or more locations at least one of: the capillary mount or the fiber mount, or the emission optical system. 36. Any of embodiments 34-35, wherein the alignment signals are produced by Raman scattering emission from water molecules within a polymer solution contained in the capillaries. 37. Any of embodiments 34-36, wherein the alignment signals comprise emissions from a single one the capillaries. 38. Any of embodiments 34-37, wherein the alignment signals comprise an average of the emissions from more than one of the capillaries. 39. Any of embodiments 34-38, further comprising:loading one or more samples containing a fluorescent molecule into the plurality of capillaries;propagating the one or more samples through the capillaries by producing an electric potential across the capillaries;illuminating each capillary within the optical detection zone with a source beam of electromagnetic radiation to produce an emitted signal from the each of the capillaries;receiving the emitted signal from the at least one spot into at least one of the optical fibers. The above presents a description of the best mode contemplated of carrying out the present invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains to make and use this invention. This invention is, however, susceptible to modifications and alternate constructions from that discussed above which are fully equivalent. Consequently, it is not the intention to limit this invention to the particular embodiments disclosed. On the contrary, the intention is to cover modifications and alternate constructions coming within the spirit and scope of the invention as generally expressed by the following claims, which particularly point out and distinctly claim the subject matter of the invention. | 65,099 |
11860123 | DETAILED DESCRIPTION In various embodiments, the electronic properties of 2D solid-state nanopore materials can be used to provide a versatile and generally applicable biosensor technology. The biosensor technology can be facilitated via use of a combination of molecular dynamics, nanoscale device simulations, and statistical signal processing algorithms. As described herein, a case study is directed to the classification of three epigenetic biomarkers (methyl-CpG binding domain 1 (MBD-1), MeCP2, and γ-cyclodextrin) attached to double-stranded DNA to identify regions of hyper-methylations and/or hypo-methylations by utilizing a matched filter. Assessed is the sensing ability of the nanopore device to identify the biomarkers based on their characteristic electronic current signatures. Such a matched filter-based classifier can enable real-time identification of the biomarkers that can be implemented on chip. In various embodiments, this integration of a sensor with signal processing architectures can enable a multipurpose technology for early disease detection. As described herein, various embodiments can provide an integrated approach that combines electronic simulation based on device physics with statistical signal processing techniques to characterize the resolution limit of solid-state nanopore sensing and to facilitate provision of algorithms for epigenetic marker classification. As described herein, various embodiments can provide a sensor technology that is capable of detecting and mapping (across the genome by utilizing bulky biomarkers) one or more regions of hyper-methylations and/or one or more regions of hypo-methylations. In various examples, these biomarkers can be further classified using electronic sheet currents resulting from electrically active 2D nanopore membranes (because each marker produces a current signature unique to its structure and spatial charge distribution). Among bulky groups to label methylated cytosines along a double-stranded DNA, utilized (in various examples) can be either methyl-CpG binding domain (MBD-1) protein or methyl CpG binding protein 2 (MeCP2) to identify regions of hypermethylation. In humans, these two proteins bind to regions of hypermethylation along the DNA and are thought to repress transcription from methylated gene promoters (see Reference 17A). Abnormal levels of MBD protein and their polymorphisms have been associated with the overall risk of lung cancer (see References 18A, 19A). Furthermore, MeCP2 mutations are thought to be responsible for Rett syndrome, a severe neurodevelopmental disorder. The expression of MeCP2 in the brain is mostly in mature neurons and therefore can play a role in the identification of neurological diseases. Analogously, to identify regions of hypomethylation, considered herein (in various examples) is the detection of unmethylated CpGs marked by γ-cyclodextrin (GCD). This synthetic biomarker can be used to identify hypomethylated sequences, similar to the approach described by Gilboa et al. (see Reference 15A). FIG.1Aillustrates, according to an embodiment, a model setup100utilized to obtain the electronic current signatures for epigenetic marker proteins. The setup consists of a 2D material (see generally call out number102) connected between two electrodes, that is, the source104and drain106to enable the flow of current through the membrane102under an applied bias. In various examples, the membrane102can comprise graphene, MoS2, or other transition-metal dichalcogenide membranes. The detection sensitivity of the membrane102is controlled via a gate electrode separated from the membrane102by a high-κ dielectric (not shown). A circularly shaped nanopore108, chosen to have a diameter (for example) of 5 nm, allows the translocation of the biomolecule through the membrane102. This dimension of 5 nm is about the smallest pore diameter through which the DNA-marker complex can translocate without any hindrance. The whole setup is (in this example) immersed in water containing a 1M electrolyte of potassium chloride (KCl). A DNA strand, complexed with a marker protein either at the methylated cytosine site (for hyper-methylation) or at unmethylated adenine (for hypo-methylation), is translocated through the nanopore108using an applied bias across the cis and trans chambers (VTC). Modulation in current flowing through the membrane (sheet current) enables calibration of the local electrostatic potential distribution within the nanopore at a given time instant. For a statistical analysis according to an embodiment, needed are signal references that are obtained from frozen DNA current signatures, where the biomolecule is artificially translocated through the pore in the absence of all-atom molecular dynamics (MD) simulations, as previously performed by Girdhar et al. (see Reference 11A) The noisy test signal is obtained from a computation scheme involving MD simulations coupled to semiconductor device models (see Reference 14A). A detailed description of MD system setup, simulation methodology, and electronic transport calculations are discussed below. In the noise-free reference signal, the observed current from the DNA-marker complexes will arise solely from the charge distribution across the proteins, which are unique to the protein structures themselves. The set of these noise-free signals will comprise a set of unique reference current signatures for epigenetic markers. Once this reference set is built, it can be used to identify the type and/or number of proteins by a statistical signal processing algorithm such as a bank of matched filters, as outlined inFIG.1B. As seen in algorithm200ofFIG.1B, reference signals for each of the marker proteins are denoted as Ri(t), where i is the marker protein. An unknown noisy signal (denoted as U), obtained by MD simulations, where the marker protein is unknown, is input into the filter bank that classifies the marker-protein type depending on correlations between the unknown current signal and reference signals (various embodiments described herein utilize data (such as electrical characteristics associated with DNA moving through a nanopore) that are obtained via simulations; such embodiments can, of course, alternatively be implemented via use of actual measured data (such as actual measured electrical characteristics associated with DNA moving through a nanopore)). In this context, it is known that the optimal filter for detecting pulses in the presence of additive white Gaussian noise is the matched filter (see References 20A-22A) (a similar model describing ionic currents in the presence of wide-band Gaussian noise was used for translocation event detection (see Reference 23A)). It can further be shown that the probability of detecting a weak signal in the presence of noise is largest when the signal-to-noise ratio is also largest (see Reference 24A). Because the matched filter output is just the correlation with the reference signal, circuit implementations (see References 22A, 25A) in nanoscale computing technologies can be implemented. In the presence of additive colored noise, for example, 1/f noise rather than white noise, one would have a whitening prefilter followed by designing the matched filter for the whitened reference signal. A noise model of one embodiment considers only the low-frequency regime of detection, that is, <100 kHz. When the sampling rate is increased toward mid- and high-frequency regimes, different noise models need to be considered and the corresponding matched filter implementations will be modified. Mid-frequency will mainly consist of thermal noise, whereas the high-frequency regime will be dominated by capacitance effects. Power spectral densities for the different frequency regions are outlined by Parkin et al. (see Reference 26A). Using such a matched filter-based detection method, provided herein is a unified framework to detect, classify, and count multiple marker proteins along the DNA (this unified framework can also build upon the dictionary of reference signals). In various embodiments, the reference signatures for each of the biomarkers were calculated for noise-free trajectories on graphene and MoS2quantum point contact nanoribbons. Additionally, another important aspect of the respective current signature is the shape of the trace because the magnitude depends on the stochastic fluctuation of the complex and its spatial orientation within the pore. In this regard, it has been previously shown that during the translocation of a methylated DNA complexed with either one or two MBD-1 protein complexes, depending on the number of methylated sites, the conductance square deviation is drastically different due to the strong dependence of the sheet conductance on the angular position of the marker protein within the pore (see Reference 13A). Once the reference current signatures are obtained, a correlation of a current signal consisting of an unknown marker with each of the reference signals will identify the type of marker protein. Given a reference signal ri(t) and a signal of an unknown marker u(t), the cross-correlation is given as ρ(t)=∫−∞∞ri(τ)u(t+τ)dτ(1) To capture just the shape of a signal and compare signals due to different marker proteins across various orientations, normalized are the correlation signals to range between −1 and 1. Given a correlation trace ρ(ti), where tiis the sampled time instant, the normalized current trace is given by ρnorm(ti)=ρ(ti)-ρ(topen)max(ρ(t))-min(ρ(t))(2) Here max(ρ(t)) and min (ρ(t)) denote the maximum and minimum values of the calculated trace during the translocation period during which the signal was acquired. This normalization allows comparison of the different correlated signals irrespective of their angular position during translocation. Specifically, the criterion used to infer the type of marker protein from the correlated signal is the Q factor defined as Q=1BWcorr(3) where BWcorris the bandwidth of correlation between the test and a particular dictionary signal. The value of correlation chosen to estimate the bandwidth is a hyperparameter (i.e., chosen by the user, based on statistics of different calculations, usually ranging from 0.6 to 0.8, as indicative from various example calculations). In these sets of simulations, the BWcorrhas been chosen to be calculated at ρi=0.70, where ρidenotes the correlation between the test and reference signal of protein i (MBD, GCD, MeCP2). These hyperparameters are nonphysical quantities that can be fine-tuned by obtaining statistics of multiple translocations of the proteins with different configurations and initial conditions. Essentially, once the correlations between the test and dictionary signals have been computed, the protein whose current signature provides the maximum Q factor is inferred to be present along the DNA. FIG.2illustrates the utility of the Q factor as a metric to infer the presence or absence of a particular protein along a translocating DNA. In these simulations, the test signal is obtained from translocating a 30 bp long DNA complexed with a single MBD1 protein at a CpG site. This test signal is noisy because it is obtained from calculating the current trace along the MoS2membrane from the resulting trajectory of an all-atom MD simulation. As shown inFIG.2, the first (leftmost) panel indicates the noisy current signature of the translocating MBD1 protein, whereas the second panel (from the left) displays the normalized current signatures (top to bottom) from MBD-DNA, MeCP2-DNA, and GCD-DNA complexes, respectively. The noisy current signal (on the leftmost panel) was obtained from a previous work (see Reference 14A). As mentioned, the second panel (from the left) consists of the calculated current traces during a noise-free translocation of a DNA complexed with a MBD1, MeCP2, and GCD marker protein, respectively. The third panel (from the left) illustrates the matched filter operation between the noisy signal and the respective reference signals normalized to the interval [−1, 1]. The Q factor is calculated at ρ=0.7, resulting in a value of 0.023 for the MBD1 reference signal. For the MeCP2 and GCD correlations, the Q factor is 0 because the maximum value of the cross-correlation signal is <0.7. It is therefore evident (in this example) that the Q factor of the correlated outcome between noisy DNA-MBD and that of current signature of MBD is the highest, indicating the presence of the MBD protein along the DNA and the corresponding absence of the GCD or MeCP2markers. To illustrate the generality of this approach, further illustrated is example classification of the marker groups with unknown and reference signals calculated from translocations in graphene nanopores.FIGS.3A,3Billustrate the normalized correlations obtained between a noisy signal of a DNA complexed with hypo-methylated (GCD) and hyper-methylated (MeCP2) epigenetic biomarkers, respectively. InFIG.3A, the reference transverse current signatures (top to bottom) for MBD1, MeCP2, and GCD are shown in the second panel (second from the left), whereas the corresponding normalized correlations between the noisy unknown signal and the reference signals are shown in the third panel (from the left). One can clearly see that the correlation of the noisy signal corresponding to the GCD biomarker with the GCD reference signal gives the sharpest peak and greatest Q factor at ρ=0.7 of ˜0.04, whereas the Q factors corresponding to correlations with the MBD1 and MeCP2 markers are ˜0.017 and 0, respectively. Similarly (seeFIG.3B), a noisy translocation of a DNA-MeCP2 complex yields the maximum Q factor of ˜0.018 at ρ=0.7 when correlated with the reference signal of a MeCP2 biomarker. Therefore, these results indicate the versatility of the approach in developing a set of reference signals and classifying the unknown epigenetic biomarker using the matched filter. The approach described above illustrates the use of the matched filter algorithm to classify particular epigenetic markers. This matched filter can be applied in any setting, but it can be proven to be the optimal linear detector in the presence of additive noise. Additionally, the Q factor alone can be used as a metric to infer the hypothesis of whether the particular type of protein is present or absent. However, to simultaneously detect, infer, and count the type and number of proteins, illustrated herein is an algorithm that is capable of automatically deciding the validity of a marker and also counting the number of surrounding markers in its vicinity recursively. This unified algorithm according to an embodiment is shown inFIG.4. In this algorithm, utilized are two hyperparameters denoting the threshold value (QTH) of the Q factor, indicating the validity of the particular hypothesis, that is, to make the decision of whether the vicinity protein is present or absent, and a threshold correlation coefficient (ρTH), indicative of the presence of the second protein in the vicinity. This algorithm according to an embodiment has two inputs: the dictionary of signals (see the block inFIG.4labeled “Ideal cancer marker current signatures.”) from the various epigenetic cancer markers (in this example, MBD1, MeCP2, and GCD) and a test signal (see the block inFIG.4labeled “Test Signal”) from a DNA complex with an unknown marker protein. These two above-mentioned blocks inFIG.4feed into the block labeled “Compute correlation of test signal with each marker signature”. Also, as shown inFIG.4, prior to the computing of the correlation, a number of hyper parameters are initialized in this example as follows: num_protien=1; QTH=0.02; ρTH=0.7. Initially, the normalized correlation and corresponding Q factors between the test and dictionary current signatures are calculated (see the block inFIG.4labeled “Detect peak(ρfirst) and compute Q factor.”), the maximum of which indicates the identity of the marker protein. As seen in the block of FIG. labeled “Q>QTH”, a “NO” leads to a “STOP” (Test and Reference signals don't match) and a “YES” leads to “Detect second peak ρsecond” (Test and reference signals match; num_protien+=1). The second peak in the calculated correlations is also simultaneously monitored (see the block inFIG.4labeled “Detect second peak ρsecond”) to infer the presence of another marker in the vicinity. If the value of the second correlation peak (ρsecond) is greater than ρTH(“YES”), then the correlation is indicative of a second marker protein of the same type present in the vicinity (if “NO”, then “STOP: Total number of proteins is ‘num_protiens’”). Next considered are the normalized correlations between the frozen single DNA-marker complex and the noisy signals from the translocation of the DNA with a single complex (known as reference correlation) and multiple complexes (of the same type such as MBD/GCD). When these correlation peaks are aligned (see the block inFIG.4labeled “Align test and reference peaks”), a difference between them (see the block inFIG.4labeled “Test signal=test signal−reference signal”) will result in the presence of a peak (num_protien+=1). From this stage onward, the value of the second peak is recursively monitored, while subtracting the reference correlation at each iteration. The value of ρsecondis used to determine the presence or absence of a marker-protein in the vicinity depending on a hyperparameter threshold value (ρTH). This process of detecting and determining the value of the second peak from the subtracted signal can be performed recursively until the hypothesis is no longer valid. The algorithm illustrated inFIG.4can be utilized to detect and count multiple proteins complexed to a DNA, as shown inFIG.5. In this simulation scenario, considered are a 60 base pair (bp) long DNA strand that consists of two methylated CpG sites that are separated by 10 bp's. Each of these individual CpG sites are complexed by MBD1 proteins. The reason that the spacing of 10 bps is chosen in this example is due to the physical dimensions of the label protein MBD1 being 10 bp's wide, making 10 bp's the minimum possible distance that the two labeled sites can be present without mutual steric hindrance from the labels. This unconstrained DNA-MBD complex is translocated to obtain the noisy signal, as shown in the leftmost column ofFIG.5. This normalized current signature of two MBD1 proteins located 10 bp's apart was previously obtained (see Reference 14A). The plot of the correlation between the noisy two-protein signal and the reference signals for DNA-GCD or DNA-MeCP2 complexes does not display a discernible peak, indicating a lack of similarity between the dictionary signal entry and the measured noisy signal. On the contrary, correlating the noisy two-protein signal with the frozen single-protein signal (third panel from the left, first row) yields two peaks that could correspond to the similarity of features between each of the individual protein signatures and the single protein dictionary entry. The second peak of the correlated signal is greater than the threshold, indicating the possible presence of the second protein. The validity of the presence of the second protein can also be determined according to the recursive matched filter algorithm shown inFIG.4. To count the number of proteins, utilized are the correlations as shown inFIG.5, where the normalized values of the frozen single-protein current signatures (see the second column from the left) with the noisy two-protein signal (see the leftmost column) are plotted. As shown in the algorithm, monitored is the value of the second peak to determine the presence of the second protein. Because ρsecond>ρTHpeaks between the two normalized signals (correlated and reference correlation signal) are aligned and subtracted, the height of ρsecondcan be examined in the resulting curve (curve “C”), as shown inFIG.6. Because the second peak of the curve “C” is less than ρTH, it is concluded that only two proteins are present. This algorithm can be performed recursively for counting multiple proteins in the vicinity. In one specific example, the real-time detection and classification of epigenetic biomarkers from a time series of current data could involve a combination of event detection similar to the approaches utilized by nanopolish in Oxford Nanopore's MinION basecaller (see Reference 27A) applied to solid-state nanopores. It is believed that the nanopolish approach can currently count the quantity of marker proteins in the vicinity only if they are of the same type. In another embodiment, the algorithm can be generalized to detect different marker proteins within the resolution limit by checking if ρsecondcorrelates with the dictionary signals (this approach is sensitive to the choice of hyperparameters, which need to be chosen specifically depending on the dictionary signals obtained and can be fine-tuned depending on the set of protein signatures available). As described herein, considering signals from solid-state nanopore devices, an algorithm has been provided to determine the type of marker proteins and to simultaneously identify the possible epigenetic markers in the vicinity. This approach has been illustrated to detect and identify the presence of different biomarkers corresponding to hypo-methylation and/or hyper-methylation within a limited set of dictionary signals. While the approach has not yet been tested on experimental traces (for which there are certain technical difficulties related to fabrication and electric measurements in the nanopore devices (see Reference 28A)), various implementations using actual traces are, of course, within the scope of this disclosure. In other embodiments, the algorithm can be expanded to include an exhaustive set of current signatures for various epigenetic markers calculated from different sensing materials. This approach can also be generalized to incorporate signals from different noise models in the matched filter algorithm. Further, an embodiment of the algorithm using matched filter banks can be implemented in hardware (see References 24A, 25A) which can enable a DNA sensor chip consisting of a highly dense array of nanopores (see Reference 29A) with sensing and inference logic realized on the same wafer. Reference will now be made to a detailed discussion of an example molecular dynamics simulation protocol to obtain noisy current signatures followed by the electronic-transport model description for graphene and MoS2nanopore membranes to obtain the sheet currents of the biomarkers translocating through the nanopores. To obtain the noisy current signatures of the DNA-marker translocations, all-atom MD simulations were run using the latest version of NAMD (see Reference 1B). Each system is built, visualized, and analyzed using VMD software (see Reference 2B). The methyl-binding proteins MBD1 (PDB code: 1IG4)(see Reference 3B), MeCP2 (PDB code: 3C2I) (see Reference 4B) for hyper-methylation and γ-cyclodextrin(GCD) (see Reference 5B) for hypo-methylation are described using CHARMM22 force field with CMAP corrections (see Reference 6B). The DNA molecule structure is obtained using 3D-DART server (see Reference 7B) and described using CHARMM27 force field (see Reference 8B). The carbon atoms comprising graphene are treated as CA atoms and described using CHARMM27 (see Reference 8B). For the MoS2membrane, the atoms are spatially fixed requiring only the non-bonded interaction parameters which are obtained from Stewart et al. (see Reference 9B). The nanopore membrane along with the DNA-marker complex is immersed in a water box, where the water molecule is modeled as TIP3P model (see Reference 10B). Ions K+and Cl−are placed randomly in the water box to achieve a uniform concentration of 1 M. The particle-mesh Ewald method is used for long-range electrostatic interactions (see Reference 11B). Van der Waals energies are calculated using a 12 Å cutoff. Each system is minimized for 5000 steps. Once the minimization converged considerably, equilibration using the minimized parameters is performed for 0.6 ns as an NPT ensemble. During the equilibration, the system is maintained at a constant pressure of 1 atm using Langevin piston (see Reference 12B). After equilibration, an external field ε=V/Lzis applied along the +z direction to drive the DNA-protein complex through the nanopore, where V is the voltage bias and Lzis the length of the water box along the z direction. During the simulation, the atoms rearrange to produce the actual non-uniform potential across the 2D membrane (see Reference 13B). The actual potential is the sum of potential induced from the simulated charges plus the applied voltage. During the simulation, a constant temperature of 300 K was maintained using a Langevin thermostat. For some cases, to ensure successful translocation of the molecule within a reasonable amount of time, a small force of 1 kcal/mol/Å in the −z direction is applied to the backbone of the DNA molecule using tclForces. While these tclForces allow for successful translocations, an embodiment of the matched filter algorithm for epigenetic detection described herein is based on the transverse sheet current signatures. These signatures remain unaffected by the force used to drive the molecule through the nanopore, as the sheet current is influenced only by the potential induced by the DNA-marker complex around the pore and not the rate of translocation. Once the translocation of the DNA-protein complex is completed using MD simulations, a trajectory file of all the molecules of the system driven through the nanopore under the influence of electric field is obtained. The transverse currents are calculated simultaneously for each of the given trajectory of the DNA-marker complex. The first step of this methodology involves calculating the electrostatic potential using a non-linear Poisson Boltzmann formulation. The rationale and detailed description of the methods used are outlined by Girdhar et al. (see Reference 11A). Briefly, given a trajectory of the DNA-marker complex described at preset time intervals, the electric potential φ(r) is calculated for each position using the Poisson equation (see References 4A and 14B-15B): ∇·[ϵ(r)∇φ(r)]=−e[K+(r)−Cl−(r)]−ρDNA(r) (4) where ρDNA(r)is the charge due to the DNA-marker complex. The charges due to the solute ions, potassium (K+(r)) and chlorine (Cl−(r)) were described assuming Boltzmann equilibrium, namely, K+(r)=C0exp[-eφ(r)kBT],(5)Cl-(r)=C0exp[eφ(r)kBT].(6) Here, K+ and Cl−are the local ion concentrations and C0is the nominal concentration in the solution which has been set to 1 M. Equations (5) and (5) were solved iteratively until convergence. Using the electric potential φ(r) calculated by Poisson Boltzmann equations, the Green's function given by equation (7) is computed using the non-equilibrium Green's function formalism. G=[(E+iη)I-H-∑α∑α]-1(7) where Σα≡VαC†[E−Hα]−1VαCis the “self energy” of lead α and I is the identity matrix. Here, H, the tight-binding Hamiltonian is used to characterize the electronic transport through each carbon atom in the defined nanopore membrane lattice (see Reference 11A). Implemented is the third nearest neighbor and three orbital interactions in the Hamiltonian. Using G, obtained is the transmission coefficientT(E) between leads 1 and 2 given as: T(E)=−Tr[(Σ1−Σ1†)G(Σ2−ρ2†)G†]. (8) Finally, the transverse conductance at the source-drain bias VSDis calculated using equation (9). G=2eVSDh∫-∞∞T_(E)[f1(E)-f2(E)]dE(9) Here, fα(E)=f(E−μα) is the probability of an electron occupying a state at energy E in the lead α, and VSD=(μ1−μ2)/e is the bias across the conductor. All conductances across graphene membranes were obtained at a Fermi energy of 0.05 eV. In order to obtain the transverse sheet currents in MoS2quantum point contact nanopore membranes, the electronic transport through MoS2is formulated as a self-consistent model based on the semi-classical thermionic Poisson-Boltzmann technique using a two-valley model within the effective mass approximation. A detailed discussion of the model and its implications on the pore size and position are outlined by Sarathy et al. (see reference 1C). Briefly, the conductance calculated from the thermionic current flowing from the source to drain through a particular mode at a given voltage is described as Gn1,2=2e2h11+exp(En1,2K-EFLkBT)+2e2h11+exp(En1,2Q-EFLkBT).(10) Here EFLis chosen as the energy level reference set up on the left side of the membrane, n1,2represents the energy mode at the particular channel, and En1,2Kand En1,2Qare the energy levels at a particular channel and mode due to the effective masses at K and Q, respectively. The total conductance is the sum of the conductances through all modes in the channels. The linear response of the electronic conductance at a particular energy mode n1,2in the presence of the DNA external potential near the pore is given by 〈Gn1,2〉=∑i=[K,Q]dGn1,2dEn1,2i〈eφDNA〉n1,2.(11) The total variation (δG) in conductance with respect to conductance of the empty pore is the sum of individual variations due to each energy mode in each channel. The quantityeφDNA1,2represents the spatially averaged value of external potential due to the DNA (φDNA) across each channel of the MoS2membrane. All the conductances across MoS2membrane were obtained for a fixed Fermi energy value corresponding to a carrier concentration of 1012/cm2. Referring now toFIG.7, this depicts an illustrative embodiment of a method700in accordance with various aspects described herein. As seen in thisFIG.7, step702comprises electronic sensing of an electrical characteristic associated with a translocation of a test DNA through a pore, wherein a feature of the test DNA comprises an epigenetic biomarker, the test DNA itself forming a conformational superstructure, or any combination thereof, and wherein the electronic sensing is performed by an electric current along the membrane that results in a sensed electrical characteristic. Next, step704comprises comparing the sensed electrical characteristic with a plurality of reference electrical characteristics, wherein each of the plurality of reference electrical characteristics is associated with a respective one of a plurality of reference features, and wherein the comparing results in a comparison result. Next, step706comprises determining, based upon the comparison result, with which of the plurality of reference features the feature of the test DNA corresponds. In various examples, the electronic sensing can be performed by an electric current along the membrane, the electronic sensing can be performed in accordance with an electric current along the membrane, or any combination thereof. While for purposes of simplicity of explanation, the respective processes are shown and described as a series of blocks inFIG.7, it is to be understood and appreciated that the claimed subject matter is not limited by the order of the blocks, as some blocks may occur in different orders and/or concurrently with other blocks from what is depicted and described herein. Moreover, not all illustrated blocks may be required to implement the methods described herein. Referring now toFIG.8, this depicts an illustrative embodiment of a method800in accordance with various aspects described herein. As seen in thisFIG.8, step802comprises electronic sensing, by a processing system including a processor, of an electrical characteristic associated with a translocation of a test DNA through a pore of a membrane, the electronic sensing being performed by an electric current along the membrane that results in a sensed electrical characteristic. Next, step804comprises obtaining, by the processing system, a first reference electrical characteristic associated with a first DNA feature. Next, step806comprises obtaining, by the processing system, a second reference electrical characteristic associated with a second DNA feature. Next, step808comprises matching the sensed electrical characteristic to one of the first reference electrical characteristic and the second reference electrical characteristic, resulting in a match. Next, step810comprises identifying, based upon the match, an epigenetic biomarker associated with the test DNA, a conformational superstructure of the test DNA, or any combination thereof. In various examples, the electronic sensing can be performed by an electric current along the membrane, the electronic sensing can be performed in accordance with an electric current along the membrane, or any combination thereof. While for purposes of simplicity of explanation, the respective processes are shown and described as a series of blocks inFIG.8, it is to be understood and appreciated that the claimed subject matter is not limited by the order of the blocks, as some blocks may occur in different orders and/or concurrently with other blocks from what is depicted and described herein. Moreover, not all illustrated blocks may be required to implement the methods described herein. Referring now toFIG.9, this depicts an illustrative embodiment of a method900in accordance with various aspects described herein. As seen in thisFIG.9, step902comprises obtaining an electrical characteristic indicative of movement of a test DNA strand through a pore of a two-dimensional thin membrane, the electrical characteristic being obtained via electronic sensing performed by an electric current along the two-dimensional thin membrane that results in a sensed electrical characteristic. Next, step904comprises comparing the sensed electrical characteristic with a plurality of reference electrical characteristics associated with a respective plurality of known DNA strands, resulting in a comparison. Next, step906comprises matching, based upon the comparison, the test DNA strand to one of the known DNA strands, resulting in a matched DNA strand. Next, step908comprises identifying, based upon the matching, an epigenetic biomarker associated with the test DNA strand, a conformational superstructure of the test DNA strand, or any combination thereof. In various examples, the electronic sensing can be performed by an electric current along the membrane, the electronic sensing can be performed in accordance with an electric current along the membrane, or any combination thereof. While for purposes of simplicity of explanation, the respective processes are shown and described as a series of blocks inFIG.9, it is to be understood and appreciated that the claimed subject matter is not limited by the order of the blocks, as some blocks may occur in different orders and/or concurrently with other blocks from what is depicted and described herein. Moreover, not all illustrated blocks may be required to implement the methods described herein. As described herein, various embodiments provide machine learning for enhanced nanopore bio-detection. As described herein, DNA methylation is one of the most common epigenetic modifications in the eukaryotic genome, occurring primarily through the addition of methyl groups at the 5th-carbon of a cytosine ring. Methylation plays a crucial role in the expression of genes in mammalian cells and therefore is related to cell development, aging and progress of diseases such as cancer. In vertebrates, methylation typically occurs in DNA sequences with a relatively high content of CpG dinucleotides (namely, 5′-CG-3′), known as CpG islands. In cancerous cells, many of the CpG islands are observed to be methylated, while normal somatic cells are free of methylation. For this reason, DNA methylation holds the potential to serve as an effective biomarker that can be used in risk assessment and early diagnosis of methylation-relevant diseases such as cancer. It is therefore of crucial importance to detect and precisely map methylation patterns in the human epigenome. As described herein, various embodiments can provide a low-cost, quick and reliable method to detect methylation on DNA. In various embodiments, the use of very thin membranes containing a pore with nanoscale feature sizes, through which DNA molecules are threaded to detect gene modifications can offer many advantages over conventional bio-chemical processes. Various embodiments can provide for the integration of solid state multi-layer nanopore membranes within a multi-functional electronic device to increase its detection sensitivity. Among the advantages of the solid state nanopore is its compatibility with semiconductor nano-electronics that favors the fabrication of compact devices, and opens the door to personalized medicine with revolutionary consequences for public health. Various embodiments can provide a methodology that combines electronic simulation based on device physics with advanced statistical signal processing to characterize the information-theoretic resolution limit of solid state nanopore sensing. Various embodiments can facilitate algorithms that approach these fundamental limits. These algorithms provide guidelines to improve the signal-to-noise ratio of the biomolecule-detecting membranes. As a study-case scenario, described herein is an assessment and achievement of the resolution limits for a nanopore device to detect methylated binding domain proteins on DNA. Identification of these proteins is critical, as their interactions with DNA have roles in breast, lung, and other kinds of cancers. Various embodiments can provide for low-cost and fast DNA sequencing methods that can be used to facilitate personalized medicine, given its role in sequence-dependent diagnosis and treatment of diseases. Certain conventional technologies for methylation detection typically utilize large and expensive machines, relying on a dated method involving bi-sulphite genome sequencing. Nanopores, on the other hand, have great potential as next-generation sequencing devices, disrupting currently widespread technologies by offering improved accuracy at reduced sizes and costs for sequencing DNA along with multiple simultaneous modalities of electronically sensing DNA. Various embodiments can use solid state nanopores for epigenetic applications. Various embodiments can facilitate a reduction in the cost of personalized medicine, enabling people to receive treatment suited to their individual DNA sequence. Various embodiments can improve upon the typical low signal-to-noise ratio some solid state nanopores provide, thus easing detection of the DNA sequence or epigenetic DNA features. In various examples, robust identification algorithms are designed for solid state nanopores. In order to detect and map epigenetic features in DNA such as methylation patterns, various embodiments can utilize transverse sheet current in solid state nanopores with molybdenum di-sulphide (MoS2) as the sensing membrane (see, e.g.,FIG.10). As demonstrated previously, transverse current offers a higher detection resolution than traditional sensing of DNA via ionic currents (see References 14A,1C). In various examples, a methodology of improving the signal-to-noise ratio is not limited to just transverse sheet current measurements in solid state nanopores but can also (or alternatively) be incorporated in the nanopore sequencing protocols which utilize ionic current signatures to potentially identify bases and methylation patterns. As described herein, as an example, is detection of the presence of a methyl-CpG binding domain protein attached to a CpG site in a ds-DNA. While a wide range of MBD proteins can be utilized for this purpose, employed (in this example) is the MBD1 protein as a biomarker for the methylation site along the DNA. Polymorphisms in the MBD1 protein have been shown to be significantly associated with lung cancer risk (see Reference 16A). Some prior work (various features of which can be utilized by one or more embodiments) relates to a multi-functional electronic device made of solid state multi-layer nanopore membranes to increase bio-detection sensitivity (see References 2C, 3C). Such devices can offer advantages over certain conventional bio-chemical techniques (the advantages can include, for instance, compatibility with semiconductor nano-electronics that favors compact devices, and enables personalized medicine with revolutionary consequences for public health). However, many conventional efforts to detect, identify and map the DNA methylation patterns using solid state nanopores have been unsuccessful because the conformational stochastic fluctuations of DNA in electrolytic solution inside the pore introduces significant noise added to the measured signal. Further, some prior work (various features of which can be utilized by one or more embodiments) proposed a scenario to improve the signal-to-noise ratio in sequencing DNA with nanopore technology by stretching the bio-molecule (see Reference 12A). Various embodiments described herein provide a methodology that will impose limit in the detection resolution of epigenetic factors. Various embodiments described herein can comprise algorithms that provide guidelines to improve the signal-to-noise ratio of the bio-detecting membranes. A drawback of certain conventional mechanisms in sensing biomolecules using nanopores has been the low signal-to-noise ratio of the measured current signals. The main source of the noise is due to fluctuations of the DNA as it translocates through the pore. Various embodiments described herein can provide for building a dictionary of “noise-free” current signatures for various proteins attached to methylated sites along the DNA. The “noise-free” current signatures are calculated (in various examples) via simulations where ideal and frozen biomolecules are translocated through the nanopore and the resulting current is calculated at each time instant. This process can be repeated for each of the MBD proteins, each of which is indicative of a different cancer (see Reference 16A). Once the dictionary of signals is built, the resulting noisy signal from (for example) a methylated DNA-MBD complex translocation can be correlated with each of the dictionary entries. The correlation yields a prominent peak when the noisy signal is correlated with the ideal current signature of the same protein. In all other cases, the correlation does not yield a significant peak. The height of the peak in the correlated signal can be used to determine the validity of a particular hypothesis test (e.g., using the likelihood ratio test). A sample test case of this procedure is shown inFIG.10where the noisy current trace during a MBD-DNA complex translocation with a single MBD protein is correlated with each of the dictionary entries corresponding to the ideal “non-noisy” current signatures of pristine un-methylated DNA and a MBD-DNA respectively. The hypothesis corresponding to the presence of a single MBD1 protein is confirmed via the presence of the correlated signal peak. An important aspect to note here is the uniform resampling of each signal to correspond to a given fixed length. In various examples, generalization of the disclosed techniques can incorporate different noise models and modification to include ionic current signatures. The technique described above in connection withFIG.10utilizes a Gaussian noise model with a simple matched filter implementation. In other embodiments, this can be generalized to incorporate many other noise models such as jitter noise, 1/f noise, and others, whether non-parametric descriptions or parametric noise models. Such other embodiments can also be generalized to incorporate factors of inter-symbol interference, as well as the possibility of memory in the epigenetic sequence itself. The likelihood ratio test can be modified for each of the different noise models used. Such other embodiments can also be generalized to be incorporated in the context of ionic current signatures. In other examples, the ideal “non-noisy” signals (e.g., for each of the methyl-CpG binding domain proteins) can be obtained by performing experiments many times over to get the statistically averaged current signature and noise characteristics. In other examples, the same procedure can be employed to identify the type of methyl-CpG protein and number of proteins as well. As described herein, various embodiments relate to classification of epigenetic biomarkers via use of atomically-thin nanopores. In one example, an algorithm is capable of calculating the electrical signatures (e.g., current signatures) of proteins and comparing them to other signatures (e.g., current signatures) in order to identify what protein is present in/on a strand of DNA. In one example, the integration of a sensor with signal processing architectures as described herein could facilitate a multipurpose technology for early disease detection. As described herein, various embodiments can provide for using a matched filter algorithm, whereby particular epigenetic markers are classified. In one example, a sensor technology is provided that is capable of detecting and mapping region(s) of hyper-methylation(s) across the genome by utilizing genetic marker(s). In one example, a sensor technology is provided that is capable of detecting and mapping regions of hypo-methylation(s) across the genome by utilizing genetic marker(s). In one example, mechanisms are provided to not only detect presence of one or more proteins, but to identify the one or more proteins (e.g., determine the type of each of the one or more proteins). In one example, the disclosed mechanisms can provide for early disease diagnosis. As described herein, various embodiments (e.g., algorithms, processing applications) relate to DNA sequencing. Unlike certain conventional nanopore sequencers (which are typically unable to identify epigenetic markers attached to methylated sites owing to size discrepancy between DNA-marker and the nanopore), various embodiments described herein are able to identify such epigenetic markers attached to methylated sites. In various embodiments, the resolution of detection of labeled sites via electronic current is limited by sizes of the labeled proteins rather than the electronic measurement quality itself. In various embodiments, nanopore sequencing can be provided with lower error rates than certain conventional technologies. As described herein, various embodiments can use unique known electronic signals of proteins, in order to sequence DNA in real time (e.g., via comparison to other signatures to determine what proteins are present at methylated sites). In one example, an unknown signature can be placed through a filter, and once correlations between test and dictionary signals have been computed, the protein whose current signature provides the maximum Q factor is inferred to be present. As described herein, an algorithm is provided that is capable of identifying the particular type of protein that exists in/on the DNA strand. As described herein, an algorithm is provided for identifying protein(s) at a methylation site in order to facilitate early disease detection. As described herein are mechanisms for the classification of epigenetic biomarkers via use of atomically-thin nanopores. In one example, provided is a low cost, fast, reliable method to access, and decode the human genome and/or epigenomes. As described herein, various embodiments can utilize solid state nanopores. In one specific example, use of a 2D solid state nanopore can offer high detection resolution. Various embodiments can improve upon certain conventional techniques to detect, identify and map DNA methylation patterns using sold state nanopores. Such conventional techniques have typically been unsuccessful because of significant noise introduced in the measured signal (such as due to the conformational stochastic fluctuations of DNA inside a pore). Various embodiments can provide improvement via use of an integrated approach that combines electronic simulations based on device physics with statistical signal processing techniques to characterize the resolution limit of solid state nanopore sensing and facilitate application of algorithms for epigenetic marker classification. Various embodiments can provide a sensor technology that is capable of detecting and mapping regions of hyper-methylation(s) and/or hypo-methylation(s) across the genome by utilizing one or more bulky biomarkers. In various embodiments, bulky groups can be used to label methylated cytosines along dsDNA (various examples can use methyl-CpG binding domain (MGD-1) protein or methyl CpG binding protein 2 (MeCP2) to identify regions of hyper-methylation. Another example can identify regions (such as sequences) of hypo-methylation, via detection of unmethylated CpGs marked by γ-cyclodextrin (GCD). In various embodiments, the detection setup can utilize a 2D material (e.g., graphene, MoS2, or other transition metal dichalcogenide membrane) connected between a source electrode and a drain electrode, enabling electron flow through the membrane. In one example, the detection sensitivity of the membrane can be controlled via a gate electrode separated from the membrane by high k dielectric. In another example, the nanopore can be circularly shaped (e.g., 5 nm). In one example, this can be the smallest size DNA marker complex that can be translocated without hindrance. In one example, the setup (e.g., the membrane with the nanopore) can be immersed in water with an electrolyte (e.g., KCl). In various embodiments, reference signals can be utilized. In one example, the reference signals can be obtained from frozen DNA current signatures. In this example, for each noise-free reference signal, the observed current from the DNA marker complexes will arise solely from the charge distribution across the proteins which are unique to the protein structures themselves. The set of noise-free reference signals can comprise a set of unique reference current signatures for epigenetic markers. In one example, once the reference set is built, it can be used to identify the type and/or number of proteins by a statistical signal processing algorithm (e.g. a bank of matched filters). As described herein, various embodiments can provide a system for detecting methylation of DNA. One process according to an embodiment can be as follows: (a) contact a nucleic acid sequence with a bulky group label of methylated cytosines (e.g., methyl-CpG binding domain (MGD-1), GCD, methyl CpG binding protein 2 (MeCP2); (b) run the DNA through a solid state nanopore placed in an aqueous solution comprising an electrolyte, the solid state nanopore comprising: a 2D membrane (e.g., graphene, MoS2, other transition metal dichalcogenide) having at least one circular pore (˜5 nm) and a source electrode and a drain electrode for applying current across the membrane; (c) measure the signals as the DNA moves through the pore; (d) compare the measured signals to at least one noise-free reference sample comprising a known signature for epigenetic markers. As described herein, various embodiments can provide for detecting and/or classifying and/or counting one or more proteins attached to DNA. In one example, multiple proteins can be detected and/or classified and/or counted simultaneously. As described herein, various embodiments can provide machine learning for enhanced nanopore bio-detection. In one example, the machine learning can be used for detecting and/or classifying and/or counting one or more proteins. As described herein, various embodiments can provide for detecting proteins without a priori looking for that specific protein. As described herein, various embodiments can be realized with hardware circuitry to carry out operations (e.g., such as operations to perform feature matching between the current signal of the test DNA strand with the corresponding feature(s) of the matched DNA strand). Such hardware circuitry can enable faster detection and identification of the test DNA. In various examples, such circuitry can be realized by a specific arrangement of silicon transistors on chip and/or by using reconfigurable circuitry such as Field Programmable Gate Arrays. Referring now toFIG.11, this depicts examples of various DNA superstructures that can be detected according to various embodiments. More particularly, thisFIG.11depicts a schematic illustration of three kinds of DNA superstructures that can be detected (according to algorithms and/or detectors of various embodiments) when the superstructures translocate through a nanopore1102in a two-dimensional membrane1104. The DNA1106(shown in the pore) is called a “hairpin”. The DNA1108(shown on the upper left) is called a “DNA loop”. The DNA1110(shown on the upper right) is called a “twisted loop” (with a binding complex in the middle). From the foregoing descriptions, it would be evident to an artisan with ordinary skill in the art that the aforementioned embodiments can be modified, reduced, or enhanced without departing from the scope and spirit of the claims described below. For example, any desired number and/or type of reference signatures can be utilized. Other suitable modifications can be applied to the subject disclosure. Accordingly, the reader is directed to the claims for a fuller understanding of the breadth and scope of the subject disclosure. FIG.12depicts an example diagrammatic representation of a machine in the form of a computer system1200within which a set of instructions, when executed, can cause the machine to perform any one or more of the methods discussed above. In some embodiments, the machine may be connected (e.g., using a network) to other machines. In a networked deployment, the machine may operate in the capacity of a server or a client user machine in server-client user network environment, or as a peer machine in a peer-to-peer (or distributed) network environment. The machine may comprise a server computer, a client user computer, a personal computer (PC), a tablet PC, a smart phone, a laptop computer, a desktop computer, a control system, a network router, switch or bridge, or any machine capable of executing a set of instructions (sequential or otherwise) that specify actions to be taken by that machine. It will be understood that a communication device of the subject disclosure includes broadly any electronic device that provides voice, video or data communication. Further, while a single machine is illustrated, the term “machine” shall also be taken to include any collection of machines that individually or jointly execute a set (or multiple sets) of instructions to perform any one or more of the methods discussed herein. The computer system1200may include a processor1202(e.g., a central processing unit (CPU), a graphics processing unit (GPU, or both), a main memory1204and a static memory1206, which communicate with each other via a bus1208. The computer system1200may further include a video display unit1210(e.g., a liquid crystal display (LCD), a flat panel, or a solid state display. The computer system1200may include an input device1212(e.g., a keyboard), a cursor control device1214(e.g., a mouse), a disk drive unit1216, a signal generation device1218(e.g., a speaker or remote control) and a network interface device1220. The disk drive unit1216may include a tangible computer-readable storage medium1222on which is stored one or more sets of instructions (e.g., software1224) embodying any one or more of the methods or functions described herein, including those methods illustrated above. The instructions1224may also reside, completely or at least partially, within the main memory1204, the static memory1206, and/or within the processor1202during execution thereof by the computer system1200. The main memory1204and the processor1202also may constitute tangible computer-readable storage media. Dedicated hardware implementations including, but not limited to, application specific integrated circuits, programmable logic arrays and other hardware devices can likewise be constructed to implement the methods described herein. Applications that may include the apparatus and systems of various embodiments broadly include a variety of electronic and computer systems. Some embodiments implement functions in two or more specific interconnected hardware modules or devices with related control and data signals communicated between and through the modules, or as portions of an application-specific integrated circuit. Thus, the example system is applicable to software, firmware, and hardware implementations. In accordance with various embodiments of the subject disclosure, the methods described herein are intended for operation as software programs running on a computer processor. Furthermore, software implementations can include, but not limited to, distributed processing or component/object distributed processing, parallel processing, or virtual machine processing can also be constructed to implement the methods described herein. While the tangible computer-readable storage medium1222is shown in an example embodiment to be a single medium, the term “tangible computer-readable storage medium” should be taken to include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) that store the one or more sets of instructions. The term “tangible computer-readable storage medium” shall also be taken to include any non-transitory medium that is capable of storing or encoding a set of instructions for execution by the machine and that cause the machine to perform any one or more of the methods of the subject disclosure. The term “tangible computer-readable storage medium” shall accordingly be taken to include, but not be limited to: solid-state memories such as a memory card or other package that houses one or more read-only (non-volatile) memories, random access memories, or other re-writable (volatile) memories, a magneto-optical or optical medium such as a disk or tape, or other tangible media which can be used to store information. Accordingly, the disclosure is considered to include any one or more of a tangible computer-readable storage medium, as listed herein and including art-recognized equivalents and successor media, in which the software implementations herein are stored. Although the present specification describes components and functions implemented in the embodiments with reference to particular standards and protocols, the disclosure is not limited to such standards and protocols. Each of the standards for Internet and other packet switched network transmission (e.g., TCP/IP, UDP/IP, HTML, HTTP) represent examples of the state of the art. Such standards are from time-to-time superseded by faster or more efficient equivalents having essentially the same functions. Wireless standards for device detection (e.g., RFID), short-range communications (e.g., Bluetooth, WiFi, Zigbee), and long-range communications (e.g., WiMAX, GSM, CDMA) are contemplated for use by computer system1200. The illustrations of embodiments described herein are intended to provide a general understanding of the structure of various embodiments, and they are not intended to serve as a complete description of all the elements and features of apparatus and systems that might make use of the structures described herein. Many other embodiments will be apparent to those of skill in the art upon reviewing the above description. Other embodiments may be utilized and derived therefrom, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Figures are also merely representational and may not be drawn to scale. Certain proportions thereof may be exaggerated, while others may be minimized. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense. Although specific embodiments have been illustrated and described herein, it should be appreciated that any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description. The Abstract of the Disclosure is provided with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in a single embodiment for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter. REFERENCES (1A) Bayley, H. Nanotechnology: holes with an edge. Nature 2010, 467, 164-165.(2A) Ehrlich, M. DNA methylation in cancer: too much, but also too little. 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11860124 | DETAILED DESCRIPTION FIG.1schematically illustrates a gas turbine engine20. The gas turbine engine20is disclosed herein as a two-spool turbofan that generally incorporates a fan section22, a compressor section24, a combustor section26, and a turbine section28. The fan section22drives air along a bypass flowpath while the compressor section24drives air along a core flowpath for compression and communication into the combustor section26, then expansion through the turbine section28. Although depicted as a turbofan in the disclosed non-limiting embodiment, it should be appreciated that the concepts described herein may be applied to other engine architectures such as turbojets, turboshafts, and three-spool (plus fan) turbofans. The engine20generally includes a low spool30and a high spool32mounted for rotation about an engine central longitudinal axis A relative to an engine static structure36via several bearings38. The low spool30generally includes an inner shaft40that interconnects a fan42, a low pressure compressor (“LPC”)44and a low pressure turbine (“LPT”)46. The inner shaft40drives the fan42directly or through a geared architecture48that drives the fan42at a lower speed than the low spool30. An exemplary reduction transmission is an epicyclic transmission, such as a planetary or star gear system. The high spool32includes an outer shaft50that interconnects a high pressure compressor (“HPC”)52and high pressure turbine (“HPT”)54. A combustor56is arranged between the high pressure compressor52and the high pressure turbine54. The inner shaft40and the outer shaft50are concentric and rotate about the engine central longitudinal axis A which is collinear with their longitudinal axes. Core airflow is compressed by the LPC44, then the HPC52, mixed with the fuel and burned in the combustor56, then expanded over the HPT54and the LPT46which rotationally drive the respective high spool32and the low spool30in response to the expansion. The shafts40,50are supported at a plurality of points by bearings38within the static structure36. With reference toFIG.2, the geared architecture48includes a sun gear60driven by a sun gear input shaft62from the low spool30, a ring gear64connected to a ring gear output shaft66to drive the fan42and a set of intermediate gears68in meshing engagement with the sun gear60and ring gear64. Each intermediate gear68is mounted about a journal pin70which are each respectively supported by a carrier74. The input shaft62and the output shaft66counter-rotate as the sun gear60and the ring gear64are rotatable about the engine central longitudinal axis A. The carrier74is grounded and non-rotatable even though the individual intermediate gears68are each rotatable about their respective axes80. An oil recovery gutter76is located around the ring gear64. The oil recovery gutter76may be radially arranged with respect to the engine central longitudinal axis A. A replenishable film of oil, not shown, is supplied to an annular space72between each intermediate gear68and the respective journal pin70. One example applicable oil meets U.S. Military Specification MIL-PRF-23699, for example, Mobil Jet Oil II manufactured by ExxonMobil Aviation, United States. Oil is supplied through the carrier74and into each journal pin70to lubricate and cool the gears60,64,68of the geared architecture48. Once communicated through the geared architecture48the oil is radially expelled through the oil recovery gutter76in the ring gear64by various paths such as oil passage78. With reference toFIG.3, an oil system80is schematically illustrated in block diagram form for the geared architecture48as well as other components which receive oil. It should be appreciated that the oil system80is but a schematic illustration and is simplified in comparison to an actual oil system. The oil system80generally includes an oil tank82, a supply pump84, an oil debris monitoring sensor86, an oil filter88, a starter90, a fuel pump92, the geared and bearing architecture48, a scavenge pump94, and an oil debris monitoring sensor96at an alternative location. The oil debris monitoring sensor86,96could be a single sensor or a set of sensors placed in branched oil paths. The oil flow to the geared and bearing architecture48may be considered an oil supply path100, and the oil flow from the geared and bearing architecture48can be considered an oil return path102. Multiple of chip collectors104may be located in the supply path100and the return path102to capture ferrous debris. The sensors86,96may utilize two field coils, excited by high frequency alternating current, to cause equal and opposing magnetic fields (M-field). The ferrous particle strength of the M-field created by one field coil after another, causes the processed signal to be a period of a sine wave. The nonferrous particle weakens the M-field created by one field coil after another, causing the similar sine wave but in opposing polarity. Generally, the signal magnitude is proportional to the size of particle and the signal width is inversely proportional to the particle speed. With Reference toFIG.4, a debris detection system110generally includes a controller120in communication with the sensors86,96. The sensors86,96may be in-line oil debris monitor sensors. The debris detection system110protects against unexpected phase angle changes which may affect individual oil debris monitors caused by replacement or redesign of other components in the system, such as a signal wire harness, that can drastically influence the phase angle. The controller120generally includes a control module122that executes logic124(FIG.4) to actively calculate and monitor the oil debris liberated in the oil system with regards to particle detection, mechanical system fault alert, and sensing system health. The functions of the logic124are disclosed in terms of functional block diagrams, and it should be appreciated that these functions may be enacted in either dedicated hardware circuitry or programmed software routines capable of execution in a microprocessor-based electronics control embodiment. In one example, the control module122may be a portion of a flight control computer, a portion of a Full Authority Digital Engine Control (FADEC), a stand-alone unit, or other system. The control module122typically includes a processor122A, a memory122B, and an interface122C. The processor122A may be any type of known microprocessor having desired performance characteristics. The memory122B may be any computer readable medium which stores data and control algorithms such as the logic124as described herein. The interface122C facilitates communication with other components such as the sensors86,96, as well as remote systems such as a ground station, Health and Usage Monitoring Systems (HUMS), or other system. The oil debris monitor phase angle is used to classify detected particle types (ferrous/nonferrous) through a mathematical transformation. The phase angle is calibrated by pulling a particle of known type and size through the sensor and using the ratio of I and Q channel amplitude and trigonometric relationships to calculate an optimum (for classification) phase angle. The I channel is the In-phase, or real component and the Q channel is the Quadrature (90° shift of real component). As will be further described below, this principle is applied to background noise in the debris detection system110by calculating the slope of the relationship between noise peaks of the oil debris monitor I and Q data channels. The background noise can be anything in the signal that is not a particle but originated from the sensor, for example, engine vibration induced noises. With reference toFIG.5, the logic124of the debris detection system110initially includes receipt of raw oil debris monitor data from either the sensors86,96into the controller for signal conversion from analog to digital (202). The raw data is stored in a controller buffer (204). The buffer for the controller is continually filled with raw data that flows as a constant stream such that a running on-board algorithm may be performed. The phase angle of the signal (206) may be calculated from the noise using the raw oil debris monitor data in the controller buffer. The phase angle may then be used for a system health assessment (208) and may be transmitted (210) for further processing in the controller as well as transmitted with system health data for off-board health monitoring (212). The mechanical system health assessment may include, for example, particle count, particle type classification, size and mass estimates, sensing system availability, debris count rates, and other metrics. The A/D converted raw oil debris monitor signals are filtered and phase angle adjusted (214) within the controller, then the particle detection algorithm executes (216). Typically, the particle signal will distribute into both I and Q channels due to phase angle misalignment between the drive signal and mixer signal as caused by system impedance in the driving and sensing circuitry. The phase angle adjustment realigns the particle signal distribution such that the ferrous particle signal is maximized in the ferrous channel and the nonferrous particle signal is maximized in then nonferrous channel. The particle classification and size data from the particle detection algorithm is then transmitted (218) for off-board health monitoring. The raw data from the conversion of analog to digital (202) is communicated to a module that calculates the particle detection threshold (220;FIG.6) based on the signal noise level to adjust the detection threshold based on the background noise in the raw ODM channel data. With reference also toFIG.6, the particle detection threshold (220) is determined from a signal root mean square (RMS) calculation (220B) using the raw I and Q data in a fixed time window. The baseline threshold from the ferrous channel and the nonferrous channel (220C) is adapted to C*RMSIQ (220D), where C is a predefined constant determined by analyzing system data to determine how the particle detection threshold should be adjusted to combat the background noises. In one example, a range for C is 2.5 to 4. The detection threshold can be further adjusted to avoid short duration, high amplitude signal anomalies. In a given system where analysis is occurring on short duration block of data on the order of less than a second, one can expect a maximum particle pass frequency during that time window (220E). For example, if processing is done on 1 second blocks, and the maximum particle rate is 2 Hz, a maximum of 4 peaks are expected in a block. If more than 4 peaks exceeding the RMS calculated detection threshold, the threshold should be adjusted to the 5th peak. This will reduce the risk of rejecting large particles, and prevent reduced availability due to signal anomalies. With reference also toFIG.7, the rate limit, or maximum number of particles allowed over a given window of time without flagging an alarm, are reduced given a high noise environment. This is necessary, because if the detection threshold of the system is increased, some portion of the failure mode is no longer visible to the sensor. An increased detection threshold alone risks missed detection of progressing failures. Making the rate limit as a function of the detection threshold, or more specifically, reducing the rate limit as the detection threshold increases, mitigates this risk. The rate limit reduction is performed by establishing a relationship between detection threshold (220F) and rate limit (222B) assuming that the calculated detection threshold was a constant in the time window where the rate limit is computed. This relation is also determined using knowledge of expected mechanical failure modes. In a dynamic system the detection threshold, however, will not be constant. Every block of data will have a unique detection threshold, and thus a unique detection threshold influence factor (rate limit for the given detection threshold multiplied by the ratio of time in a given block over the total evaluation period). These unique threshold influence coefficients are summed (222B-D) such that every block of data has an equal influence on the net rate limit. Given the expected particle distributions for a failure mode, there will be a minimum acceptable calculated rate limit (222E) to reliably detect failure modes without creating a nuisance. If the calculated rate limit falls below the minimum acceptable limit, an alert will be issued to indicate maintenance of the sensing system is required and one or more components in the sensing systems86,96needs to be replaced. With reference also toFIG.8, if the noise in the debris detection system110is very high, detection capability is reduced. To prevent the debris detection system110from spending too much time at the elevated noise levels, a critical detection threshold should be established. The amount of time spent above this threshold will be accumulated (224A). Detectability is then calculated as the ratio of time spent with a calculated threshold below the critical detection threshold. The time above the critical threshold (224B) can also be weighted (224C) so that a significant threshold exceedance impacts the detectability more than a minor exceedance (i.e., if the limit were 120 millivolts, the calculation can be weighted such that 1 second with a threshold at 250 millivolts is equivalent to 10 seconds with a threshold at 125 millivolts, since the former condition is much more severe). If the calculated detectability (224D) falls below the minimum acceptable limit (224E), an alert will be issued to drive maintenance of the sensing system. The updated detection threshold is also communicated to the particle detection algorithm ((220F);216), a rate limit adjustment algorithm ((222F);222;FIG.7), detectability algorithm ((224E)224;FIG.8), particle estimation algorithm (400;FIG.13), and mass accumulation algorithm (500;FIG.15,FIG.16). With continued reference toFIG.5, the controller monitors the debris detection system110background noise levels in real-time and dynamically calculates a particle detection threshold such that an adequate signal to noise ratio is enforced. This has the effect of ensuring relatively large particles are counted rather than missed entirely due to a detection threshold falling below background noise levels. In other words, the particle detection threshold based on the signal noise level (220) shifts the focus from debris (particle) detection to event (fault/failure) detection. The system monitors the debris detection system110background noise levels in real-time and dynamically calculates a particle detection threshold. System availability (226) is calculated over time using a signal rejection time determined from the particle detection algorithm (216) and is compared to a minimum limit which results in a sensor health warning (228) if below the predetermined limit. Alternative to rate limited reduction described in [0038], the amount of debris/particles that are unable to be detected as a result of detection threshold increase can be estimated in the time window of interest as a function of the adaptive detection threshold. The sum of such estimate and the rate of particles actually detected make the total rate of particle release (230). That is, the particle release rate (232) may be estimated utilizing the actual particle release rate increased as a function of the adaptive detection threshold to estimate missed debris. The total rate of particle release is compared to a predefined, constant minimum limit, which results in a mechanical failure alert if the limit is exceeded. The detectability may be calculated utilizing the time history data of the adaptive threshold. The detectability (224) is also calculated for comparison to a minimum limit which results in a sensor health warning (234) if below the predetermined limit. With reference toFIG.9, for example, a baseline detection threshold300may be buried in background noise304. When a fixed detection threshold leads to a signal to noise ratio of less than 1, the debris detection system110may lose the ability to detect debris, mechanical failures, and may generate nuisance particle counts. That is, the current state uses a fixed detection threshold in a particle detection algorithm to detect the particle so the detection threshold has to be crossed for the particle detection algorithm, so if there is noise consistently in excess of the detection threshold the debris detection system110will spend time processing and rejecting the noise even when particles are coming through the system. The availability of the debris detection system110with a baseline detection threshold300is thus essentially 0% which cannot properly detect particles. An adaptive threshold310as provided by the logic124of the debris detection system110based on the signal304is nearly 100% above the signal and can detect particles with adequate signal to noise ratio. The threshold change is limited data frame to data frame to prevent the debris detection system110from reacting to an asymmetric particle, leading to a missed detection. Additional threshold change is based on the multiple peaks that are still above the detection threshold, typical of a connection issue. The further adjustment will set the detection threshold at n-th peak with n being derived from the possible particle release of a mechanical failure. If majority of the peaks are one-sided, an alert can be transmitted to indicate a possible connection issue. With reference toFIG.10, the rate limit adjustment algorithm (222;FIG.5) includes a rate limit influence factor (240;FIG.11). The rate limit influence factor240is calculated by assigning a rate limit to a detection threshold, and multiplying by the ratio of the time of the processing block for which the detection threshold is computed relative to the time of the window in which the rate is computed (long term, short term). The rate limit influence factor240are then summed over the number of processing blocks in the window. The sum is then the new rate limit for the particle detection algorithm (216;FIG.5). Utilizing a noise measure to adjust the particle detection threshold, for example Root Mean Square (RMS), is effective when a particle quickly passes through the sensor, resulting in a high frequency signal that encompasses only a small portion of monitored signal but has minimal impact on the RMS algorithm. If, on the other hand, a particle passes slowly through the sensor, the impact on the RMS algorithm can be significant, presenting a risk that the threshold would be set above the particle and result in misdetection. To mitigate this risk, the change in detection threshold can be limited based on knowledge of system behavior, or a limited threshold change rate. To prevent setting the detection threshold too low, peak counting over an RMS based threshold can be applied to set the final detection threshold. That is, the RMS based threshold will filter out the majority of the background noise without affecting the possible particle signals standing out of the noise, and the peak counting algorithm will fine-tune the detection threshold so that the final detection threshold will be above all but possible particle signals. For example, if the expected particle release rate is 5 Hz, a maximum of 2 peaks may be expected within a 200 millisecond window to be a particle. The 3rd peak can be assumed as noise, and the threshold can be set above that peak. Once the detection threshold is adjusted, the fundamental detection capability of the debris detection system110is changed. To prevent late detection of a failure event, the failure alert is changed accordingly. For example, if in an ideal noiseless system, the debris rate to trigger an alert is 50 particles per hour, and in a high noise system, the detection threshold is adjusted and the debris detection system110has lost 20% detectability, then the debris rate alert limit may be adjusted to 40 particles per hour to compensate for the detection capability loss. With reference toFIG.12, the debris detection system110includes algorithms for detectability (224;FIG.5) and availability (226;FIG.5) that are calculated to assess the time that the debris detection system110spends above a critical detection threshold. The detectability algorithm can be weighted by level of detection threshold (i.e. a very high detection threshold will impact detectability more than a lower but still elevated threshold). A health fault alert for sensor replacement250may be provided if the rate limits are calculated critically low and/or if the debris detection system110spends too much time rejecting noise. This detection methodology facilitates system integrity for failure detection, namely, the debris detection system110compensation for background noise does not result in a detection capability loss that is too excessive to detect failure. A critical detection capability may be determined by utilizing knowledge of failure modes and retained over the engine operation. If the debris detection system110reduces detection capability beyond that critical point, an alert is generated such that the degraded hardware can be replaced. Furthermore, with an adaptive threshold that adapts to background noise, a rejected threshold crossing can be assumed as signal anomalies. That is, if signal anomalies lead to the debris detection system110spending too much time rejecting particles, the hardware should also be replaced. As an alternative to adjustment (reduction) of the rate limit in response to the elevated detection threshold for mechanical fault alert, the rate limit could remain fixed, but the number of particles that missed being detected as a result of elevated detection threshold, and hence, the total number of particles liberated, must be estimated for comparison with the fixed rate limit. With reference toFIG.13, the background noise levels may also be utilized to predict mechanical systems failures through the augmentation of the particles counted rather than adjustment of the particle release rate limits. That is, the particle release rate may be estimated (232;FIG.5). In this embodiment, knowledge of particle size distributions of expected system failure modes with respect to the calculated detection threshold ((400);220;FIG.6) can be used to determine a ratio of particles detected at various detection threshold levels (402;FIG.14). Every block of data will have a unique detection threshold, and thus a unique ratio of particles detected. The ratio is defined as the number of particles actually detected with the associated detection threshold over the total number of particles supposedly detectable by the sensing system (the sum of the number of particles detected and the possible number of particles missed being detected as a result of the elevated detection threshold). For any given time window of interest that contains N blocks of samples, the average ratio of particles detected is calculated (404), and the total number of particles supposedly detected is computed as a function of the number of particles actually detected and the average ratio of particles detected. This total count can be estimated as the number of particles actually detected divided by the average ratio of the particles detected (406), and the result will be compared directly to a fixed rate limit as an alternative to the process outlined inFIG.7for communication to the limit comparison algorithm (410). Detailed knowledge of the expected mechanical system and particle size distributions of failure modes allow for rate limit compensation to be optimized. While the rate alert limit is adjusted lower to compensate for the detection capability loss due to noises, the robustness of failure detection may be weakened as fewer particles are counted in the decision making. Such robustness reduction will be made up by examining particle accumulation over a longer time period of time. In other words, a failure event is detected if the rate alert limit is exceeded and a large number of particles have been accumulated in the past. In one embodiment, window sizes of 1 hour or less are utilized to calculate release rates. This may be adequate for quick failures; however, this may be inadequate to detect slow progressing failures over multiple flights. Furthermore, particle count may not be a good metric for mechanical failure when tracked over multiple flights as size of particles matters. The corresponding mass loss of the particles is a normalized measure, and hence, setting a multi-flight mass loss accumulation and comparing it to a fixed limit provides an additional failure annunciation. Mass loss will be accumulated in the controller memory over a fixed window of time or cycles. If the limit is crossed a fault will be annunciated. If the failure is troubleshot, the capability to reset the counter is advantageous. With reference toFIG.15, in one embodiment, an adjusted detection threshold is then transmitted to the particle detection and accumulation algorithms (500) to identify a ratio of particles detected, δRDi, with the detection threshold (502). Then, the number of particles counted using δRDi is estimated (504), and the particle count is converted (506) to mass loss. The accumulated mass loss within a flight (508) and the accumulated mass loss across multiple flights may be stored within the nonvolatile memory (510;FIG.16). The multi-flight accumulation is then compared to a limit and an alert is issued if required (512). The nonvolatile memory is then reset when the source of fault is resolved (514). The mass loss is thereby estimated utilizing the actual particle release increased as a function of the adaptive detection threshold to include debris missed, and converted to mass. If the reset accumulation flag is set to true, the multiple flight accumulation in the nonvolatile memory is set to zero. The accumulation can be continuous, or begin at a defined onset of mechanical failure defined by system knowledge via the accumulation flag. The debris detection system110thereby adapts oil debris monitor or other monitor technology to high background noise jet engine applications. Although particular step sequences are shown, described, and claimed, it should be appreciated that steps may be performed in any order, separated or combined unless otherwise indicated and will still benefit from the present disclosure. The foregoing description is exemplary rather than defined by the limitations within. Various non-limiting embodiments are disclosed herein; however, one of ordinary skill in the art would recognize that various modifications and variations in light of the above teachings will fall within the scope of the appended claims. It is therefore to be appreciated that within the scope of the appended claims, the disclosure may be practiced other than as specifically described. For that reason, the appended claims should be studied to determine true scope and content. | 27,190 |
11860125 | In the drawings, reference numbers may be reused to identify similar and/or identical elements. DETAILED DESCRIPTION FIGS.1A and1Bshow examples of an encoder, wheel speed sensors, and a wheel speed measuring system. The speed of a wheel of a vehicle is sensed using an encoder100and one or more wheel speed sensors102,104. InFIG.1A, the encoder100includes a magnetic material arranged around a rim of the encoder100. The magnetic material is arranged such that a series of north and south poles (examples identified at110,112) are radially disposed around the rim of the encoder100. The magnetic material is laminated for protection from dirt, water, and other elements that can damage the magnetic material. The encoder100is fitted to the wheel bearing. InFIG.1B, one or more wheel speed sensors102,104are mounted proximate to the rim of the encoder100. As the wheel rotates, the encoder100rotates at the speed of the wheel. The wheel speed sensors102,104detect the magnetic poles on the encoder100and generate outputs. A wheel speed measuring system118comprises one or more brake control modules (e.g., first and second brake control modules120,122). The first and second brake control modules120,122receive the outputs of the wheel speed sensors102,104, respectively. Each of the first and second brake control modules120,122independently calculates the speed of the wheel based on the output of the wheel speed sensors102,104, respectively. Each of the first and second brake control modules120,122is connected to a Controlled Area Network (CAN) bus130in the vehicle. Each of the first and second brake control modules120,122provides the calculated speed of the wheel to other modules such as an ABS module132, a TCS module134, and a stability control module136of the vehicle via the CAN bus130. In many vehicles, redundancy in wheel speed sensing is provided by using two wheel speed sensors (e.g., elements102,104shown inFIG.1B) and respective brake control modules (e.g., elements120,122shown inFIG.1B) that calculate the wheel speed based on data received from the respective wheel speed sensors. However, only one encoder is used. Although laminated, encoders are susceptible to degradation due to wear and tear. If an encoder fails, the wheel speed cannot be sensed. Loss of wheel speed sensing can degrade performance of autonomous vehicles. Inability to proactively detect failures in wheel speed sensing systems can impact safety and drivability of the vehicle and can lower customer experience. Relatively high amount of noise in a wheel speed signal output by the wheel speed sensors due to defects in the sensor-encoder interface can deteriorate performance of ABS, TCS, and stability control of the vehicle. The present disclosure provides a prognostics system that monitors the health of the encoder, proactively detects degradation in the encoder's health, and provides alerts regarding servicing the encoder before the encoder fails. The prognostics system combines health indicators from a noisy wheel speed signal to detect a health state of the sensor/encoder interface. Based on the measured state of health (SOH) of the sensor/encoder interface, the prognostics system generates a noise-tolerant wheel speed signal using an adaptive Kalman filter to maximize the availability of vehicle stability control features such as ABS and TCS. Throughout the present disclosure, reference is made Germany's Verband der Automobilindustrie (VDA), which defines standards for automotive industry. The prognostics system of the present disclosure leverages the ability of the wheel speed sensors to classify dynamic changes in the sensor/encoder interface through a VDA signal to estimate a state of health (SOH) of the encoder. The prognostics system combines the noise determined from envelope- and FFT-based detection processes with the magnetic strength of the encoder derived from a VDA signal to improve the SOH estimate. The prognostics system uses an adaptive Kalman filter to correct the high-noise wheel speed signal to allow an autonomous vehicle to perform its functions. Specifically, any defect in the wheel speed sensor-encoder interface increases the noise in the wheel speed signal. The prognostics system identifies the noise in the wheel speed signal by combining three different health indicators: a VDA signal, an envelope based process, and a fast Fourier transform (FFT) based process, to obtain a robust SOH estimate. A noise-tolerant wheel speed signal is produced using an adaptive Kalman filter that allows an autonomous vehicle to perform its operations in the event of mild degradation in wheel speed sensing. These and other features of the prognostics system of the present disclosure are now described below in further detail. The present disclosure is organized as follows. The prognostics system is shown and described with reference toFIGS.2A and2B. A method performed by the prognostics system is shown and described with reference toFIG.3. Examples of various methods performed by a weigh adjustment module of the prognostics system are shown and described with reference toFIGS.4A-4D. Various signals received, processed, and generated by the prognostics system are shown inFIGS.5A-5Eand are described during the discussion ofFIGS.2A,2B, and3. FIGS.2A and2Bshow a prognostics system200for determining a state of health (SOH) of the encoder100that is used to sense the wheel speed.FIG.2Ashows a block diagram of the prognostics system200in its entirety.FIG.2Bshows a noise detection module of the prognostics system200in detail. The prognostics system200can be implemented in each of the first and second brake control modules120,122. InFIG.2A, the prognostics system200comprises the encoder100, the wheel speed sensor102(or104), and a signal processing module202. The prognostics system200further comprises a noise detection module204, an SOH estimation module206, an adaptive Kalman filter208, and a weight adjustment module210. The prognostics system200communicates with an infotainment subsystem212, the ABS module132, the TCS module134, and the stability control module136of the vehicle. The signal processing module202also communicates with a rough road sensor220and other sensors222of the vehicle. The signal processing module202processes the data received from the wheel speed sensor102and generates a wheel speed signal230. The signal processing module202also outputs serial data called a VDA bit stream (explained below with reference toFIG.2B) along with the wheel speed signal230. The sensor102generates the VDA bit stream. The signal processing module202decodes and parses the VDA bit stream. Additionally, the signal processing module202processes data received from the rough road sensor220and the other sensors222of the vehicle and outputs respective signals232to the weight adjustment module210, which is described below in further detail with reference toFIG.2B. The noise detection module204estimates the amount of noise in the wheel speed signal230using various techniques described below in detail with reference toFIG.2B. The SOH estimation module206estimates the SOH of the encoder100based on the amount of noise in the wheel speed signal230estimated by the noise detection module204as described below in detail with reference toFIG.3. The SOH estimation module206provides an alert (e.g. an audiovisual alert) via the infotainment subsystem212of the vehicle when the SOH of the encoder100degrades severely. The adaptive Kalman filter208filters the noise in the wheel speed signal230depending on whether the amount of noise in the wheel speed signal230is relatively low or high. When the amount of noise in the wheel speed signal230is relatively low (e.g., below a first threshold), the adaptive Kalman filter208filters the noise lightly (i.e., using a relatively low filter constant). When the amount of noise in the wheel speed signal230is relatively high (e.g., above a second threshold), the adaptive Kalman filter208filters the noise using a relatively high filter constant. Accordingly, the adaptive Kalman filter208tailors its filter constant to the amount of noise in the wheel speed signal230and therefore to the SOH of the encoder100. The adaptive Kalman filter208provides a noise tolerant wheel speed signal240to the ABS module132, the TCS module134, and the stability control module136of the vehicle. FIG.2Bshows the noise detection module204in further detail. The noise detection module204employs three independent noise detection techniques to detect the amount of noise in the wheel speed signal230, which comprises a VDA bit stream230-1and a wheel speed signal230-2.FIG.5Ashows an example of the wheel speed signal230-2, which is shown as a graph of wheel speed500relative to time502.FIG.5Bshows an example of the VDA bit stream230-1, which is shown as a graph of a normalized amplitude504of pulses of the VDA bit stream230-1relative to time506. The noise detection module204comprises a VDA noise detector250, an envelope filter252, and an FFT module254. The VDA noise detector250detects noise in the VDA bit stream230-1. The envelope filter252determines the amount of noise in the wheel speed signal230-2. The FFT module254detects peaks in the wheel speed signal230-2(e.g., due to defects in the encoder100). The VDA noise detector250, the envelope filter252, and the FFT module254are described below in turn. The VDA bit stream230-1comprises a set of nine bits that are serially output by the wheel speed sensor102upon sensing magnetic pole pairs110,112on the encoder100. AsFIG.5Bshows, in the VDA bit stream230-1, a first bit510-1indicates whether an air gap limit is reached, where the air gap refers to a gap between the encoder100and the wheel speed sensor102. A second bit510-2indicates a mode of operation of the encoder100and the wheel speed sensor102(calibrated or un-calibrated). A third bit510-3provides an indication of a protocol (standard or advanced) used by the encoder100and the wheel speed sensor102to provide the VDA bit stream230-1. A fourth bit510-4indicates whether the direction of motion of the wheel indicated by the encoder100is valid. A fifth bit510-5indicates the direction of motion of the wheel indicated by the encoder100(clockwise or counterclockwise). The sixth, seventh, and eight bits510-6,510-7, and510-8(collectively shown as510-6,7,8) indicate a magnetic strength (air gap) of the magnetic poles on the encoder100sensed by the wheel speed sensor102. A ninth bit510-9is a parity bit. The nine bits510-1to510-9are collectively called the VDA bits510. The VDA noise detector250detects the amount of noise in the VDA bits510, which can be used to estimate the health of the encoder100. The VDA bits510include noise depending on the vehicle's operation and road conditions. For example, the fourth and fifth bits510-4,510-5can include jitter that can indicate wear in the encoder100. For example, if the sixth, seventh, and eighth bits510-6,510-7, and510-8indicate that the magnetic strength (air gap) is increasing and decreasing frequently, such an inconsistent pattern can indicate wear in the encoder100. In general, the content as well as the pattern of the VDA bits510detected by the VDA noise detector250can be indicative of the health of the encoder100. The envelope filter252determines a normalized amount of noise in the wheel speed signal230-2.FIG.5Cshows a graph of wheel speed500relative to time502and shows an envelope520of the wheel speed signal230-2. The envelope filter252determines a normalized noise522in the envelope520. The FFT module254converts the wheel speed signal230-2into frequency domain and detects peaks in the wheel speed signal230-2.FIG.5Dshows a graph of power spectral density530of the wheel speed signal230-2relative to frequency532. The FFT module254detects a peak534in the wheel speed signal230-2having a magnitude greater than a predetermined threshold. For example, the peak534may occur due to faults in the encoder100, which can occur due to deposition of contaminants and/or other wear and tear of the encoder100. The weight adjustment module210adjusts the weights of the VDA noise detector250, the envelope filter252, and the FFT module254. The noise in the wheel speed signal230varies depending on various factors. For example, the noise varies based on the vehicle's operation (e.g., vehicle speed, whether the vehicle is turning, etc.), which can be sensed by the other sensors222of the vehicle. Additionally, the noise varies depending on road conditions. For example, rough road conditions may include potholes, rumble strips, etc. encountered by the wheel, which can be sensed by the rough road sensor220. Various other factors related to the vehicle's operation and road conditions are sensed by the other sensors222of the vehicle. The weight adjustment module210adjusts the weights of the VDA noise detector250, the envelope filter252, and the FFT module254depending on these factors. For example, at relatively low vehicle speeds, the wheel speed signal230can include a relatively high amount of noise. Accordingly, at relatively low vehicle speeds, the envelope filter252may detect the relatively high amount of noise, which may not reliably indicate the health of the encoder100. For example, at relatively low vehicle speeds, the SOH estimation module206may misinterpret the relatively high amount of noise detected by the envelope filter252in the wheel speed signal230-2as an indication wear in the encoder100. To avoid such a skewed determination or detection of a false positive by the SOH estimation module206, the weight adjustment module210can reduce the weight of the envelope filter252at relatively low vehicle speeds. On the other hand, at lower vehicle speeds, the VDA bits can include relatively low amount of noise than at higher vehicle speeds. Accordingly, the weight adjustment module210can increase the weight of the VDA noise detector250at relatively low vehicle speeds. Further, when the vehicle is turning, the vehicle's speed is typically relatively low, and the VDA bits510can include relatively low amount of noise. Accordingly, the weight adjustment module210can increase the weight of the VDA noise detector250when the vehicle is turning, which can be detected by the other sensors222. Conversely, at relatively high vehicle speeds, the VDA bit stream is generally truncated (i.e., not all of the VDA bits510are output with the wheel speed signal230). Therefore, potentially incorrectly inferring wear on the encoder100based on the truncated VDA bit stream can generate false positives. Accordingly, the weight adjustment module210can reduce the weight of the VDA noise detector250and increase the weight of the envelope filter252and the FFT module254at relatively high vehicle speeds. As another example, in rough road conditions, the FFT module254and the envelope filter252may detect noise in the wheel speed signal230-2. Therefore, incorrectly inferring wear on the encoder100based on the noise detected by the FFT module254and the envelope filter252in the wheel speed signal230-2can also generate false positives. Accordingly, the weight adjustment module210can reduce the weight of the FFT module254and the envelope filter252when rough road conditions are detected. In general, the weight adjustment module210can dynamically adjust the weights of the VDA noise detector250, the envelope filter252, and the FFT module254depending on factors such as the vehicle's speed, whether the vehicle is turning, road conditions, and so on to prevent the SOH estimation module206from detecting false positives and skewing the estimation of the health state of the encoder100. The SOH estimation module206determines the health of the encoder100based on the amount of noise estimated by the noise detection module204as follows. FIG.3shows a method300performed by the prognostics system200. For example, one or more components of the prognostics system200can perform the steps of the method300. Accordingly, the term control used in the following description refers to one or more components of the prognostics system200. At302, control (e.g., the signal processing module202) generates the wheel speed signal230based on the data received from the wheel speed sensor102that is coupled to the encoder100. At304, control (e.g., the noise detection module204) detects and analyzes the noise in the wheel speed signal230. At306, control (e.g., the SOH estimation module206) estimates the health of the encoder100based on the noise analysis. At308, control (e.g., the SOH estimation module206) determines if the noise in the wheel speed signal230is less than a first threshold (Th1). If the noise is less than a first threshold (Th1), at310, control (e.g., the SOH estimation module206) determines that the encoder100is healthy (i.e., has no defects or wear and is operating normally). At312, control (e.g., the adaptive Kalman filter208) lightly filters the wheel speed signal230(i.e., using a relatively low filter constant) and provides the lightly filtered wheel speed signal230to one or more control systems (e.g., the ABS module132, the TCS module134, and the stability control module136) of the vehicle. Control returns to302. If the noise in the wheel speed signal230is greater than the first threshold (Th1), at314, control (e.g., the SOH estimation module206) determines if the noise in the wheel speed signal230is less than a second threshold (Th2), where Th2>Th1. If the noise in the wheel speed signal230is greater than the first threshold (Th1) but less than the second threshold (Th2), at316, control (e.g., the SOH estimation module206) determines that the encoder100is degrading (i.e., the encoder100has some amount of wear or defects) but the errors due to degradation are recoverable (i.e., the amount of the wear is less than a predetermined threshold). At318, control (e.g., the adaptive Kalman filter208) increases the filter constant and filters the wheel speed signal230with a relatively high amount of filtering (i.e., using the relatively higher filter constant). Control (e.g., the adaptive Kalman filter208) provides the relatively highly filtered wheel speed signal230to one or more control systems (e.g., the ABS module132, the TCS module134, and the stability control module136) of the vehicle. Control returns to302. If the noise in the wheel speed signal230is greater than the second threshold (Th2), at320, control (e.g., the SOH estimation module206) determines that the encoder is severely or significantly degraded (i.e., the amount of the wear is greater than the predetermined threshold). Control (e.g., the SOH estimation module206) generates an alert (e.g., displays a message to schedule service on the infotainment subsystem212). Control returns to302. FIG.5Eshows an example of the health state of the encoder determined by the SOH estimation module206.FIG.5Eshows the health state in terms of a graph of the amount of noise540in the wheel speed signal230detected by the noise detection module204relative to the wheel speed542. In the graph shown inFIG.5E, a region544indicates a severely degraded health state of the encoder100, where the errors due to the degradation are irrecoverable. A region546indicates a moderately degraded health state of the encoder100, where the errors due to degradation are recoverable. A region548indicates a healthy state of the encoder100, where the error rate is relatively low (e.g., less than a predetermined threshold). FIGS.4A-4Dshow various examples of methods performed by the weight adjustment module210. The weight adjustment module210performs these methods concurrently to dynamically adjust the weights of the VDA noise detector250, the envelope filter252, and the FFT module254depending on factors such as the vehicle's speed, whether the vehicle is turning, road conditions, and so on, to prevent the SOH estimation module206from detecting false positives and skewing the estimation of the health state of the encoder100. InFIG.4A, the weight adjustment module210performs a method400as follows. At402, the weight adjustment module210determines if the wheel speed is relatively low (e.g., less than a first speed). If the wheel speed is relatively low, at404, the weight adjustment module210reduces the weight of the envelope filter252. At406, the weight adjustment module210increases the weight of the VDA noise detector250. The method400ends. InFIG.4B, the weight adjustment module210performs a method420as follows. At422, the weight adjustment module210determines if the wheel speed is relatively high (e.g., greater than a second speed, which is greater than the first speed). If the wheel speed is relatively high, at424, the weight adjustment module210reduces the weight of the VDA noise detector250. At426, the weight adjustment module210increases the weights of the envelope filter252and the FFT module254. The method420ends. InFIG.4C, the weight adjustment module210performs a method450as follows. At452, the weight adjustment module210determines if the vehicle is turning (e.g., based on one of the signals232received from the signal processing module202). If the vehicle is turning, at454, the weight adjustment module210increases the weight of the VDA noise detector250, and the method450ends. InFIG.4D, the weight adjustment module210performs a method480as follows. At482, the weight adjustment module210determines if rough road condition is detected (e.g., based on one of the signals232received from the signal processing module202). If rough road condition is detected, at484, the weight adjustment module210decreases the weight of the envelope filter252and the FFT module254, and the method450ends. Accordingly, the prognostics system200provides two levels of controls to detect the wear in the encoder100and to mitigate effects of the wear in the encoder100. A first level of control is provided by the weight adjustment module210, which dynamically adjusts the weights of the VDA noise detector250, the envelope filter252, and the FFT module254to correctly detect the SOH and therefore wear in the encoder100as described above. A second level of control is provided by the adaptive Kalman filter208, which selectively filters the wheel speed signal230based on the amount of the noise detected by the noise detection module204to mitigate the effects of the wear in the encoder100. Further, the prognostics system200proactively provides an alert when the wear in the encoder100becomes greater than a predetermined threshold, which allows servicing the encoder100before it fails, which in turn prevents the vehicle stability features (e.g., ABS, TCS, etc.) from being disabled. The foregoing description is merely illustrative in nature and is not intended to limit the disclosure, its application, or uses. The broad teachings of the disclosure can be implemented in a variety of forms. Therefore, while this disclosure includes particular examples, the true scope of the disclosure should not be so limited since other modifications will become apparent upon a study of the drawings, the specification, and the following claims. It should be understood that one or more steps within a method may be executed in different order (or concurrently) without altering the principles of the present disclosure. Further, although each of the embodiments is described above as having certain features, any one or more of those features described with respect to any embodiment of the disclosure can be implemented in and/or combined with features of any of the other embodiments, even if that combination is not explicitly described. In other words, the described embodiments are not mutually exclusive, and permutations of one or more embodiments with one another remain within the scope of this disclosure. Spatial and functional relationships between elements (for example, between modules, circuit elements, semiconductor layers, etc.) are described using various terms, including “connected,” “engaged,” “coupled,” “adjacent,” “next to,” “on top of,” “above,” “below,” and “disposed.” Unless explicitly described as being “direct,” when a relationship between first and second elements is described in the above disclosure, that relationship can be a direct relationship where no other intervening elements are present between the first and second elements, but can also be an indirect relationship where one or more intervening elements are present (either spatially or functionally) between the first and second elements. As used herein, the phrase at least one of A, B, and C should be construed to mean a logical (A OR B OR C), using a non-exclusive logical OR, and should not be construed to mean “at least one of A, at least one of B, and at least one of C.” In the figures, the direction of an arrow, as indicated by the arrowhead, generally demonstrates the flow of information (such as data or instructions) that is of interest to the illustration. For example, when element A and element B exchange a variety of information but information transmitted from element A to element B is relevant to the illustration, the arrow may point from element A to element B. This unidirectional arrow does not imply that no other information is transmitted from element B to element A. Further, for information sent from element A to element B, element B may send requests for, or receipt acknowledgements of, the information to element A. In this application, including the definitions below, the term “module” or the term “controller” may be replaced with the term “circuit.” The term “module” may refer to, be part of, or include: an Application Specific Integrated Circuit (ASIC); a digital, analog, or mixed analog/digital discrete circuit; a digital, analog, or mixed analog/digital integrated circuit; a combinational logic circuit; a field programmable gate array (FPGA); a processor circuit (shared, dedicated, or group) that executes code; a memory circuit (shared, dedicated, or group) that stores code executed by the processor circuit; other suitable hardware components that provide the described functionality; or a combination of some or all of the above, such as in a system-on-chip. The module may include one or more interface circuits. In some examples, the interface circuits may include wired or wireless interfaces that are connected to a local area network (LAN), the Internet, a wide area network (WAN), or combinations thereof. The functionality of any given module of the present disclosure may be distributed among multiple modules that are connected via interface circuits. For example, multiple modules may allow load balancing. In a further example, a server (also known as remote, or cloud) module may accomplish some functionality on behalf of a client module. The term code, as used above, may include software, firmware, and/or microcode, and may refer to programs, routines, functions, classes, data structures, and/or objects. The term shared processor circuit encompasses a single processor circuit that executes some or all code from multiple modules. The term group processor circuit encompasses a processor circuit that, in combination with additional processor circuits, executes some or all code from one or more modules. References to multiple processor circuits encompass multiple processor circuits on discrete dies, multiple processor circuits on a single die, multiple cores of a single processor circuit, multiple threads of a single processor circuit, or a combination of the above. The term shared memory circuit encompasses a single memory circuit that stores some or all code from multiple modules. The term group memory circuit encompasses a memory circuit that, in combination with additional memories, stores some or all code from one or more modules. The term memory circuit is a subset of the term computer-readable medium. The term computer-readable medium, as used herein, does not encompass transitory electrical or electromagnetic signals propagating through a medium (such as on a carrier wave); the term computer-readable medium may therefore be considered tangible and non-transitory. Non-limiting examples of a non-transitory, tangible computer-readable medium are nonvolatile memory circuits (such as a flash memory circuit, an erasable programmable read-only memory circuit, or a mask read-only memory circuit), volatile memory circuits (such as a static random access memory circuit or a dynamic random access memory circuit), magnetic storage media (such as an analog or digital magnetic tape or a hard disk drive), and optical storage media (such as a CD, a DVD, or a Blu-ray Disc). The apparatuses and methods described in this application may be partially or fully implemented by a special purpose computer created by configuring a general purpose computer to execute one or more particular functions embodied in computer programs. The functional blocks, flowchart components, and other elements described above serve as software specifications, which can be translated into the computer programs by the routine work of a skilled technician or programmer. The computer programs include processor-executable instructions that are stored on at least one non-transitory, tangible computer-readable medium. The computer programs may also include or rely on stored data. The computer programs may encompass a basic input/output system (BIOS) that interacts with hardware of the special purpose computer, device drivers that interact with particular devices of the special purpose computer, one or more operating systems, user applications, background services, background applications, etc. The computer programs may include: (i) descriptive text to be parsed, such as HTML (hypertext markup language), XML (extensible markup language), or JSON (JavaScript Object Notation) (ii) assembly code, (iii) object code generated from source code by a compiler, (iv) source code for execution by an interpreter, (v) source code for compilation and execution by a just-in-time compiler, etc. As examples only, source code may be written using syntax from languages including C, C++, C #, Objective-C, Swift, Haskell, Go, SQL, R, Lisp, Java®, Fortran, Perl, Pascal, Curl, OCaml, Javascript®, HTML5 (Hypertext Markup Language 5th revision), Ada, ASP (Active Server Pages), PHP (PHP: Hypertext Preprocessor), Scala, Eiffel, Smalltalk, Erlang, Ruby, Flash®, Visual Basic®, Lua, MATLAB, SIMULINK, and Python®. | 30,496 |
11860126 | DETAILED DESCRIPTION Systems and methods are disclosed to use eddy current NDE to detect flaws in a sample. In some embodiments, an NDE device may be used to detect defects. The NDE device may include a component housing and/or a probe head coupled with a motor. In some embodiments, the component housing and the probe head may be coupled together. The motor may be configured to rotate the component housing a fixed or variable rotational speed. The probe head may include one or more permanent magnets and/or one or more pickup coils. The one or more pickup coils may include a plurality of loops of wire. As discussed in more detail, these pickup coils may detect magnetic fields induced within a sample at the site of a defect. In some embodiments, the component housing may include one or more integrator circuits, a data acquisition unit, and/or a wireless transmitter. In some embodiments, the component housing may include one or more batteries. In some embodiments, the component housing may include one or more accelerometers, one or more rotation sensors, and/or one or more position sensors. In some embodiments, the component housing may also include a data storage and/or retrieval system such as, for example, flash, DRAM, SRAM, etc. In some embodiments, the component housing may include a processor such as, for example, a microprocessor, FPGA, etc. In some embodiments, the component housing may also include a rotating electrical interface such as, for example, a Mercury contactor or connector, that can allow data signals to be transmitted from the probe head and/or the component housing. In some embodiments, the NDE device may be effective for surface crack detection and/or defect detection under surface layers and/or paints. Additional sensitivity can be obtained at high AC frequencies but deep measurements in metals are limited by skin depth penetration, which are well characterized for many materials. Several methods to increase measurement sensitivity for eddy current NDE, including direct measurement of the magnetic flux density using Hall sensors, giant magnetoresistive (GMR) sensors, and superconducting quantum interference devices (SQUIDS), have been attempted. High temperature SQUIDS have shown promise for NDE and provide very sensitive detection capabilities as low as 1 pT/Hz1/2. Since skin depth is inversely proportional to the square root of the excitation frequency of the AC coil, the increased sensitivity of SQUIDS may allow for deeper flaw detection. Sub-surface flaws up to 10 mm in Al have been demonstrated. The disadvantage of SQUID-based NDE systems is the need to cool the probe head to cryogenic temperatures. This is true even for high temperature (77 K) SQUIDS, leading to complex and rather large probe heads not easily used for rapid investigation of various geometries needed for spacecraft NDE. However, the principle of using more sensitive magnetic probes demonstrates the potential ability to advance the method of eddy current NDE to allow for detection of deep subsurface cracks and corrosion. Embodiments of the invention include a simple, robust, and low-cost method for making highly sensitive magnetic measurements with similar precision to SQUIDS but without the added complications of cryogenically cooled superconductors. Some embodiments may include methods for making sensitive magnetic measurements in noisy environments through the use of small inductive pick-up loops coupled to very high gain active integrator circuits. One possible advantage of this method is simplicity, since precision measurements of small-scale magnetic perturbations can be made using simple high gain active integrator circuits that may include operational amplifiers and passive circuit components. Inductive pick-up loops also work in high noise environments and in the presence of large magnetic fields that would render active measurement devices like Hall and GMR probes useless. Active integration may allow for signal gains on the order of 106or larger, which enables the measurement of very small signal levels. In some embodiments, to convert the direct voltage measurements from the inductive pickup coil to a magnetic field measurement, the coil voltage may be integrated. In principle, direct integration of the signal should not pose a significant challenge, but in practice several factors make the integration difficult, especially when there are many orders of magnitude difference between the fast and slow magnetic signals and where high gain integrator circuits are being used. It is fairly easy to make a high gain integrator circuit stable over many RC times. For example, it is routine to make integrator circuits with gains of over 106(RC<1 μs) that can remain stable for up to millisecond timescales. However, the ability to keep high gain integrator circuits stable over millions or billions of RC times suitable long duration NDE measurements can be challenging. For NDE utilization, for example, it may be beneficial for the integrator circuit to remain stable during typical measurement periods, which can be characterized by the scan frequency of the NDE device. For deep defect detection, for example, slower scan frequencies may be needed to offset the skin depth effect with target scan speeds on the order of 10 Hz desirable for deep penetration. This may, for example, require integrator circuit stability for time periods on the order of seconds to minutes to ensure sufficient resolution, which may amount to tens of millions of RC times for gains necessary to measure defect induced eddy current perturbations. Some embodiments include an ultra-stable high gain integrator (HGI). In some embodiments, an integrator circuit can take advantage of very fast (<10 ns) digital control. In some embodiments, when gated on, the integrator circuit may begin integrating. Then, on a regular interval set by an external clock, a processor may reset the integrator circuit back to zero output. An example integrator circuit is shown inFIG.1. Other examples of integrator circuits are described in U.S. Pat. No. 9,495,563, the contents of which are incorporated herein in its entirety for all purposes. In some embodiments, the integrator circuit (and/or the sample and hold circuit reset) process may be very fast, for example, at speeds less than about 10 ms, 1 ms, 100 ns, etc. During this fast time interval, data may not be recorded, however, the fast time interval should be fast enough to allow signal measurements with a bandwidth of 5 MHz or greater, which should be sufficient for most applications and should be much faster than any relevant timescale necessary for an NDE device. The time between resets can, for example, be controlled by an external clock signal. During the fast time interval, for example, the integrator circuit may be gated on, and data may be output to the digitizer. The data may be recombined in software to produce the full signal. In some embodiments, the absolute error/noise may grow in time, but at a very slow rate. Since the integrator circuit can be reset on timescales fast compared to thermally induced drift and resultant instabilities, this error source can be significantly reduced. Random error/noise, which may always be present, can be reduced to approach its theoretical limits by decreasing time between resets. In some embodiments, the integrator circuit may comprise a passive RC integrator that does not include, for example, any op amps or digital control elements. In some embodiments, the integrator circuit may include multiple integrators that are stable on relatively short time scales, and that also may be utilized for relatively long time scale integration. Over long periods of time, for example, integrators may drift, which may result in integration error. To overcome this drift, among other things, the integrators may be switched between active and passive loads so that, while one integrator is integrating the active load, the other integrator may be reset when integrating the passive load. The resistance, inductance, and/or capacitance of the active load and the passive load may be identical or substantially identical (within 1%, 2%, 5%, or 10% of each other, or within manufacturing tolerances), while the active load provides a voltage and/or current signal and the passive load does not. In some embodiments, an additional circuit may be placed at the output of the integrator pair such that an output from multiple integrator circuit pairs may be averaged to achieve better or increased performance. In some embodiments, the integrator circuit switches may be selected so that most or all charge injection and/or leakage currents are balanced, both during and between switching events. Accordingly, respective switches may be paired and balanced with each other. In part, this may be realized by the use of what would otherwise appear to be switches without purpose but are switching between a pair of resistors each tied to circuit ground. In some embodiments, all integrators, as well as, for example, the integrator input coil, may see identical input loads and/or output loads so, from the perspective of the integrators, those integrators do not respond or change states as if they normally would when being switched. Rather, everything is balanced and appears constant in time. Part of this may include balancing any delay(s) generated in gate/drive logic, and may involve the use of additional drive logic and gates that would otherwise appear to be without purpose. Some embodiments may include an integrator circuit that enables a method for eddy current NDE with expanded detection depth over standard eddy current techniques. In some embodiments, the minimum magnetic field perturbation that can be detected with an integrator circuit can be determined as follows. From Faraday's law: Vcoil(t)=dΦBdt=NAdB∥dt, where ΦBis the magnetic flux, A is the area of a single loop in a coil, B is the magnetic flux through a single turn, N is the number of loops, and t is time. This equation can be integrated to obtain ∫Vcoil(t)dt=NA∫dBdtdt=NAB. The transfer function for an ideal integrator is Vout(t)=1RC∫Vcoil(t)dt=NABRC, where RC is the integration time constant of the integrator circuit and 1/RC is the gain. After the second equal sign, the equation for the integral of the coil voltage (Vcoil) has been substituted. This equation can be rewritten B(t)=RCNAVout(t) to determine the minimum detectable magnetic field that can be measured by this method. For example, a typical off-the-shelf data acquisition system operating at a reasonable signal-to-noise ratio may be able to detect signals of 1 mV or less. The disclosed integrator circuits typically operate with a gain of 106and can be operated with gains as high as 108. In some embodiments, a coil for detecting defect sizes is on order of 0.5 cm2, 0.75 cm2, 1 cm2, 1.25 cm2, 1.5 cm2, etc. but can be any size. It may be reasonable to make pickup coils with up to about 104loops (or turns). Combining these numbers into the equation above provides an estimate of the minimum measurable magnetic field of 1 nT, using a gain of 106. The actual minimum magnetic field that can be detected may be an order of magnitude smaller and/or may be achieved by operating at higher gain. Statistical methods could be employed and would likely increase the peak sensitivity by at least another order of magnitude through oversampling and averaging. More sensitive data acquisition systems may also improve the sensitivity by an order of magnitude or more. The integrator circuit's signal-to-noise ratio may be dominated by random noise, so it averages away. In some embodiments, the minimum field sensitivity may be on order of 10 pT, which may be comparable to the low temperature SQUIDs. The sensitivity of the disclosed integrator circuit can be compared to the magnitude of the magnetic field perturbation likely to be produced when detecting a defect to determine its potential as an NDE device. In the case where a permanent magnet is being moved over a conductive surface, such as a metal sheet, at a constant velocity, thus generating eddy currents in the metal sheet. As with standard eddy current measurements, changes in magnetic flux due to thickness changes, cracks, corrosion, and/or voids can be detected by the measurement coil.FIG.2, for example, is a schematic of an NDE device based on a permanent magnet and a high gain integrator circuit. The permanent magnet field lines and eddy currents produced by the field's movement are also shown. The advantage of using the permanent magnet is that very large magnetic fields can be produced at very low cost with little complexity. Since the amplitude of the produced eddy current is proportional to dB/dt, the larger magnetic field also allows for lower scanning rates while producing similar signal amplitudes on the pickup coil. The lower frequency scanning can allow for deeper depth penetration into the metal. This coupled with the increased sensitivity of the integrator circuit, for example, may allow for much deeper detection depths with a very simple system. Eddy currents may be induced in a conductive sheet by a moving permanent magnet. The penetration depth of the eddy currents is proportional to the skin depth of the material. Some embodiments may leverage the large magnetic fields of permanent magnets to reduce the required time rate of change of the flux and therefore significantly increases depth of penetration into the material. Once the NDE device velocity becomes constant there is no change in flux seen by a pickup coil unless there is a change in the material, which will modify the eddy current generation. FIGS.3A,3B and3Cillustrate how changes in magnetic flux can be used to detect defects according to some embodiments.FIG.3Ashows a magnet and coil305on the surface of sample310with a constant velocity creating an opposing magnetic field. The magnet and coil305is coupled with an integrator circuit315. Magnetic filed lines320are created by the magnet and no eddy currents are created in the sample310. FIG.3Bshows the magnet and coil in motion and eddy currents325created in the sample310.FIG.3Cshows a defect in sample310. As the magnet passes over the defect the eddy currents in the sample310change causing an opposing magnetic field330. The pickup coil detects the opposing magnetic field330as a dipole335. In some embodiments, a high gain integrator circuit may be coupled with one or more permanent magnets moving over a conductive surface at a constant velocity. A pickup coil may be used with each magnet to measure changes in magnetic flux due to defects as shown inFIGS.3A,3B, and3C. The voltage that the integrator circuit will output due to a magnetic field perturbation may be: Vout(t)=NABdipRC,(1) where A is the area of a single loop in the pickup coil, N is the number of loops of wire, RC is the integration time constant of the integrator circuit, and Bdipis the magnetic field strength due to the defect being approximated as a dipole. In some embodiments, a defect can be approximated as a dipole because of the change in magnetic flux due to a change in eddy currents in the material. As a permanent magnet moves over a conducting surface, the induced eddy currents within the material will alter the magnetic field amplitude at the location of the pickup coil. The change in the magnetic field due to eddy currents everywhere in a material except for at the location of the defect, is equivalent to current being nowhere in the material and only in the defect. The former may be a dip in signal and the latter may be a peak in signal. Thus, the defect can be assumed as a dipole creating a magnetic field that is detected by a pickup coil. The strength of the magnetic field due to an axially magnetized rod magnet along its symmetry axis can be calculated using, Bm=Br2(h+zRm2+(h+z)2-zRm2+z2),(2) where Br, also called Brmax, is the residual flux density given for the magnetic material, h is the height or length of the magnet, Rmis the radius of the magnet, and z is the distance away from the magnet. This equation can be used to calculate the strength of the magnetic field at the location of the defect. When the permanent magnet moves over a defect in the material, it is assumed that an opposing magnetic field will be produced due to the eddy currents in the material. The current required to produce the opposing magnetic field can be approximated using the Biot-Savart Law derived for a current-carrying loop, B=∫μ04πIdlsinθr2=μ0I2Rd.(3) This equation can be rewritten, and substitute Bmfrom equation 2 to determine the maximum current generated in the material due to eddy currents: Imax=2RdBmμ0,(4) where Rdis the radius of the defect and μ0is the permeability of free space. The following equation can be used to approximate the effective current at the location of the defect, Ieff=Imax(13)(DdDm),(5) where Ddis the thickness of the defect and Dmis the thickness of the material. The ratio of defect thickness to material thickness is applied to account for the current flowing only through the defect, and not the current flowing through the whole thickness of material. To make this approximation, the magnetic field gradient may be assumed to be constant throughout the material, thus a simple ratio can be used to approximate the current only in a fraction of the material thickness. The factor of one third is applied to account for attenuation due to skin depth. The velocity of the magnet will be calculated so that the skin depth of the eddy currents is equal to the thickness of the material being tested. Since skin depth is defined as the depth at which the current density is approximately 1/e, about 0.37, of the surface current, then using a factor of one third in equation 5 is an approximation for the loss in current due to the depth of penetration. The equation used to calculate skin depth in a conductive material due to an electromagnet can be written as δ=2ρωμ,(6) where ρ is the resistivity of the material, μ is the permeability of the material, and ω is the angular frequency of the current. Since eddy currents will be induced via a moving a permanent magnet, not an electromagnet, equation 6 can be varied to approximate the velocity at which the permanent magnet should move to achieve a desired skin depth. The velocity required to make the skin depth approximately equal to the material thickness can be called vsdand will be approximated as follows. In equation 6, the angular frequency can be converted into frequency and then into a period: ω=2πf=2π4T=π2T,(7) where T is the quarter period. A factor of one fourth can be applied after the second equals sign. By combining equations 6 and 7, the quarter period for a desired skin depth can be calculated using T=δ2πμ4ρ.(8) The quarter period should be the time it takes for the eddy currents from a moving magnet to reach a desired skin depth. This can be seen by visualizing a sine wave, it only takes a quarter period for the wave to reach its first maximum, so the current will reach its maximum depth in only one fourth of the period. The magnet can be moved at a velocity that allows the eddy currents enough time, T, to reach the skin depth before the magnet's diameter has completely swept over that specific surface area of material. Thus, the velocity can be approximated as vsd=dmT,(9) where dmis the diameter of the permanent magnet being swept over the surface. If the operating velocity of the magnet, vo, is not equal to vsd, then another factor, vsd/vo, can be applied to equation 5. Skin depth decreases with increase in velocity. This factor accounts for the change in current density at the depth of the defect due to a change in velocity of the permanent magnet. By changing the velocity of the probe head, the depth of the defect can be mapped out as long as maximum depth of measurement for any particular probe diameter and velocity follows equation 9. In some embodiments, the defect acts as a dipole with a current value, Ieff, calculated from equation 5. The dipole can create an additional magnetic field which can be detected by a pickup coil back at the surface of the material. The equation for the magnetic field due to a dipole may be Bdip=μ04πr3[3(m·rˆ)rˆ-m],(10) where r is the radial distance from the center of the dipole, or defect, and m is the magnetic moment. The magnetic field on axis for permanent magnet is well known but no solution exists for the magnetic field off axis created by a permanent magnet. An analytic solution for the field strength of a physical magnetic dipole may be known. The permanent magnet, for example, may be assumed to be a simple current loop of radius a. In cartesian coordinates the z-component of the magnetic field may be given by the following equation. Bz=μ0I2π[(a+x)2+z2]-12[K+a2-x2-z2(a-x)2+z2E] Where K and E are solutions of the complete elliptic integrals of the first and second kind, respectively and given by the following expansions. K=π2(1+k24+964k4+⋯),E=π2(1-14k21-964k43-⋯) The substitution parameter is written as follows: k2=4ax(a+x)2+z2 With the magnetic field component of interest as a function of (x,z) the eddy current generated can be calculated by the defect once the pertinent time scale is defined. From Faraday's law, the magnet can be swept past the defect very quickly to maximize the time rate of change of magnetic flux through the defect. In some embodiments, the skin depth may have a greater attenuating effect at higher speeds. The skin depth may be defined as the depth at which the current density is approximately 1/e of the surface current and the equation used to calculate the skin depth in a conductive material due to an electromagnet may be: δ=2ρωμ. Where ρ is the resistivity of the material, μ is the permeability of the material, and w is the angular frequency of the current. Since eddy currents may be induced by a moving permanent magnet, not a pulsed electromagnet, the velocity at which the permanent magnet should move to achieve a desired skin depth can be approximated. For a conductive material of some thickness defined by h, the velocity required to make the skin depth equal to the material thickness can be approximated as follows. Let Trbe the quarter period such that, ω=2πf=2πT=2π4Tr The quarter period may be the time it takes for the eddy currents from the moving magnet to reach a desired skin depth. This can be seen by visualizing a sine wave, it only takes a quarter period for the wave to reach its first maximum, so the current will reach its maximum depth in only one fourth of the period. This means the magnet should be moved at a velocity that allows the eddy currents enough time to reach the skin depth before the magnet's diameter has completely swept over that specific surface area of material. Thus, the skin depth velocity can be approximated as vsd=RmTr. Where Rmis the radius of the magnet being swept over the surface and the quarter period can be written as function of the material thickness because δ=h at skin depth velocity vsd. Tr=h2πμ4ρ As the skin depth is the location in a conductive material at which the magnitude of the surface field decreases by 1/e then this attenuation must be applied to the z-component of the dipole magnetic field when that field is swept at velocity vsd. If the operating velocity of the magnet, vo, is not equal to vsd, the factor (e−(vo/vsd)) may be applied to the scanning magnetic field. To estimate the voltage on a pickup coil, due to a small defect in a conducting material, moving with the scanning magnet, the magnetic field at the defect may be calculated.FIG.4is an example plot of the magnetic field strength at a depth of ¼″ in aluminum created by a ¼″ diameter physical dipole moving at the ¼″ skin depth velocity. To elucidate the effect that changes to a given parameter (e.g., defect size, defect depth, operating velocity, etc.) has, the maximum magnetic field strength located on axis at the surface is normalized to 1 T, for example, regardless of the magnet size. In some embodiment, the electromagnetic induction at the defect can be calculated from the magnitude of the magnetic field and the speed at which the magnet is moving using Faraday's law. This electromotive force drives a current in the vicinity of the defect. Determining the magnitude of this current may be difficult. It can be assumed, for example, that the current flows in a ring around the defect and/or the cross-sectional area of that current ring is equivalent to half the area of the defect. The length of the current ring can be assumed to be the circumference of the defect plus the radius of the current ring. This can enable an estimate of the resistance in the current ring. The actual current may then be calculated from Ohm's law. Although the area of the defect can be used to calculate the voltage around the defect, an additional attenuation factor can be applied to the defect current to account for the diminishing pickup coil signal strength as the defect size decreases. In some embodiments, this can be a weighted attenuation factor which is essentially the integral of the section of the dipole magnetic field directly over the defect at a given time divided by the total integral of the dipole field. In some embodiments, as the magnet sweeps over the defect, a new section of the dipole magnetic field curve, like the one shown inFIG.4, may be integrated. This value can be divided by the total integral of the magnetic field curve which is then applied to the current around the defect. In some embodiments, this can result in a substantial attenuation and/or it may yield results similar to measured values. In one example, the defect eddy current as a function of time is shown inFIG.5. Once the defect eddy current is determined, for example, the resultant dipole magnetic field can be calculated as shown inFIG.6. In some embodiments, the voltage on the pickup coil that is swept with the magnet can be calculated using Faraday's law from the estimated magnetic field from the defect. In some embodiments, a larger pickup coil may receive a greater induced voltage. In some embodiments, a larger diameter magnet may generate a greater signal on the pickup. In some embodiments, the estimated voltage on a ¼″ diameter pickup coil with 300 turns, for example, is shown inFIG.7for a 0.10″ diameter defect with a height of 0.050″ located 0.25″ below the surface of a solid aluminum plate.FIG.7shows that a larger magnet may generate a greater voltage on the pickup coil. FIG.8shows the pickup coil voltage as a function of probe sweep velocity according to some embodiments. FIG.9Ais a top view of a rotating head900of a precision eddy current NDE device according to some embodiments.FIG.9Bis a side view of a rotating head900of a precision eddy current NDE device according to some embodiments. The rotating head900may include a disc905that includes permanent magnets910A,910B. The rotating head905may have a translational stage that can be used to move the rotating head905. The rotating head905, for example, may comprise an aluminum disc. The rotating head905may include two permanent magnets910A,910B disposed on opposite sides of the disc905(e.g., glued to the disc), for example, along a diameter of the disc905. In some embodiments, the permanent magnets910A,910B may include bobbins glued to the outer surfaces of the disc905. In some embodiments, the permanent magnets910A,910B may include a pickup coil915A,915B (e.g., a 30 AWG pickup coil) wound around each respective permanent magnets910A,910B. Each of the two permanent magnets910A,910B with a pickup coils may be coupled with or within a probe head body. For example, two permanent magnets910A,910B may be glued to the disc905on opposite ends of a diameter. In some embodiments, the permanent magnets910A,910B may include a bobbins930A,930B and pickup coils915A,915B. In some embodiments, the pickup coil915A,915B may not be glued with the rotating head905. In some embodiments, for stability, an empty bobbin may be attached to the side of the permanent magnet facing the disc905that may result in a wider glue base. In some embodiments, a second disc940(e.g., 1/16″ polycarbonate disc) may be attached (e.g., glued) on the other side of the permanent magnets910A,910B where the coil is disposed. FIG.10is a photograph of an example eddy current NDE device. The eddy current NDE device may allow for a controlled, adjustable rate of rotation, and/or may allow the material samples clamped relative to the probe head for a stable configuration during prototype testing. Additionally, it may allow for the vertical distance between the probe head and the material sample to be precisely controlled. FIG.11illustrates an assembly of an example NDE device1100according to some embodiments. NDE device1100includes a rotating body1110coupled with motor1105. The rotating body1110may be a probe head. A permanent magnet moving over a conductive surface generates eddy currents in the material that may counteract the changing magnetic flux. The magnetic fields created by the eddy currents in the material produces a force that pushes against the oncoming magnet in the front and pulls at the magnet as it is moving away. These forces are both in the direction opposite the motion of the magnet, and the result is known as eddy current braking. The motor1005may be selected to counteract any eddy current braking such as, for example, the Nanotec Electronic brushless electric motor model DB42M02 or model DB42C01, which are small, lightweight, and rotate at a high speed. In some embodiments, the motor1105may rotate the rotating body1110at a known rotation rate. In some embodiments, motor1105may spin at speeds up to 500 RPM, 1000 RPM, 2000 rpm, etc. In some embodiments, the rotating body1110may include one or more permanent magnets1115. In some embodiments, the one or more permanent magnets1115may include a Neodymium magnet or any other type of permanent magnet or electromagnet. In some embodiments, the one or more permanent magnets1115may have a 0.25″ diameter, but this diameter may range from 0.01″ to 2″. The one or more permanent magnets1115may have any shape such as, for example, cylindrical, rectangular, toroidal, etc. In some embodiments, the rotating body1110may include one or more pickup coils1125. The one or more pickup coils1125, for example, may be wound by hand or by machine, and/or may have one or more turns, up to several thousand. In some embodiments, the one or more pickup coils1125may be wrapped around the magnet or bobbin. In some embodiments, the one or more pickup coils1125may be mounted near the one or more permanent magnets1115. In some embodiments, the one or more pickup coils1125may be fixed relative to the one or more permanent magnets1115when the rotating body1110is rotating. In some embodiments, the one or more pickup coils1125may not be fixed with respect the one or more permanent magnets1115when the rotating body1110is rotating. In some embodiments, each of the one or more pickup coils1125may include any number of loops of wire such as, for example, between 20 and 3000 loops of wire or between 200 and 2000 loops of wire, or between 500 and 1000 loops of wire. In some embodiments, each of the one or more pickup coils1125may include 0.25″ diameter 40 AWG magnet wire looped or wound around a bobbin or the permanent magnets1115. In some embodiments, the one or more pickup coils1125and the one or more permanent magnets1115may be coupled with the bottom portion of the rotating body1110. In some embodiments, the motor1105may include a precision brushless electric motor (e.g., a model DB42M02 and/or a model N5-2-3 motor controller from Nanotec Electronic Inc.). Use of a brushless motor, for example, may reduce any noise pickup on the signal of interest since brushless motors may produce lower levels of electromagnetic interference than brushed electric motors. In some embodiments, the rotating body1110may include various electronics that are mounted separately from the one or more permanent magnets1115and/or the one or more pickup coils1125. In some embodiments, some of the electronics may be disposed on a nonconductive (e.g., polyimide) plate1175, which may separate the electronics from the one or more permanent magnets1115and/or the one or more pickup coils1125. These electronics may include, for example, an integrator circuit1130, a wireless transmitter1145, batteries1140, digital storage, a microprocessor, a data acquisition unit1135, etc. In some embodiments, the integrator circuit1130may include a two-channel, ultra-stable high gain integrator circuit such as, for example, those produced by Eagle Harbor Technologies. An example integrator circuit is shown inFIG.1.FIGS.12A, and12Bare photographs of example electronics. In some embodiments, the data acquisition unit1135may receive analog integrated voltage signals from the integrator circuit1130. In some embodiments, the data acquisition unit1135may include an analog to digital converter that may digitize analog integrated voltage signals from the integrator circuit. In some embodiments, the data acquisition unit may detect 20 μV per step per reading. In some embodiments, the digitized integrated voltage data may feed into a microprocessor and/or transmitted wirelessly via the wireless transmitter. In some embodiments, the integrator circuit1130may not be used. Instead, the data acquisition unit1135may receive and/or record voltage data directly from the pickup coils. In some embodiments, the rotating body1110may also include a wireless transmitter1145that may be used to communicate data from the rotating body1110to an external device. In some embodiments, the wireless transmitter1145may include Wi-Fi bridge circuit. In some embodiments, the wireless transmitter1145may include Bluetooth circuitry. In some embodiments, the rotating body1110may include batteries1140, rotational sensor1150, and/or various other electronics. In some embodiments, the batteries1140may power the various electronics within the rotating body1110. In some embodiments, the batteries1140, for example, may include lithium polymer, 250 mAh batteries. In some embodiments, the batteries may be coupled with battery charging connectors. In some embodiments, the battery charging connectors may be attached to one side of the integrator circuit1130or rotating housing or shroud. In some embodiments, the rotational sensor1150(e.g., accelerometer) that may record the degree of rotation of the motor. The angle of rotation may be saved with the integrated voltage data to ensure the angle of rotation of the integrated voltage data is recorded, which can be translated into spatial data relative to the sample or another fiducial. In some embodiments, the rotating body1110may include positional tracking system (e.g., a laser system) that may give it spatial awareness. In some embodiments, the rotating body1110may be disposed within a nonrotating shroud. In some embodiments, the shroud may comprise a shatter-resistant polycarbonate material. In some embodiments, the shroud may be designed to protect the components of the rotating body1110while in operation. In some embodiments, the shroud may also protect the device from direct contact with the test material under evaluation. In some embodiments, the data acquisition unit may be coupled with the integrator circuit and/or the wireless transmitter. In some embodiments, leads from the various electronics can be connected to an electrical rotary connector. This may be used in lieu or in conjunction with a wireless transmitter. Since the signal may be very small, a low noise electrical rotary connector may be used. For example, the electrical rotary connector may include a Mercotac Model 430 unit. A picture of the mount setup is shown inFIG.10. In this example, the top of the electrical rotary connector may protrude from the mount piece and/or may have stationary wires, which may carry the voltage signal to a high gain integrator circuit. The NDE device may include a rigid spinning assembly that houses the electronics and/or batteries and the probe head, which contains the magnets and pickup coils. A polycarbonate shroud, not shown inFIG.11,12A or12B, may attach to the shroud support and/or provide support for the motor and spinning assembly and/or may isolate the moving frame from contact with stationary objects. The motor, motor mount, shroud, and/or shroud support may be stationary and/or allow the operator to handle the probe safely. The spinning assembly may be rigid, light, and/or balanced about the motor shaft. The frame of the spinning assembly may be constructed from 6061 aluminum, and/or brass fasteners may be used to minimize the distortion of the magnetic field. In some embodiments, the motor shaft attachment, the first disk below the motor mount inFIG.12B, may be press fit onto the motor shaft. While the motor shaft extends 21 mm from the bottom of the motor, the shape of the motor may be such that the shaft extends 24 mm from the motor mounting location. Thus, the motor shaft attachment part is designed to press fit over the bottom 0.725″ of the motor shaft for maximum alignment and stability. The four large extension rods are screwed to the motor shaft attachment, and the probe head with the magnets and pickup coils are mounted to the end of the extension rods. The extension rods are center bored to the cutting diameter for #6-32 UNC threads to reduce weight. This design allows for easily changing the probe head to characterize different magnet and pickup coil sizes and configurations. In some embodiments, a two-channel dip switch may be included. The electronics for this initial NDE device may be activated by simply setting the sliders in the dip switch to the on position. When the device is not in use or it needs to be charged, the dip switch sliders may be set to off. Other embodiments may use other methods to generate the needed velocity/motion of the permanent magnet and/or the pickup coils relative to the sample. This may include linear or reciprocating motion either along the plane of the surface or perpendicular to the plane of the surface. Other embodiments may include motion of a non-motorized probe head by hand by a technician. Yet other embodiments may include a stationary probe head/assembly, with a sample that is moved relative to it, for example for NDE of materials that are being unrolled/extruded/etc. such as wires, pipes, tubes, etc. In other embodiments, a miniaturized version of the probe may be dropped or guided through narrow tubes, holes, pipes, etc. In some embodiments, the magnet and the pickup coil may be physically attached and co-moving. This simplifies the mathematics of reconstructing the defect shape and location. In other embodiments, it may be advantageous to locate the magnet and pickup coil separately. Alternatively, the magnetic fields could be generated by other means, such as an electromagnet, current flow through the sample, or other means. In some embodiments, there may be multiple pickup coils. Multiple pickup coils may allow for improved spatial resolution or reduced scanning time. In other embodiments, a single pickup coil may be optimal to allow for the smallest possible probe design. The size of the pickup coil may be varied to adjust the sensitivity of the system to defects of varying sizes and at varying depths. In some embodiments, the output data may be dominated by a large sinusoidal signal due to the movement of the magnet and pickup coil relative to the Earth's (or any other) magnetic field as the probe head is moved. Various schemes may be used to eliminate or reduce this sinusoidal signal. For example, two matched coils connected differentially (e.g., opposite polarity, opposite sides of the rotating disk) can be used to cancel out the sinusoidal signal. As another example, the sinusoidal signal can be removed mathematically in post-processing. A sinusoidal signal like this, for example, can be extracted by removing the relevant frequency components with an FFT, since the timescale of the probe head's rotation is slower than the timescale associated with passing over a small defect feature. In some embodiments, the very sensitive nature of the pickup coil can pose problems. For example, due to its proximity to the motor and other moving parts, the motor operation could also be detected by the coil. As another example, coupling to the long output cables going from the coil to the integrator circuit may induce more features into the signal that were not related to the sample being studied. These problems can, for example, be addressed by improving the shielding and/or grounding of the setup. One of the advantages of the rotating probe head, for example, may be the ability to mitigate signal noise by averaging the output of many rotations together. For example, if the probe head is rotated at 100-400 rpm, then scanning for several tens of seconds generates on the order of one hundred samples. These samples, for example, can be synchronized together and/or averaged to allow the real signals to be amplified while the noise and random components can be averaged out. This may improve the signal to noise ratio. As shown inFIG.14A, the raw data may include a repetitive waveform, with each repetition corresponding to one rotation. To synchronize the repetitive waveforms, all of their peaks may be lined up. The largest signal, for example, may be from a fiducial feature. The fiducial feature may, for example, be a non-rotating object (e.g., a metal object) placed close to the probe head. A fiducial, for example, may be a metal object placed on the sample plate near the probe head. Alternately or additionally, the fiducial could be a metal object built into the probe head. The fiducial, for example, may be located at a fixed location relative to where the magnet and/or pickup coil pass on each turn such as, for example, on the probe head housing or shroud or placed on the sample under test. The use of the fiducial may, for example, be useful to help ensure that the signal from each rotation can be aligned (e.g., precisely aligned) to enable statistical averaging of many rotations together. Other techniques could also be used to synchronize the signals from each rotation such as, for example, measuring the rotation mechanically or optically and processing this information together with the signal. In some embodiments, using the fiducial may allow information to be processed with just a single channel of data acquisition. The resulting signal to noise improvement is shown inFIG.8B. The different colored traces show the data from each individual pass, while the black line is the averaged data. This technique, for example, may greatly improve the signal to noise ratio, allowing the method to detect smaller perturbations corresponding to smaller defect sizes. In some embodiments, the integrator circuit inside the NDE device head may include a droop effect which may be achieved by placing a droop resistor across the integration capacitor, shown in the integrator circuit shown inFIG.1(R8and R9). This droop effect can be used to achieve long duration stability of the integrator circuit. The droop RC timescale can be set, for example, to be longer than the RC integration timescale but shorter than the rotation timescale. For example, the integration RC timescale might be 10 μs, the rotation timescale might be 50 ms (1200 rpm), and the RC droop timescale might be 500 μs. In this case, any background effects such as the Earth's magnetic field or the curvature of a curved sample that is being scanned would be subjected to droop, eliminating their contribution to the output signal, while the signal of interest (abrupt edges of cracks, small holes, etc.), would be detected. In some embodiments, an NDE device can be tested using a variety of test samples as well as NDE standards provided by NASA (shown inFIG.15). These test samples may, for example, include flat aluminum plates of varying thicknesses with through holes of varying sizes. These test samples may, for example, include flat aluminum plates of varying thicknesses with holes of varying sizes going part way through the thickness (e.g., not all the way through). These test samples may, for example, include Aluminum plates set side by side, so that the boundary between the two plates forms a “crack”. In some embodiments, the NDE device head may also be tested with other samples including flat plates and sheets, pressure vessels, tubes, pipes, rods, extrusions, and non-uniform structures. These test samples may, for example, include rib-stiffened plates, represented by a typical geometry from Russian International Space Station (ISS) hardware with a small-rib wall separation (˜3-inch square, 1/16-inch thin wall). This wall type is found on the Russian SM (Zvezda) and FGB (Zarya) modules. In the case of the SM, the waffle pattern was on the inside of the module; for the FGB, the pattern was on the outside of the module. Standard 1A had flaws generated on the waffle side of the plate, while Standard 1B had the flaws place on the smooth side. To emulate cracks and pits in the FGB and SM standards, EDM notches 0.100 inch long and either 0.010 or 0.020 inch deep were manufactured. To emulate pits, partial through-the-thickness holes were drilled with a 3/64-inch-diameter drill to depths of 0.010 and 0.020 inch. These flaws were mostly located adjacent to the ribs of the waffle pattern. These test samples may, for example, include a flat aluminum 3/16-inch plate that contained a row of EDM notches two inches from one edge and a row of partial through-the-thickness holes 2 inches from another edge. The EDM notches may range from depths of 0.025 to 0.125 inch and lengths of 0.032 to 0.094 inch. The holes had diameters of 1/32 to 3/32 inch and depths of 0.025 to 0.100 inch. These test samples may, for example, include plates with a radius of curvature of 25 inches. The purpose of these standards was to demonstrate the ability of the NDE method to correctly handle the small radii of curvature found in some parts of the ISS modules. The flat plates are relatively simple to evaluate as the probe head can be placed close to the surface and maintain a constant separation distance throughout the scan. This was accomplished with simple flat plates produced by the sample flat plates. For the curved plate, difficulties arise because the probe head is not small compared to the scale of the plate's curvature. Therefore, as the magnet and pickup coil rotate around the axis of the probe head, their distance relative to the plate will change. This will impose a large sinusoidal signal on the output. However, this signal can be ignored since its timescale will vary significantly from any expected defects, as with the sinusoid due to the Earth's magnetic field described above. The other issue imposed by the plate's curvature is that the probe head will be farther away from the plate's surface through much of its rotation, attenuating the output signal. Therefore, it is simply a matter of verifying that there is still a sufficient signal to noise ratio to detect the defects of interest. The ribbed plate posed the greatest difficulty because the plate is fundamentally not uniform. As the probe head rotates over the surface, the ribs and rib intersections will each generate unique signals, which will overlay with the signals arising from any defects. Without an accurate simulation of the expected signal as a function of the sample geometry, the simplest way to try to identify defects is to scan a defect-free reference plate at the exact same location and save that signal, then subtract it from the signal obtained from scanning a plate with a suspected defect. Any difference between the test signal and the reference signal will therefore correspond to a defect, but it will be necessary to have accurate reference data at each location where the sample is to be evaluated. Alternatively or additionally, in some embodiments, if the spatial resolution of the NDE device can be made high enough, it may be possible to image the plate and simultaneously see the ribs in the plates as well as defects near them. Bench testing included scanning for simple through hole defects, scanning for divots as in the NDE sample plates, scanning for cracks, and evaluating the effect of rotation speed on defect detection depth. Through hole defects, along with cracks, are among the easiest kind to detect since they generate the largest signals. Therefore, through holes were the first type of defects that were used to test the system. The signal amplitude changes both as a function of the hole diameter and the plate thickness.FIG.16shows a waveform where the probe head was passed over a ¼″, ⅛″, 1/16″, and 1/32″ hole in succession. The plots inFIG.17AandFIG.17Bshow the scaling of the signal-to-noise ratio as a function of the hole size and the plate thickness. Clearly, larger holes are easier to detect than smaller ones. Reduced signal-to-noise ratio occurs with thicker plates. While the signal amplitude remained approximately constant regardless of plate thickness, the observed noise increased greatly with thicker plates. The source of the noise remains uncertain; however, it could be that the noise is not really “noise” but real signals resulting from variations/imperfections in the plate material, which have a greater impact for a thicker plate. This effect can be investigated further and may be useful in determining impurity or thickness variations in the manufacturing process. Waveforms showing how a defect signal scales with the depth are shown inFIG.18. The large synchronization signal in each waveform is a detection of a fiducial used for synchronization, which may be held at a fixed position relative to the probe head. The smaller feature later in time is a defect. In some embodiments, the defect signal can have a progressively smaller amplitude for deeper depths.FIG.19is a plot showing the scaling of signal amplitude as a function of the defect depths in the plate. In some embodiments, there may be a dependence on the rotation rate of the probe head. For example, faster rotation rates generate larger signals due to the increased dB/dt while background noise and effects may have a fixed amplitude, thus improving signal to noise ratio. Faster rotation rates may generate larger eddy currents. However, as the rotation rate is increased, eventually the magnetic fields may not be able to penetrate all the way through the material due to the skin depth limitation. By adjusting the rotation rate, the probe head can sweep through a range of depths that the signals are able to penetrate, providing information about the defect depth and/or the thickness of the sample as shown inFIG.20. FIG.20is a plot that shows the effect of rpm on signal size for through hole defects. It may be possible to achieve greater rotation rates (e.g., up to 4000 rpm or more) by improving probe head balance and/or avoiding the use of a mercury contactor by either storing data locally onto an SD card on the probe head and then retrieving it afterwards or by transmitting the data wirelessly as it is collected. FIG.21shows an example plot of a crack detected in a sample according to some embodiments. The signal waveform when passing over a hole defect looks like approximately 2 periods of an oscillation, while the waveform when passing over the crack is more like 1 period. FIG.22shows two example plots of a crack detected in a sample according to some embodiments. The plot on the left shows the detection of a crack defect measured at the sample surface. The plot on the right shows the detection of a crack through a ½″ thick Aluminum plate. As shown in this example, going from detecting the crack at the sample surface to detecting the crack through a ½″ plate reduced the signal amplitude by less than a factor of 2. FIG.23shows two plots detecting two different standard divots of different sizes. These standard divots include a flat plate with a series of divots of decreasing size (e.g., 213A12N00551 NDE Standard 2). The large features show the NDE device detecting the edges of the plate. The small perturbation between the two large features is the defect. Despite the defect appearing small on this scale, it appears with excellent signal to noise ratio. An imaging algorithm may compute the position of the pickup coil as a function of time throughout its rotation or translation. After one set of rotations or movements is complete, the probe head may be moved relative to the sample by a fixed increment, and another set of movements may be carried out. In this way, large areas can be scanned. In some embodiments, the position tracking could be accomplished with an optical system or an encoder wheel. FIG.25shows the imaging results from the ⅛″ thick aluminum plate shown inFIG.24having a series of through holes of progressively smaller size from ¼″ to 1/16″ diameter. The top image inFIG.25shows the signal amplitude as a function of x,y position while the bottom image shows the derivative of the signal amplitude, which may highlight the defects more clearly. There are some interesting artifacts present. For example, the circular or linear path over which the probe head rotates is discernible. In some embodiments, scanning of defects under a pressure wall repair kit (PWRK) patches can occur. These patches typically consisted of a section of metal tape with a rubber seal in the middle, which covers the damaged region and prevents the tape from being cut against the potentially sharp defect edges. In some embodiments, an NDE device can detect and image defects while rotating at a rate of only several hundred rpm, meaning that magnetic fields can pass much deeper into the material without reaching the skin depth limitation. FIG.26shows a “mock-up” of a PWRK patch that includes Aluminum foil with a ⅛″ thick rubber seal. This patch was adhered on the surface of a 1/16″ thick Aluminum plate, centered over a ⅛″ through hole. In some embodiments, the spatial resolution of a system may be low compared to conventional eddy current tools. In some embodiments, an NDE device can detect and image a defect through the PWRK patch, as shown inFIG.27. In some embodiments, the defect shape may be better resolved after the spatial resolution is enhanced. In some embodiments, the system may include an imaging algorithm based on 2D and 3D finite element solvers. The finite element solvers may simulate the induced voltage on a pickup coil travelling under a permanent magnet over a defect in a conductive material. These simulation results can be used to predict the actual signal from the NDE device for a given defect. In conjunction with a database of resultant signals obtained from the NDE device for known defect sizes and depths. In some embodiments, a machine learning algorithm may be used to accurately estimate the size and location in three dimensions of an unknown defect.FIG.28is a plot of a 2D finite element simulation of a surface defect in aluminum. In some embodiments, an NDE device can include a stand-alone and/or handheld device. Data, for example, may be transmitted wirelessly to avoid the issue of feeding out signals from a rotating probe head to non-rotating electronics or data may be fed out through a data cable.FIG.29andFIG.30show an example handheld device. Some embodiments may provide increased spatial resolution of the NDE device as well as improved sensitivity to smaller defects. This may be achieved by reducing the size of the magnet and/or pickup coil, and/or reducing the rotation radius. Decoupling the magnetic field source from the detector may enable other possibilities in NDE device design. For example, another way to increase spatial resolution may be to attach multiple small pickup coils to a single large permanent magnet. By varying the size of the pickup coils, down to about 0.020″ diameter, separately from the size of the magnet, it may be possible to maintain deep scanning penetration depths (since the depth is dependent on the shape of the dipole field generated by the permanent magnet) while enhancing the spatial resolution, which depends on the size of the pickup coil. In some embodiments, an NDE device may have faster rotation, which may aid in improving spatial resolution. In some embodiments, a NDE device that oscillates back and forth may be used, for example, using a piezo-electric actuator. In some embodiments, a position encoding system to the NDE device may also be included so that scans of samples over large areas can be performed. In some embodiments, an optical position encoding system that does not require a reaction force may be considered. FIGS.29and30show an example design of a handheld NDE device. In some embodiments, the interior may include a rotation apparatus to which varying probe heads may be attached. The varying probe heads may, for example, allow optimization for different applications as needed, depending on the sample material, sample thickness, and/or the nature of the defects of interest. In some embodiments, the magnets may rotate at a constant, trackable speed generating desired eddy currents within the material. In some embodiments, the supports may allow the NDE device to evenly rest on the surface, which may eliminate any motion other than the rotating magnets. This handheld NDE device may track the motion of NDE devices being swept over a surface. Additionally or alternatively, by sweeping the NDE devices at a constant rate over the same test spot over and over again multiple samples may be acquired in a short period of time allowing for a wide range of statistical analysis to be conducted providing for higher resolution measurements. In some embodiments, a probe head may be designed to not require rotation and can simply be moved by hand, when a quicker inspection with less setup time is desired. Methods and systems for non-destructive evaluation (NDE) of structures are disclosed. Some embodiments may include eddy-current based systems and/or method that are able to circumvent some of the limitations of other NDE based tools. For instance, conventional eddy current methods use a single coil which may be driven by an AC waveform which induces eddy currents in the sample under test. The changing inductance of the sample at the locations of discontinuities affects the amplitude and phase relations of the current and voltage waveforms in the AC coil, and it is this information that is used to infer the presence and geometry of defects. In some embodiments, the physical movement of a permanent magnet may be used to generate eddy currents in the sample, and a separate inductive pickup coil, which may be fixed in location relative to the permanent magnet, may be used to measure changes in the magnetic field. The signal from the inductive pickup coil may be integrated by a High Gain Integrator, which may allow for very small signals to be measured. Because the pickup coil may be fixed relative to the permanent magnet, the large signals due to the movement of the permanent magnet are not measured. In some embodiments, only the signals that are caused by a discontinuity of the sample being tested are picked up by the coil. In some embodiments, decoupling the source of the magnetic field from the detector in this way may allow for increased detection depth compared to conventional eddy current methods. In some embodiments, the performance of an NDE device can detect defects at greater than a ¼″, ⅜″, ½″, etc. depth. Some embodiments may be able to image a through hole defect directly through a PWRK patch. Unless otherwise specified, the term “substantially” means within 5% or 10% of the value referred to or within manufacturing tolerances. Unless otherwise specified, the term “about” means within 5% or 10% of the value referred to or within manufacturing tolerances. Numerous specific details are set forth herein to provide a thorough understanding of the claimed subject matter. However, those skilled in the art will understand that the claimed subject matter may be practiced without these specific details. In other instances, methods, apparatuses or systems that would be known by one of ordinary skill have not been described in detail so as not to obscure claimed subject matter. Some portions are presented in terms of algorithms or symbolic representations of operations on data bits or binary digital signals stored within a computing system memory, such as a computer memory. These algorithmic descriptions or representations are examples of techniques used by those of ordinary skill in the data processing arts to convey the substance of their work to others skilled in the art. An algorithm is a self-consistent sequence of operations or similar processing leading to a desired result. In this context, operations or processing involves physical manipulation of physical quantities. Typically, although not necessarily, such quantities may take the form of electrical or magnetic signals capable of being stored, transferred, combined, compared or otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to such signals as bits, data, values, elements, symbols, characters, terms, numbers, numerals or the like. It should be understood, however, that all of these and similar terms are to be associated with appropriate physical quantities and are merely convenient labels. Unless specifically stated otherwise, it is appreciated that throughout this specification discussions utilizing terms such as “processing,” “computing,” “calculating,” “determining,” and “identifying” or the like refer to actions or processes of a computing device, such as one or more computers or a similar electronic computing device or devices, that manipulate or transform data represented as physical electronic or magnetic quantities within memories, registers, or other information storage devices, transmission devices, or display devices of the computing platform. The system or systems discussed herein are not limited to any particular hardware architecture or configuration. A computing device can include any suitable arrangement of components that provides a result conditioned on one or more inputs. Suitable computing devices include multipurpose microprocessor-based computer systems accessing stored software that programs or configures the computing system from a general purpose computing apparatus to a specialized computing apparatus implementing one or more embodiments of the present subject matter. Any suitable programming, scripting, or other type of language or combinations of languages may be used to implement the teachings contained herein in software to be used in programming or configuring a computing device. Embodiments of the methods disclosed herein may be performed in the operation of such computing devices. The order of the blocks presented in the examples above can be varied—for example, blocks can be re-ordered, combined, and/or broken into sub-blocks. Certain blocks or processes can be performed in parallel. The use of “adapted to” or “configured to” herein is meant as open and inclusive language that does not foreclose devices adapted to or configured to perform additional tasks or steps. Additionally, the use of “based on” is meant to be open and inclusive, in that a process, step, calculation, or other action “based on” one or more recited conditions or values may, in practice, be based on additional conditions or values beyond those recited. Headings, lists, and numbering included herein are for ease of explanation only and are not meant to be limiting. While the present subject matter has been described in detail with respect to specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily produce alterations to, variations of, and equivalents to such embodiments. Accordingly, it should be understood that the present disclosure has been presented for purposes of example rather than limitation, and does not preclude inclusion of such modifications, variations and/or additions to the present subject matter as would be readily apparent to one of ordinary skill in the art. | 64,800 |
11860127 | DETAILED DESCRIPTION The present disclosure is described in detail below in combination with specific examples. Specific Example I According toFIG.1toFIG.2, the present disclosure provides an eddy current probe based on a Halbach array coil. The present disclosure proposes an eddy current probe based on a Halbach array coil. For the deficiencies of an existing coil structure, the present disclosure provides an eddy current probe design based on a Halbach array coil in the field of high-precision measurement of a micro displacement. On the basis of parallel cylindrical single coils, a coil arrangement in the form of a Halbach array is creatively adopted; by means of arranging permanent magnets in different magnetizing directions according to a certain rule, magnetic lines can be gathered on one side of the magnets, and magnetic lines can be weakened on the other side, so as to obtain an ideal single-sided magnetic field, so that the magnetic field of one side is obviously enhanced; a stronger eddy current effect can be generated; and the measurement precision of the micro displacement and the resolution can be improved. The technical solution adopted by the present disclosure is an eddy current probe design based on a Halbach array coil, and the design is characterized in that firstly, coils are arranged according to a direction of a single-sided magnetic field to be obtained (namely, a displacement measurement direction of a eddy current sensor), as shown inFIG.1. 5 coils with the same winding directions are arranged, as shown in the figure. A first coil1, a third coil3and a fifth coil5are horizontally placed; a second coil2and a fourth coil4are vertically placed; currents are made to different terminals (a current is made to different terminals of the first coil1and the third coil3; a current is made to different terminals of the second coil2and the fourth coil4; and a current is made to the same terminals of the first coil1and the fifth coil5); and a magnetic field in a direction as shown in the figures is generated according to the Faraday's theorem of electromagnetic induction. This coil placement method will generate a strong magnetic field on the lower side (the displacement measurement direction) of the figure, and a simulation result of the magnetic field is as shown inFIG.2. The magnetic field on one side in this arrangement method will be obviously enhanced and can be used as a measurement end of the eddy current probe. The magnetic field intensity enhanced by means of the coil design can generate enhanced eddy current induction inside a measured conductor, thereby improving the measurement precision and resolution of the eddy current sensor. The present disclosure mainly has the advantages that the eddy current probe based on the Halbach array coil used for enhancing the intensity of the magnetic field in the displacement measurement direction is designed; a coil will generate a magnetic field when a current is made to the coil, and the direction of the magnetic field corresponds to a magnet polarization direction of a Halbach array; and the coil is correspondingly placed to achieve an effect of the Halbach array. By means of the designed structure of this coil, the intensity of the magnetic field in the displacement measurement direction can be enhanced, so that the intensity of the magnetic field can be maximized by the smallest number of coils; and the number of magnetic fields on the other side is smaller, so that the influence caused by this side during measurement of a micro displacement can be reduced. By adopting the above-mentioned structural design, the intensity of the magnetic field in the displacement measurement direction is greatly enhanced. In the measurement process, the magnetic field will induce a stronger eddy current effect inside the measured conductor, greatly improving the measurement precision and resolution of the eddy current sensor. As shown inFIG.2, one side with dense magnetic induction lines is used as the displacement measurement direction of the eddy current probe. The eddy current probe is fixed. When the measured conductor moves towards or away from the probe, an eddy current effect may be caused inside the measured conductor. The intensity of the eddy current effect is related to the intensity of the magnetic field at this place. When the measured conductor moves towards the probe, the intensity of the magnetic field increases, and the eddy current effect is enhanced. When the measured conductor moves away from the probe, the intensity of the magnetic field is relatively low, and the eddy current effect is weakened. Since an eddy current is an alternating current, the eddy current may generate an induced magnetic field. The direction of the induced magnetic field is opposite to the direction of the original magnetic field according to the Lenz's law, so as to weaken the original magnetic field of the coil. The size of the induced magnetic field is affected by the size of the eddy current. A displacement of a measured metal object and internal defects may affect the size of the eddy current, thereby affecting the size of the induced magnetic field, and this finally causes the impedance of the coil to change. According to the principle of the eddy current, a displacement of the measured conductor is related to an equivalent impedance of a detection coil; the equivalent impedance Z of the detection coil is related to a geometric parameter of the coil, a frequency f of an exciting signal and a displacement x of the measured conductor and is related to an electrical conductivity σ and a magnetic conductivity μ of the measured conductor; and therefore, the equivalent impedance of the detection coil may be written as: Z=f(Rb,Ra,h,N,f,x,σ,μ) where Rbis an external radius of the coil; Rais an internal radius of the coil; h is a thickness of the coil; and N is the number of turns of the coil. According to the above formula, if the geometric parameter of the coil and the frequency of the exciting signal are fixed, and the electrical conductivity and magnetic conductivity of the measured conductor are also kept unchanged, the equivalent impedance of the coil is only related to the displacement of the measured conductor. The control variate method is used to fix all the parameters except for the displacement of the measured conductor; and a signal conditioning system of the sensor is designed by using a relation that the displacement of the measured conductor and the equivalent impedance of the coil are univalent functions, so as to complete the design of an eddy current displacement sensor. The above is only a preferable implementation of the eddy current probe based on the Halbach array coil, and the protection scope of the eddy current probe based on the Halbach array coil is not limited to the above example. All technical solutions under this idea fall within the protection scope of the present disclosure. It should be pointed out that for those skilled in the art, several improvements and changes made without departing from the principle of the present disclosure shall also be regarded as the protection scope of the present disclosure. | 7,222 |
11860128 | DETAILED DESCRIPTION In an embodiment, the present invention provides, for controlling a process relying on the knowledge of the inhomogeneity status of a liquid medium in a vessel, a system and a method that allows to determine the inhomogeneity of a medium in a simple, robust, and cost-effective way. The described embodiments similarly pertain to the method for measuring an inhomogeneity of a medium, the measurement system, the control unit, the use of the measurement system, the computer program element and the computer-readable medium. Synergetic effects may arise from different combinations of the embodiments although they might not be described in detail. Further on, it shall be noted that all embodiments of the present invention concerning a method, might be carried out with the order of the steps as described, nevertheless this has not to be the only and essential order of the steps of the method. The herein presented methods can be carried out with another order of the disclosed steps without departing from the respective method embodiment, unless explicitly mentioned to the contrary hereinafter. Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used. In this disclosure, the terms “ultrasound emitter” and “ultrasound receiver” are used. If no distinction between emitter and receiver is necessary, also the more general terms “sensor” or “transducer” are used. Sensors or transducers may be devices that emit and/or receive ultrasound signals. According to a first aspect, a measurement system for measuring an inhomogeneity of a medium in a vessel is provided. The system comprises a first ultrasound emitter configured to send a first ultrasound signal along a first path, a second ultrasound emitter configured to send a second ultrasound signal along a second path different from the first path, a first ultrasound receiver to receive the first ultrasound signal sent by the first ultrasound emitter, and to measure a first measurement parameter p1of the received first ultrasound signal, a second ultrasound receiver to receive the second ultrasound signal sent by the second ultrasound emitter, and to measure a second measurement parameter p2of the received second ultrasound signal. The measurement system further comprises a control unit, which is configured to receive the first measurement parameter p1from the first ultrasound receiver, receive the second measurement parameter p2from the second ultrasound receiver, determine the ratio p1/p2of the first measurement parameter p1to the second measurement parameter p2, compare the ratio p1/p2with a ratio h/D of a distance h covered by the first ultrasound signal along the first path to the distance D covered by the second ultrasound signal along the second path. The control unit is further configured to control a process, e.g. a mixing process or a chemical process, based on the comparison of the ratio p1/p2with the ratio h/D. The non-invasive measurement system allows thus to measure the homogeneity of the medium with only few measurements and only few mathematical operations by using one reproducible quantity. The distances h and D may be known from the known specifications of the vessel, e.g., the diameter of the vessel, known co-ordinates of the positions of the ultrasound transducers or sensors, respectively, or a known level height in case that the signal is reflected at the surface of the medium. Advantageously, the ratio h/D may be determined by a calibration measurement, which is performed using a homogeneous medium. If h/D does not change from one measurement to another, e.g. if they depend only on fixed vessel and geometry data, the ratio h/D may be stored in a non-volatile memory. Otherwise, e.g., when h depends on a level height that may vary from one measurement to another, the calibration may be performed as needed. It is to be noted, that “a second path” means, that the system may comprise also a third path. Generally, n different paths may be provided. According to an embodiment, the control unit is further configured to determine a comparison value based on the ratio p1/p2and the ratio h/D and to control a process, as for example a mixing process, if the comparison value or a suitable measure characterizing the deviation of the two values is above a predetermined threshold. The process may be continued if the comparison value is above the threshold. If the comparison value is equal or below the threshold, the mixing process may be considered to be finished and may thus be stopped. The comparison value may be determined, for example, by subtracting p1/p2from h/D, e.g., diff=p1/p2−h/D, or by determining a value k the ratio in t1/t2=k*h/D, and comparing k to 1. If k=1, the velocities of sound along the two paths are equal. The threshold may then be compared to diff or to k. If the distances D and h are constant, also their relation h/D is constant. In this case, instead of comparing p1/p2and h/D, the difference between p1/p2of a measurement and a subsequent measurement may be determined and compared to a threshold. This means that in this case, h and D have not to be known. However, due to the flat, asymptotic curve, this variant is not as accurate as the comparison with respect to h/D. The control unit has preferably a calculating unit. For example, the calculating unit calculates the ratios p1/p2and h/D, subtracts the ratios from each other, and compares the absolute value of the subtraction with a configurable threshold. If the value is smaller than the threshold, the control unit may stop the process, e.g., a mixing process, and indicate this event, for example optically, acoustically, or by sending a signal to a server that is connected to the control unit. Alternatively, the control unit sends the measurement data to, e.g., a central server that performs the calculations so that the control unit can be kept simple and at low costs. Such an arrangement may be useful when, for example, a large number of processes has to be performed in parallel. Thus, the control unit is responsible for starting, performing, and stopping the measurements, and eventually for communication and indication. Further, it may control the process itself, i.e. the controlling of the motor that drives, e.g., the rotating blades. “Controlling a process” in this context may be, for example, controlling a mixing process with respect to a phase change in the medium, or a temperature difference, or a chemical reaction. Controlling a process implies also the monitoring of the corresponding parameters, as for example the inhomogeneity status, the temperature differences, etc. A material has some specific characteristics with respect to ultrasound, as for example velocity and attenuation. The velocity in turn is temperature dependent, so that the times of flight are indicative of inhomogeneity and of temperature differences. Therefore, the following parameters are usable as measurement parameters. According to an embodiment, the first measurement parameter p1is a first time of flight t1in the medium, and the second parameter p2is a second time of flight t2in the medium. Since the velocity of the ultrasound waves depend on the material and the temperature of the medium, the temperature should preferably be equal in the tank or vessel when measuring the inhomogeneity. Vice versa, when measuring temperature differences, the medium should be in a homogeneous state. The term “time of flight” relates to the time of flight in the medium and is used in this sense in the present disclosure. Other effects, e.g., the time needed for the signal to pass through the wall of the vessel and runtimes inside the sensors have to be taken into account in the measurement, i.e., for example, have to be subtracted from the measurement value. The first emitter and the second emitter may be included in one single transducer or in different transducers. Similarly, the first receiver and the second receiver may be included in one single transducer or in different transducers. Further, any combination thereof may be possible. Especially, in an example, the first and the second emitter, and the first and the second receiver are integrated in one single transducer. Furthermore, different signals and paths may be realized by different excitation modes of the ultrasound waves and/or frequencies. According to a further embodiment, the first measurement parameter p1is a first attenuation a1of the ultrasound signal related to the emitted and the received first ultrasound signal, and the second parameter p2is a second attenuation a2of the second ultrasound signal related to the emitted and the received second ultrasound signal. The attenuation therefore may be defined as the ratio of the excitation voltage amplitude to the received voltage amplitude. The attenuation is experienced by the ultrasound when hitting transitions from one substance of the medium to a further substance or one of the further substances, respectively. Here again, it may be necessary to subtract the attenuation in the transducer and the vessel wall to correct the measured values. According to an embodiment, the control unit is further configured to monitor and control a chemical reaction, a phase change in the medium, a mixing process, and/or a temperature difference of the medium. Further, it can be used for heating or cooling the medium at different locations, which may be important for chemical reactions. It may further be used for adaptation of the process time, e.g., in diffusive processes, etc. For that, the control unit may, for example, drive the motor at a higher or a lower rotation speed, dependent on the requirements, such as processing time. The controlling of, e.g. the rotation speed, may be supported by a feedback loop. The feedback loop may comprise data or signals from further sensors. For example, a further ultrasound sensor may be used to monitor the distances h or D. Further sensors may monitor the temperature, pressure or other parameters. According to an embodiment, the first path is a path in vertical direction and the second path is a path in horizontal direction. Other directions and additional measurements in these or other directions are possible. In the case of a vertical and horizontal direction, the emitter and the receiver of one of these directions can be co-located, i.e., they may be arranged at the same position, e.g. in the same housing using the same piezo (same transducer is used) or different piezos (same transducer, but different emitter/receiver). Therefore, according to an embodiment, the first ultrasound emitter and the first ultrasound receiver are co-located, and the first ultrasound signal is reflected at the wall of the surface of the medium. Further, the second emitter and the second receiver are co-located, and the second ultrasound signal is reflected at the wall of the vessel. The evaluation can be generalized to a larger number of sound paths along the same or further directions. In one example, instead of measuring the time-of-flight between two transducers, two sensors may be arranged at further locations, opposite of each other. The first sensor emits the ultrasound signal. If a first time of flight is measured by the second sensor between the first and second sensor, and a second time of flight of the echo of the reflected pulses is measured by the first sensor, the propagation times in the transducer and the wall can be eliminated. According to a second aspect, as explained further above, the described measurement system is used to determine temperature differences in the medium, density differences, and/or sedimentation. The system therefore further allows for quantifying, e.g. sedimentation or temperature differences which also lead to different times of flight of the two beams. Furthermore, the system is capable of measuring the composition, temperature, grain size or other information by analyzing the response signal in a usual way, e.g. using one of the sound paths. According to a third aspect, a usage of a measurement system for monitoring and controlling a chemical reaction, a phase change in the medium, a mixing process, and/or a temperature difference in a vessel as described above is provided. Some examples for controlling a process are given in the description above. According to a fourth aspect, a method for measuring an inhomogeneity of a medium in a vessel is provided, wherein measurement parameters of a first and a second ultrasound signal running through a medium inside a vessel are measured. In a first step, a first measurement parameter p1of the first ultrasound signal along a first path is measured. In a second step, a second measurement parameter p2of the second ultrasound signal along a second path different from the first path is measured. In a third step, the ratio p1/p2of the first measurement parameter p1to the second measurement parameter p2of the second ultrasound signal is determined. In a fourth step, the ratio h/D of a distance h covered by the first ultrasound signal along the first path to the distance D covered by the second ultrasound signal along the second path is determined. In a fifth step, the ratio p1/p2is compared with the ratio h/D, and in a last step a process is controlled based on the comparison of the ratio p1/p2with the ratio h/D. The comparison of the ratio p1/p2with the ratio h/D may further comprise determining the difference between or the ratio of the ratio p1/p2and the ratio h/D, and comparing the difference or the ratio to a threshold. Further embodiments of the method correspond to the measurement system described above. The proposed method does not require specific system properties as an input as, for example material properties of the vessel or the vessel content. According to a further aspect, a control unit is provided. The control unit is configured to receive a first measurement parameter p1from a first ultrasound receiver detecting a first ultrasound signal, which has been travelling along a first path, to receive a second measurement parameter p2from a second ultrasound receiver detecting a second ultrasound signal, which has been travelling along a second path different from the first path, to determine the ratio p1/p2of the first measurement parameter p1to the second measurement parameter p2, to determine the ratio h/D of a distance h covered by the first ultrasound signal along the first path to the distance D covered by the second ultrasound signal along the second path, to compare the ratio p1/p2with the ratio h/D and to control a process based on the comparison of the ratio p1/p2with the ratio h/D. According to a further aspect, a program element is provided which when being executed by the processor of a measurement system instructs the measurement system to perform the steps of the method described above. According to a further aspect, a computer readable medium is provided on which the program element is stored. The computer program element may be part of a computer program, but it can also be an entire program by itself. For example the computer program element may be used to update an already existing computer program to get to the present invention. The computer readable medium may be a storage medium, such as for example, a USB stick, a CD, a DVD, a data storage device, a hard disk, or any other medium on which a program element as described above can be stored. These and other features, aspects and advantages of the present invention will become better understood with reference to the accompanying figures and the following description. The figures are described in detail in the following. In the examples, the system is explained using time-of-flight as measurement parameter. FIG.1shows a measurement system100according to an embodiment. The measurement system100is arranged at a tank or vessel102which encloses a medium140. For purpose of illustration, the medium140is drawn inFIG.1to be more dense at the bottom of the tank102and less dense near the surface representing an inhomogeneity with different speeds of sound. The measurement system100comprises a first ultrasound emitter104which is co-located with the first ultrasound receiver108, a second ultrasound emitter106which is co-located with the second ultrasound receiver110, and a control unit112. The control unit112is connected to the ultrasound receivers106,110and emitters104,106. The control unit112controls the measurement, for example, by giving a signal to the emitters104,108to emit an ultrasound signal, e.g., in form of one or several short pulses. Emitter104emits a first signal in a first path120in a vertical direction, which is reflected at the surface142of the medium140in the vessel102. The reflected ultrasound signal is detected by the receiver108, which provides the measured time-of-flight t1of the signal to the control unit112. Note that also chirps in frequency can be used and comparison between emitted and received signal frequency allows to determine the time of flight. The measurement may be supported by, e.g., timestamps indicating the exact emitting and receiving time. The timestamps may be inserted into the data stream, or a message, which is sent from the emitter/receiver pair104,108to the control unit112. Similarly, the emitter/receiver pair106,110measures the time-of-flight t2of a second ultrasound signal in a second path122in a horizontal direction. The distances D132and h130may be known, e.g., from manufacturing data of the tank, from other measurements, as for example, time of flight measurements with a homogeneous medium, or from the filling process when filling the medium into the tank. Due to the exemplary density gradient of the substances to be mixed, the velocity of the ultrasound signal in the horizontal direction is constant while the velocity of the first signal in the vertical direction is increasing with height. The effect of different velocities applies to any inhomogeneous mixture or density distribution and is not restricted to the density distribution shown inFIG.1. It has to be noted that the velocity is an average along the path, so that it may happen in some density distributions that the average in one direction is by chance the same as the average in the other direction. To determine an inhomogeneity in such a case, either further paths may be introduced, or a time dependent variation may be detected. FIG.2shows a diagram illustrating the development of the inhomogeneity during a mixing process over time. If the substances would be perfectly mixed, the velocity of the ultrasound signal would be equal in each direction, so that the ratio t1/t2is equal to the ratio h/D resulting in a single value, represented by line202. If, however, the substances are distributed, e.g., as shown in the example ofFIG.1, the velocities in one direction would differ from the velocity in the other direction, so that the time of flight-ratio for a measurement would not correspond to the ratio h/D, resulting, e.g. in point208inFIG.2. The more the medium gets homogeneous during the mixing process, the more t1/t2approaches the constant ratio h/D, which is a asymptote for t1/t2. Therefore, there is no need to determine a fluctuating quantity as, for example, variance, but only the ratios t1/t2and h/D, which simplifies the evaluation. Furthermore, no temporal variation is measured. Also, in the case of oscillations, single values deviating from the absolute h/D-constant indicate an inhomogeneity, so that no long time series for statistical evaluation have to be performed. For example, the difference between t1/t2and h/D can be monitored in a configurable window comprising a few single difference values, in which the probability to detect a value indicating that an inhomogeneity is nearly 100%. FIG.3shows a flow diagram300of the method for measuring an inhomogeneity of a medium. Measurement parameters of a first and a second ultrasound signal running through a medium inside a vessel are measured, comprising the following steps: In302, a first measurement parameter p1of the first ultrasound signal along a first path is measured. In304, a second measurement parameter p2of the second ultrasound signal along a second path different from the first path is measured. In306, the ratio p1/p2of the first measurement parameter p1to the second measurement parameter p2of the second ultrasound signal is determined. In308, the ratio h/D of a distance h covered by the first ultrasound signal along the first path to the distance D covered by the second ultrasound signal along the second path is determined. This determination may be obtained, for example, by a measurement or by retrieving the ratio h/D or the values h and D from a memory. In310, the ratio t1/t2is compared with the ratio h/D. Finally, in312, a process is controlled based on the comparison of the ratio t1/t2with the ratio h/D. If the corresponding path lengths are unknown, the change of the propagation time ratio t1/t2during the process can also be considered as a measure for inhomogeneity. The method may also be applied using the attenuation of the amplitude instead of the time of flight. FIG.4shows a diagram of a first variant of the measurement arrangement depicted inFIG.1, with a single transducer150comprising two emitters and two receivers. Transducer150emits a first signal, which travels along a first path120. The signal is reflected at the surface142of the medium140, the wall of the vessel102, and the bottom of the vessel. Finally, the transducer150receives the first signal again. Furthermore, transducer150emits a second signal, which travels along a second path122. The signal is reflected back at the wall at a point opposite of the transducer and received by transducer150again. FIG.5shows a diagram of a second variant of the measurement arrangement depicted inFIG.1, with two transducers160and162. Transducer160comprises two emitters and transducer162comprises two receivers. The first signal is emitted by transducer160along a path to the receiving transducer162. The second signal is emitted by transducer160along a path to the bottom of the vessel102, where it is reflected such that it arrives at transducer162. FIG.6shows a diagram of a third variant of the measurement arrangement depicted inFIG.1with a single transducer170. In this example, two different paths120and122are realized in a horizontal plane. Each of the two signals is reflected at the wall of the vessel102, and arrives again at transducer170, which receives the signal again. The measurement arrangements in the examples show that many variants, i.e. also many further variants are possible. It is clear, that instead of two different paths, any number of different paths may be realized. In case of more than two paths, a more accurate image of the distribution of the substances of the medium and a more reliable determination, whether the medium is homogeneous or not, can be obtained. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from the study of the drawings, the disclosure, and the appended claims. In the claims the word “comprising” does not exclude other elements or steps and the indefinite article “a” or “an” does not exclude a plurality. A single processor or other unit may fulfill the functions of several items or steps recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. A computer program may be stored/distributed on a suitable medium such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems. Any reference signs in the claims should not be construed as limiting the scope of the claims. While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. It will be understood that changes and modifications may be made by those of ordinary skill within the scope of the following claims. In particular, the present invention covers further embodiments with any combination of features from different embodiments described above and below. Additionally, statements made herein characterizing the invention refer to an embodiment of the invention and not necessarily all embodiments. The terms used in the claims should be construed to have the broadest reasonable interpretation consistent with the foregoing description. For example, the use of the article “a” or “the” in introducing an element should not be interpreted as being exclusive of a plurality of elements. Likewise, the recitation of “or” should be interpreted as being inclusive, such that the recitation of “A or B” is not exclusive of “A and B,” unless it is clear from the context or the foregoing description that only one of A and B is intended. Further, the recitation of “at least one of A, B and C” should be interpreted as one or more of a group of elements consisting of A, B and C, and should not be interpreted as requiring at least one of each of the listed elements A, B and C, regardless of whether A, B and C are related as categories or otherwise. Moreover, the recitation of “A, B and/or C” or “at least one of A, B or C” should be interpreted as including any singular entity from the listed elements, e.g., A, any subset from the listed elements, e.g., A and B, or the entire list of elements A, B and C. | 25,969 |
11860129 | The schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar. DETAILED DESCRIPTION In the following detailed description several specific embodiments of compounds, compositions, products and methods are disclosed. It is to be understood that other embodiments are contemplated and may be made without departing from the scope or spirit of the present disclosure. The following detailed description, therefore, is not to be taken in a limiting sense. All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” encompass embodiments having plural referents, unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements. As used herein, “have”, “having”, “include”, “including”, “comprise”, “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to”. It will be understood that “consisting essentially of”, “consisting of”, and the like are subsumed in “comprising” and the like. As used herein, “consisting essentially of,” as it relates to a composition, product, method or the like, means that the components of the composition, product, method or the like are limited to the enumerated components and any other components that do not materially affect the basic and novel characteristic(s) of the composition, product, method or the like. The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure, including the claims. Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc. or 10 or less includes 10, 9.4, 7.6, 5, 4.3, 2.9, 1.62, 0.3, etc.). Where a range of values is “up to” a particular value, that value is included within the range. Any direction referred to herein, such as “top,” “bottom,” “left,” “right,” “upper,” “lower,” and other directions and orientations are described herein for clarity in reference to the figures and are not to be limiting of an actual device or system or use of the device or system. Devices or systems as described herein may be used in a number of directions and orientations. Disclosed devices advantageously combine high sensitivity with a large dynamic range. The sensitivity of disclosed devices can be enhanced through a number of protocols. One of which is disclosed in U.S. patent application Ser. No. 16/285,304 entitled “THIN FILM BULK ACOUSTIC RESONATOR WITH SIGNAL ENHANCEMENT”, filed on Feb. 26, 2019 and published as United States Patent Publication Number 2019/0187098, the disclosure of which is incorporated herein by reference thereto. Sensors, Devices and Systems The sensors disclosed herein include at least two thin film resonator sensor, such as bulk acoustic wave (BAW) resonator sensors. In disclosed devices the at least two BAW resonator sensors have different sensitivities to the analyte of interest. One sensor can be referred to as the high sensitivity sensor and the other sensor can be referred to as the low sensitivity sensor. It is noted that this terminology is only meant to convey relative sensitivity of the two sensors and does not indicate the sensitivity of the two sensors to any external sensor or device. A BAW resonator sensor includes a piezoelectric layer, or piezoelectric substrate, and input and output transducer. BAW resonator sensors are small sensors making the technology suitable for use in handheld devices. Accordingly, a handheld device for detecting target analytes comprising a sensor described herein is contemplated. Turning now to the drawings with reference toFIGS.1A and1B, general operating principles of an embodiment of a bulk-acoustic wave piezoelectric resonator20used as a sensor to detect an analyte are shown. The resonator20typically includes a planar layer of piezoelectric material bounded on opposite sides by two respective metal layers which form the electrodes of the resonator. The two surfaces of the resonator are free to undergo vibrational movement when the resonator is driven by a signal within the resonance band of the resonator. When the resonator is used as a sensor, at least one of its surfaces is adapted to provide binding sites for the material being detected. The binding of the material on the surface of the resonator alters the resonant characteristics of the resonator, and the changes in the resonant characteristics are detected and interpreted to provide quantitative information regarding the material being detected. By way of example, such quantitative information may be obtained by detecting a change in the insertion or reflection coefficient phase shift of the resonator caused by the binding of the material being detected on the surface of the resonator. Such sensors differ from those that operate the resonator as an oscillator and monitor changes in the oscillation frequency. Rather such sensors insert the resonator in the path of a signal of a pre-selected frequency and monitor the variation of the insertion or reflection coefficient phase shift caused by the binding of the material being detected on the resonator surface. Of course, sensors that monitor changes in oscillation frequency may also be employed in accordance with signal amplification described herein. In more detail,FIG.1Ashows the resonator20before the material being detected is bound to its surface26. The depicted resonator20is electrically coupled to a signal source22, which provides an input electrical signal21having a frequency f within the resonance band of the resonator. The input electrical signal is coupled to the resonator20and transmitted through the resonator to provide an output electrical signal23. In the depicted embodiment, the output electrical signal23is at the same frequency as the input signal21, but differs in phase from the input signal by a phase shift ΔΦ1, which depends on the piezoelectric properties and physical dimensions of the resonator. The output signal23is coupled to a phase detector24which provides a phase signal related to the insertion phase shift. FIG.1Bshows the sensing resonator20with the material being detected bound on its surface26. The same input signal is coupled to the resonator20. Because the resonant characteristics of the resonator are altered by the binding of the material as a perturbation, the insertion phase shift of the output signal25is changed to ΔΦ2. The change in insertion phase shift caused by the binding of the material is detected by the phase detector24. The measured phase shift change is related to the amount of the material bound on the surface of the resonator. FIG.1Cshows an alternative to measuring the insertion phase of the resonator. A directional coupler27is added between the signal source22and the resonator20with the opposite electrode grounded. A phase detector28is configured to measure the phase shift of the reflection coefficient as a result of material binding to the resonator surface. Other BAW resonator phase-shift sensors that may be employed with the signal amplification aspects described herein include those described in, for example, U.S. Pat. No. 8,409,875, which patent is hereby incorporated herein by reference in its entirety to the extent that it does not conflict with the disclosure presented herein. For example, sensor apparatuses may include (i) a sensing resonator comprising binding sites for an analyte; (ii) actuation circuitry configured to drive the sensing resonator in an oscillating motion; (iii) measurement circuitry arranged to be coupled to the sensing resonator and configured to measure one or more resonator output signals representing resonance characteristics of the oscillating motion of the sensing resonator; and (iv) a controller operatively coupled with the actuation and measurement circuitry. The controller can be interfaced with data storage containing instructions that, when executed, cause the controller to adjust the frequency at which the actuation circuitry drives the sensing resonator to maintain a resonance point of the sensing resonator. Accordingly, sensing may be accomplished by actuating the BAW resonator into an oscillating motion; measuring one or more resonator output signals representing resonance characteristics of the oscillating motion of the BAW resonator; and adjusting the actuation frequency of the sensing resonator to maintain a resonance point of the BAW resonator. In embodiments, the frequency at which the actuation circuitry drives the sensing resonator is a frequency of maximum group delay. Such phase detection approaches can be advantageously used with piezoelectric resonators of different resonant frequencies. In various embodiments, BAW resonators for use with the methods, devices, and system described herein have resonance frequencies of about 500 MHz or greater, such as about 700 MHz or greater, about 900 MHz or greater, about 1 MHz or greater, 1.5 GHz or greater, about 1.8 GH or greater, about 2 GHz or greater, 2.2 GHz or greater, 2.5 GHz or greater, about 3 GHZ or greater, or about 5 GHZ or greater can provide enhanced sensitivity when used with amplification element mediated mass loaded, which is described in more detail below. In embodiments, the BAW resonators have resonance frequencies of from about 500 MHz to about 5 GHz, such as from about 900 MHz to about 3 GHz, or from about 1.5 GHz to about 2.5 GHz. Some of such frequencies are substantially higher than frequencies of previously described piezoelectric resonators. The sensing resonators described herein are thin-film resonators (TFR). Thin film resonators comprise a thin layer of piezoelectric material deposited on a substrate, rather than using, for example, AT-cut quartz. The piezoelectric films typically have a thickness of less than about 5 micrometers, such as less than about 2 micrometers, andmay have thicknesses of less than about 100 nanometers. Thin-film resonators are generally preferred because of their high resonance frequencies and the theoretically higher sensitivities. Depending on the applications, a thin-film resonator used as the sensing element may be formed to support either longitudinal or shear bulk-acoustic wave resonant modes. Preferably, the sensing element is formed to support shear bulk-acoustic wave resonant modes, as they are more suitable for use in a liquid sample. Additional details regarding sensor devices and systems that may employ TFRs are described in, for example, U.S. Pat. No. 5,932,953 issued Aug. 3, 1999 to Drees et al., which patent is hereby incorporated herein by reference in its entirety to the extent that it does not conflict with the disclosure presented herein. TFR sensors may be made in any suitable manner and of any suitable material. By way of example, a resonator may include a substrate such as a silicon wafer or sapphire, a Bragg mirror layer or other suitable acoustic isolation means, a bottom electrode, a piezoelectric material, and a top electrode. Any suitable piezoelectric material may be used in a TFR. Examples of suitable piezoelectric substrates include lithium tantalate (LiTaO3), lithium niobate (LiNbO3), Zinc Oxide (ZnO), aluminum nitride (AlN), plumbum zirconate titanate (PZT) and the like. Electrodes may be formed of any suitable material, such as aluminum, tungsten, gold, titanium, molybdenum, or the like. Electrodes may be deposited by vapor deposition or may be formed by any other suitable process. Any suitable device or system may employ a thin film resonator and amplification as described herein. By way of example and with reference toFIG.2, a system for detecting an analyte may include a container10(or more than one container), the thin film resonator20, actuation circuitry22, measurement circuitry29, and control electronics30. A fluid path couples the one or more containers10to the resonator20. The control electronics30are operably coupled to the actuation circuitry and the measurement circuitry. In embodiments, control electronics30are configured to modify the frequency at which the actuation circuitry22oscillates the resonator20based on input from the measurement circuitry29. Still with reference toFIG.2, the container10(or more than one container) may house an amplification molecule, an amplification element-linked second recognition component or components thereof, and optionally one or more of a tag, an analyte molecule, and a first recognition component. Each of these reagents is described in more detail below. Control electronics30may control the flow of such reagents from container10to resonator20; e.g. via a pump, vacuum, or the like. Any suitable control electronics30may be employed. For example, control electronics may include a processor, controller, memory, or the like. Memory may include computer-readable instructions that, when executed by processor or controller cause the device and control electronics to perform various functions attributed to device and control electronics described herein. Memory may include any volatile, non-volatile, magnetic, optical, or electrical media, such as a random access memory (RAM), read-only memory (ROM), non-volatile RAM (NVRAM), electrically-erasable programmable ROM (EEPROM), flash memory, or any other digital media. Control electronics30may include any one or more of a microprocessor, a controller, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or equivalent discrete or integrated logic circuitry. In some examples, control electronics30may include multiple components, such as any combination of one or more microprocessors, one or more controllers, one or more DSPs, one or more ASICs, or one or more FPGAs, as well as other discrete or integrated logic circuitry. The functions attributed to control electronics herein may be embodied as software, firmware, hardware or any combination thereof. Molecular Recognition and Signal Amplification Molecular recognition of a sample comprising a significant background signal may be facilitated by amplification of the signal. The sensors, systems and methods described herein employ a second recognition component comprising an amplification element such as a linked enzyme. The BAW resonator sensors, at the higher frequency ranges described herein, responded very efficiently to mass increase of the sensor surface due to precipitation of a substrate cleaved by an enzyme. Referring now toFIGS.3A-D, schematic drawings illustration enzyme amplification on a BAW resonator are shown. As depicted inFIG.3A, a molecular recognition component100configured to bind to an analyte is immobilized on a surface26of a resonator20. The resonator20having immobilized molecular recognition component100may be contacted with a composition comprising an analyte110, which may bind molecular recognition component100(seeFIG.3B). The resonator20having immobilized molecular recognition component100to which analyte110is bound may be contacted with a composition comprising a second molecular recognition component120linked to an amplification element130such as an enzyme. The second molecular recognition component120is configured to bind to analyte110such that the second molecular recognition component120and linked amplification element130are immobilized relative to the surface26(seeFIG.3C). In the depicted embodiments, a soluble substrate140may be converted by amplification element130to an insoluble product150, which precipitates and accumulates on the surface26of the sensor20, thereby amplifying the mass signal as a function of amount or concentration of bound analyte110(seeFIG.3D). It will be understood that the series of events depicted inFIGS.3A-3Dare shown for purposes of illustration and that any other suitable sequence of events may be employed. For example, the analyte110may be contacted with the second molecular recognition component120(and bound amplification element130) before the analyte (with bound second molecular recognition component) is contacted to the surface26of the resonator20relative to which the molecular recognition component100is immobilized. The substrate140may be present at the time the second molecular recognition component120—amplification element130is added or may be added later. In any case, washing may be performed prior to amplification. Non-limiting examples of target analytes include nucleic acids, proteins, peptides, antibodies, enzymes, carbohydrates, chemical compounds, or infectious species such as bacteria, fungi, protozoa, viruses and the like. In certain applications, the target analyte is capable of binding more than one molecular recognition component. Any suitable molecular recognition component (e.g.,100inFIG.3) may be bound to the surface of a resonator. The molecular recognition component preferably selectively binds to the analyte of interest. By way of example, the molecular recognition component may be selected from the group consisting of nucleic acids, nucleotide, nucleoside, nucleic acids analogues such as PNA and LNA molecules, proteins, peptides, antibodies including IgA, IgG, IgM, IgE, lectins, enzymes, enzymes cofactors, enzyme substrates, enzymes inhibitors, receptors, ligands, kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronic acid, thiol, heparin, polysaccharides, coomassie blue, azure A, metal-binding peptides, sugar, carbohydrate, chelating agents, prokaryotic cells and eukaryotic cells. Any suitable method for immobilizing a molecular recognition component on a surface of a BAW resonator may be used. By way of example, a uniform coating of epoxy silane may be deposited on the sensor surface using a vapor deposition process. Test and reference molecular recognition components, such as antibodies, may then be deposited onto the test and reference resonators using, for example, piezo based nanodispensing technology. Primary amines on the antibodies react with the epoxide groups covalently binding the antibody to the sensor surface. By way of further example, a thiol group, if present, of the molecular recognition component bind to a surface of the BAW RESONATOR. The surface of the BAW RESONATOR may be modified, as appropriate or necessary, to permit binding of the molecular recognition component. Any suitable molecular recognition components, such as those described above, may be used as the second molecular recognition component (e.g.,120inFIG.3). The second molecular recognition component may be linked to any suitable amplification element, such as an enzyme. Preferably, the second molecular recognition component is an antibody and the amplification element is an enzyme. Any suitable amplification element may be linked to the second molecular recognition component. In embodiments, the amplification element is an activatable polymerization initiator, such as a photoinitiator, a chemical initiator, or a thermoinitiator. The polymerization initiator may be activated in the presence of one or more monomers to cause a polymer to graft from the second molecular recognition component. In embodiments, the amplification element is an enzyme. In embodiments, the enzyme is capable of converting a substrate that is soluble in the assay environment to an insoluble product that precipitates on the surface of the sensor. Examples of suitable enzymes include alkaline phosphatase (ALP), horse radish peroxidase (HRP), beta galactosidase, and glucose oxidase. Examples of enzyme/substrate systems that are capable of producing an insoluble product which is capable of accumulating on a surface of a BAW resonator include alkaline phosphatase and 5-bromo-4-chloro-3-indolylphosphate/nitro-blue tetrazolium chloride (BCIP/NBT). The enzymatically catalyzed hydrolysis of BCIP produces an insoluble dimer, which may precipitate on the surface of the sensors. Other analogous substrates having the phosphate moiety replaced with such hydrolytically cleavable functionalities as galactose, glucose, fatty acids, fatty acid esters and amino acids can be used with their complementary enzymes. Other enzyme/substrate systems include peroxidase enzymes, for example horse radish peroxidase (HRP) or myeloperoxidase, and one of the following: benzidene, benzidene dihydrochloride, diaminobenzidene, o-tolidene, o-dianisidine and tetramethyl-benzidene, carbazoles, particularly 3-amino-9-ethylcarbazole, and various phenolic compounds all of which have been reported to form precipitates upon reaction with peroxidases. Also, oxidases such as alphahydroxy acid oxidase, aldehyde oxidase, glucose oxidase, L-amino acid oxidase and xanthine oxidase can be used with oxidizable substrate systems such as a phenazine methosulfate-nitriblue tetrazolium mixture. It will be understood that any type of competition assay may be employed. It will be further understood that the analyte may be modified to include a tag recognizable by the first or second recognition complex, such as a streptavidin tag; biotin tag; a chitin binding protein tag; a maltose binding protein tag; a glutathione-S-transferase tag; a poly(His) tag; an epitope tag such as a Myc tag, a HA tag, or a V5 tag; or the like. It will be further understood that the tag-linked analyte may include a variant or derivative of the analyte. The variant or derivative is a variant or derivative that is selectively recognizable by the first or second molecular recognition component that is configured to recognize the analyte. In some situations, it may be desirable that the variant or derivative analyte have an affinity for the first or second molecular recognition component that is different than the affinity of the non-tag-linked analyte. The variant or derivative of the analyte may be a variant or derivative that allows for ease of manufacture of the tag-linked analyte. For example, the tag-linked analyte may comprise a recombinant polypeptide, etc. When competition assays employing tag-linked analyte molecules are performed, the tag-linked analyte molecule, rather than or in addition to the analyte, may bind a first molecular recognition component immobilized on a surface of a resonator. In disclosed devices the at least two BAW resonator sensors have different sensitivities to the analyte of interest: the high sensitivity sensor and the low sensitivity sensor. The sensitivity of the low sensitivity sensor can be tailored with respect to the high sensitivity sensor by immobilizing on the low sensitivity sensor a molecular recognition component with a lower affinity, a molecular recognition component with a lower capacity, or a combination thereof than is present on the high sensitivity sensor. This can of course also be explained with respect to the high sensitivity sensor, which would have immobilized thereon a molecular recognition component with a higher affinity, a molecular recognition component with a higher capacity or a combination thereof than is present on the low sensitivity sensor. The affinity, capacity, or both of the molecular recognition component can be tailored by modifying the coating (amount, concentration, etc.) of the same molecular recognition component, using a different molecular recognition component, or a combination thereof. In some embodiments, where the coating is modified various parameters can be utilized to affect the relative sensitivities, including for example the concentration of the molecular recognition component in the coating for example. In some embodiments, a filler molecule, which could also be referred to as a non-specific component can be mixed in the coating composition along with the molecular recognition component. The relative amounts of the non-specific component and the molecular recognition component can then be chosen to determine the sensitivity level of the low sensitivity sensor. In some embodiments, different molecular recognition components can be utilized to affect the relative sensitivities. For example, molecular recognition components with different sensitivities to the analyte of interest can be included on the two sensors. For example, a molecular recognition component with a lower affinity for the analyte of interest than that on the high sensitivity sensor can be coated on the low sensitivity sensor and a molecular recognition component with a higher affinity than that on the low sensitivity sensor can be coated on the high sensitivity sensor. The devices, systems, and methods described herein may be employed to detect a target analyte in a sample. The devices may find use in numerous chemical, environmental, food safety, or medial applications. By way of example, a sample to be tested may be, or may be derived from blood, serum, plasma, cerebrospinal fluid, saliva, urine, and the like. Other test compositions that are not fluid compositions may be dissolved or suspended in an appropriate solution or solvent for analysis. Non-limiting examples of target analytes include nucleic acids, proteins, peptides, antibodies, enzymes, carbohydrates, chemical compounds, or infectious species such as bacteria, fungi, protozoa, viruses, pesticides and the like. In certain applications, the target analyte is capable of binding more than one molecular recognition component. The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, assumptions, modeling, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein. Device for Detecting Thyroid Stimulating Hormone (TSH) Table 1 shows the dynamic range of a few central-lab based human TSH tests currently available. Normal range levels for children and adults have been shown to be 0.27-4.2 uIU/ml (95th percentile) (Roche Elecsys TSH Factsheet). TABLE 1Measurement ranges of a few central-lab TSH tests. Only Access TSH (3rd IS) isbased on the 3rd WHO reference TSH preparation. All others are based on the 2nd WHOreference TSH preparation. 6.8 IU/ng conversion is based on the scripps TSH antigen used tomake the BAW sensor assay measurements.MeasuringApproximate rangerange (uIU/ml)(pg/ml) (6.8 IU/mg)Roche Elecsys TSH0.005-1000.735-14705Beckman Coulter Access TSH0.005-500.735-7353(3rdIS)Beckman Coulter Access0.015-1002.21-14705HYPERsensitive hTSHBeckman Coulter Access Fast0.030-1004.41-14705hTSH FIG.4shows a calibration curve of a BAW sensor immunoassay for human TSH. At the upper limit the 1000 pg/ml the frequency is shifting over 350 kHz/sec. At higher concentrations the rapid shift of the BAW sensor becomes difficult to track and the precipitate significantly dampens the resonator. In order to compare with the dynamic range of the Access TSH (3rd IS) a 7 fold increase in dynamic range is needed. While some improvements can be made with improved electronics and software algorithms, this may only amount to a 2 fold increase. FIG.5shows that at high concentrations the tracking algorithm is unable to track the high level of frequency shift employing a system generally as described in U.S. 2015/0377834 A1. Frequency tracking is lost at about 12 seconds. Even if the first 10 seconds are fit, a large amount of variability is observed from part to part due to the large signals and extreme dampening of the resonators. By incorporating two resonators, the dynamic range of the BAW immunoassay may be increased. The first resonator is a high sensitivity resonator capable of generating a response similar to that shown inFIG.4. The second resonator is a lower sensitivity resonator capable of measuring at higher concentration levels without saturating the measurement system. The second resonator is made lower in sensitivity by immobilizing a mixture of specific capture ligand and non-specific filler molecule at a fixed ratio such as 1:4 (or any other suitable ratio). This is effective because, in the enzyme enhanced BAW system, the limit in the dynamic range is not the immunoassay itself, but the ability of the system to track the rapidly shifting resonators. In many cases the immunoassay binds much less than 10% of the total surface capacity. Therefore, when immobilizing a 1:4 mixture of specific to non-specific capture molecules, the resultant signal produced from the resonator should be scaled by the mixture ratio, and therefore increase the dynamic range by the same ratio. When the concentrations exceed the dynamic range of the high sensitivity resonator, the instrument then uses the lower sensitivity resonator to report the concentration value. For intermediate concentrations both resonators could be used to improve precision of the result. As an alternative to mixing, the lower sensitivity resonator can be prepared using a lower affinity antibody. This would effectively reduce the sensitivity by the ratio of the antibody affinities and thereby increase the dynamic range similarly. Thus, embodiments of BULK ACOUSTIC WAVE RESONATOR WITH INCREASED DYANMIC RANGE are disclosed. The implementations described above and other implementations are within the scope of the following claims. One skilled in the art will appreciate that the present disclosure can be practiced with embodiments other than those disclosed. The disclosed embodiments are presented for purposes of illustration and not limitation. | 30,668 |
11860130 | DETAILED DESCRIPTION In one or more embodiments of the disclosed subject matter, an energy storage device, such as a battery cell (e.g., a lithium-ion battery cell), can be non-destructively assessed using ultrasound in order to determine a state of health or other information regarding the interior structure of the energy storage device. In some embodiments, the ultrasonic assessment may occur while the energy storage device is in use (e.g., during charging or discharging of the battery cell) so as to provide real-time information regarding the state of health of the energy storage device in the field. Control or use of the energy storage device may be altered in response to the real-time information, for example, to take corrective action to address imminent failure of the energy storage device. Alternatively or additionally, the information can be used to predict reliability or determine the remaining useful life of a system incorporating the energy storage device, for example, a battery pack with multiple battery cells. In other embodiments, the ultrasonic assessment may occur when the energy storage device is not yet in service, for example, as part of a field return evaluation or manufacturing quality control. In any of the embodiments, health monitoring can be accomplished by monitoring confidence values computed by applying statistical pattern recognition techniques to the transient behavior of battery cells, transient responses, and correlation of the responses with models and validated with experimental data. To perform the ultrasonic assessment of the energy storage device, ultrasonic pulses generated by an ultrasound source (e.g., an ultrasound pulser or transducer) can be focused at specific depths within the energy storage device to be assessed. As the acoustic wave approaches an interface within the energy storage device, the acoustic wave may be partially or totally reflected. Such interfaces may be associated with changes in the internal volume of the energy storage device caused by delamination, voiding, or other phenomena. The intensity of the reflected wave and/or transmitted wave is a function of the acoustic impedance of the interface. The reflected and/or transmitted waves can thus provide a measure of changes in the internal structure of the energy storage device that may be indicative of imminent failure or increasing degradation. Lithium Ion Batteries In one or more embodiments of the disclosed subject matter, the energy storage device is a battery cell, for example, a lithium-ion battery cell. Lithium-ion battery cells are free of many of the deficiencies from which other rechargeable battery cells suffer, such as high self-discharge rates and memory effects due to partial charge and discharge. In addition, because of their high energy density, long cycle life, and battery cell voltage, lithium-ion cells have been adopted for use in portable electronics as well as automotive applications, for example, as an energy storage device for all-electric or hybrid vehicles. FIGS.1A-1Bare schematic diagrams of discharging and charging configurations, respectively, to illustrate various structures associated with a lithium-ion battery cell100. In general, a lithium-ion battery cell100can include one or more cathode layers104, one or more anode layers102, respective current collectors on which the layers102,104are disposed, a separator106, and electrolytes108filling the interior volume of the battery cell100. The electrolytes108can include a mixture of organic carbonate solvents (e.g., ethylene carbonate and dimethyl carbonate) and polymer salts (e.g., LiPF6), which provide conductivity for the transport of lithium ions110between the electrodes102,104. Electrical contact between the internal electrodes102,104is made via anode terminal112and cathode terminal114, respectively, which are disposed external to the interior volume of the battery cell. During the charging process (FIG.1B), a current118is supplied to the cathode terminal114via a charging power source126. Electron flow120is from the cathode terminal114to the charging power source126. The cathode104has a high standard redox potential. As a result the current118and electron flow120, the transition metal of the cathode104is oxidized and lithium ions110bdiffuse through the separator106to the anode102, where they are intercalated into layers of, for example, carbon graphite to form LixC6. During the discharging process (FIG.1A), a load116is connected between the cathode terminal114and the anode terminal112. As a result, the electron flow120is from the anode terminal112to the cathode terminal114while the current118is in the opposite direction. As a result, the transition metal of the cathode104is reduced. In addition, lithium ions110aare deintercalated from the anode102and move though the electrolyte108and the separator106back to the cathode104. It is to be appreciated thatFIGS.1A-1Bare simplified illustrations of the inner workings of a lithium-ion battery cell and that practical embodiments of a lithium-ion battery cell will include more complex arrangements than those illustrated. For example, the cathode and electrode layers can be arranged in an interdigitated fashion, with alternating layers of anode and cathode electrodes in a thickness direction of the battery cell. Such a battery cell200is illustrated schematically inFIG.2A. Thus, the anode layers202are arranged between adjacent cathode layers204, and vice versa. Each anode layer202and each cathode layer204may be connected (in parallel or in series) to the anode terminal212and the cathode terminal214, respectively, which provides electrical connection to a load or charging device located external to the battery cell. A separator layer (not shown) is provided between each adjacent cathode204and anode202. The multi-layered electrode separator structure220sits in an electrolyte solution within an interior volume of the battery cell200and operates in a similar manner as described above with respect toFIGS.1A-1B. For simplicity of illustration and explanation, the electrodes and separator (including their arrangement and number) in the following drawings have been simplified as a number of parallel lines and referred to generally as220. However, other arrangements and configurations for the structure of the battery cell beyond those specifically illustrated and discussed are also possible according to one or more contemplated embodiments. For example, the battery cell may have a so-called button configuration. In the button configuration, the battery cell may be substantially cylindrical with a top surface of the cylinder serving as one electrode terminal and a bottom surface of the cylinder serving as the other electrode terminal. The electrode layers within the button cell can be arranged perpendicular to an axis of the button cell, as annular layers extending along the axis of the button cell, or in any other arrangement. In another contemplated embodiment, the battery cell need not have clearly delineated electrode layers within the interior volume thereof. For example, the interior volume of the battery cell may be filled with a slurry and electrode terminals can extend into the interior volume to provide electrical contact thereto. In another example, the interior volume of the battery cell may comprise lumps of material rather than planar electrodes. Such configurations may suffer from similar degradation as the planar electrode configurations discussed above. One of ordinary skill in the art will appreciate that other battery cell configurations are also possible and will benefit from assessment using the disclosed ultrasonic health monitoring devices and techniques. Despite advances in materials, packaging technologies and state monitoring solutions, many challenges regarding reliable use of lithium-ion battery cells remain. Over the lifecycle of a lithium-ion battery cell, various thermal, mechanical, and electrochemical processes contribute to the degradation of the cell's performance. Lithium-ion batteries generally begin to degrade almost immediately after completion of manufacturing and continue to degrade during storage and use. Degradation due to self-discharge, which occurs during storage as well as during charge/discharge cycling, may depend on the ambient temperature, storage time, and the state of charge of the cell (e.g., fully charged, partially charged, fully discharged, etc.). Additional degradation during charge/discharge cycles can be accelerated based on high-temperature exposure, frequent charge/discharge cycles, deep discharge (i.e., approaching fully discharged before recharging), and overcharge (i.e., charging beyond a designated capacity of the battery cell). Vital interfaces where degradation may occur inside a lithium-ion battery cell include the interface between the metallic current collector and the active material for both the anode and cathode. Internal resistance can increase with complex chemical reactions between the active materials, electrodes, and the electrolyte. Battery materials may also be susceptible to expansion during charging. As a result, the anode or cathode layers within the battery cell may delaminate from their respective current collector, thereby causing a shift in charge and discharge properties of the battery. FIG.2Bdepicts a battery cell200ain an as-manufactured state. Battery cell200athus has a set initial thickness and an ordered arrangement of electrode layers220within the internal volume of the battery cell. However, with continued charge and discharge cycles, swelling, delamination, or other degradation can occur. FIG.2Cshows a battery cell200bthat has degraded after multiple charging/discharging cycles. In particular, the electrode layers220have deformed resulting in electrode ruffling. Volumetric expansion of the electrode particles can cause localized stress concentrations that ruffle the electrode. This can cause a loss of connectivity between the electrode active material particles and the electrically conductive particles included in the electrode matrix. Separation or delamination of the electrode material and the current collector can occur. As a result, the useful capacity of the battery cell decreases due to its reduced charge-transfer capabilities. FIG.2Dshows another battery cell200cthat has degraded after multiple charging/discharging cycles. For example, localized heating and abusive operating conditions can result in the release of gas species that can cause volumetric expansion and/or venting of the cell to relieve internal pressure. If the battery cell is overcharged, the cathode can become unstable and the electrolyte can decompose. Alternatively or additionally, if the battery cell is overdischarged, the current collector (e.g., made of copper) can dissolve and cause internal short circuiting. As a result, local hotspots within the cell may be created that cause a variety of chemical reactions that release gas as a by-product. The gas release can cause ruffling of the electrode layers220, similar to that illustrated inFIG.2C, and may further cause a swelling of a housing of the battery cell200c, thereby resulting a change in thickness of the battery cell200cand/or localized deformation of one or more surfaces of the housing of the battery cell200c. In some cases, swelling of the housing of the battery cell200dmay be present without significant ruffling of the electrode layer220, as illustrated schematically inFIG.2E. In worst case scenarios, rapidly increasing temperatures and excessive gas generation can cause the cell to explode and catch fire. However, many cells exhibit less severe levels of gas generation over their lifetime. This gas generation contributes to internal structural changes to the cell that can degrade the battery over its life cycle. For example, gas pockets in cycled cells can lead to a degradation due to displacement of the electrodes, thereby making it more difficult to transport ions through the electrolyte. Additionally, porosity in the electrode can increase thereby lowering connectivity between adjacent electrode material particles. A significant consequence of this degradation is a drop in the capacity of the battery cell as it is charged and discharged. As used herein, capacity of a battery cell refers to the maximum current that a battery cell can supply over a given time period and is commonly expressed in ampere-hours (Ah). For example, if a battery cell is rated for 1000 mAh, then it should be capable of supplying 1000 mA over a one-hour period before it needs recharging. While capacity rating provides a basis for what type of battery can best meet the needs of a particular system, it only provides a measure of the battery's initial capabilities. Any decrease in capacity due to degradation of the battery cell during storage and/or use will compromise the battery's ability to deliver effectively store and deliver energy. Given the chemical, mechanical and thermo-dynamic processes that occur within the battery cell and contribute to the degradation, the battery cell can be monitored to provide real-time information. For example, metrics of interest for real-time monitoring include amount of degradation in battery capacity as well as, but not limited to, amount of remaining charge in a battery and remaining useful battery life. Knowledge of the internal state of the battery cell can help determine charging times, appropriate discharge strategies, balancing between different cells in a battery pack, and/or thermal management within a battery pack. The current capacity of a battery cell over the as-manufactured or initial capability can be used to define one metric for a battery cell's state of health. The state of health metric reflects the degradation of the battery and/or the battery pack and can be based on a decrease in the available capacity that a battery can deliver or an increase in the internal resistance thereof. The state of health metric can be determined using data obtained by measuring the voltage and current during the charge and discharge stages and estimating, for example, the decay in capacity and state of health based on current, voltage, and temperature measurements. In general, a 20% decrease in deliverable capacity can represent a threshold beyond which the battery performance begins to deteriorate rapidly. While a decrease in the deliverable capacity or an increase in internal resistance may reflect degradation in a lithium-ion battery system, this only relates to reduced performance and does not address safety. Gas generation and changes to the structure of the cell also capture performance-related information as well as information related to unsafe conditions that could be exacerbated as the battery cell is further stressed. Thus, investigation of the internal cell structure can help provide a more complete state of health evaluation that includes decreases in both performance and safety. Battery Health Monitoring Device Embodiments of the disclosed subject matter can use ultrasonic assessment of the internal structure of an energy storage device, for example, a battery cell, in order to provide information regarding a state of health of the assessed battery cell. Such information can be used alone or in conjunction with other state of health metrics to provide a more complete picture of the state of health of a battery cell or a battery pack including multiple battery cells. Ultrasonic assessment detects changes in acoustic impedance, such as an interface between two materials, through measurement of reflected and/or transmitted acoustic signals. Modes of assessment can include a pulse-echo mode, a through-transmission mode, or both. In the pulse-echo mode, a sensor is arranged to detect ultrasound reflected from the interior of the battery cell. In the through-transmission mode, a sensor is arranged to detect ultrasound transmitted through the interior of the battery cell (e.g., by being located opposite the ultrasonic source or on a side of the battery cell opposite the ultrasonic source). In either mode, the source and sensor are configured to generate and detect, respectively, sound having a frequency in the ultrasound range, for example, greater than 1 MHz. These assessment modes can be used to assess, among other things, swelling, electrode expansion, electrode delamination, voiding, and/or electrode ruffling within an assessed battery cell. These degradation mechanisms may be related to degradation resulting from, for example, typical charge/discharge cycling, intermittent operation at an elevated temperature that may be within or above specified operating limits, or mechanical and thermomechanical stresses acting on the cell during its operation. Based on the detected ultrasound, a metric can be provided for the degradation within the battery cell. For example, the amplitude of the reflected pulse and/or transmitted pulse can be used as a metric to assign a degradation level to the electrodes within the battery. Alternatively or additionally, a controller can make a determination about the state of health of the battery cell based on the detected ultrasound alone or along with other information regarding operating characteristics of the battery cell. The ultrasonic source (e.g., a pulser or transducer) can emit pulses of ultrasonic energy at a specific frequency. Selection of the appropriate ultrasonic source and the corresponding assessment frequency can be based on the battery cell to be investigated, including among other things, the thickness of the battery cell, the geometry of the battery cell, and the temperature of assessment. Generally, a lower frequency transducer can be used to penetration into thick, more attenuating, and/or highly scattering battery cell materials, and a higher frequency transducer can be used for thinner, lower attenuating, and/or lower scattering battery cell materials. The ultrasonic source can be provided adjacent to an external surface of the battery cell and arranged to direct the ultrasonic energy into the interior of the battery cell. For example, an emission direction of the ultrasonic energy may be perpendicular to the plane of one or more electrode layers within the interior of the battery cell. The ultrasonic sensor (e.g., a transducer) can detect ultrasonic energy in a broad range of ultrasound frequencies or limited to the specific frequency emitted by the ultrasonic source. As with the ultrasonic source, the ultrasonic sensor can be provided adjacent to an external surface of the battery cell and arranged to detect ultrasonic energy coming from the interior of the battery cell, e.g., reflected or transmitted ultrasound. In some embodiments, the ultrasonic source and the ultrasonic sensor can be parts of a single transducer, for example, for use in a pulse-echo mode configuration. In other embodiments, the source and sensor can be separate components disposed at different portions on the battery cell. The ultrasonic source and/or the ultrasonic sensor can be disposed on the battery cell through a respective couplant. For example, the couplant may be disposed between and in contact with an external surface of the battery cell and a corresponding active surface of the ultrasonic source and/or the ultrasonic sensor. Thus, ultrasound traveling to or from the battery cell would pass through the couplant. The couplant provides a pathway to/from the battery cell for the ultrasound in order to avoid attenuation due to exposure of the ultrasound to air or other high attenuation mediums, which may otherwise compromise assessment of the battery cell. The couplant may be a separate component, for example, an encapsulated gel or gel pad, that is placed between the battery cell and the source or sensor, or attached to the source, sensor, or battery cell surface. Alternatively, the couplant may be integrated with the source or the sensor, e.g., as the material of an emission or detection face thereof. Appropriate couplants can be selected based on acoustic velocity, impedance, and attenuation, as well as other factors, for example, as discussed in “Approximate Material Properties in Isotropic Materials,” published inIEEE Transactions on Sonics and Ultrasonics, May 1985, SU-32(3): pp. 381-94, which is hereby incorporated by reference herein. In some embodiments, the couplant is a hydrocarbon grease. In other embodiments, the couplant is a pad of encapsulated gel. For example, the gel pad includes one or more polymers having an attenuation with respect to the applied ultrasonic frequencies similar to or approaching that of water. The gel can be a dry couplant elastomer comprising a blend of isomers of branched homopolymers with an attenuation of 5 dB relative to water, for example, as described in “Ultrasonic Properties of a New Low Attenuation Dry Couplant Elastomer,” by Ginzel et al., April 1994, which is also incorporated by reference herein in its entirety. Other examples of couplants include but are not limited to the Water Gel Ultrasound Solid Gel Pad by BlueMTech (BlueMTech, Korea) and the AquaFlex® Ultrasound Gel Pad (Parker Laboratories, USA). Other couplants can also be used according to one or more contemplated embodiments. The detected ultrasound from the battery cell may be evaluated as either an A-scan or C-scan. In general, A-scans can provide a determination of the state of health of the battery cell, and C-scan can be used to image an interior of the battery cell, for example, to visualize and locate regions of degradation. As used herein, A-scan refers to an amplitude modulation scan, i.e., the actual waveform of the acoustic signal obtained by the sensor. After application of an ultrasonic pulse to the battery cell, the resulting detected signal can be graphed where the horizontal axis is time and the vertical axis is amplitude. In contrast, C-scan refers to scanning the transducer in one or two dimensions, e.g., raster-scanning the source/sensor over the entire surface area of the battery cell to produce a digitized image. After application of an ultrasonic pulse at each scanned position, the resulting detected signal at a particular time (corresponding to a particular depth in the battery cell, for example, as determined by placement of the data gate) is graphed to provide a map of the interface at that depth. The data gate targets an interface of interest within the thickness of the sample through the assessment of a portion of the corresponding A-scan waveform. For example, the interface of interest can be defined by positioning a rectangular frame over the portion of the A-scan that corresponding with the interface of interest using appropriate image processing software. The larger the time value associated with the data gate on the time axis of the A-scan, the greater the depth of interface of interest within the battery cell. By moving the gate position in time on the A-scan, different depths within the sample can be imaged. If air, gas, or another medium is present at a particular interface, all of the ultrasonic signal from that particular region may be reflected back, which results in detectable areas on a C-scan. The presence of air or gas pockets due to inner delaminated regions or voids will also cause the shape of the A-scan waveforms, which are taken at different points over the interface of interest, to be different in the data-gate region. In cases of extreme delamination or separation between active material and corresponding current collector, a phase inversion of the A-scan signal occurs at the data-gate region. In one or more embodiments, a battery health monitoring device uses transmitted ultrasound to assess the internal volume of a battery cell, as described above, to make a determination regarding the state of health of the battery. For example, the battery health monitoring device300can be configured in a through-transmission mode for interrogating battery cell200, as shown inFIG.3AandFIG.3B. The battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. An ultrasound source304is disposed on a first side of the battery cell200with a couplant306between the battery cell200and the source304. For example, an ultrasonic pulse308from the source304may be directed substantially perpendicular to the plane of one or all electrode layers220within the interior of the battery cell. Alternatively or additionally, at least an emission face of the source304is arranged substantially parallel to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200. The ultrasound sensor310can be disposed on a second side of the battery cell200opposite to the first side and arranged to receive the ultrasonic pulse308transmitted through the interior volume of the battery cell200. Although not shown, the sensor310may also be disposed with a couplant between the battery cell200and the sensor310. The sensor310can be arranged directly opposite to the source304so as to receive ultrasound308traveling in a straight line through the thickness of the battery cell200, i.e., from a first side of the battery cell200facing the source304to a second side of the battery cell200facing the sensor310. A controller312(i.e., control unit) can be provided to control operation of the ultrasound source304and the ultrasound sensor310and to determine a state of health of the battery cell200based on signals from the ultrasound sensor310. For example, the controller312may command the ultrasound source304to apply an ultrasonic pulse to the battery cell200. The ultrasound sensor310can generate a signal based on detected ultrasound and convey the signal to the controller312, which may use the signal to compose an A-scan. The resulting A-scan can be evaluated based on timing and/or amplitude of the detected pulse, for example, to make a determination regarding state of health of the battery cell200. Such evaluation can include, but is not limited to, a comparison of the resulting A-scan with a previously obtained A-scan, which may be an A-scan of the battery cell200that was taken when the battery cell was in an as-manufacture or as-delivered non-cycled state. The controller312can also be configured to control the battery cell200, for example, to alter charging, discharging, or other operations of the battery cell based on its determined state of health. The source304and the sensor310can be disposed to assess one portion of the interior of the battery cell200, for example, where structures within the interior of the battery cell200may be especially susceptible to degradation. Alternatively or additionally, the source304(and couplant306) and the sensor310can move along a length (i.e., from left to right inFIG.3A) and/or a width (i.e., into or out of the page inFIG.3A) of the battery cell200to assess different portions of the battery cell200. For example, the source304and the sensor310can move in order to obtain a C-scan of the battery cell200in order to image a particular defect or degradation. Controller312may control the source304and/or the sensor310, for example, through an appropriate displacement mechanism, to provide the desired movement. In one or more additional embodiments, a battery health monitoring device uses reflected ultrasound to assess the internal volume of a battery cell, as described above, to make a determination regarding the state of health of the battery. For example, the battery health monitoring device400can be configured in a pulse-echo mode for interrogating battery cell200, as shown inFIG.4. As with the previously described embodiment, the battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. A transducer402is disposed on a first side of the battery cell200with a couplant306between the battery cell200and the transducer402. The transducer402includes both an ultrasound source portion404and an ultrasound sensing portion410. Although shown as separate portions, it is contemplated that the source portion404and the sensing portion410can be the same structure, for example, an active portion of the transducer402that serves an emitter when the ultrasound is emitted and then serves as a sensor after the ultrasound pulse is emitted. For example, the ultrasound308(e.g., an ultrasonic pulse) from the source portion404may be directed substantially perpendicular to the plane of one or all electrode layers220within the interior of the battery cell. Alternatively or additionally, at least an emission/detection face of the transducer402is arranged substantially parallel to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200. The sensing portion310receives ultrasound408reflected from structures (e.g., electrode layers220) within the interior volume of the battery cell200. A controller312can be provided to control operation of the transducer402and to determine a state of health of the battery cell200based on signals from the transducer402. For example, the controller312may command the transducer402to apply an ultrasound308to the battery cell200. The transducer402can generate a signal based on detected ultrasound and convey the signal to the controller312, which may use the signal to compose an A-scan. The resulting A-scan can be evaluated based on timing and/or amplitude of the detected energy, for example, to make a determination regarding state of health of the battery cell200. Such evaluation can include, but is not limited to, a comparison of the resulting A-scan with a previously obtained A-scan, which may be an A-scan of the battery cell200that was taken when the battery cell was in an as-manufacture or as-delivered non-cycled state. The controller312can also be configured to control the battery cell200, for example, to alter charging, discharging, or other operations of the battery cell based on its determined state of health. The transducer402can be disposed to assess one portion of the interior of the battery cell200, for example, where structures within the interior of the battery cell200may be especially susceptible to degradation. Alternatively or additionally, the transducer402(and couplant306) can move along a length (i.e., from left to right inFIG.4) and/or a width (i.e., into or out of the page inFIG.4) of the battery cell200to assess different portions of the battery cell200. For example, the transducer402can move in order to obtain a C-scan of the battery cell200in order to image a particular defect or degradation. Controller312may control the transducer402, for example, through an appropriate displacement mechanism, to provide the desired movement. Alternatively or additionally, the transducer402may be fixed while the battery cell200is moved with respect to the transducer402to assess different portions of the battery cell200. For example, the transducer can include a roller or wheel-shaped contact portion, such as but not limited to the Olympus Roller Ultrasonic Transducer (e.g., part number RT-0105-16SY by Olympus Corporation). In one or more additional embodiments, a battery health monitoring device can use transmitted and reflected ultrasound, either simultaneously or sequentially, to assess the internal volume of the battery cell to make a determination regarding the state of health of the battery cell. For example, the battery health monitoring device500, as shown inFIG.5, can combine the through-transmission assessment features of the embodiment ofFIGS.3A-3Band the pulse-echo assessment features of the embodiment ofFIG.4. As with the previously described embodiments, the battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. In addition, as with the previously described embodiments, the device500can be disposed to assess a single portion of the battery cell200, or the device500and/or the battery cell200can be moveable to assess different portions of the battery cell200, for example, to obtain a C-scan. The controller312controls operation of the transducer402and sensor310and determines a state of health of the battery cell200based on signals from the transducer402and sensor310. For example, the controller312may command the transducer402to apply an ultrasonic pulse308to the battery cell200. Transducer402generates a signal based on detected, reflected ultrasound408and conveys a first signal to the controller312, while sensor310generates a signal based on detected, transmitted ultrasound308and conveys a second signal to the controller312. The controller312may use the first and second signals to determine a state of health of the battery cell, for example, by evaluating timing and/or amplitude in the detected signals and/or by comparison to previously obtained A-scans. For example, the controller312can use reflected ultrasound signals to localize degradation planes within the battery cell200while the transmitted ultrasound signals are used to measure the degree of degradation within the battery cell200. The controller312can also be configured to control the battery cell200, for example, to alter charging, discharging, or other operations of the battery cell based on its determined state of health. In one or more additional embodiments, a battery health monitoring device600uses reflected ultrasound to assess the internal volume of a battery cell200, as shown inFIG.6. Similar to the embodiment ofFIG.4, battery health monitoring device600is configured in a pulse-echo mode for interrogating battery cell200. However, the ultrasound source604and the ultrasound sensor610are separate from each other and spaced on a first side of the battery cell200. The ultrasound source604is disposed with an angled couplant606between the battery cell200and the source604. The couplant606supports the source604in an angled configuration with respect to the surface of the battery cell200and/or the electrode layers220therein. Thus, the ultrasonic pulse308from the source604may be directed at an angle (i.e., not perpendicular or parallel) with respect to one or all electrode layers220within the interior of the battery cell. Alternatively or additionally, at least an emission face of the source604is arranged at angle with respect to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200. The ultrasound sensor310can be arranged to receive ultrasound608reflected from structures (e.g., electrode layers220) within the interior volume of the battery cell200. The ultrasound sensor310can be arranged with an active face thereof parallel to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200, as shown inFIG.6. Alternatively, the ultrasound sensor310may have an angled configuration similar to that of ultrasound source604, but shifted to be aligned with a direction of the reflected ultrasound608. As with the previously described embodiments, controller312can be provided to control operation of the source604and the sensor610and to determine a state of health of the battery cell200based on signals from the sensor610. The battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. The source604and the sensor610can be disposed to assess one portion of the interior of the battery cell200, for example, where structures within the interior of the battery cell200may be especially susceptible to degradation. Alternatively or additionally, the source604(and couplant606) and the sensor610can move along a length (i.e., from left to right inFIG.6) and/or a width (i.e., into or out of the page inFIG.6) of the battery cell200to assess different portions of the battery cell200. For example, the source604and the sensor610can move in order to obtain a C-scan of the battery cell200in order to image a particular defect or degradation. Alternatively or additionally, the angle of the source604(or the angle of the sensor610, when angled) can be varied to assess different portions of battery cell200. Controller312may control the source604and/or the sensor610, for example, through an appropriate angling and displacement mechanism, to provide these desired movements. In one or more additional embodiments, a battery health monitoring device700uses transmitted ultrasound to assess the internal volume of a battery cell200, as shown inFIG.7. Similar to the embodiment ofFIG.3A, battery health monitoring device700is configured in a through-transmission mode for interrogating battery cell200. However, the ultrasound source604and the ultrasound sensor710are spaced from each other in a length direction of the battery cell. The ultrasound source604is disposed with an angled couplant606between the battery cell200and the source604. The couplant606supports the source604in an angled configuration with respect to the surface of the battery cell200and/or the electrode layers220therein. Thus, the ultrasonic pulse308from the source604may be directed at an angle (i.e., not perpendicular or parallel) with respect to one or all electrode layers220within the interior of the battery cell. Alternatively or additionally, at least an emission face of the source604is arranged at angle with respect to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200. The ultrasound sensor710can be arranged to receive ultrasound308transmitted through the interior volume of the battery cell200. The ultrasound sensor710can be arranged with an active face thereof parallel to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200, as shown inFIG.7. Alternatively, the ultrasound sensor710may have an angled configuration parallel to that of ultrasound source604so as to be aligned with a direction of the transmitted ultrasound608. As with the previously described embodiments, controller312can be provided to control operation of the source604and the sensor710and to determine a state of health of the battery cell200based on signals from the sensor710. The battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. The source604and the sensor710can be disposed to assess one portion of the interior of the battery cell200, for example, where structures within the interior of the battery cell200may be especially susceptible to degradation. Alternatively or additionally, the source604(and couplant606) and the sensor710can move along a length (i.e., from left to right inFIG.7) and/or a width (i.e., into or out of the page inFIG.7) of the battery cell200to assess different portions of the battery cell200. For example, the source604and the sensor710can move in order to obtain a C-scan of the battery cell200in order to image a particular defect or degradation. Alternatively or additionally, the angle of the source604(or the angle of the sensor710, when angled) can be varied to assess different portions of battery cell200. Controller312may control the source604and/or the sensor710, for example, through an appropriate angling and displacement mechanism, to provide these desired movements. In one or more additional embodiments, a battery health monitoring device800uses transmitted ultrasound to assess the internal volume of a battery cell200, as shown inFIG.8. Similar to the embodiment ofFIG.3A, battery health monitoring device800is configured in a through-transmission mode for interrogating battery cell200. However, an array810of ultrasound sensors310is provided on the opposite side of the battery cell200from the ultrasound source304. For example, the ultrasonic pulse308from the source304may be directed substantially perpendicular to the plane of one or all electrode layers220within the interior of the battery cell. Alternatively or additionally, at least an emission face of the source304is arranged substantially parallel to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200. An ultrasound sensor310can be arranged to directly opposite the ultrasound source304so as to receive the ultrasound pulse308transmitted through the interior volume of the battery cell200. The remaining ultrasound sensors310of the array810can be arranged to receive any spreading808of the ultrasound308, for example, due to expansion of the ultrasound wave in the interior of the battery cell200and/or scattering or deflection by internal structures (e.g., electrode layers220) of the battery cell. Each ultrasound sensor310can be arranged with an active face thereof parallel to an external surface of the battery cell200and/or the plane of one or all electrode layers220within the interior of the battery cell200, as shown inFIG.8. As with the previously described embodiments, controller312can be provided to control operation of the source304and the sensors310and to determine a state of health of the battery cell200based on signals from the sensors310. The battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. The source304can be disposed to assess one portion of the interior of the battery cell200, for example, where structures within the interior of the battery cell200may be especially susceptible to degradation. Alternatively or additionally, the source304(and couplant306) and, optionally one or more of sensors310of the array, can move along a length (i.e., from left to right inFIG.8) and/or a width (i.e., into or out of the page inFIG.8) of the battery cell200to assess different portions of the battery cell200. For example, the source304can move with respect to the sensor array810, with different sensors310of the array serving as the sensor directly opposite the source304as the source304moves, in order to obtain a C-scan of the battery cell200and to image a particular defect or degradation. Controller312may control the source304and/or the sensor array810, for example, through an appropriate displacement mechanism, to provide these desired movements. In an alternative embodiment, multiple sources304can be provided as an array904similar to the array810of sensors310, as illustrated in the battery health monitoring device900ofFIG.9. In such a configuration, each source304of the source array904may be arranged opposite to and correspond to a particular sensor310of the sensor array810. To prevent cross-talk between adjacent sensors310, each source304may be activated separately such that the corresponding through pulse308is detected by the corresponding sensor310before the next source304in the array is activated. Alternatively, sources304with sufficient spacing between each other may be activated at the same time, where the spacing minimizes the amount of cross-talk that may be detected by the corresponding sensors310. For example, every other source304in array810may be actuated at the same time. In yet another alternative, sources304may be simultaneously activated and the sensors310of the array810simultaneously sampled and the resulting signals used in combination by the controller312to determine a state of health. In still another alternative embodiment, multiple sources can be provided as a source array similar to that illustratedFIG.9, but with multiple sensors corresponding to each source of the source array. Thus, the number of sensors in the sensor array may be more than the number of sources in the source array. For example, each source can have a primary sensor disposed directly opposite thereto and secondary sensors with the primary sensor therebetween, but the secondary sensors do not have a source disposed directly opposite thereto. Activation of each source may be sequential or simultaneous, for example, as described above with respect toFIG.9. In one or more additional embodiments, a battery health monitoring device1000uses reflected ultrasound to assess the internal volume of a battery cell200, as shown inFIG.10. Similar to the embodiment ofFIG.4, battery health monitoring device1000is configured in a pulse-echo mode for interrogating battery cell200. However, a first array1002aof ultrasound transducers402is provided on a first side of the battery cell200, and optionally, a second array1002bof transducers402can be provided on a second side opposite to the first side. Each transducer402may emit a pulse308into the interior of the battery cell200and can detect the resulting reflection from internal structures (e.g., electrode layers220) within the battery cell200. To prevent cross-talk between adjacent transducers402, each transducer402may be activated separately such that the corresponding reflected pulse408is detected by the same transducer402before the next transducer402in the array1002aor1002bis activated. Alternatively, transducers402with sufficient spacing between each other may be activated at the same time, where the spacing minimizes the amount of cross-talk that may be detected by the transducers402. For example, every other transducer in each array1002a,1002bmay be actuated at the same time. In yet another alternative, transducers402on one side of the battery cell200may be simultaneously activated. For example, the transducers402in array1002amay be simultaneously activated and signals from transducers402in array1002asimultaneously sampled. In another example, transducers402in array1002bcan serve as through ultrasound sensors for the pulses emitted by transducers402in array1002a. For example, each transducer402in array1002bcan receive any spreading808of the ultrasound308, for example, due to expansion of the ultrasound wave in the interior of the battery cell200and/or scattering or deflection by internal structures (e.g., electrode layers220) of the battery cell. Alternatively, each transducer402in array1002bmay be arranged directly opposite to a corresponding transducer402in array1002ain order to receive transmitted pulse308directly. Thus, such a configuration may allow simultaneous pulse-echo and through-transmission detection with assessment from opposite sides of the battery cell. As with the previously described embodiments, controller312can be provided to control operation of the source304and the sensors310and to determine a state of health of the battery cell200based on signals from the sensors310. The battery cell200may be connected to a load or charger302via terminals212,214such that the battery cell200may be assessed while the battery cell200is in use, for example, during discharging or charging. Other configurations and arrangements of components of a battery health monitoring device beyond those specifically discussed above are also possible according to one or more contemplated embodiments. For example, the embodiment ofFIG.10can be modified to include an array1002aof transducers402only a single side of the battery cell200. Other variations and combinations will be apparent to one of ordinary skill in the art, and embodiments of the disclosed subject matter are not limited to the specific embodiments illustrated in the drawings and described herein. Although shown and described separately, it is contemplated that elements of the health monitoring device may be provided as one or more integral units. For example, the control unit can be integrated with the ultrasound source and/or the ultrasound sensor. In another example, the source, the sensor, and the control unit may comprise a single transducer with appropriate integrated circuitry for controlling operation of the source and sensor and processing the resulting signals. In yet another example, the control unit is separate from an integrated unit of the ultrasound source and the ultrasound sensor, and the control unit receives the signals from the integrated unit, for example, a hard-wired connection, over the Internet, or via a wireless connection. In still another example, the control unit comprises a separate module within a common housing of the source and the sensor. In any of the contemplated embodiments and examples, the health monitoring device can be provided as a handheld unit, for example, with individual manually positioned parts, as shown inFIG.3B, or as an integrated unit that a user manually brings into contact with a particular battery cell. EXAMPLES Tests were performed on commercial lithium-ion batteries having a nominal voltage of 3.7 V. The electrodes were in a stacked configuration, and the separator was folded over in an accordion-like fashion so as to separate the stacked anode and cathode electrodes from each other. The anode and cathode materials were thus contained in alternating folds of the separator. Additionally, the anode and cathode materials were connected in series. Cell failure was defined as a drop in capacity of less than 80% (e.g., less than 75% or less than 72.5%) of the manufacturer-specified nominal capacity. Continuous battery charge/discharge cycling tests were performed using a commercial battery tester (e.g., a Cadex C8000) having four independent channels. The continuous cycling test was performed at a rate of 0.5 C. In addition to the cells that were charged and discharged at 0.5 C, a few uncycled, as-received cells were used as controls for periodic physical evaluation and comparisons. In accordance with the protocols described in UL 1642 and IEEE 1725, the batteries were cycled at room temperature to the specification of the manufacturer. Baseline capacity measurements were taken to ensure that full rated capacity was used during charging and discharging cycles.FIG.11shows an example of a constant current/constant voltage protocol that was used to charge and discharge the batteries. While the discharge mode occurred as a constant current load, actual operating conditions resulted in a variable current being applied to the battery. In one example of a battery under test, a sharp drop in capacity was seen after the 76thcycle of the continuous charge/discharge cycling profile, as illustrated inFIG.12A. There were no additional stresses placed on the cell, such as overcharging, overdischarging, or an increase in ambient temperature. Despite having this sudden drop in capacity during the 76thcycle, the cell did not reach the predefined failure threshold (e.g., 75% capacity) until after 133 cycles. A visual examination of the cell showed the cell had an increased external thickness as compared to the control battery cells. The change in thickness that was observed in the cycled cell was attributed to electrode ruffling and gas evolution within the cell. It is to be noted that the change in capacity illustrated inFIG.12Ais only an example, and other battery cells may have faster capacity loss rate (e.g., Battery 3, Battery 4) or slower capacity loss rate (e.g., Battery 1, Battery 2) until a predefined failure threshold (e.g., 72.5% capacity) is reached, as illustrated inFIG.12B. Ultrasonic assessment was performed on cycled and uncycled cells. The ultrasonic transducers were able to detect changes in acoustic impedance, such as at an interface between two materials, where a portion of the ultrasonic signal would be reflected back while the remainder would be transmitted through the interface and detected via through transmission. A portable digital ultrasonic sensor instrument was used to obtain A-scans of the acoustic signal. A handheld assessment setup is shown inFIG.3B. The cell was first disconnected from the Cadex C8000 battery tester and then connected to the ultrasonic detection setup. A 5 MHz, ¼-in. diameter ultrasonic pulser transducer was placed on top of the cell, and a 5 MHz, ¼-in. diameter ultrasonic receiver was placed on the bottom, as shown inFIG.3B. A hydrocarbon-based grease was used as a couplant at the interfaces of the pulser and receiver with the outer casing of the cell. The through-transmission parameters are shown in Table 1 below. TABLE 1Examples of Parameters for UltrasonicAssessment of Lithium Ion Battery CellParameterValueUltrasound Source (e.g., Pulser)5 MHz, ¼-in diameterUltrasound Sensor (e.g., Receiver)5 MHz, ¼-in diameterUltrasound Frequency5MHzEnergy400VDamping400ΩReceiver Filter1.5 MHz to 8.5 MHzGain50dB After the cell was set up for through transmission, an A-scan representation was obtained on the display of the portable ultrasonic sensor. An A-scan from a non-cycled, as-manufactured control cell is shown inFIG.13. As is apparent fromFIG.13, a strong through-pulse was detected by the receiver transducer, which pulse is consistent with the fact that the cell was uncycled and thus had not yet experienced any degradation.FIG.14shows an A-scan of the cycled cell that exhibited the drop in capacity after the 76thcycle. As is apparent fromFIG.14, the cycled cell transmitted only a very weak, delayed pulse. The weakening of the input ultrasonic pulse amplitude sensed by the receiver transducer suggests that the interfaces within the cell degraded due to at least one of electrode expansion, gas evolution, and residual stress developing along the interfaces as the cell is cycled. Thus, information from ultrasonic assessment of a cell can be used to evaluate the internal condition of structures of the cell and thereby provide a measure of the state of health of the cell. Systems with Battery Health Monitoring A battery-management system (BMS) can be incorporated into a host system that uses single cells or banks of cells arranged in series, parallel, or combinations thereof. A BMS enables safer and reliable operation by performing, among other things, state monitoring, charge control, and cell balancing (in multi-cell pack systems). Since certain battery operations (e.g., over-discharge) can reduce cell capacity, the BMS can monitor and control the battery cells based on safety circuitry incorporated within the battery pack to avoid such damaging operations. For example, whenever any abnormal conditions are detected, such as overvoltage or overheating, the BMS can notify the user and/or execute the predetermined corrective procedures. The BMS can use one or more sensors to monitor battery conditions and can determine a state of health of individual batteries or the entire battery back responsive to signals from the sensors. Cells connected together in a battery pack may not be easily accessible once assembled. Thus, physical examination of individual cells for structural changes may require disassembly of the battery pack, which, in general, may not be safely performed by an end user. However, the disclosed ultrasonic health monitoring device provides information on the internal structural changes of a monitored battery cell. Thus, a BMS that incorporates information from the ultrasonic health monitoring device can improve overall safety and/or reliability of the battery pack. Early fault detection can help the BMS inform the user when to implement repair and maintenance strategies to prolong the life of the battery pack and/or avoid further degradation that could result in imminent or eventual battery failure. As noted above, the battery cell being monitored can be a part of a larger battery pack that includes multiple battery cells connected in series, parallel, or any combination thereof. The ultrasonic transducers can be used to nondestructively and noninvasively monitor and assess the internal state of vital battery interfaces, e.g., the interface between the current collector and the corresponding anode and cathode materials. The ultrasonic data can be used to determine the instantaneous safety and health of the battery pack, for maintaining the battery system, and/or for evaluating the state of health over the course of the battery pack's lifetime. In one or more embodiments, a battery system employs a battery health monitoring device, for example, one or more of the battery health monitoring devices described above, to monitor individual battery cells within a battery pack. For example, the battery system1500can have a battery pack1504with a plurality of battery cells200, each with a corresponding ultrasonic health monitoring device1502, as shown inFIG.15. The battery pack1504can be connected to a load or charging device (not shown). The ultrasonic health monitoring devices1502can thus assess and monitor the individual battery cells200while the battery pack1504is in use (e.g., charging or discharging). Signals from the respective ultrasonic health monitoring devices1502can be conveyed to controller (Ctrl)312, where a determination of the state of health of each battery cell (BC)200can be made, for example, as described above. The battery system1500can also include a battery control module (BCM)1506, which regulates operation of the individual battery cells200within the battery pack1504. Controller312may communicate state of health determinations to the battery control module1506for use in controlling operation of the individual battery cells200. For example, battery control module1506may control charging and/or discharging profiles of each cell200and/or shut-down particular cells200in response to safety or reliability concerns. Together with controller312, battery control module1506may form a battery management system (BMS)1508, which may receive additional information regarding the battery cells200in determining appropriate operation or a state of health of the battery pack1504. For example, in response to signals from the ultrasonic health monitoring devices1502indicative of the states of health for the various battery cells, the BMS1508can control charging/discharging within the battery pack1504to avoid defective or degraded cells200, can determine an overall state of health of the battery pack1504or remaining useful lifetime for the battery pack1504, and/or can provide an external alert regarding a degraded or dangerous condition of one of the battery cells200or the battery pack1504. Additionally, the BMS1508can receive signals from other sensors (not shown) that monitor one or more performance characteristics of the battery cells200and/or the battery pack1504in determining the state of health of the cells and/or the battery pack. For example, the performance sensors can be configured to measure battery cell internal resistance, battery cell discharge profile, battery cell charging time, battery cell current or voltage, battery cell temperature, battery cell strain, battery cell dimensions, or gas venting from the battery cell and to generate a measurement signal responsively thereto. Using the information from the performance sensors in combination with the information from the ultrasonic health monitoring devices1502can provide a more complete picture of the state of health of each individual battery cell200and the battery pack1504overall. Alternatively or additionally, health monitoring can be accomplished by monitoring confidence values computed by applying statistical pattern recognition techniques to the transient behavior of battery cells, transient responses, and correlation of the responses with models and validated with experimental data. As with the ultrasonic health monitoring devices, performance sensors may be provided for each device. Alternatively, one or only some of the battery cells200are provided with performance sensors. For example, the performance sensors may be provided to one or more battery cells200within the pack1504that are more susceptible to degradation. Alternatively or additionally, one or more of the performance sensors may be shared among multiple battery cells200. For example, a single temperature sensor may be provided for the entire battery pack1504or a subset of battery cells200within the battery pack1504and can measure a temperature that is associated with each of the battery cells200. In yet another alternative, one or only some of the battery cells200can be provided with ultrasonic health monitoring devices, for example, as with battery system1600inFIG.16. In contrast to the embodiment ofFIG.15, only the subset1602of the plurality of battery cells200are provided with an ultrasonic health monitoring device1502. The number of battery cells200within subset1602that receive an ultrasonic health monitoring device may be limited, for example, to no more than one-third of the total battery cells200within pack1504, and may be even further limited to less than 10%. The controller312can use information from the ultrasonic health monitoring devices1502associated with the subset1602to infer or predict the state of health of the remaining cells200within the battery pack200. For example, the ultrasonic health monitoring devices may be provided to one or more battery cells200within the pack1504that are more susceptible to degradation. In such an example, presumably the degradation of battery cells200outside the subset1602would be less than the degradation of battery cells200within the subset1602, such that information from the ultrasonic health monitoring devices1502represents a worst-case scenario for the battery pack1504. In one or more embodiments, a battery system with in-situ ultrasonic health monitoring is provided in an automotive application, for example, as an energy source for a hybrid-electric or all electric vehicle. For example, an automobile system1700can have a battery pack1504with a plurality of battery cells200, as shown inFIG.17. The battery pack1504can be connected to an electric motor1716, which drives wheels1703of the vehicle1701. An engine controller1706monitors and regulates performance of the electric motor1716, for example, in response to drive conditions or user input. Ultrasonic health monitoring devices1502are provided to a subset1602of the battery cells200for interrogating and monitoring a state of health of the cells200and the battery pack1504, as described above. In addition, performance sensors1704are provided to some of the battery cells200for interrogating and monitoring performance characteristics of the battery cells200. A performance sensor signal processor (Sig. Prc.)1702receives signals from the performance sensors and conveys information regarding the performance characteristics to the battery control module1506responsively thereto. For example, the performance sensors can be configured to measure at least one of battery cell discharge profile, battery cell charging time, battery cell current or voltage, and battery cell temperature and to generate a measurement signal responsively thereto. One or more performance sensors may be associated with a same battery cell200as one of the ultrasonic health monitoring devices1502, for example, performance sensor1704bmonitoring battery cell200in subset1602. Alternatively or additionally, one or more performance sensors may be associated only with a battery cell200that is not monitored by one of the ultrasonic health monitoring devices1502, for example, performance sensor1704a. Selection of the subset1602of cells200that are to receive an ultrasonic health monitoring device and/or a performance sensor may be based on, for example, susceptibility to degradation or exposure to degrading conditions. The most vulnerable cells in the battery pack may be a result of the particular arrangement of the cell200in the battery pack1504, for example, cells200that are arranged closer to an engine1716or that may see higher temperatures than other cells200in the pack1504. The subset1602of sampled cells200can be used to predict a state of health of entire battery pack1504, or can be used to provide a fault indication if one of the monitored cells in the subset1602catastrophically fails or is in danger of imminent failure. For example, sampling may be such that less than 5% of cells200are monitored. In an example, the number of battery cells200in battery pack1504is three-hundred and only between five and ten, inclusive, of the total number of battery cells200in the pack1504are monitored. The output signals from the ultrasonic health monitoring devices1502can be integrated into the an automotive battery management system (aBMS)1708, which can include, among other things, ultrasonic health monitoring controller312, performance sensor signal processor1702, and battery control module1506. In the case of an automobile, by selective placement of such ultrasonic health monitoring devices1502on the battery cells200within the battery pack1504, the onboard aBMS1708can provide indicators that show the state of the health, usage, performance, and longevity of the battery pack1504. Using the techniques described herein, the aBMS1708can provide real-time, in-situ monitoring of representative batteries (e.g., subset1602) within the battery pack1504. When an abnormal condition is detected, such as an electrode delamination beyond a set threshold or battery cell swelling due to overheating, the aBMS1708can notify onboard safety systems (e.g., via engine controller1706or other onboard controllers), execute a set of corrective procedures (e.g., via battery control module1506), and/or notify a user, operator, manufacturer or other external entity (e.g., via user interface1710). For example, the user interface1710may comprise an on-board dashboard indicator. Alternatively or additionally, the user interface1710is a communication device that allows transmission of data to an external computer or system, for example, a wireless connection to a user's smartphone or an Internet transmission to the automobile manufacturer. In addition to providing alarms due to adverse events, incorporation of this technique allows real-time recording of degradation within the representative battery cell1502of the battery pack1504. In one or more embodiments, the ultrasonic health monitoring device can be arranged on a surface of the battery cell200or battery pack1504. For example, a battery configuration1800can have the ultrasonic health monitoring device mounted on an exterior surface1802of battery cell200, as shown inFIG.18. The exterior surface1802can include a mounting portion1804that retains the ultrasonic source304and the couplant306to the exterior surface1802. A similar mounting portion may be provided for the sensor310(not shown), when necessary for a through-transmission configuration. As noted above, the couplant306can comprise an encapsulated gel pad or insert. Such gel pads may enjoy a relatively long lifetime and may be reusable depending on the application. The couplant306can be pre-attached to or integral with the ultrasonic source304so that the combination of the source304and couplant306are inserted into the mounting portion1804at the same time. Alternatively, the couplant306may be a separate piece and inserted into the mounting portion1804before the source304. For example, the mounting portion1804may comprise a screw mechanism, locking mechanism, epoxy, glue, or any other retaining mechanism that can rigidly couple the ultrasonic health monitoring device to the surface. As the exterior surface1802moves, for example, due to swelling or other internal deformations, the mounting portion1804allows the ultrasonic health monitoring device to follow the movement of the exterior surface1802. Alternatively, the ultrasonic health monitoring device can be flexibly mounted so as to follow movement of the exterior surface1802, as shown in the configuration1900ofFIG.19. For example, the ultrasonic source or transducer402can be provided with an annular lip1902. A spring1906can provide an axially biasing force between lip1902and mounting support1904(e.g., a portion of the automotive body or other support structure independent of the particular battery cell200) that urges the transducer402and couplant306into contact with the exterior surface1802of the battery cell200. Movement of the exterior surface1802is accommodated by corresponding compression of spring1906. Additionally, the movement of the transducer402, which may be monitored by displacement sensors, for example, can provide an additional indicator of state of health of the battery cell200, i.e., by providing a measure of the swelling of surface1802. A similar configuration may be provided for the sensor310(not shown), when necessary for a through-transmission configuration. Battery Health Testing Systems In one or more embodiments, a testing system employs a battery health monitoring device, for example, one or more of the battery health monitoring devices described above, to test individual battery cells, as part of a field return evaluation or quality control of a manufacturing process. For example, a battery testing system2000can have a testing platform2002with a first support2004for a first portion of an ultrasound health monitoring device and a second support2006for a second portion of the ultrasound health monitoring device, as shown inFIG.20. For example, the first support2004may support the ultrasound source304and couplant306above the battery cell200and the second support2006may support the ultrasound sensor310below the battery cell200, or vice versa. In some configurations, only one of the first and second supports2004,2006can be provided, for example, when only a pulse-echo mode is employed for testing the battery cells200. Alternatively or additionally, an additional support (not shown) may be provided to hold battery cell200for assessment by the ultrasonic health monitoring device. One or more of the supports (when provided) may be configured to move in at least one dimension, for example, to bring the couplant306and ultrasound source304into contact with a first surface of the battery cell200and to bring the ultrasound sensor310into contact with a second surface of the battery cell200. The couplant306can be, for example, a gel pad attached to an end of the ultrasonic source304, which is brought into contact with each individual battery cell200conveyed to the testing platform2002. A controller2012can control the testing platform2002to move the support portions2004,2006and/or battery cell200to perform ultrasonic assessment thereof. For example, the controller2012can control a conveying device (not shown) to move a battery cell200from a batch of cells to the testing platform2002for assessment. The controller2012can then control the testing platform2002to bring the couplant306and/or the sensor310into contact with the battery cell200and to subject the battery cell200to an ultrasonic pulse from the source304. Alternatively or additionally, the source304and/or the sensor310may comprise a roller transducer, such as the Olympus Ultrasonic Roller Transducer referenced above. In such a configuration, the battery cell200may be linearly displaced between the rolling contact surfaces of the roller transducer to perform an assessment. Alternatively or additionally, the roller transducers can be displaced with respect to the battery cell200in order to perform an assessment. The controller2012can receive a signal from sensor310indicative of the detected ultrasound and can provide an indication of the state of health, as described above. The controller2012can direct the battery cell200from the testing platform2002and/or provide an indication (e.g., a visual or auditory signal) based on a result of the assessment. Alternatively or additionally, the controller2012can move the ultrasonic health monitoring device and/or the battery cell200to allow assessment of more than one location within the interior of the battery cell200, for example, by raster scanning across the surface of the battery cell200. In some embodiments, the battery cell200can be manually placed within the testing platform2002for evaluation. The controller can then control the testing platform2002to bring the couplant306into contact with the battery cell200. In other embodiments, the testing platform2002can be actuated manually, for example, by a user moving one or more of the supports2004,2006to contact the ultrasonic health monitoring device with the battery cell. Alternatively or additionally, the testing system2000can be configured as a handheld testing unit where a user can bring the testing platform2002into contact with the battery cell200, for example, by inserting battery cell200into a receptacle of a handheld testing platform2002. In still other embodiments, the testing platform2002may have one or more of the supports2004,2006that can passively move in response to a thickness of the battery cell200arranged between the supports2004,2006. For example, at least one of the supports2004,2006can be spring mounted with a spacing between a surface of couplant306and a facing surface of the couplant (not shown) associated with sensor310being less than a thickness of the battery cell200. Insertion of the battery cell200between the couplants biases the source304and sensor306against the respective surfaces of the battery cell200. In one or more embodiments, a testing system2100can optionally include a selection device that selects individual battery cells from a plurality of battery cells for respective assessment by the ultrasound source and sensor. For example, the selection device can include a conveying device2106that moves the battery cell200to a testing platform2104, as shown inFIG.21. For example, the conveying device2106can comprise a conveyor belt on which multiple battery cells200to be tested are disposed. As described above, the testing platform2104can ultrasonically test each individual battery cell200by bringing the ultrasonic health monitoring device2102into contact with the battery cell200. The controller2112receives signals from the ultrasonic health monitoring device2102indicative of an internal condition of the assessed battery cell200and can control conveying device2106to direct the assessed battery cell200from the testing platform2104based thereon. For example, battery cells200that do not meet predetermined criteria can be directed to a defect or reject bin while those that do meet the predetermined criteria can be directed to an acceptable bin for further processing. A redirection unit2108can have an arm2110that pushes a rejected battery cell200as it moves from the testing platform2104in order to move the rejected battery cell200via a different conveyor path2114to the reject bin. Acceptable battery cells200can continue along conveyor path2116to the acceptable bin. Other mechanisms for conveying the battery cells200to/from the testing platform2104and for redirecting the battery cells based on measured status are also possible according to one or more contemplated embodiments. For example, the conveying device can comprise a reel. When the battery cell is a returned or reprocessed battery cell (i.e., one that has already undergone multiple charge/discharge cycles and/or has been stored for a significant period of time after manufacture), the controller (e.g., controller2012inFIG.19or controller2112inFIG.20) may determine based on the detected ultrasound signal if the state of health of the battery cell200is sufficient for reuse (e.g., that the capacity of the battery cell has not degraded below 80%). Such state of health assessment can evaluate for degradation due to gas generation, active material delamination, electrode buckling, lithium ion diffusion, separator shrinkage, lithium plating, and tab shifting, for example. Those battery cells that are determined to be insufficient for reuse may be directed, for example, to a waste bin for proper disposal. When the battery cell is a new battery cell (i.e., one that has not undergone multiple charge/discharge cycles and/or has been stored for a short period of time after manufacture), the controller (e.g., controller2012inFIG.19or controller2112inFIG.20) may determine based on the detected ultrasound signal if the battery cell meets certain quality control criteria. Such quality control assessment can include determining the presence and location of metal particle inclusions, excessive current collector overhang, poor tab welds, agglomeration of active material, uneven thickness of active material, for example. Those battery cells that are determined to have quality control flaws may be directed, for example, to a defect bin for reprocessing. Embodiments of the disclosed subject matter have been described above with respect to lithium-ion battery cells having a stacked electrode configuration and a substantially rectangular exterior shape. However, this discussion is merely intended to illustrate the principles and techniques of the disclosed systems, methods, and devices. The disclosed principles and techniques are also applicable to other battery cell configurations and other energy storage devices, and the above description should not be understood as limiting the present disclosure to lithium-ion batteries. For example, the energy storage device may have an interior volume comprised of a slurry without a well-defined electrode configuration. In another example, the energy storage device may have an exterior shape that is substantially spherical, oval, elliptical, or any other shape. In such configurations, the source and sensor may be disposed on the same surface, for example, at the same location (e.g., as part of the same transducer) or at different positions on the same surface (e.g., as separate transducers). Other structures and shapes are also possible according to one or more contemplated embodiments. In addition, although embodiments have been described where each ultrasonic health monitoring device assesses a single battery cell, embodiments of the disclosed subject matter are not limited thereto. Rather, more than one battery cell can be disposed for assessment by a single ultrasonic health monitoring device. For example, in a through-transmission mode configuration, more than one battery cell can be stacked in a thickness direction thereof, with the ultrasound source disposed on a surface of the upper-most battery cell and the ultrasound sensor disposed on a surface of the lower-most battery cell. In another example, in pulse-echo mode configuration, more than one battery cell can be stacked in a thickness direction thereof, with a transducer disposed on an upper-most battery cell such that reflected ultrasound from battery cells in the stack can be received by the transducer. In still another example, in pulse-echo mode configuration, more than one battery cell can be stacked in a thickness direction thereof, with a first transducer disposed on a upper-most battery cell and a second transducer disposed on a lower-most battery cell so as to be able to assess the battery cell stack from both sides thereof. Furthermore, although specific applications of the ultrasonic health monitoring device have been described with respect to battery management systems, automotive systems, battery field testing, and quality control assessment, embodiments of the disclosed subject matter are not limited thereto. Rather, the ultrasonic health monitoring device can employed in a wide array of applications beyond those specifically disclosed herein, such as, but not limited to, home or office back-up battery system monitoring, non-automotive electric vehicles (e.g., battery powered planes), battery warehouse inventory monitoring, etc. In one or more first embodiments, a battery health monitoring device comprises an ultrasound source, a couplant, an ultrasound sensor, and a controller. The ultrasound source is configured to generate ultrasonic pulses having a frequency greater than 1 MHz. The couplant is arranged to convey the ultrasonic pulses from the ultrasound source to a monitored battery cell. The ultrasound sensor is configured to detect ultrasound having a frequency greater than 1 MHz and is arranged to detect ultrasound reflected from or transmitted through the monitored battery cell. The controller is configured to determine a state of health of the monitored battery cell based on a signal from the ultrasound sensor indicative of the detected ultrasound. In the first embodiments or any other embodiment, the ultrasound source and the ultrasound sensor are part of a single transducer disposed on a same side of the monitored battery cell. The ultrasound sensor can be arranged to detect ultrasonic pulses reflected from an interior of the monitored battery cell. In the first embodiments or any other embodiment, the ultrasound source is disposed on a first side of the monitored battery cell, and the ultrasound sensor is disposed on a second side of the monitored battery cell opposite from said first side. The ultrasound sensor can be arranged to detect ultrasonic pulses transmitted through an interior of the monitored battery cell. In the first embodiments or any other embodiment, the battery health monitoring device further comprises a second ultrasound sensor configured to detect ultrasound having a frequency greater than 1 MHz. The second ultrasounds sensor is arranged to detect ultrasound reflected from or transmitted through the monitored battery cell. The second ultrasound source can be disposed on said first side of the monitored battery cell. In the first embodiments or any other embodiment, the battery health monitoring device further comprises at least one additional ultrasound source, at least one additional couplant, and at least one additional ultrasound sensor. Each additional ultrasound source is configured to generate ultrasonic pulses having a frequency greater than 1 MHz. Each additional couplant corresponds to a respective additional ultrasound source and is arranged to convey the ultrasonic pulses from the respective additional ultrasound source to the monitored battery cell. Each additional ultrasound sensor is configured to detect ultrasound having a frequency greater than 1 MHz and is arranged to detect ultrasound reflected from or transmitted through the monitored battery cell. The controller is further configured to determine the state of health of the monitored battery cell based on signals from the ultrasound sensor and the at least one additional ultrasound sensor. In the first embodiments or any other embodiment, the battery health monitoring device further comprises a plurality of additional ultrasound sensors. Each additional ultrasound sensor is configured to detect ultrasound having a frequency greater than 1 MHz and is arranged to detect ultrasound reflected from or transmitted through the monitored battery cell. The ultrasound sensor and the plurality of additional ultrasound sensors are arranged in an array on a same side of the monitored battery cell. In the first embodiments or any other embodiment, the controller is configured to control the ultrasound source and the ultrasound sensor to perform an A-scan and to determine the state of health based on at least one of amplitude of the detected ultrasound and timing of the detected ultrasound. In the first embodiments or any other embodiment, the couplant comprises hydrocarbon grease or an encapsulated gel. In the first embodiments or any other embodiment, the battery health monitoring device further comprises a testing platform and a conveying device. The testing platform supports one or more of the ultrasound source, the couplant, and the ultrasound sensor. The conveying device moves individual battery cells from a plurality of battery cells to the testing platform for respective assessment by the ultrasound source and the ultrasound sensor. In the first embodiments or any other embodiment, the controller is further configured to control the conveying device to direct assessed battery cells from the testing platform responsive to the determined state of health from the respective assessment. In the first embodiments or any other embodiment, the battery health monitoring device further comprises a performance sensor. The performance sensor is configured to measure at least one of battery cell discharge profile, battery cell charging time, battery cell current or voltage, and battery cell temperature and to generate a measurement signal responsively thereto. In the first embodiments or any other embodiment, the battery performance sensor is arranged to monitor a different battery cell from that monitored by the ultrasound sensor at a same time. In the first embodiments or any other embodiment, the performance sensor and the ultrasound sensor monitor the same battery cell. In the first embodiments or any other embodiment, the controller comprises a battery management system for a battery pack including a plurality of individual battery cells. The controller is further configured to determine a state of health of the battery pack based on the measurement signal from the performance sensor and the signal from the ultrasound sensor. In the first embodiments or any other embodiment, the monitored battery cell comprises a lithium-ion battery cell with multiple electrode layers. The ultrasound source is arranged so as to direct the generated ultrasonic pulses perpendicular to a plane of one or more of the electrode layers. In the first embodiments or any other embodiment, at least the ultrasound source and the couplant are mounted on a surface of the monitored battery cell. In the first embodiments or any other embodiment, at least the ultrasound source and the couplant are coupled to a surface of the monitored battery cell so as to move with said surface. In one or more second embodiments, a method of monitoring battery cell state of health comprises applying one or more ultrasonic pulses through a couplant to a first side of a lithium-ion battery cell, each ultrasonic pulse having frequency greater than 1 MHz. The method further comprises detecting ultrasound having a frequency greater than 1 MHz that is reflected from or transmitted through the lithium-ion battery cell using one or more ultrasound sensors coupled to the lithium-ion battery cell, and generating a signal indicative of the detected ultrasound. In the second embodiments or any other embodiment, the method further comprises determining a state of health of the lithium-ion battery cell based at least in part on the generated signal indicative of the detected ultrasound. In the second embodiments or any other embodiment, the determining a state of health is based on at least one of amplitude of the detected ultrasound and timing of the detected ultrasound. In the second embodiments or any other embodiment, the lithium-ion battery cell is one of a plurality of cells in lithium-ion battery pack. In the second embodiments or any other embodiment, the method comprises measuring at least one of battery cell discharge profile, battery cell charging time, battery cell current or voltage, and battery cell temperature of one of the lithium-ion battery cells. The method further comprises generating a measurement signal indicative of a result of said measuring, and determining a state of health of the lithium-ion battery pack based on the measurement signal and the signal indicative of the detected ultrasound. In the second embodiments or any other embodiment, the lithium-ion battery cell has multiple electrode layers, and the applied one or more ultrasonic pulses are directed perpendicular to a plane of at least one of the electrode layers. In the second embodiments or any other embodiment, at least one of the one or more ultrasound sensors is part of a transducer that generates said one or more ultrasonic pulses. In the second embodiments or any other embodiment, the detecting ultrasound that is reflected from or transmitted through the lithium-ion battery cell comprises detecting one or more ultrasonic pulses reflected from an interior of the monitored battery cell. In the second embodiments or any other embodiment, the detecting ultrasound that is reflected from or transmitted through the lithium-ion battery cell comprises detecting one or more ultrasonic pulses transmitted through an interior of the monitored battery cell. In the second embodiments or any other embodiment, at least one of the one or more ultrasound sensors is positioned on the first side of the lithium-ion battery cell. In the second embodiments or any other embodiment, at least one of the one or more ultrasound sensors is positioned on a side of the lithium-ion battery cell opposite from the first side. In the second embodiments or any other embodiment, the applying one or more ultrasonic pulses and the detecting ultrasound are such that an A-scan is performed on the lithium-ion battery. In the second embodiments or any other embodiment, the couplant comprises hydrocarbon grease or an encapsulated gel. In the second embodiments or any other embodiment, the couplant comprises a gel pad. In the second embodiments or any other embodiment, the method comprises attaching the couplant to an ultrasonic source configured to generate the one or more ultrasonic pulses. The method further comprises, prior to said applying, contacting the couplant to the first side of the lithium-ion battery cell and arranging the one or more ultrasound sensors to receive ultrasound reflected from or transmitted through the lithium-ion battery cell. In the second embodiments or any other embodiment, the method comprises mounting the couplant and an ultrasonic source configured to generate the one or more ultrasonic pulses on an external surface of the lithium-ion battery cell. The mounting can be such that the couplant and the ultrasonic source move with the external surface of the lithium-ion battery cell. In the second embodiments or any other embodiment, the method comprises, after the applying and detecting, repeating the applying and the detecting on a second lithium-ion battery cell and generating a second signal indicative of the detected ultrasound from the lithium-ion battery cell. In the second embodiments or any other embodiment, the method comprises, before the repeating, at least one of: moving the second lithium-ion battery cell to a testing platform supporting an ultrasound source that generates the one or more ultrasonic pulses and the one or more ultrasound sensors, and moving the testing platform supporting the ultrasound source and the one or more ultrasound sensors to the second lithium-ion battery cell. In the second embodiments or any other embodiment, the method comprises, after the repeating the applying and the detecting on the second lithium-ion battery cell, directing the second battery cell from the testing platform based on the second signal. In the second embodiments or any other embodiment, the method comprises, at a same time as the applying one or more ultrasonic pulses and detecting ultrasound, at least one of charging the lithium-ion battery cell, discharging the lithium-ion battery cell, and repeatedly charging and discharging the lithium-ion battery cell. In one or more third embodiments, a battery system with state of health monitoring comprises a battery pack, one or more ultrasonic health monitoring devices, and a battery management system. The battery pack comprises a plurality of individual lithium-ion battery cells. Each ultrasonic health monitoring device is arranged to assess one of the lithium-ion battery cells. Each ultrasonic health monitoring device comprises an ultrasound source that directs ultrasound at the respective lithium-ion battery cell. Each ultrasonic health monitoring device further comprises an ultrasound sensor that detects ultrasound reflected from or transmitted through the respective lithium-ion battery cell and generates a signal responsive thereto. The battery management system is configured to receive the signal from each ultrasound sensor and to determine a state of health of the battery pack based at least in part on said signal. In the third embodiments or any other embodiment, each ultrasonic health monitoring device further comprises a couplant arranged between the ultrasound source and a surface of the respective lithium-ion battery cell. In the third embodiments or any other embodiment, the couplant comprises a gel pad. In the third embodiments or any other embodiment, each ultrasonic health monitoring device is mounted on a surface of the respective lithium-ion battery cell so as to move with said surface. In the third embodiments or any other embodiment, each ultrasound source is configured to generated ultrasonic pulses having a frequency greater than 1 MHz, and each ultrasound sensor is configured to detect ultrasound having a frequency greater than 1 MHz. In the third embodiments or any other embodiment, only some of the battery cells in the battery pack are provided with one of the ultrasonic health monitoring devices. In the third embodiments or any other embodiment, up to 5% of the battery cells in the battery pack are provided with one of the ultrasonic health monitoring devices. In the third embodiments or any other embodiment, each battery cell in the battery pack is provided with one of the ultrasonic health monitoring devices. In the third embodiments or any other embodiment, the battery system further comprises one or more performance sensors. Each performance sensor is arranged to assess one or more of the lithium-ion battery cells. Each performance sensor is configured to measure battery cell internal resistance, battery cell discharge profile, battery cell charging time, battery cell current or voltage, battery cell temperature, battery cell strain, battery cell dimensions, or gas venting from the battery cell and to generate a measurement signal responsively thereto. In the third embodiments or any other embodiment, the battery management system is configured to receive the measurement signal from each performance sensor and to determine the state of health of the battery pack based at least in part on said measurement signal. In the third embodiments or any other embodiment, only a subset of the battery cells in the battery pack are provided with one of the ultrasonic health monitoring devices, and at least one performance sensors assesses one of the lithium-ion battery cells different from said subset. In the third embodiments or any other embodiment, only a subset of the battery cells in the battery pack are provided with one of the ultrasonic health monitoring devices, and at least one performance sensors assesses one of the lithium-ion battery cells within said subset. In the third embodiments or any other embodiment, the ultrasound source and the ultrasound sensor in each ultrasonic health monitoring device are part of a single transducer disposed on a same side of the respective lithium-ion battery cell. In the third embodiments or any other embodiment, at least one ultrasound sensor is arranged to detect ultrasound reflected from an interior of the respective lithium-ion battery cell. In the third embodiments or any other embodiment, at least one ultrasound sensor is arranged to detect ultrasound transmitted through an interior of the respective lithium-ion battery cell. In the third embodiments or any other embodiment, the battery pack is constructed for use in an automotive vehicle. In one or more fourth embodiments, a health monitoring device comprises an ultrasound source and an ultrasound sensor. The ultrasound source is configured to generate and direct ultrasound at an energy storage device. The ultrasound sensor is configured to detect ultrasound reflected from or transmitted through the energy storage device and to generate a signal responsive to the detected ultrasound from the energy storage device. In the fourth embodiments or any other embodiment, the health monitoring device further comprises a couplant arranged between the ultrasound source and the energy storage device, and/or a couplant arranged between the energy storage device and the ultrasound sensor. In the fourth embodiments or any other embodiment, the ultrasonic source comprises a couplant that contacts a surface of the energy storage device, and/or the ultrasonic sensor comprises a couplant that contacts a surface of the energy storage device. In the fourth embodiments or any other embodiment, the couplant comprises hydrocarbon grease or an encapsulated gel. In the fourth embodiments or any other embodiment, the ultrasound source and the ultrasound sensor are disposed on a same surface of the energy storage device and spaced from each other. In the fourth embodiments or any other embodiment, the surface of the energy storage device is spherical, elliptical, oval, or rectangular. In the fourth embodiments or any other embodiment, the ultrasound source and the ultrasound sensor are part of a single transducer, and the ultrasound sensor is arranged to detect ultrasound reflected from an interior of the energy storage device. In the fourth embodiments or any other embodiment, the ultrasound source is disposed opposite to the ultrasound sensor with the energy storage device therebetween, and the ultrasound sensor is arranged to detect ultrasound transmitted through an interior of the energy storage device. In the fourth embodiments or any other embodiment, a second ultrasound sensor is configured to detect ultrasound reflected from the energy storage device, and the ultrasound source and the second ultrasound sensor are part of a single transducer. In the fourth embodiments or any other embodiment, the health monitoring device further comprises a plurality of additional ultrasound sensors. Each additional ultrasound sensor is configured to detect ultrasound reflected from or transmitted through the energy storage device, the ultrasound sensor. The plurality of additional ultrasound sensors are arranged in an array. In the fourth embodiments or any other embodiment, the health monitoring device further comprises at least one additional ultrasound source and at least one additional ultrasound sensor. Each additional ultrasound source is configured to generate and direct ultrasound at the energy storage device. Each additional ultrasound sensor is configured to detect ultrasound reflected from or transmitted through the energy storage device and to generate a signal responsive to the detected ultrasound from the energy storage device. In the fourth embodiments or any other embodiment, the health monitoring device further comprises a control unit configured to determine a state of health of the energy storage device based on signals from the ultrasound sensor and the at least one additional sensor. In the fourth embodiments or any other embodiment, the health monitoring device further comprises a control unit that receives the signal from the ultrasound sensor. The control unit is configured to determine a state of health of the energy storage device responsive to said signal. In the fourth embodiments or any other embodiment, the energy storage device comprises a battery cell and the control unit is configured to determine the state of health of the battery cell. In the fourth embodiments or any other embodiment, the energy storage device comprises a lithium-ion battery cell and the control unit is configured to determine the state of health of the lithium-ion battery cell. In the fourth embodiments or any other embodiment, the ultrasound source or the ultrasound sensor comprises the control unit. In the fourth embodiments or any other embodiment, the ultrasound source and the ultrasound sensor are separate from the control unit. In the fourth embodiments or any other embodiment, the energy storage device is a battery cell within a battery pack that includes a plurality of individual battery cells. The control unit comprises a battery management system for the battery pack, and the control unit is further configured to determine a state of health of the battery pack based on the signal from the ultrasound sensor. In the fourth embodiments or any other embodiment, the control unit is configured to control the ultrasound source and the ultrasound sensor to perform an A-scan and to determine the state of health based on at least one of amplitude of the detected ultrasound and timing of the detected ultrasound. In the fourth embodiments or any other embodiment, the generated and detected ultrasound comprises one or more ultrasonic pulses having a frequency greater than 1 MHz. In the fourth embodiments or any other embodiment, the health monitoring device further comprises a testing platform and a selection device. The testing platform comprises the ultrasound source and the ultrasound sensor. The selection device selects individual energy storage devices from a plurality of energy storage devices for respective assessment by the ultrasound source and the ultrasound sensor of the testing platform. In the fourth embodiments or any other embodiment, the selection device comprises a conveying system that moves the individual energy storage devices from the plurality of energy storage devices to the testing platform for the respective assessment. In the fourth embodiments or any other embodiment, the conveying system comprises a conveyor belt or reel. In the fourth embodiments or any other embodiment, the health monitoring device further comprises a control unit that receives the signal from the ultrasound sensor and determines a state of health of the energy storage device responsive to said signal. The control unit controls the conveying system to direct energy storage devices from the testing platform responsive to the determined state of health from the respective assessment. In the fourth embodiments or any other embodiment, the health monitoring device is constructed as a handheld unit with the ultrasound source and the ultrasound sensor disposed therein. In the fourth embodiments or any other embodiment, the energy storage device comprises a battery cell. The health monitoring device further comprises a second sensor configured to measure battery cell internal resistance, battery cell discharge profile, battery cell charging time, battery cell current or voltage, battery cell temperature, battery cell strain, battery cell dimensions, or gas venting from the battery cell and to generate a measurement signal responsively thereto. In the fourth embodiments or any other embodiment, the energy storage device is one of a plurality of battery cells, and the second sensor is arranged to monitor a different battery cell from that monitored by the ultrasound sensor at a same time. In the fourth embodiments or any other embodiment, the energy storage device is one of a plurality of battery cells, and the second sensor and the ultrasound sensor monitor the same battery cell. In the fourth embodiments or any other embodiment, the energy storage device comprises a lithium-ion battery cell with multiple electrode layers, and the ultrasound source is arranged so as to direct the generated ultrasound perpendicular to a plane of one or more of the electrode layers. In the fourth embodiments or any other embodiment, at least the ultrasound source is coupled to a surface of the energy storage device so as to move with said surface. In one or more fifth embodiments, a method of monitoring an energy storage device comprises applying ultrasound to an energy storage device, detecting ultrasound reflected from or transmitted through the energy storage device, and generating a signal indicative of the detected ultrasound. In the fifth embodiments or any other embodiment, the applying ultrasound to the energy storage device is via an ultrasonic source through a first couplant or the detecting ultrasound from the energy storage device is via an ultrasound sensor through a second couplant. In the fifth embodiments or any other embodiment, the first couplant or the second couplant comprises hydrocarbon grease, an encapsulated gel, or a gel pad. In the fifth embodiments or any other embodiment, the applying and detecting ultrasound comprises applying and detecting one or more ultrasonic pulses having a frequency greater than 1 MHz. In the fifth embodiments or any other embodiment, the energy storage device comprises a battery cell, for example, a lithium-ion battery cell. In the fifth embodiments or any other embodiment, the method further comprises determining a state of health of the energy storage device based at least in part on the generated signal indicative of the detected ultrasound. In the fifth embodiments or any other embodiment, the determining a state of health is based on at least one of amplitude of the detected ultrasound and timing of the detected ultrasound. In the fifth embodiments or any other embodiment, the energy storage device is a battery cell within a battery pack that includes a plurality of individual battery cells. The method further comprises measuring battery cell internal resistance, battery cell discharge profile, battery cell charging time, battery cell current or voltage, battery cell temperature, battery cell strain, battery cell dimensions, or gas venting of one of the battery cells. The method additionally comprises generating a measurement signal indicative of a result of said measuring, and determining a state of health of the battery pack based on the measurement signal and the signal indicative of the detected ultrasound. In the fifth embodiments or any other embodiment, the measuring of one of the battery cells is of a different battery cell than the detecting ultrasound. In the fifth embodiments or any other embodiment, the measuring of one of the battery cells is of a same battery cell as the detecting ultrasound. In the fifth embodiments or any other embodiment, the energy storage device comprises a battery cell with multiple electrode layers, and the applying ultrasound comprises directing ultrasound perpendicular to a plane of at least one of the electrode layers. In the fifth embodiments or any other embodiment, the detecting ultrasound that is reflected from or transmitted through the energy storage device comprises detecting ultrasound reflected from an interior of the energy storage device. In the fifth embodiments or any other embodiment, the detecting ultrasound that is reflected from or transmitted through the energy storage device comprises detecting ultrasound transmitted through an interior of the energy storage device. In the fifth embodiments or any other embodiment, the applying ultrasound and the detecting ultrasound are such that an A-scan is performed on the energy storage device. In the fifth embodiments or any other embodiment, the applying and the detecting ultrasound comprises disposing an ultrasound source and an ultrasound sensor on a same surface of the energy storage device. In the fifth embodiments or any other embodiment, the ultrasound source and sensor are part of a same transducer. In the fifth embodiments or any other embodiment, the ultrasound source and sensor are spaced from each other on the same surface. In the fifth embodiments or any other embodiment, the surface of the energy storage device is spherical, elliptical, oval, or rectangular. In the fifth embodiments or any other embodiment, the applying and the detecting ultrasound comprises disposing an ultrasound source and an ultrasound sensor opposite from each other with the energy storage device therebetween. In the fifth embodiments or any other embodiment, the method further comprises attaching a couplant to an ultrasonic source configured to generate ultrasound or to the energy storage device, and arranging one or more ultrasound sensors to receive ultrasound reflected from or transmitted through the energy storage device. In the fifth embodiments or any other embodiment, the method further comprises mounting an ultrasonic source with an integral couplant on an external surface of the energy storage device. The ultrasonic source is configured to generate ultrasound. The mounting is such that the couplant and the ultrasonic source move with the external surface of the energy storage device. In the fifth embodiments or any other embodiment, the method further comprises, after the applying and detecting, repeating the applying and the detecting on a second energy storage device and generating a second signal indicative of the detected ultrasound from the second energy storage device. In the fifth embodiments or any other embodiment, the method further comprises, before the repeating, at least one of moving the second energy storage device to a testing platform supporting an ultrasound source that generates ultrasound and one or more ultrasound sensors, and moving the testing platform supporting the ultrasound source and the one or more ultrasound sensors to the second energy storage device. In the fifth embodiments or any other embodiment, the method further comprises, after the repeating the applying and the detecting on the second energy storage device, directing the second energy storage device from the testing platform based on the second signal. In the fifth embodiments or any other embodiment, the method further comprises, at a same time as the applying and the detecting ultrasound, at least one of charging the energy storage device, discharging the energy storage device, and repeatedly charging and discharging the energy storage device. In the fifth embodiments or any other embodiment, the method further comprises measuring at least one of discharge profile of the energy storage device, charging time of the energy storage device, current or voltage of the energy storage device, temperature of the energy storage device, strain levels on the energy storage device, dimensions or change in dimensions of the energy storage, internal resistance of the energy storage device, and venting of gas from the energy storage device via a gas vent sensor or strain measurements. In the fifth embodiments or any other embodiment, the energy storage device is a battery cell within a battery pack that includes a plurality of individual battery cells. The method further comprises determining a state of health of the battery pack based at least in part on the generated signal. In the fifth embodiments or any other embodiment, the applying and the detecting ultrasound comprise supporting by hand an ultrasound source or an ultrasound sensor with respect to the energy storage device. In the fifth embodiments or any other embodiment, the energy storage device comprises a battery cell with multiple electrode layers, and the applying ultrasound comprises directing generated ultrasound perpendicular to a plane of one or more electrode layers. In any of the embodiments, a system can be configured to perform any method disclosed herein. In any of the embodiments, a non-transitory computer-readable storage medium is embodied with a sequence of programmed instructions, and a computer processing system executes the sequence of programmed instructions embodied on the computer-readable storage medium to cause the computer processing system to perform any of the methods disclosed herein. It will be appreciated that the modules, processes, systems, and devices described above, for example, the control unit, can be implemented in hardware, hardware programmed by software, software instruction stored on a non-transitory computer readable medium or a combination of the above. For example, a method for determining a state of health of one or more battery cells using ultrasonic assessment can be implemented, for example, using a processor configured to execute a sequence of programmed instructions stored on a non-transitory computer readable medium. For example, the processor can include, but is not limited to, a personal computer or workstation or other such computing system that includes a processor, microprocessor, microcontroller device, or is comprised of control logic including integrated circuits such as, for example, an Application Specific Integrated Circuit (ASIC). The instructions can be compiled from source code instructions provided in accordance with a programming language such as Java, C++, C#.net or the like. The instructions can also comprise code and data objects provided in accordance with, for example, the Visual Basic™ language, LabVIEW, or another structured or object-oriented programming language. The sequence of programmed instructions and data associated therewith can be stored in a non-transitory computer-readable medium such as a computer memory or storage device which may be any suitable memory apparatus, such as, but not limited to read-only memory (ROM), programmable read-only memory (PROM), electrically erasable programmable read-only memory (EEPROM), random-access memory (RAM), flash memory, disk drive and the like. Furthermore, the modules, processes, systems, and devices, for example, the control unit, can be implemented as a single processor or as a distributed processor. Further, it should be appreciated that the steps mentioned herein may be performed on a single or distributed processor (single and/or multi-core). Also, the processes, modules, and sub-modules described in the various figures of and for embodiments herein, for example, the control unit, may be distributed across multiple computers or systems or may be co-located in a single processor or system. Structural embodiment alternatives suitable for implementing the modules, systems, or processes described herein, for example, the control unit, are provided below. The modules, processes, systems, and devices described above, for example, the control unit, can be implemented as a programmed general purpose computer, an electronic device programmed with microcode, a hard-wired analog logic circuit, software stored on a computer-readable medium or signal, an optical computing device, a networked system of electronic and/or optical devices, a special purpose computing device, an integrated circuit device, a semiconductor chip, and a software module or object stored on a computer-readable medium or signal, for example. Embodiments of the methods, processes, modules, devices, and systems (or their sub-components or modules), for example, the control unit, may be implemented on a general-purpose computer, a special-purpose computer, a programmed microprocessor or microcontroller and peripheral integrated circuit element, an ASIC or other integrated circuit, a digital signal processor, a hardwired electronic or logic circuit such as a discrete element circuit, a programmed logic circuit such as a programmable logic device (PLD), programmable logic array (PLA), field-programmable gate array (FPGA), programmable array logic (PAL) device, or the like. In general, any process capable of implementing the functions or steps described herein can be used to implement embodiments of the methods, systems, or computer program products (software program stored on a non-transitory computer readable medium). Furthermore, embodiments of the disclosed methods, processes, modules, devices, systems, and computer program product, for example, the control unit, may be readily implemented, fully or partially, in software using, for example, object or object-oriented software development environments that provide portable source code that can be used on a variety of computer platforms. Alternatively, embodiments of the disclosed methods, processes, modules, devices, systems, and computer program product, for example, the control unit, can be implemented partially or fully in hardware using, for example, standard logic circuits or a very-large-scale integration (VLSI) design. Other hardware or software can be used to implement embodiments depending on the speed and/or efficiency requirements of the systems, the particular function, and/or particular software or hardware system, microprocessor, or microcomputer being utilized. Embodiments of the methods, processes, modules, devices, systems, and computer program product, for example, the control unit, can be implemented in hardware and/or software using any known or later developed systems or structures, devices and/or software by those of ordinary skill in the art from the function description provided herein and with knowledge of battery assessment or health monitoring systems and/or computer programming arts. Furthermore, the foregoing descriptions apply, in some cases, to examples generated in a laboratory, but these examples can be extended to production techniques. For example, where quantities and techniques apply to the laboratory examples, they should not be understood as limiting. In addition, although specific chemicals and materials have been disclosed herein, other chemicals and materials may also be employed according to one or more contemplated embodiments. Features of the disclosed embodiments may be combined, rearranged, omitted, etc., within the scope of the invention to produce additional embodiments. Furthermore, certain features may sometimes be used to advantage without a corresponding use of other features. It is thus apparent that there is provided in accordance with the present disclosure, system, methods, and devices for monitoring a state of health of an energy storage device, such as a lithium-ion battery cell. Many alternatives, modifications, and variations are enabled by the present disclosure. While specific embodiments have been shown and described in detail to illustrate the application of the principles of the present invention, it will be understood that the invention may be embodied otherwise without departing from such principles. Accordingly, Applicants intend to embrace all such alternatives, modifications, equivalents, and variations that are within the spirit and scope of the present invention. | 118,720 |
11860131 | DETAILED DESCRIPTION The present invention is generally directed to systems and methods of non-destructive testing using ultrasonic transducers. In one embodiment, the present invention is directed to a system for ultrasonic testing of composite materials, including a central housing, having a front end and a back end, defining an interior sealed chamber, a transducer, wherein the transducer is located within the interior sealed chamber of the central housing, a fluid connector attached to the central housing, wherein the fluid connector is operable to connect with a fluid pump to introduce coupling fluid into the sealed chamber, wherein the front end of the central housing is sealed by a membrane, wherein the membrane is acoustically translucent to the coupling fluid, and wherein the transducer is operable to emit and receive ultrasonic waves. In another embodiment, the present invention is directed to a method for ultrasonic testing of composite materials, including providing a transducer housing assembly including a central housing, having a front end and a back end, defining an interior sealed chamber, a transducer, wherein the transducer is located within the interior sealed chamber of the central housing, a fluid connector attached to the central housing, wherein the fluid connector is operable to connect with a fluid pump to introduce coupling fluid into the sealed chamber, wherein the front end of the central housing is sealed by a membrane, wherein the membrane is acoustically translucent to the coupling fluid; and the transducer emitting ultrasonic waves into a test material, and the transducer housing assembly receiving ultrasonic waves reflected from the test material. In yet another embodiment, the present invention is directed to a system for ultrasonic testing of composite materials, including a central housing, having a front end and a back end, defining an interior sealed chamber, a coupling element sealingly engaged with the back end of the central housing and coupled with a transducer, wherein adjustment of the coupling element moves the transducer relative to the central housing, a fluid connector attached to the central housing, wherein the fluid connector is operable to connect with a fluid pump to introduce coupling fluid into the sealed chamber, wherein the front end of the central housing is sealed by a membrane, wherein the membrane is acoustically translucent to the coupling fluid, and wherein the transducer is operable to emit and receive ultrasonic waves. Due to their high strength-to-weight ratios, composites are becoming increasingly common, particularly for structural applications in the aerospace, automotive, sporting goods, rail, petrochemical, defense and other industries. Manufacturing defects as well as damage caused during use of the composite can have a significant impact on the laminate's structural performance and sometimes lead to structural failure. Damage to a composite can frequently occur as a result of, for example, hail strikes, lightning, bird collisions, mishandling of the part, or general fatigue. Examples of defects that can lead to the failure of a composite material during use include foreign objects within the material, insufficient bonding between the layers of the composite, wrinkling of the layers of the composite, delamination within at least one layer of the composite, incomplete curing of the composite, improper conformance to manufacturing specifications, and excessively large pores within the composite. Therefore, properties such as the bond line thickness, porosity, ply type, delaminations, local failure points, and weave type of the composite can have significant effects on the overall material properties and performance of a composite structure, and they may even serve as crack initiation points. Ultrasonic inspection is one of the primary NDT techniques currently used in industry to evaluate composite performance and conformity with industry standards. Ultrasonic NDT techniques rely on the propagation and measurement of high-frequency sound waves through the thickness of a structure in order to detect the physical or material properties of the structure. Traditionally, ultrasonic methods have been divided between contact methods and immersion-based methods. In contact methods, transducers are directly applied to the test material to be tested, with a thin film of coupling fluid (e.g. water, gel, grease, oil, etc.) disposed in between the transducer and the test material. Immersion methods do not involve contact between the transducer and the test material, but instead utilize a large tank of coupling fluid, typically water, in which both the material and transducer are placed. The coupling fluid allows the waves produced by the transducer to easily travel to the test material and data can be recorded from the resulting reflections and refractions of the wave. Alternatives to the immersion method include running a continuous jet of coupling fluid over the test material for the duration of the test in order to allow the transducer to be coupled to the material without a large and often expensive tank. Transducers used during immersion type testing are often spherically focused, which allows the transducers to achieve improved resolution and sensitivity relative to contact transducers. Both contact transducers and immersion type transducers traditionally include one of two different configurations for operation. In pulse-echo, or reflection, ultrasonic configurations, a transducer generates high-frequency ultrasonic energy, which is introduced to and transmitted through the surface of the test material in waves. The use of such systems typically requires an acoustic medium (e.g., water, gel) to bridge the gap between the transducer and the test material. As the waves propagate through the thickness of the test material, discontinuities within the test material (due to material changes, cracks, delaminations, foreign objects, etc.) cause a reflection of the wave, which can then be detected by the transducer and displayed or characterized. In contrast, in through transmission, or attenuation, ultrasonic configurations, a transducer generates high frequency ultrasonic energy, which is transmitted through one side of a test material and then received by a corresponding receiver on the opposite side of the test material. As the waves propagate through the thickness of the test material, discontinuities within the test material may cause waves in some areas to be slowed or fully attenuated before they reach the receiver. The receiver can then characterize the test material by measuring the degree of attenuation of the ultrasonic wave. In selecting ultrasonic inspection systems, the constraints of portability and robustness are often inversely proportional to high resolution and fidelity. When attempting to optimize both portability and robustness, most inspectors currently select a contact transducer. Contact transducers allow an inspector to quickly place a thin gel on a part to be inspected and place the transducer in intimate contact with the part. Data can be quickly gathered and at the same time, the system can operate on a variety of surfaces and environmental conditions. The primary downside of this approach is the resolution of the acquired data. The planar resolution of the transducer is dictated by the physical footprint of the transducer. This footprint can be mitigated by fabricating smaller and smaller transducers. The through-thickness resolution of the transducer, however, is determined by the frequency of the transducer and the power with which it can be fired. As a transducer's planar dimension is reduced, both the power that can be sent to fire the transducer and the frequency is simultaneously reduced. Thus, improvements to planar resolution are in direct conflict with improvements in through thickness resolution. An alternative to the contact transducer is a spherically focused transducer. These transducers are capable of operating at high frequencies (25-50 MHz) and have a planar resolution as fine as can be machined into the transducer housing lens, which may be less than 1/10th of a millimeter. However, spherically focused transducers can only operate when the transducer is acoustically coupled to the surface of a component being inspected. Acoustically coupling the transducer to the test material requires immersing the transducer in an acoustic medium while ensuring the transducer has a viable acoustic path between it and the surface to be tested. Water is the most widely used acoustic medium for immersion transducers, as the difference in acoustic impedance between the water and transducer lens is minimal. Currently, there are two main techniques to achieve a viable acoustic coupling water path: full immersion tank testing or water jets. The full immersion tank requires the part to be submerged in water, thus preventing many larger components, such as aircraft wings and fuselages, from being tested without a substantial (and often impractical) infrastructure investment. Water jets, on the other hand, require water to be spraying in all directions, which causes water to pool under the component being scanned. Water jets therefore also require infrastructure investments, often in the form of grates to collect the sprayed water, pumps to circulate the water, and framing to protect equipment in the area that can be damaged by water. One alternative to the use of traditional water jets is a “bubbler”. To use a bubbler, a temporary watertight box is built around a region of a component to be scanned for inspection. Water is then poured into a column that houses the transducer and is allowed to slowly leak out from a base of the box. This approach requires a new box to be installed at every new location for the scan. In order for a bubbler to work, the membrane must be pressed tightly against the object to be tested, as water leaks out slowly enough from the bubbler that it cannot maintain the blast pressure necessary to allow the device to be placed at an offset from the test material. Not only does this create the risk of impact between the bubbler and the test material, which may cause damage, but it also greatly reduces the resolution of the device. Bubblers rely on permeable membranes that slowly allow the water to leak out, but when the bubbler is operating and the membrane is pressed firmly against the test surface, the systems cannot effectively distinguish between waves in the membrane and in the test material, rendering the devices less effective if not entirely inoperable. Furthermore, bubblers suffer from similar drawbacks as traditional water jets, in that they require water to be pumped in constantly and require a means to catch leaking water. Additionally, full immersion, traditional water jets, and the bubbler all require the test material to be exposed to water, which is undesirable in some situations. Traditional water jets and bubblers also suffer similar problems of being unable to scan hard to reach areas of a device. Hard to reach areas are often semi-contained within a device or component to be tested and therefore, the use of traditional water jets is highly likely to cause water to pool within the device, which may cause damage or be difficult to pump out. Furthermore, both traditional water jet systems and bubblers require the continuous pumping in of water via a water column. However, the need for this water column eliminates the ability of those devices to effectively navigate to hard-to-reach areas of a device or component to be tested. As of yet, no other efforts to produce a robust functional ultrasonic scanner utilizing water-filled chambers have been successful. Some existing systems require the chamber to leak around a rolling ball, through a permeable membrane, or otherwise, to span the gap between the chamber and the surface to be measured. Such systems still require a flow of water into the chamber to replenish the water loss to maintain the acoustic coupling and require contact between the water and the test material. Other systems sacrifice the use of a spherically focused transducer and therefore have decreased resolution. Still other systems have fixed focal lengths, and other systems have fixed lengths of lens housing and lens that preclude reaching portions of surfaces for scanning. Therefore, there remains a need to provide a more simplified and versatile system, including a housing for an ultrasonic spherically focused transducer that can be operated independently of a full immersion tank and yet be used to scan omnidirectionally from a sealed fluid-tight housing for acoustic coupling with a component surface. Additionally, traditional ultrasonic testing devices utilize calibration blocks. Before testing, the testing device is used on one or more calibration blocks, which typically are either an exemplary form of the material to be tested or a material with known defects. Traditional ultrasonic inspection systems use this calibration method as a means of comparison in determining whether the signals reflected from the test material match or differ from those of the calibration block. However, reliance on calibration blocks weakens the ability to specifically indicate important properties of a test material. For example, during porosity testing, traditional systems may recognize calibration blocks with porosities of 0.2, 0.4 and 0.6, but a test material which most closely aligns with the 0.4 porosity calibration block may still have a porosity of anywhere between 0.3 and 0.5, with further specificity being limited. Furthermore, testing using calibration blocks may be hindered by unknown flaws in the calibration blocks or unconsidered confounding variables that differ between the calibration blocks and the actual test material. Therefore, a system is needed that is capable of directly determining qualities of a material, such as ply orientation, porosity, bond line thickness, the presence of wrinkles, unevenness in the bond line, or other important physical properties of a composite material without reference to a calibration block. The present disclosure provides a system with a transducer housing assembly that maintains an acoustic coupling path needed for the spherically focused transducers while allowing placement of the housing at any angle relative to a vertical plane. The system enables the use of higher-resolution immersion-type ultrasonic transducers without the complete water immersion or water jets typically required for both the transducer and the component to be scanned with the transducer. This invention extends the use of the spherically focused transducers into portable systems and can significantly reduce operational costs and complexity. The system with the transducer housing assembly features a lens housing with an opening sealed by a replaceable fluid-tight membrane. The membrane forms an acoustic window with acoustic properties similar to those of the fluid in the housing and therefore acoustically transparent or at least acoustically translucent to the transducer and causing minimal signal loss. The transducer housing also includes the ability to coarsely and finely adjust the focal point of the transducer relative to the component surface. This feature allows adjustments for individual transducers having different focal points relative to the transducer housing, and allows an operator to focus at different depths within the part. Another feature is the ability to quickly replace the fluid-tight membrane coupled to the lens housing, if the film becomes damaged during use. The transducer housing assembly in operation contains a small volume of fluid, so that even if the film becomes damaged, the spilled fluid can simply be cleaned up with a small typical shop rag, without requiring inconvenient or expensive set ups to catch leaked fluid. In at least one embodiment, the transducer housing assembly, with the transducer, can fit within a 5 cm×5 cm×15 cm volume. The transducer housing assembly with the acoustic transducer can be connected to a variety of translation devices, including robotic arms. The system allows for non-destructive scanning in a pulse echo configuration using immersion-type ultrasonic transducers without requiring typical full immersion tank testing or water jets. By implementing high-resolution immersion transducers, the technology overcomes the resolution limitations of current portable scanners that conventionally have relied on contact transducers. The transducer housing assembly is capable of operating on a variety of composite materials, especially those commonly used in automotive and aerospace applications, including carbon fiber, fiberglass, concrete, and other composite materials. In one embodiment, the transducer housing assembly is used to test materials with a thickness between 1/16 of an inch and ½ of an inch. In another embodiment, the transducer housing assembly is used to test materials with a thickness greater than ½ of an inch, including laminates with a thickness of greater than 2 inches. The present invention utilizes the transducer housing assembly is used to determine qualities of a material, including the porosity of the material, the ply orientation of the material, whether material layers are a weave or unidirectional, the presence of wrinkles in the layers of the material, bond line thickness, inconsistencies in the bond line, the presence of foreign objects in the material, and the presence of internal defects within the material without use of a calibration block. Therefore, the transducer housing assembly is able to directly measure these quantities and provide a quantifiable output via a display means, instead of merely determining whether the sample matches some previously scanned control material. The transducer housing assembly is able to used in conjunction with other testing devices for testing of larger structures. In one embodiment, a separate phase array scanner is used to scan over a large area of a test material and identify potential problem areas in the material. Subsequently, the transducer housing assembly is used to more precisely scan the identified potential problem areas. In another embodiment, a thermographic scan of a material is first performed before the transducer housing assembly is used to more precisely scan areas identified by the thermographic scan. In yet another embodiment, a thermographic scan is performed first on a component, followed by a phase array scan of a subarea of the component, and finally the transducer housing assembly is used to scan individual parts of the subarea of the component. FIG.1illustrates an orthogonal side view of a transducer housing assembly4according to one embodiment of the present invention. The transducer housing assembly4includes a central housing6with a front portion10and a back portion8. In one embodiment, the front portion10and back portion8are hollow cylindrical pieces and are integrally formed with each other. Alternatively, the front portion10and back portion8are not integrally formed but are separately formed and are joined together via any chemical and/or mechanical means known in the art. In another embodiment, the front portion10and back portion8are another shape, such as rectangular prisms. In one embodiment, the diameter of the front portion10is greater than that of the back portion8, with the diameter of the central housing6tapering down between the front portion10and back portion8at a midsection9. The central housing6is attached to a fluid connector24. In one embodiment, the fluid connector24is attached to the front portion10of the central housing6, while in another embodiment, the fluid connector24is attached to the midsection9or back portion8of the central housing6. In one embodiment, a mounting bracket26extends from the front portion10of the central housing6. In another embodiment, the mounting bracket26extends from the midsection9or back portion8of the central housing6. The front portion10of the central housing6is connected to a lens housing20, which extends outwardly from the front end of the central housing6. The front end of the lens housing20includes an opening22. In one embodiment, at least one surface offset element28extends from the front end of the central housing6. In another embodiment, the surface offset elements28extend outwardly directly from the lens housing20. Transducer is disposed within the central housing6. In some embodiments, the transducer is directly attached to an elongate member52. The elongate member52is attached to the central housing6by means of a coupling element16. In one embodiment, the position of the elongate member52, and therefore the transducer, can be adjusted relative to the central housing6by rotating or otherwise adjusting the coupling element16. In one embodiment, the elongate member52and coupling element16include a metal material, such as, but not limited to, steel or aluminum. In another embodiment, the elongate member52and coupling element16are formed of the same metal material. Forming both the elongate member52and coupling element16from the same metal material is advantageous, as it prevents one of the elements acting as a cathode or an anode, which would allow for galvanic cell activity in the transducer housing assembly4, shortening the useful life of the device. In one embodiment, the central housing6is formed from a plastic, such as polycarbonate or polyethylene. In another embodiment, the central housing6is formed via3D printing of the device using an ultraviolet (UV) curable polymer, which is then cured after formation. In one embodiment, as shown inFIG.1, the mounting bracket26includes a first plane262extending away from the central housing6at an angle and a second plane263extending from the end of the first plane262in a direction substantially parallel to a central axis of the transducer housing assembly4. In another embodiment, as shown inFIG.5, the mounting bracket26is a substantially rectangular piece disposed between and orthogonal to the front portion10and back portion8of the central housing6. As can be seen inFIG.2, in other embodiments, the mounting bracket26takes different shapes, depending on the device to which it is to be attached. FIG.3illustrates an isometric view of the transducer housing assembly4shown inFIG.1.FIG.4illustrates a top view of the transducer housing assembly shown inFIG.1. As can be seen inFIGS.3and4, in one embodiment, the transducer housing assembly4includes three surface offset elements28. In one embodiment, the mounting bracket26includes at least one attachment bore261 FIG.5illustrates an isometric exploded view of a transducer housing assembly4according to another embodiment of the present invention. In one embodiment, the fluid connector24is attached to the central housing6of the transducer housing assembly4by connecting to a connection port34. In one embodiment, the fluid connector24connects to the connection port34by means of threading located on the outside surface of the fluid connector24and the interior surface of the connector port34. In one embodiment, the coupling element16is a hollow cylinder and the elongate member52extends through the coupling element16. The elongate member52and the coupling element16are held together by frictional contact between the outside surface of the search tube52and the interior surface of the coupling element. As shown inFIG.6, in another embodiment, the elongate member52is secured to the coupling element16by a securing element54. In one embodiment, the securing element54is a screw, bolt, or compressible pin. In one embodiment, the elongate member52is a hollow cylinder with the transducer50being frictionally engaged within a front end of the elongate member52. In one embodiment, the coupling element16connects to the central housing6by means of threading on part of the surface of the coupling element16and on the inner surface of a first opening12in the back portion8of the central housing6. In another embodiment, when the coupling element16is engaged with the central housing6, the coupling element16can be rotated so as move the coupling element16and the elongate member52longitudinally relative to the central housing6. In yet another embodiment, the securing element54can be removed, compressed or otherwise altered, which allows the coupling element16and the elongate member52to move longitudinally relative to the central housing6. By moving the elongate member52longitudinally relative to the central housing6, the position of the transducer50is able to be changed, which allows for accommodation of a range of sizes for transducers50, as well as greater precision in the focusing on the transducer. In one embodiment, the first opening12includes sealing elements, which prevent fluid leakage through the first opening12. In one embodiment, the sealing elements include 0-rings lining the inner surface of the first opening12. In another embodiment, the chamber within the central housing6is not fully sealed during operation, with either the back end of the central housing6or the interface with the fluid connector24being left unsealed. The option to use the transducer housing assembly4without sealing the chamber of the central housing provides flexibility in the parts used to construct the device, including allowing for the reduction of manufacturing cost. However, for use of the transducer housing assembly4that involves putting the transducer housing assembly4at an angle, it is advisable to sealed the interior chamber to prevent fluid leakage, which could cause decoupling of the transducer to the test material. The front portion10of the central housing6further includes a second opening18. The lens housing20is inserted into the second opening18in order to engage the lens housing20with the central housing6. In one embodiment, the lens housing20and central housing6are engaged by means of threading on the exterior surface of the lens housing20and on the interior surface of the second opening18. In another embodiment, the lens opening20includes annular or helical grooves58, within which sealing elements are attached. When the lens opening20placed into the second opening18, the sealing elements engage with the interior surface of the second opening18and form a fluid-tight seal. In one embodiment, the sealing elements are 0-rings. In yet another embodiment, the second opening18includes at least one engagement notch32and the lens housing20includes at least one engagement protrusion30, as shown inFIG.7. As shown inFIGS.8-11, in order for the lens housing20to be placed within the second opening18, the at least one engagement protrusion30of the lens housing20must align with the at least one engagement notch32of the second opening18. After the lens housing20is placed within the second opening18, the lens housing20is turned such that the at least one engagement protrusion30no longer aligns with the at least one engagement notch32. In one embodiment, the lens housing20is easily separated from the central housing6by twisting the lens housing20and pulling it out. This is advantageous in the event that the lens housing20becomes damaged and needs to be replaced, or where lens housings20of different sizes are needed in order examine different parts of a component. FIG.6illustrates an orthogonal exploded view of components of the transducer housing assembly shown inFIG.5. The fluid connector24is able to be connected to one end of a conduit36, such as a hose or a pipe. In on embodiment, the other end of the conduit36is connected to a fluid pump or fluid reservoir, from which fluid is able to be introduced through the conduit36and the fluid connector24into the sealed chamber. In another embodiment, the transducer housing assembly4is not connected to a fluid pump and fluid is added to the central housing6by other means, such as manual pouring. In one embodiment, the fluid connector24includes a pressure relief valve, which allows fluid to escape when the volume of fluid exceeds the volume of the sealed chamber. The pressure relief valve therefore advantageously provides an adjustable volume of fluid into the sealed chamber, depending on the distance between the transducer50and the front end of the central housing6. In one embodiment, the fluid connector24is able to be connected to a pump and air is pumped out of the sealed chamber before or while filling the chamber with a coupling fluid. Pumping out air helps to assure a lack of bubbles in the fluid, which improves the acoustic coupling path between the transducer50and a component to be tested. Furthermore, after testing has completed, the air pump is able to be used to pump air into sealed chamber, which assists in removing remaining fluid, reducing prolonged exposure to the coupling fluid, which could cause damage to the transducer housing assembly, such as corrosion. FIG.12illustrates an orthogonal view of a lens housing20according to one embodiment of the present invention. A membrane38is placed over the front end of the lens housing20. The membrane38creates a fluid-tight seal on the front end of the lens-housing20. When the lens housing20with the membrane38is placed into the central housing6, a sealed chamber is formed within the central housing6. The sealed chamber is a fluid-tight chamber, which is sealed by a combination of the interface between the coupling element16and first opening12of the back portion8of the central housing6, the interface between the lens housing20and the second opening18of the front portion10of the central housing6, the membrane38, and the fluid connector24. In one embodiment, the membrane38is secured to the lens housing20by at least one retainer40. In one embodiment, the at least one retainer40includes at least one O-ring surrounding a portion of the lens housing20and pressing the membrane38tightly against the lens housing20. Advantageously, in the event that membrane38is punctured or otherwise is unable to effectively seal the sealed chamber, it may easily be replaced by removing the retainer40, refitting a new membrane, and then reapplying the retainer40. The membrane38is acoustically transparent or translucent with respect to fluid in the sealed chamber. The material used for the membrane38is selected to have a similar acoustic impedance, and therefore similar stiffness and density, as the fluid in the sealed chamber. In one embodiment, the fluid is water or another fluid with an index of refraction approximately equal to 1. In another embodiment, the index of refraction of the membrane38is between 0.9 and 1.2. In yet another embodiment, the membrane is made from AQUALENE. As the frequency of the transducer50increases, the temporal resolution quality of the transducer increases. However, as the frequency of the transducer50increases, the depth of a material visible to the system decreases due to high frequency attenuation. In one embodiment, the transducer50is able to operate at frequencies between 1 and 50 MHz. In a preferred embodiment, the transducer50operates between 5 and 15 MHz. An external couplant can be used to fill the gap between the transducer housing assembly4and the test material. In one embodiment, the external couplant is an acoustic gel, such as glycerin, couplant D12, couplant H, a shear wave couplant, or another suitable acoustic gel. FIG.13illustrates an orthogonal side view of a surface offset element according to one embodiment of the present invention. In one embodiment, the surface offset elements28include pins281attached to a biasing member282. The biasing member282allows the surface offset elements28to retract when pressed against the surface of a test material. Furthermore, when the pin281is pressed against a test material, the biasing member282is able to absorb some of the displacement that would otherwise be imparted to the test material through a force or the transducer housing assembly4, preventing potential damage to both the transducer housing assembly4and the test material. In one embodiment, the degree to which the surface offset elements28are able to retract is limited by a stop. When the front of the transducer housing assembly4is pressed against a component to be tested, the surface offset elements28contact the component first, which prevents damage to the component or to the transducer housing assembly4that can be caused by quick and direct contact between the lens housing20and the component. Furthermore, by providing a stop to limit the retraction of the surface offset elements28, the lens housing20is able to stay at a fixed and known distance from the component, which allows for improved accuracy during the testing process. In another embodiment, the surface offset elements28are threadably connected to the front portion10of the central housing6and can be manually adjusted before use with different test materials. In one embodiment, the transducer housing assembly4operates at an offset distance from the test material approximately equal to one half the thickness of the test material. In one embodiment, the distance that the transducer housing assembly4is offset from the test material is determined using a calibration wave. An initial wave is transmitted via the transducer into the test material. Time of flight data is gathered regarding ultrasonic waves reflecting off of a membrane covering the opening22of the lens housing20, waves reflecting off the front surface of the test material, and waves reflecting off the back surface of the test material. Without the need to input material properties or dimensions of the test material, the transducer housing assembly4is able to automatically offset by a fixed distance from the test material based on the results of the time of flight data. In another embodiment, the material properties of the test material, such as the speed of sound, and dimensional data of the test material, such as the thickness, is manually entered, allowing the transducer housing assembly4to automatically offset by a fixed distance from the test material without the need for a calibration wave. FIG.14illustrates an orthogonal side view of a transducer housing assembly mounted on a robotic arm. The attachment bore261is able to receive a screw, bolt, pin, or other affixing means attached to a robotic arm48. The robotic arm48both allows the transducer housing assembly4to reach tighter spaces and allows the device to be held steadily for the duration of the testing, increasing the accuracy of the test. In another embodiment, the mounting bracket26is attached to a translation stage. The translation stage operates to move the transducer housing assembly4to different positions along an X-Y plane. This is especially advantageous in situations wherein the operator desires to scan large sections of a relatively flat test material. In one embodiment, the transducer housing assembly4is attached to an array element. In another embodiment, the array element includes attachment points for more than one transducer housing assembly4, allowing multiple transducer housing assemblies4to be attached to a single array element, which acts as an array of transducers. The array of transducers is therefore able to scan multiple points of a test material simultaneously, with each individual transducer housing assembly4being adjustable, so as to allow the array of transducers to scan components with uneven surfaces or scan components having multiple different material types. In another embodiment, the transducer housing assembly4is manually operated. By way of example, the transducer housing assembly4may be placed into an assembly attached to the test material. An operator is then able to manually slide the transducer housing assembly4within the assembly while the assembly ensures that the transducer housing assembly4remains at a substantially fixed distance from the test material. In still another embodiment, the transducer housing assembly4is able to automatically move to a plurality of different points on the test object based on preset position data entered into a computer or attached display. In one embodiment, the elongate member52is attached to a connection receiving end. In another embodiment, the connection receiving end attached to the elongate member52is connected to a first end of a cable. The second end of the cable is connected to an output display device. Examples of an output display device include a pulser receiver. In one embodiment, the pulser receiver is able to connect to a multiple transducer housing assemblies simultaneously. In one embodiment, the connection receiving end is a UHF connector, a Bayonet Neill-Concelman (BNC) connector, or a Universal Serial Bus (USB) connector. In another embodiment, the connection receiving end is a wireless adapter, allowing the transducer housing assembly4to wireless connect with the pulser receiver. The pulser receiver is connected to a computer, having a processor and memory. Furthermore, the computer includes display means for outputting graphical results of ultrasonic testing performed using the transducer housing assembly4. In yet another embodiment, the computer is also connected with the robotic arm, translation stage, or array element to which the transducer housing assembly4is attached and is operable to issue control instructions to the robotic arm, translation stage, or array element. The computer is connected to a display means able to display a graphical user interface (GUI), which is able to display the results of the testing after processing by the pulser receiver. In another embodiment, a display is directly mounted to the transducer housing assembly4, which allows results to be displayed to the user of the transducer housing assembly4without the operator needing to step away to check the computer. The GUI is able to accept a variety of input factors before each test, including the operator's name, the time, and material properties including the speed of sound of the material to be tested, the thickness of the material to be tested, the stiffness of the material to be tested, or the type of material to be tested. In one embodiment, the GUI is also able to accept a range of locations and a run time, indicating where the robotic arm, the array element, or the translation stage should position itself for testing. The GUI is capable of displaying information regarding a variety of factors of a laminate, including the location and depth of foreign objects within the laminate, the ply orientation of the laminate, the location of wrinkles within the laminate, the thickness of the bond line of the laminate, areas of incomplete bonding along the bond line of the laminate, the porosity of the laminate, and the location, depth, and size of internal defects and areas of delamination within the laminate. FIG.15is a schematic diagram of an embodiment of the invention illustrating a computer system, generally described as800, having a network810, a plurality of computing devices820,830,840, a server850, and a database870. The server850is constructed, configured, and coupled to enable communication over a network810with a plurality of computing devices820,830,840. The server850includes a processing unit851with an operating system852. The operating system852enables the server850to communicate through network810with the remote, distributed user devices. Database870is operable to house an operating system872, memory874, and programs876. In one embodiment of the invention, the system800includes a network810for distributed communication via a wireless communication antenna812and processing by at least one mobile communication computing device830. Alternatively, wireless and wired communication and connectivity between devices and components described herein include wireless network communication such as WI-FI, WORLDWIDE INTEROPERABILITY FOR MICROWAVE ACCESS (WIMAX), Radio Frequency (RF) communication including RF identification (RFID), NEAR FIELD COMMUNICATION (NFC), BLUETOOTH including BLUETOOTH LOW ENERGY (BLE), ZIGBEE, Infrared (IR) communication, cellular communication, satellite communication, Universal Serial Bus (USB), Ethernet communications, communication via fiber-optic cables, coaxial cables, twisted pair cables, and/or any other type of wireless or wired communication. In another embodiment of the invention, the system800is a virtualized computing system capable of executing any or all aspects of software and/or application components presented herein on the computing devices820,830,840. In certain aspects, the computer system800is operable to be implemented using hardware or a combination of software and hardware, either in a dedicated computing device, or integrated into another entity, or distributed across multiple entities or computing devices. By way of example, and not limitation, the computing devices820,830,840are intended to represent various forms of electronic devices including at least a processor and a memory, such as a server, blade server, mainframe, mobile phone, personal digital assistant (PDA), smartphone, desktop computer, netbook computer, tablet computer, workstation, laptop, and other similar computing devices. The components shown here, their connections and relationships, and their functions, are meant to be exemplary only, and are not meant to limit implementations of the invention described and/or claimed in the present application. In one embodiment, the computing device820includes components such as a processor860, a system memory862having a random access memory (RAM)864and a read-only memory (ROM)866, and a system bus868that couples the memory862to the processor860. In another embodiment, the computing device830is operable to additionally include components such as a storage device890for storing the operating system892and one or more application programs894, a network interface unit896, and/or an input/output controller898. Each of the components is operable to be coupled to each other through at least one bus868. The input/output controller898is operable to receive and process input from, or provide output to, a number of other devices899, including, but not limited to, alphanumeric input devices, mice, electronic styluses, display units, touch screens, signal generation devices (e.g., speakers), or printers. By way of example, and not limitation, the processor860is operable to be a general-purpose microprocessor (e.g., a central processing unit (CPU)), a graphics processing unit (GPU), a microcontroller, a Digital Signal Processor (DSP), an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA), a Programmable Logic Device (PLD), a controller, a state machine, gated or transistor logic, discrete hardware components, or any other suitable entity or combinations thereof that can perform calculations, process instructions for execution, and/or other manipulations of information. In another implementation, shown as840inFIG.15, multiple processors860and/or multiple buses868are operable to be used, as appropriate, along with multiple memories862of multiple types (e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core). Also, multiple computing devices are operable to be connected, with each device providing portions of the necessary operations (e.g., a server bank, a group of blade servers, or a multi-processor system). Alternatively, some steps or methods are operable to be performed by circuitry that is specific to a given function. According to various embodiments, the computer system800is operable to operate in a networked environment using logical connections to local and/or remote computing devices820,830,840through a network810. A computing device830is operable to connect to a network810through a network interface unit896connected to a bus868. Computing devices are operable to communicate communication media through wired networks, direct-wired connections or wirelessly, such as acoustic, RF, or infrared, through an antenna897in communication with the network antenna812and the network interface unit896, which are operable to include digital signal processing circuitry when necessary. The network interface unit896is operable to provide for communications under various modes or protocols. In one or more exemplary aspects, the instructions are operable to be implemented in hardware, software, firmware, or any combinations thereof. A computer readable medium is operable to provide volatile or non-volatile storage for one or more sets of instructions, such as operating systems, data structures, program modules, applications, or other data embodying any one or more of the methodologies or functions described herein. The computer readable medium is operable to include the memory862, the processor860, and/or the storage media890and is operable be a single medium or multiple media (e.g., a centralized or distributed computer system) that store the one or more sets of instructions900. Non-transitory computer readable media includes all computer readable media, with the sole exception being a transitory, propagating signal per se. The instructions900are further operable to be transmitted or received over the network810via the network interface unit896as communication media, which is operable to include a modulated data signal such as a carrier wave or other transport mechanism and includes any delivery media. The term “modulated data signal” means a signal that has one or more of its characteristics changed or set in a manner as to encode information in the signal. Storage devices890and memory862include, but are not limited to, volatile and non-volatile media such as cache, RAM, ROM, EPROM, EEPROM, FLASH memory, or other solid state memory technology; discs (e.g., digital versatile discs (DVD), HD-DVD, BLU-RAY, compact disc (CD), or CD-ROM) or other optical storage; magnetic cassettes, magnetic tape, magnetic disk storage, floppy disks, or other magnetic storage devices; or any other medium that can be used to store the computer readable instructions and which can be accessed by the computer system800. In one embodiment, the computer system800is within a cloud-based network. In one embodiment, the server850is a designated physical server for distributed computing devices820,830, and840. In one embodiment, the server850is a cloud-based server platform. In one embodiment, the cloud-based server platform hosts serverless functions for distributed computing devices820,830, and840. In another embodiment, the computer system800is within an edge computing network. The server850is an edge server, and the database870is an edge database. The edge server850and the edge database870are part of an edge computing platform. In one embodiment, the edge server850and the edge database870are designated to distributed computing devices820,830, and840. In one embodiment, the edge server850and the edge database870are not designated for distributed computing devices820,830, and840. The distributed computing devices820,830, and840connect to an edge server in the edge computing network based on proximity, availability, latency, bandwidth, and/or other factors. It is also contemplated that the computer system800is operable to not include all of the components shown inFIG.15, is operable to include other components that are not explicitly shown inFIG.15, or is operable to utilize an architecture completely different than that shown inFIG.15. The various illustrative logical blocks, modules, elements, circuits, and algorithms described in connection with the embodiments disclosed herein are operable to be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application (e.g., arranged in a different order or partitioned in a different way), but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention. Other and further embodiments utilizing one or more aspects of the invention described above can be devised without departing from the spirit of Applicant's invention. For example, various seals and seal configurations can seal the components to form the chamber in the transducer housing assembly; various translation devices can be used to move the transducer housing assembly along a component surface in space; various quick disconnect configurations can be used to attach the lens housing; various ultrasonic signal generation and receive devices (combined or separate) can be used to send and/or receive signals from the transducer; and the like can be used to form the transducer housing assembly and the other system equipment, along with other variations can occur in keeping within the scope of the claims. The above-mentioned examples are provided to serve the purpose of clarifying the aspects of the invention, and it will be apparent to one skilled in the art that they do not serve to limit the scope of the invention. By nature, this invention is highly adjustable, customizable and adaptable. The above-mentioned examples are just some of the many configurations that the mentioned components can take on. All modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the present invention. | 50,078 |
11860132 | DETAILED DESCRIPTION FIG.1provides a good overview of the material analysis device1according to the invention. The material analysis device1comprises a vibration exciter2. The vibration exciter2applies vibrations to a loading shaft3. The latter transmits these vibrations to a sample4, which is sketched only very generally here. The material analysis device1also comprises a sample chamber5, which is shown only diagrammatically inFIG.1. This sample chamber5is substantially closed by means of a cover6during operation. The desired test temperatures can be established in the sample chamber. Optionally, it is also possible to apply radiation, for example UV radiation, to the test object. If necessary, misting or steam application with liquids which, for example, have a corrosive effect or attack or influence the plastic material in another way is also possible. Similar conditions can optionally also be implemented with the aid of immersion in a suitable immersion bath. It can also be easily seen inFIG.1that in this exemplary embodiment a region outside the sample chamber, between the sample chamber and the measurement system, is automatically cooled by a cooling system based on heat pipe technology. To this end, a support plate7, which is preferably in the form of a cooling plate, lies above the insulation8and thus between the sample chamber and the measurement system. The support plate7can be a layer of the multi-layered cover6, as sketched here, but can also be completely independent of the cover6, which is not shown in the figures here. The cooling plate7eliminates or reduces the loading on the measurement system, in this case situated above the sample chamber, by heat from the sample chamber. Fluid coolant preferably flows through the cooling plate7and is cooled back down via a cooling device34. However, the cooling plate7can also function in the manner of a “heat pipe”, as is used in laptop construction. It is particularly favourable to implement the cooling such that the cooling plate7is provided with a mostly radial bore. A tubular heat pipe is inserted into this bore, usually using a thermally conductive paste in order to produce an optimal heat transfer. The heat pipe used is longer than the bore in the cooling plate receiving it. Therefore, it protrudes laterally out of the cooling plate7. As can be seen easily inFIG.1, for example, the part protruding laterally out of the cooling plate7extends into the cooling device34. There, it is accommodated by a groove or bore in a typically finned cooling body, which for its part acts as a heat sink and is typically subjected to cooling air by a fan. Such a “heat pipe” of the type used here is typically hermetically sealed and cannot be opened without being destroyed. The heat pipe is typically designed such that a fluid circulates in it, driven solely by the temperature difference, possibly supported by capillary action, said fluid absorbing heat at one end of the heat pipe, transporting it to the other end of the heat pipe, and then emitting it outwards there. To go into somewhat more detail in respect of the heat pipe used according to the invention, the following can be said about the heat pipes to be used by preference: The cooling plate7inevitably conducts a certain heat flow, specifically the heat loss which the insulation8was able to overcome. The heat input in the region of the cooling plate increases the temperature of the vessel forming the heat pipe, typically a copper pipe, and of the working medium therein until the boiling point of the working medium is reached. The working medium then begins to evaporate. The temperature does not increase any more, instead, all the energy conducted further is converted into evaporation heat. As a result, the pressure in the heat pipe is locally increased above the liquid level, which results in a slight pressure gradient within the heat pipe. The steam produced begins to spread out in the entire available volume, i.e., it flows to wherever the pressure is lower; at the points where its temperature falls below the boiling point of the working medium, it condenses. To do this, the steam must emit energy to the vessel, and the vessel must emit energy to the surroundings. This happens the most at the point where the condenser is situated, in which active cooling can take place, that is, in the region of the cooling device34. The temperature then does not fall any more, until all the latent heat, the condensation heat, has been emitted to the surroundings. The liquid fraction of the working medium returns to the evaporator as a result of the capillary forces developed by the metal mesh typically installed in the pipe used here as the heat pipe. Alternatively, the heat pipe used can also be a pipe which does not have a smooth inner side but is provided with fins which run in the direction of the pipe longitudinal axis and enclose free spaces between them, which can be regarded as capillary grooves. Preferably, the insulation8is a high-temperature-resistant plate of inorganic material, usually based on dispersed amorphous silica. This plate will often also have special infra-red opacifiers, so that any infra-red radiation produced in the sample chamber5also cannot easily overcome the insulation. On one side of the insulation8, in the sample chamber, there is usually so-called cover heating9, which is used for temperature control of the sample chamber. The sample is held in position in the sample chamber5with the aid of a sample holder10. The sample holder10usually consists of a type of sample table or sample cross member11. This is in turn held in position with the aid of pillars12. To this end, a pillar anchoring means13is arranged on the outside of the cover. This preferably comprises a synchronous actuation reproduction mechanism, as is described in more detail below. The pillars protrude through through-openings, assigned to them, in the cover into the region of the pillar anchoring means13. It can also easily be seen that the loading shaft3applies the vibrations communicated to it by the vibration exciter2to the sample not directly but via a probe shaft14coupled thereto. The coupling which couples the loading shaft to the probe shaft14in this case has reference sign15. The coupling15is shown only roughly inFIGS.1and2and is explained in more detail below. The probe shaft14projects through a through-opening assigned to it, in the cover into the region of the sample chamber5. It is notable that the probe shaft14is hollow-bored, preferably at least in the region with which it protrudes into the sample chamber5. Typically, it also has a number of radial windows. In this way, the cross-section available for heat conduction on the probe shaft14, via which heat can flow out of the sample space along the probe shaft and into a region outside the sample space, is kept small. The pillars12holding the sample table or sample cross member11are preferably also designed similarly in respect of their reduced thermal conductivity. This has the same reason as explained for the probe shaft. FIG.3shows the coupling15and presents in detail how the probe shaft14is coupled to the loading shaft3. The loading shaft3, which is hollow at least at its end facing the probe shaft, can easily be seen here. This hollow end forms a bushing16. In the present case, the probe shaft14bears an in this case male coupling piece17at its end. This design is particularly expedient because the key does not then have to be pulled completely out of the window assigned to it for decoupling. Expediently, the coupling piece17is screwed onto the probe shaft14. Unlike the probe shaft14, the coupling piece17will generally be solid. The coupling piece17is inserted into the bushing16of the loading shaft3for coupling. As can be seen, the coupling piece17has a groove18which is accessible radially from the side. In the fully coupled state, the groove18comes to lie behind a window19in the bushing16. A key20is inserted through the window. The key20can be in the form of a round key or, which is clearly preferred, a flat key. The flat key is shown schematically here. In this case, the flat key has a planar, purely radially oriented lower sliding face21. Opposite this, it has a usually likewise planar, obliquely running key face22. The key face22interacts with a counter key face23on the coupling piece17. The key20is preloaded in the radial direction towards the coupling piece17by a spring element, which in this case is preferably formed by the leaf spring24. This means that the leaf spring24forces the key20into the groove18. By the key face22of the key20and the counter key face23of the coupling piece sliding on one another on one side and the key20being supported by its lower sliding face21on the edge of the window19on the other side, the coupling piece17tends to be drawn deeper into the bushing16of the loading shaft. Centring occurs automatically as a result. This is because the outer taper25of the coupling piece17is thereby pulled into the taper seat26right at the end of the bushing16of the loading shaft3. In this way, the play, although usually small, which the cylindrical shank27of the coupling piece17must naturally have in relation to the inner surface of the bushing16is made safe. It can also be easily seen that this type of coupling enables the loading shaft3to transmit vibrations to the probe shaft14both in one direction along its longitudinal axis L and in the opposite direction without losses. It can also be easily seen that the key20is fixed interlockingly in the region of its radially outer end to the leaf spring24. To this end, the leaf spring24can have a window. The key protrudes through this window. The leaf spring24is in this case characterised in that only one of its ends is clamped, as can be seen. Preferably, a type of pipe clip28is used to clamp its end. Said pipe clip fits locally around the loading shaft3and fixes said end of the leaf spring24immovably on one side. The opposite end of the leaf spring24preferably forms a section29which is angled or in any case runs obliquely relative to the longitudinal axis L of the loading shaft. Its function is explained in more detail below. FIGS.4and4ashow clearly, when compared withFIG.1, how the loading shaft can be detached in a remote-controlled, motorised manner here.FIG.1shows the material analysis device, as mentioned, in its ready-to-operate state. The vibration exciter2can be moved up and down in a motorised, usually remote-controlled manner, by means of vertical guides (not shown). This movability is actually used to be able to position the end of the probe shaft exactly so that it can be coupled precisely to the sample4. However, this movability is now “diverted from its use” or subjected to a secondary use according to the invention. As can be seen easily fromFIGS.4and4a, the vibration exciter2is moved downwards to undo the coupling15. In the process, the obliquely running section29of the leaf spring comes up against a stop30at some point. As the vibration exciter continues to move downwards, the obliquely running section29is bent outwards precisely because of its slope acting in a wedge-like manner. This means that the leaf spring is pivoted, in this case clockwise. In the process, it pulls the key20out of the groove18. In this way, the probe shaft14is unlocked. It can then be pulled out of the loading shaft3, as shown inFIG.4a. FIGS.5and6show more detail. The leaf spring24, held at its upper end preferably clamped by the pipe clip28, can be seen very well here. The stop30is in this case formed favourably by the shackle31, which is mounted in a stationary manner on the material analysis device and bears a roller32. FIG.5shows the whole thing in the ready-to-operate position.FIG.6shows the whole thing after the vibration exciter2has been moved down far enough. As can be seen, the obliquely running section29of the leaf spring24then rolls on the roller32of the stop. The leaf spring24is thereby drawn outwards in an almost frictionless manner. It is notable that the connection between the key20and the leaf spring24can also be seen easily in these figures. As can be seen here, the leaf spring24bears a window33. The end of the key20is fixed interlockingly to the window or preferably between two jamb sides of the window. Ideally, the key will have an end which is grooved on two opposing sides for this purpose, as can be seen inFIGS.5and6. Preferably, the cross-section of the key is not square but rectangular at its end. The window33reproduces the same rectangular cross-section but rotated 90°. In this way, the key can be inserted into the window33during assembly until its two grooved sides are at the same height as the line of the window33. The key20is then rotated 90° into its end position. In this way, two mutually opposing jambs of the window33then engage interlockingly in the two grooves at the end of the key. In this way, the key can be forced to and fro by the leaf spring, perpendicular to the longitudinal axis L of the loading shaft. Of course, other fastening types are also conceivable, such as a screw-fastening of the key to the leaf spring. The manner in which the pillars of the sample holder are fastened to the cover so that the sample holder hangs down into the sample space from the cover of the sample space is shown best inFIGS.7to12. FIG.11provides an overview of the pillar anchoring means13, which forms the outermost part of the cover6facing away from the sample chamber, as shown inFIG.1. The pillars to be fastened protrude through cut-outs in the cover into the region of the outside of the cover, where the pillar anchoring means is attached. The whole thing has the advantage that the pillar anchoring means remains substantially cold, approximately at room temperature. Blind holes are entirely or partially provided in this usually planar pillar anchoring means. Each of these blind holes receives a coupling piece35of the respective pillar12. As can be seen, the term blind hole in this case means a hole which forms a stop for the upper end face of the coupling piece35. The coupling piece35has a lateral groove36. The groove36has at least one groove flank which faces the stop of the blind hole and forms a key face37. The coupling piece35is fixed in that a movable key38is inserted. It could be a round key. However, a flat key, as shown schematically here, is much more suitable. By means of its preferably upper key face, which interacts with the key face37on the groove flank, the coupling piece35is clamped between the key38and the stop of the blind hole. In this way, the relevant pillar12is immobilised in and counter to the direction of its longitudinal axis LS. Movements transverse or oblique to the longitudinal axis LS of the pillar12are prevented by the circumferential walls of the blind hole. The key38slides on its side facing the sample space on a planar face of the pillar anchoring means13. It generally also has planar sides on which it is guided laterally. Each pillar12is individually assigned such a key coupling. An optional special feature is that the four or more keys are actuated synchronously. A synchronous actuation reproduction mechanism is provided to this end. This is capable of retracting the keys in the manner shown inFIGS.9and10. The key tip is then completely disengaged from the groove36. The sample table11can then be removed downwards together with the pillars12. The structure of the synchronous actuation reproduction mechanism, which is part of the pillar anchoring means here, is shown best inFIGS.11and12. The reader should turn toFIG.12first, for the sake of better understanding. Various components which can still be seen inFIG.11and block the view of the critical parts are cut away inFIG.12. As can be seen, the keys38run to and fro in a groove or a limited depression in the plate46. The plate46is part of the pillar anchoring means13. To this end, preferably lateral guide strips39are provided next to each key, as can be seen relatively well. A joint actuating slide switch40is assigned to each pair of keys38. On close examination, it can be seen that each key is connected to the actuating slide switch40via an elongate cylindrical element. The elongate cylindrical element ends at one of the long, narrow side faces of the key. The elongate cylindrical element is a spring element, preferably in the form of a flexible bar spring. In addition, a double eccentric41is mounted pivotably in the plate46. With the aid of its pivot handle42, the double eccentric41can be rotated about the eccentric axis43. As soon as the pivot handle42is rotated clockwise to actuate the double eccentric41, one eccentric, in this case the eccentric facing the viewer, of the double eccentric41presses on the actuating slide switch40. The actuating slide switch40is thereby pushed to the left in the present case. As a result, the keys38which are each connected to the actuating slide switch40via a flexible bar spring44are pushed out of their open position into their closed position. IfFIGS.11and12are viewed side by side, it can be seen how the synchronous actuation reproduction mechanism functions. Specifically, the synchronous actuation frame45belongs to it. The second eccentric of the double eccentric41bears against the end, on the right-hand side in this case, of the synchronous actuation frame from the inside. The same movement of the double eccentric which moves the actuating slide switch40facing it to the left in the present case causes the synchronous actuation frame45to be pulled by the second eccentric to the right in the case shown schematically here. This pulling movement continues beyond the entire pillar anchoring means13into the region of its left-hand actuating slide switch40. This left-hand actuating slide switch40is then pulled from the left to the right by the synchronous actuation frame. In the process, it pushes the keys38connected to it likewise via flexible bar springs from their open position to the right into their closed position. In this way, a sample table or a sample cross member11can be changed simply and manageably. After the removal of the compartment bounding the sample chamber5, the sample table or sample cross member11is held in one hand while the other hand turns the pivot handle4290°. The sample table or sample cross member11can then be removed downwards with one hand. | 18,493 |
11860133 | Explanation of numbers marked in the figure: 1. Base;101. Vertical support plate;102. Positioning shaft;103. Vertical bracing plate;104. Stepping frame;105. I-shaped installation plate;106. Six-edge positioning shaft;107. Vertical short shaft;2. Hydraulic cylinder;3. Square press plate;301. Jacking pillar;4. Bracket plate;401. Rotating shaft;402. Driven gear;5. Fence frame;501. Foot frame;502. F-shaped sliding bar;503. Gear rack;504. Wedge;6. L-shaped insert;601. Connecting rod. DETAILED DESCRIPTION OF THE EMBODIMENTS The technical solutions of the embodiments of the present invention will be described expressly and integrally in conjunction with the appended figures of the embodiments of the present invention. It is clear that the described embodiments are some but not all of the embodiments of the present invention. Referring toFIGS.1to9, the invention presents an embodiment: a multi-shaft pressurized rock mechanics tester comprising a base (1), wherein the base (1) is of an integral rectangular structure, and welded six vertical support plates (101) symmetrically at the front and back ends, a-shaped bracket plate is horizontally welded at the top of the six vertical support plates (101) (“” is a Chinese character, read as “Ri”), and a bracket plate (4) is rotatably installed on the top section of the three rear vertical support plates (101), pressed against the-shaped bracket plate, and used to support the rock to be tested; the left and right sides of the base (1) are symmetrically welded with four vertical bracing plates (103), an I-shaped installation plate (105) is welded on the top of the four vertical bracing plates (103), four hydraulic cylinders (2) are locked, fixed and hoisted on the bottom of the I-shaped installation plate with screws, a square press plate (3) is locked, fixed and hoisted at the bottom of four piston shafts on the four hydraulic cylinders (2) with screws, and a jacking pillar (301) is welded and fixed at the bottom center of the square press plate (3) and slides down to contact the rock block to be tested; two of four vertical strip grooves are set respectively on the front and back ends of the vertical support plates (101), a positioning shaft (102) is welded in each of the four vertical strip grooves, and a rectangular fence frame (5) is mounted on the four positioning shafts (102) in a sliding way; two six-edge positioning shaft (106) are symmetrically welded on the middle section of the front and back vertical support plates (101) located in the middle position, two L-shaped inserts (6) are installed on the two six-edge positioning shafts (106) by pushing and sliding a first set of spring on the top, the four hydraulic cylinders (2) can push the square press plate (3) and the jacking pillar (301) down through the four piston shafts on the cylinders to test the rock mechanical properties, the fence flame (5) during the test can slide up to baffle the top of the bracket plate (4) to intercept the broken rocks to prevent the rocks from jumping and injuring people, and the fence flame (5) can slide down and hidden under the bottom of the bracket plate (4) before and after the test so as not to block the rocks or affect rock unloading; two-shaped foot frames (501) (“” a Chinese character, read as “Kan”) are symmetrically welded on the bottom of the left and right panels of the fence frame (5) and used in conjunction with the springs on the four positioning shafts (102), the fence frame (5) can be driven by foot to slide up and down to switch between blocking and idle states, making it more labor-saving compared to manual operation; the four F-shaped sliding bars (502) are symmetrically welded at the bottom of the front and rear panels of the fence frame (5), and four sliding sleeves are welded at the bottom of the four F-shaped sliding bars (502), and the four sliding sleeves are pushed by a second set of springs to slide with the four positioning shafts (102); the head ends of the two L-shaped inserts (6) both have an oblique section structure, and two protruding support rods are welded in opposite directions on the vertical support sections of the two L-shaped inserts (6), and the tails of the two protruding support rods are rotatably connected to two connecting rods (601); a vertical short shaft (107) is welded to the middle section of the horizontal brace connecting rod located on the left side inside the base (1), and a stepping frame (104) is mounted on the vertical short shaft (107) in a sliding way. As shown inFIG.6, a rotating shaft (401) is welded at the rear of the bracket plate (4) and two driven gears (402) are symmetrically sleeved on the left and right ends of the rotating shaft (401); as the bracket plate (4) is rotatably connected, it can be turned up and set in an inclined state after the test is completed to unload the rock slag, which saves the trouble of manually pushing, scraping and unloading the rock slag with the help of external tools, making the cleaning and unloading at the top of the bracket plate (4) convenient and quick. As shown inFIG.4, two gear racks (503) are symmetrically welded in both sides on the top of the rear panel of fence frame (5) and will get engaged and contacted with the two driven gears (402) while the fence frame (5) slides down with interference; through the power transmission by the two racks (503), when being lowered, shrunk and hidden after the test is completed, the fence frame (5) can slide downward with interference and interlinked and engaged to drive the two driven gears (402) to control the bracket plate (4) to overturn and unload the materials, which solves the problem of driving the bracket plate (4) to overturn with the help additional manual effort, which is convenient to operation, saving both time and labor; As shown inFIG.3, two wedges (504) are symmetrically welded on the inner side of the middle section of the front and rear panels of fence frame (5) and will slide down and come into contact with the oblique sections of the head ends of the two L-shaped inserts (6), and the two L-shaped inserts (6) can be inserted to position two wedges (504) and limit the fence frame (5) to a sliding idle state, but the fence frame (5) can still continue to slide downward with interference after being blocked by the two L-shaped inserts (6), which ensures the fence frame (5) is interlinked to overturn the bracket plate (4) normally; As shown inFIG.8, the stepping frame (104) has a rectangular rear with opening structure, and the tails of the two side support shafts of the stepping frame (104) are rotatably connected together with the tails of the two connecting rods (601); two connecting rods (601), two L-shaped inserts (6) and a stepping frame (104) are connected together to form a double-crank sliding bar mechanism, through which the stepping frame (104) can be driven by foot to slide downward to extract and release the two L-shaped inserts (6), saving the trouble of bending over and holding the stepping frame (104) compared with manual operation. Working principle: the rock to be tested is first placed in the top center position of the bracket plate (4), and the press frame (104) is driven by foot to slide down; since the two connecting rods (601), the two L-shaped inserts (6) and the stepping frame (104) are connected together to form a double-crank sliding bar mechanism, the stepping frame (104) can slide down to drive the two L-shaped inserts (6) to slide and release the fence frame (5); after being released positioning, the fence frame (5) is jacked by the spring on the four positioning shafts (102) and can slide up to baffle the top of the bracket plate (4), and then the four hydraulic cylinders (2) are started and push the square press plate (3) and the jacking pillar (301) downward through the four piston shafts on the cylinders to test the rock mechanical properties; after the test is completed, the square press plate (3) is lifted up by the four hydraulic cylinders (2), and finally the fence frame (5) is driven to slide down and hidden by the dynamic output of the foot frame (501); through the power transmission by the two racks (503), when being lowered, shrunk and hidden after the test is completed, the fence frame (5) can slide downward with interference and interlinked and engaged to drive the two driven gears (402) to control the bracket plate (4) to overturn and unload the materials; the inclined bracket plate (4) can be used to unload the rock slag on it, and can be driven back to its horizontal position when the fence frame is restored upward by sliding with interference, preparing for the next test. It is apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention is capable of being realized in other specific forms without departing from the spirit or essential features of the present invention. Accordingly, the embodiments shall be regarded as exemplary and non-limiting in every point of view, and the scope of the present invention is limited by the appended claims and not by the foregoing specification, and is therefore intended to encompass all variations falling within the meaning and scope of the equivalent elements of the claims. Any appended markings in the claims shall not be regarded as a limitation to claims of the present invention. | 9,436 |
11860134 | DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The term “workpiece” as used herein includes any object, thing, structure, piece of machinery, piece of equipment, padeye, hook, shackle, bar, beam, tubular member, or any other thing which is or has at least a portion thereof subject to tension and/or compressive forces while being used, transferred, lifted, or subjected to any other activity. Referring first toFIG.1, one embodiment of the apparatus of the present invention, shown generally as10, comprises first and second spaced side frame members or columns12and14, and frame assembly16, which is connected to the upper ends of side members12and14. Apparatus10can also include a base18to which side members12and14are connected. As shown hereafter, there are a series of anchors connected to base18, albeit that in its simplest version a single anchor could be employed. Secured to frame assembly16is a force generator or tensioning assembly shown generally as20and described more fully below. Turning now toFIGS.2-5, it can be seen that frame assembly16comprises a sub-structure16A comprising a beam17on which is mounted a superstructure16B, a tensioning assembly20being secured to superstructure16B. Superstructure16B, as best seen inFIG.3, generally comprises plates27and29welded to I-beams32, which in turn are welded to the top of beam17. Force generator/tensioning assembly20is operative to apply an upward force to a workpiece positioned between and connected to tensioning assembly20and base18and can be comprised of spaced I-beams22and24, I-beams22and24being attached to one another by a top plate26, there being bottom plates28and30connected to the bottoms of I-beams22and24, respectively. A pair of hydraulic cylinders40and42having piston rods40A and42A are secured to plates28and30, respectively, the hydraulic cylinders40and42being oriented such that piston rods40A and42A move upwardly out of the cylinders40and42when hydraulic pressure (from a source not shown) is supplied below the pistons to which rods40and42are connected. Thus, the entire piston area is subject to the force of the hydraulic pressure. Piston rods40A and42A are connected to a T-bar44which in turn is connected via a coupling46to a hook assembly48, hook assembly48having a hole50for cable or the like. As shown inFIG.2, a workpiece70having a padeye72is connected by a cable74to hook assembly48. Workpiece70is also provided with padeyes76and78which in turn are connected by cables76A and78A to anchors80respectively. It will thus be appreciated that when hydraulic pressure from a source (not shown) is applied to the pistons of cylinders40and42, piston rods40A and42A will be moved upwardly in the direction of arrow A tensioning cables74,76A and78A. The cables are flexible in the sense that they can be formed into loops or the like but generally have ultimate tensile strength sufficient to accommodate the loads to which they are subjected pursuant to their use in the present invention. In practice, the cables, wires, ropes, etc. will be chosen as to size, minimum breaking strength, and other relevant parameters so as to withstand the loads to which they will be subjected. In practice, the cables are tensioned to a predetermined amount sufficient to ensure that padeye72will not shear or break away from workpiece70, thus ensuring that padeye72and its connection by welding or the like to workpiece70, has sufficient structural integrity to withstand lifting forces applied to padeye72while workpiece70is being lifted. As best seen inFIGS.4and5, tensioning assembly20is provided with two rack and pinion assemblies90and92. The pinions of rack and pinion assemblies90,92are mounted on a shaft94which is journaled in flanges44A and44B of T-bar44. Thus, the pinions and the connecting shaft94are movable with T-bar44. The racks of the pinion assemblies90and92are fixedly connected to beams22and24and have equally spaced teeth. Since the pinions and the piston rods40and42are connected to the T-bar44, movement of the piston rods40and42results in movement of the pinions on the racks. This is an important feature as it ensures that as piston rods40and42extend, they move at the same speed because of engagement during movement of the equally spaced teeth of the racks with the pinion teeth, thus ensuring that the pistons do not become canted. Also connected to T-bar44are rollers100and102which engage and move on I-beams22and24as T-bar44moves up and down. Turning now toFIGS.6,7, and8, a base and anchor assembly usable in the apparatus of the present invention is shown. The base shown generally as18is fixed and can include a concrete pad104on which is positioned a platform shown generally as106. By “fixed” is meant that the base is capable of withstanding upward tension forces applied by the apparatus of the present invention without moving. Platform106comprises end beams108and110and center I-beams112and113having webs114and116. Webs114and116have registering holes for receipt of a load bar or shaft120, load bar or shaft120having a handle121. There is an access opening124in plate111having a hinged door126, which when opened as shown inFIG.7, allows the handle121of load bar120to be accessed and moved into and out of the registering holes in webs114and116. Accordingly, an eye hook or any similar connecting device can be placed in the access opening124when load shaft120is removed. Shaft120can then be repositioned in the registering holes through an eye or opening in the eye hook whereby the eye hook is in a position secured to platform106by load shaft120. In addition to the anchor formed by load shaft120in connection with platform106as described above, there are a plurality of other anchors80formed by padeyes secured to platform106. FIG.9depicts how the height of the apparatus of the present invention can be raised or lowered. In this regard, side frame members12and14can be modular in that they can have sections12A and14A which can be selectively added or removed as desired. To this end, the upper ends of side frame members12and14have a projecting formation130having chamfered sides132which can be received in a complementary shaped socket formed in the bottom plates134and136of modular sections12A and14A. In other words, the sections fit together much like a ball and socket arrangement. Turning now toFIG.10there is shown a reel140suspended by a hook141and cable143from tensioning apparatus20. As can be seen, reel140has been removed from the cradle C on the bed of a truck T. In this regard, the vertical design of the apparatus of the present invention allows a truck carrying a workpiece to be driven onto the base, whereby the workpiece can be unloaded, tested, and then reloaded. Reel140is of typical design, having side rims only one of which142is shown, and a center, hollow core144. Reel140can be of a type designed to carry long lengths of flexible material such as steel cable, fiber optic cable, etc. Such reels can have a drum diameter up to ten feet and greater. It will be appreciated that when a cable or the like is wound onto the core144until the reel140is full, the weight of the cable on the core144can be considerable. Again, this is a function of the size of cable, the diameter of the core, and other factors. In any event, it is known that the force from the weight of the cable on the drum, can collapse the core144. Accordingly, it is desirable that the core be tested to ensure it can withstand the forces exerted on the core by the wound cable. To this end, reference is now made toFIG.11. A hoisting cable section150is connected between hook48via link149and second link152. A loop of cable154encircles core144and is also connected to link152. A second loop of cable155encircles core144and is connected to anchors156and158connected to platform106. When the cylinders in tensioning assembly120are activated, cable150moves upwardly in the direction shown by the arrow, tensioning cable loop154around drum144. At the same time, loop155is also tensioned around core144. The net result is that there is substantially a radially inwardly directed force exerted substantially circumferentially around core144. The tensioning assembly can be used to tension the loops154and155and exert the desired predetermined amount of collapse force on core144. Turning now toFIG.12, the apparatus of the present invention is shown testing the connection holes on a triangular spreader beam shown generally as170. Spreader beam170has a plate portion172and a bottom rib174provided with a plurality of attachment holes173to which a connection link, e.g., a hook, can be attached. In testing spreader beams, it is necessary that the lifting holes be tested in pairs. In this regard, each of the pairs is spaced an equal distance from the centerline of the spreader bar. As can be seen in the depiction ofFIG.12, four pairs of connecting holes are being tested, indicated by the lines180,182,184, and186. When the pistons in tensioning assembly20are actuated as shown by the arrow, tension is transferred through cable190and through spreader bar170to the various sets of cable180-186to test the structural integrity of the attachment holes173and specifically the structural integrity of the rib174. Turning now toFIGS.13and14, there is shown a spreader bar200being tested by the apparatus of the present invention. Spreader bar200has two upper lifting eyes202and204and two lower lifting eyes206and208. A cable210is connected between hook48and lifting eye202, while a second cable211is connected to lifting eye204and hook48. Cables210and211are connected to lifting eyes202and204, respectively, by bolt type anchor shackles202A and204A. Lifting eye206is connected to an anchor80via cable section212and a bolt type anchor shackle206A. In like fashion, lifting eye208is connected to an anchor80via cable section214and bolt type anchor shackle208A. It will be appreciated from the above discussion that tensioning assembly20can apply a tensioning force to all of the lifting eyes on spreader bar200to the desired degree. FIG.15shows the apparatus of the present invention testing padeyes welded to a large cylindrical workpiece such as a section of pipeline pipe P. Hook48is connected to a spreader bar220. A pair of linkages222and224connect spreader bar220to padeyes226and228, respectively, which are welded to pipe P. Cable loops230and232are connected to anchors80. In the manner described above, when the piston rods of the cylinders, are extended, linkages222,224, and cable loops230,232are placed in tension, applying a tensile force to padeyes226and228, the force being applied to a predetermined level. It will be understood that in measuring various forces on a workpiece that can be exerted using the apparatus of the present invention, a variety of devices for measuring force/weight can be employed. In particular load cells which measure compression, tension, bending, or shear forces can be employed. Non-limiting examples of load cells that can be employed include compression load cells, tension load cells, tension and compression load cells, beam load cells, load measuring shackles, load measuring pins, load monitoring links, etc. The type of load cell or other force/weight measuring device will be dependent upon the type of workpiece, or portion thereof, which is being tested. Although specific embodiments of the invention have been described herein in some detail, this has been done solely for the purposes of explaining the various aspects of the invention, and is not intended to limit the scope of the invention as defined in the claims which follow. Those skilled in the art will understand that the embodiment shown and described is exemplary, and various other substitutions, alterations and modifications, including but not limited to those design alternatives specifically discussed herein, may be made in the practice of the invention without departing from its scope. | 11,943 |
11860135 | DETAILED DESCRIPTION OF THE EMBODIMENTS In the embodiment of the present disclosure, a three-dimensional dynamic and static load test system for simulating deep roadway excavation, as shown inFIGS.1-15. A three-dimensional dynamic and static load test system for simulating deep roadway excavation is provided, which includes a mobile platform7, a box body8, a support frame, a roadway excavation device, a data monitoring unit and etc. The mobile platform7is provided with a support platform28, the support platform28is capable to move relative to the mobile platform7, and the box body8is placed on the support platform28. The support platform28moves relative to the mobile platform7to drive the box body8to move. Specifically, the mobile platform7includes a support base30, a long platform29, and guides rails27, along platform29is arranged in the middle of the support base30, the long platform29extends along a front-rear direction of the support base30, the guide rails are arranged on the left side and the right side of the support base, and the guide rails extends along the front-rear direction of the support base. The edge positions of the front side and the rear side of the support platform28are respectively provided with a first support seat281. The first support seat281is provided with a first lifting oil cylinder2401, a telescopic end of the first lifting oil cylinder2401faces downwards, and the end of the telescopic end of the first lifting oil cylinder2401is provided with a wheel seat241. The wheel seat241is rotationally connected with a roller26, and the roller26is located above the long platform29. After the telescopic end of the first lifting oil cylinder2401is extended, the roller26contacts the long platform29. The edge positions of the left side and the right side of the support platform are respectively provided with a second support seat282. The second support seat282is provided with a second lifting oil cylinder2402, a telescopic end of the second lifting oil cylinder2402faces downwards, and the end of the telescopic end of the second lifting oil cylinder2402is provided with a slider242. The slider242is slidably connected to the guide rail. The cylinder body of a telescopic oil cylinder31is connected to the support base30, and the telescopic end of the telescopic oil cylinder31is connected to the support platform28via a connection piece25. The box body8is a steel structure with a large volume, and the weight of similar material model placed inside the box body8is heavy, so it is time-consuming and laborious to move them in manual. The movement process of the support platform28relative to mobile platform7is as follows:1. When it needs the box body8to be moved forward with the support platform28relative to the mobile platform7, the telescopic end of the second lifting oil cylinder2402on the second support seat282is extended to drive the support platform28to lift upward; after the support platform28is lifted at the set height, the telescopic end of the first lifting oil cylinder2401on the first support seat281is extended, and the roller26contacts the long platform29; the telescopic end of the second lifting oil cylinder2402on the second support seat282is retracted to a set distance, so that the slider242no longer exerts pressure on the guide rail27, keeping the slider connected to the guide rail27in sliding; the telescopic end of the telescopic oil cylinder31is extended to push the roller26to roll forward along the long platform29, to drive the support platform28to move forward relative to the mobile platform7. During the process of moving out, the slider242and the guide rail27slide together to achieve guidance. After the support platform28is moved out, the telescopic ends of the lifting oil cylinders on the first support seat281and the second support seat282are retracted, so that the support platform28is pressed onto the long platform29, to facilitate the model laying and the placement of sensors by the testing personnel.2. When it needs the box body8to be moved backward with the support platform28relative to the mobile platform7, the telescopic end of the second lifting oil cylinder2402on the second support seat282is extended to drive the support platform28to lift upward; after the support platform28is lifted at the set height, the telescopic end of the first lifting oil cylinder2401on the first support seat281is extended, and the roller26contacts the long platform29; the telescopic end of the second lifting oil cylinder2402on the second support seat282is retracted to a set distance, so that the slider242no longer exerts pressure on the guide rail, keeping the slider242connected to the guide rail27in sliding; the telescopic end of the telescopic oil cylinder31is retracted to push the roller26to roll backward along the long platform29, so as to drive the support platform28to move backward relative to the mobile platform7. During the process of moving in, the slider242and the guide rail27slide together to achieve guidance. After the support platform28is moved in, the telescopic ends of the lifting oil cylinders on the first support seat281and second support seat282are retracted, so that the support platform28is pressed onto the long platform29for subsequent testing. The box body8is in a rectangular structure, which is symmetrically distributed in front and back, and left and right. The box body8is formed by a plurality of blocks21, and the plurality of blocks21are detachable spliced. The block21is made of steel material, with a hollow structure in the middle. Assembly holes are set on the side walls of the block21, and the adjacent blocks21are connected by high-strength bolts22. In this way, several different specifications and sizes of box body8can be formed by splicing the blocks21according to the experimental needs. A similar material model is placed inside the box body8. A observation window19is detachably arranged on the box body8, which is used to observe the deformation and failure mode of surrounding rock through the observation window19, and the observation window19is made of toughened glass material. The lateral bearing plates23are arranged at the left end and the right end of the box body8, and an axial bearing plate17is arranged at the top inside of the box body8. The lateral bearing plate23is used to transfer force between the loading end of the lateral loading cylinder18and the similar material model, while the axial bearing plate17is used to transfer force between the loading end of the axial loading cylinder15and the similar material model. The support frame includes an outer frame5and an inner frame6, both of which are in an n-shaped structure. The support frame is erected at the left, right, and top ends of the mobile platform7and the box body8. The left end of the support frame is provided with a plurality of pendulum impact units. Each of the plurality of the pendulum impact units includes a first impact rod91, a swing rod56, a pendulum12, a first fixed pulley591, and a first pull rope57. The first impact rod91passes through the box body8and is capable to move along horizontal direction. One end of the first impact rod91contacts the lateral bearing plate23at the left end inside of the box body8. The support frame is provided with a support rack501, the upper end of the swing rod56is hinged to the support rack501, and the lower end of the swing rod56is provided with the pendulum12. The first fixed pulley591is arranged on the support rack501, one end of the first pull rope57is connected to the pendulum12, and the other end of the first pull rope57is led out through the first fixed pulley591. After pulling the first pull rope57and releasing the first pull rope57, the swing rod57swings relative to the support rack501, and the pendulum12strikes the other end of the first impact rod91. One end of the first impact rod91impacts the lateral bearing plate23at the left end inside the box8. By changing the swing height of the pendulum12through the first pull rope57, the force of the pendulum12hitting the first impact rod91is adjusted. The left end of the support frame is provided with a plurality of lateral actuators10, each of the lateral actuators10is connected to one end of the second impact rod92, the second impact rod92passes through the box body8, the second impact rod92is capable to move in a horizontal direction, and the other end of the second impact rod92contacts the lateral bearing plate23at the left end inside the box body8. The right end of the support frame is provided with a plurality of lateral loading cylinders18, the loading end of each of the lateral loading cylinders18passes through the box body8, the loading end of each of the lateral loading cylinders18conducts loading along the horizontal direction, and the loading end of each of the lateral loading cylinders18contacts the lateral bearing plate23at the right end inside the box body8. The top of the support frame is provided with a plurality of drop hammer impact units, each of drop hammer impact units includes a third impact rod93, a drop hammer13, a second fixed pulley592, and a second pull rope58. The third impact rod93passes through the box body8and is capable to move along vertical direction, one end of the third impact rod93contacts the axial bearing plate17at the top inside of the box body8, the second fixed pulley592is arranged on the outer frame5of the support frame, one end of the second pull rope58is connected to the drop hammer13, and the other end of the second pull rope58is led out through the second fixed pulley592. After pulling the second pull rope58and releasing the second pull rope58, the drop hammer13hits the other end of the third impact rod93, so that one end of the third impact rod93impacts the axial bearing plate17at the top of the inside of the box body8. By changing the lifting height of the drop hammer13through the second pulling rope58to adjust the force of the drop hammer13hitting the third impact rod93. The top of the support frame is provided with a plurality of axial actuators14, each of the axial actuators14is connected to one end of the fourth impact rod94, the fourth impact rod94passes through the box body8, the fourth impact rod94is capable to move along vertical direction, and the other end of the fourth impact rod94contacts the axial bearing plate17at the top inside the box body8. A hollow transmission cylinder11is arranged between the support frame and the box body8, and the first impact rod91, the second impact rod92, the third impact rod93, and the fourth impact rod94are all located inside the hollow transmission cylinder11. In the figures, the hollow transmission cylinder11at the positions of the second impact rod92, the third impact rod93, and the fourth impact rod94is omitted for clearly expression of the second impact rod92, the third impact rod93, and the fourth impact rod94. The guidance of each impact rod is achieved by the hollow transmission cylinder11. Wherein a threaded hole is provided on the support frame, an external thread is provided on the outer surface of the hollow transmission cylinder11, and the hollow transmission cylinder11is threaded connected to the threaded hole. The hollow transmission cylinder11is rotated relative to the threaded hole to adjust the position of the hollow transmission cylinder11according to the specifications and dimensions of the box body8. The top of the support frame is provided with a plurality of axial loading cylinders15, wherein the loading end of the axial loading cylinder15passes through the box body8, the loading end of the axial loading cylinders15conducts loading in vertical direction, and the loading end of the axial loading cylinder15contacts the axial bearing plate17at the top inside of the box body8. Both the lateral actuator10and the axial actuator14are set as electro-hydraulic servo actuators, and the lateral actuators10, the axial actuators14, the lateral loading cylinders18, and the axial loading cylinders15share a high-pressure pump box4. The high-pressure pump box4provides hydraulic power for the lateral actuators10, the axial actuators14, the lateral loading cylinders18, and the axial loading cylinders15. The static load control units201are installed in the static load control cabinet2. The static load control units201are respectively connected to the lateral loading cylinder18and the axial loading cylinder15through signal connections, hydraulic sensors16are provided in the lateral loading cylinder18and the axial loading cylinder16, and the static load control units201and the hydraulic sensors16are connected to the main control unit3through signal connections. The main control unit3controls the static load control units201to perform static loading, unloading, and load holding on the loading ends of the lateral loading cylinder18and the axial loading cylinder15, and can also achieve servo control, displacement control, stress control, and other loading methods. The hydraulic sensors16provide real-time feedback on the oil pressure inside the lateral loading cylinder18and the axial loading cylinder15. The hydraulic sensors16will realize safety warning during the loading process if the oil pressure inside the oil cylinder is over the preset value. The dynamic load control unit is connected to the lateral actuator10and the axial actuator14through signals, and the dynamic load control unit is connected to the main control unit3through signals. The main control unit3controls the dynamic load control unit to apply a set impact form of dynamic loads to the lateral actuator10and the axial actuator14, so as to change the force and frequency of impact. The lateral loading cylinder18and the axial loading cylinder15apply static load to the similar material model to simulate initial crustal stress. The lateral actuator10and the axial actuator14apply vibration to the similar material model to simulate continuous disturbances in surrounding roadway excavation and mining engineering of surrounding working faces. The pendulum impact unit and the drop hammer impact unit impact the similar material model to simulate the instantaneous disturbances of geological structure sudden change caused by fault slip and roof failure. A power supply1is used to supply power to the main control unit3, the static load control units201, the dynamic load control unit, and the data monitoring unit. The roadway excavation device is configured to excavate simulated roadway20in the similar material model. Specifically, the roadway excavation device includes a mobile chassis39, a driving mechanism, a rotary table37, a rotation driving mechanism, a cantilever rack35, a multi-stage oil cylinder34, a support oil cylinder36, a rotary drill bit32, a rotary driving mechanism, a stress sensor33, and an excavation control unit. The mobile chassis39is driven to move by a driving mechanism, the rotary table37is rotatably connected to the mobile chassis39, the rotary table37is driven to rotate by the driving mechanism, the cantilever rack35is installed on the rotary table37, the cantilever rack35is arranged on the rotary table37, and a cylinder end of the multi-stage oil cylinder34is hinged with the cantilever rack35. One end of the support oil cylinder36is hinged with the cantilever rack35, and the other end of the support oil cylinder36is hinged with the cylinder end of the multi-stage oil cylinder34. The end of the telescopic end of the multi-stage oil cylinder34is provided with the rotary drill bit32, the rotary drill bit32is driven to rotate by a rotary driving mechanism, and a stress sensor33is arranged on the rotary drill bit32. The excavation control unit38is respectively connected to the driving mechanism, the rotation driving mechanism, the multi-stage oil cylinder34, the support oil cylinder36, the rotary driving mechanism, and the stress sensor33through signals, and the excavation control unit38is connected to the main control unit3through signals. The main control unit3controls the driving mechanism through the excavation control unit38to drive the mobile chassis39forward, backward, stop, or turn; controls the rotation driving mechanism to drive the rotary table37to rotate at a set angle relative to the mobile chassis39to adjust the excavation angle; controls the expansion and contraction of the multi-stage oil cylinder34to drive the rotary drill bit32forward or backward; controls the expansion and contraction of the support oil cylinder36to drive the swing of the multi-stage oil cylinder34to adjust the pitch angle of the multi-stage oil cylinder34; controls the rotation driving mechanism to drive the rotation of the rotary drill bit32to excavate the simulated roadway20, and monitor the drilling pressure during the excavation of the simulated roadway20in real-time through the stress sensor33. The data monitoring unit is configured to monitor the parameters of the similar material model during the process of excavating the simulated roadway20. Specifically, the data monitoring unit includes an acoustic emission monitoring unit, a stress monitoring unit, a strain monitoring unit, a displacement monitoring unit and a deformation monitoring unit. The acoustic emission monitoring unit includes an acoustic emission monitoring control host40and an acoustic emission probe45, The acoustic emission probe45is set on the steel nail46, and the steel nail46is inserted into the similar material model to arrange the acoustic emission probe45at different positions in the similar material model (including any position inside the simulated roadway20). The acoustic emission probe45is connected to the acoustic emission monitoring control host40through a signal cable. Using the acoustic emission monitoring unit to monitor and simulate the development of cracks in the surrounding rock of roadway20before, during, and after excavation. The stress monitoring unit includes a stress monitoring control host41, a strain gauge47, and a soil pressure box48. The strain gauge47and the soil pressure box48are arranged at different positions in the similar material model, and the strain gauge47and the soil pressure box48are connected to the stress monitoring control host41through a signal cable. The stress monitoring unit is used to monitor the stress changes in local areas of the similar material model, wherein the strain gauge47is used to monitor the stress situation in the local X, Y, and Z directions of the model, and the soil pressure box48is used to monitor the stress situation in a single direction of the model. The strain monitoring unit includes a strain monitoring control host42and an optical fiber sensor53, the optical fiber sensor53is arranged at different positions in the similar material model, the optical fiber sensor53is connected to the strain monitoring control host42through a signal cable. The strain monitoring unit is used to monitor the strain changes in the model area and local range. The optical fiber sensor53is buried around the model or roadway to monitor the strain (one-dimensional strain) of a certain measuring line, and can be arranged vertically or bent according to the monitoring requirements. The displacement monitoring unit includes a displacement monitoring control host43and a grating displacement sensor49, the grating displacement sensor49is arranged at different positions in the similar material model, and the grating displacement sensor49is connected to the displacement monitoring control host43through a signal cable. The displacement monitoring unit is used to monitor the displacement changes in local areas of the model. The grating displacement sensor49can be placed around the roadway to measure the displacement of the surrounding rock of the roadway, or the grating displacement sensor49can be placed at the edge of the model to measure the overall deformation of the model. The deformation monitoring unit includes a deformation monitoring control host44, a speckle camera51and a 3D scanner50, and the speckle camera51and the 3D scanner50are connected to the deformation monitoring control host44through a signal cable. The deformation monitoring unit is used to monitor the deformation of the model area or the internal deformation of the roadway. The scattered spots52is uniformly sprayed on the model surface, and the speckle camera51is used to monitor the evolution process of the model surface deformation (two-dimensional strain) field through the observation window19. The 3D scanner50is used to scan the inner wall of the roadway and analyze the deformation (3D strain) of the roadway. In the embodiment of the present disclosure, a three-dimensional dynamic and static load test method for simulating deep roadway excavation is further provided, which applies the three-dimensional dynamic and static load test system for simulating deep roadway excavation mentioned above, the method including: Step 1: Experimental Scheme Design Firstly, developing a experimental scheme for this experiment, including the volume of box body8, the type of excavation roadway, the material of the model, the loading plan, and the monitoring plan. After determining the experimental plan, assemble box body8using blocks21. After the assembly of box body8is completed, placing the lateral bearing plates23on the left and right sides inside the box body8, and installing the observation windows19in front and behind the box body8. Step 2: Model Laying and Sensor Layout Moving the box body8forward with the support platform28relative to the mobile platform7, adjusting a material ratio based on mechanical parameters and a similarity ratio of different rock layers, and laying materials to form a similar material model inside the box body8; specifically, manual laying is used to adjust the material ratio based on the mechanical parameters and similarity ratio of different rock layers; layered laying is used, and each layer of material is compacted to ensure that the similar material model has sufficient stiffness to maintain uniform force transmission; burying the acoustic emission probe45, the strain gauge47, the soil pressure box48, the optical fiber sensor53, and the grating displacement sensor49in corresponding positions within the similar material model during the material laying process; after the material laying is completed, an axial bearing plate17is placed above the similar material model to shape the model, and the similar material model is placed for a set time (2-3 weeks); after the similar material model is air dried and formed, removing the observation windows19, spraying scattered spots52on a simulated roadway excavation position of the similar material model, then reinstalling the observation windows19on the box body8, and then moving the box body8backward with the support platform28relative to the mobile platform7. Step 3. Initial Crustal Stress Simulation According to the experimental scheme, loading the similar material model to simulate an initial crustal stress state of roadway excavation, wherein the loading method is hierarchical loading under stress control, using the lateral loading cylinders18and the axial loading cylinders15to hierarchical synchronized load to the similar material model, and maintaining loading for a set time (about 30 min) after completing each level of loading. Step 4: Roadway Excavation Simulation Using the roadway excavation device to excavate a simulated roadway20in the similar material model. After excavating one footage of the simulated roadway20by the excavation device, different lengths of steel sticks54are used to simulate anchor rod (anchor cable) support. A force gauge55is installed at the bottom of the steel stick54to monitor the force status of the anchor rod (anchor cable) in real-time through the force gauge55. Step 5: Dynamic Load Simulation Setting up the impact form (including parameters such as waveform, wavelength, frequency, amplitude, etc.) of the axial actuator14after the excavation of the simulated roadway20is completed, and then applying the axial dynamic load to the similar material model through the axial actuator14; setting up the impact form (including parameters such as waveform, wavelength, frequency, amplitude, etc.) of the lateral actuator10and then applying lateral dynamic load to the similar material model through the lateral actuator10; applying axial impact to the similar material model through the drop hammer impact unit; applying lateral impact to the similar material model through the pendulum impact unit. There are several axial actuators14, lateral actuators10, drop hammer impact units, and pendulum impact units, all of which transmit dynamic loads to the internal model through each impact rod, and the impact position can be changed. During steps 3 to 5, the data monitoring unit is used to monitor the parameters of the similar material model. Specifically, the acoustic emission monitoring unit is used to monitor the development of surrounding rock fissures before, during and after the excavation of the simulated roadway20, the stress monitoring unit is used to monitor the stress change in the local area of the model, the strain monitoring unit is used to monitor the strain change in the model area and local area, the displacement monitoring unit is used to monitor the displacement change in the local area of the model, and the deformation monitoring unit is used to monitor the deformation in the model area or the internal deformation of the roadway. In addition, the long-term load holding function of static loading (the lateral loading cylinder18and the axial loading cylinder15) is used to apply crustal stress to the model, and the monitoring system is used to observe the stress, displacement and deformation of the surrounding rock of the roadway for a long time to achieve creep testing. At this point, a detailed description of this embodiment has been provided in conjunction with the accompanying drawings. Based on the above description, those skilled in the art should have a clear understanding of the three-dimensional dynamic and static load test system used to simulate deep roadway excavation of the present disclosure. The three-dimensional dynamic and static load test system and method for simulating deep roadway excavation can reproduce the whole process of roadway excavation, simulate the multi-directional loading of deep roadway, and restore the real stress state of deep roadway under the influence of dynamic and static load superimposed disturbance. The problem of insufficient research under the condition of unidirectional static loading and lack of multi-directional dynamic and static loading in current large-scale experimental devices has been solved. In addition, the stress and deformation of the surrounding rock of the roadway are reflected in real-time through the data monitoring unit. | 26,956 |
11860136 | DETAILED DESCRIPTION OF THE INVENTION Hereinafter, an embodiment of the gas chromatography analysis method and system according to the present invention will be described with reference to the drawings. As shown inFIG.1, the gas chromatography analysis system of the present embodiment includes an injector2, a separation column4, a detector6, a column oven8, a gas generator10, a control device12, an advanced flow controller (AFC)20, and an advanced pressure controller (APC). The separation column4may be, for example, a capillary column coated or filled with a separation medium for separating components included in sample gas. The separation column4has an inlet end fluidly connected to the injector2and an outlet end fluidly connected to the detector6. The separation column4is housed in the column oven8in which the temperature of the internal space is adjusted to a set temperature. By the way, a packed column may be used as the separation column4instead of the capillary column. In particular, in a case where the detector6is a TCD, it is preferable that a packed column is used as the separation column4. The injector2is for generating sample gas and introducing the generated sample gas into the separation column4by carrier gas. In the present embodiment, hydrogen gas generated by the gas generator10is used as the carrier gas. The detector6is for detecting components included in the sample gas that has passed through the separation column4. As the detector6, any one of an FID, an FPD, an FTD, and a TCD can be used. When the detector6is any of an FID, an FPD, and an FTD, hydrogen gas and oxygen gas generated by the gas generator10are supplied to the detector6as detector gas. Further, when the detector6is a TCD, only hydrogen gas generated by the gas generator10is supplied to the detector6as detector gas (that is, a flow rate of oxygen gas introduced into the detector6is adjusted to zero). The gas generator10is configured to be able to generate hydrogen gas and oxygen gas by electrolysis of water, and to take out each of the generated hydrogen gas and oxygen gas in a separated manner.FIG.4schematically shows the gas generation principle in the gas generator10. An anode electrode and a cathode electrode are provided in the gas generator10with an ion exchange film sandwiched between them. As DC voltage is applied between the anode electrode and the cathode electrode, oxygen is generated by the electrolysis reaction of the following formula on the anode electrode side: 2H2O-4e=O2+4H+ and oxygen is generated by the electrolysis reaction of the following formula on the cathode electrode side: 4H++4e=2H2. The gas generator10is configured to be able to take out the oxygen gas generated at the anode electrode and the hydrogen gas generated at the cathode electrode individually. Note that an electrolysis device configured to be able to individually take out hydrogen gas and oxygen gas generated by electrolysis of water is known (for example, see JP-A-2020-066796, JP-A-2018-178231). The gas generator10has a hydrogen gas outlet and an oxygen gas outlet, and a flow path13is connected to the hydrogen gas outlet and a flow path18is connected to the oxygen gas outlet. The flow path13is branched into a flow path14leading to the injector2and a flow path16leading to the detector6. The flow path18leads to the detector6. The flow path16constitutes a first flow path for guiding hydrogen gas generated by the gas generator10to the detector6as detector gas. The flow path18constitutes a second flow path for guiding oxygen gas generated by the gas generator10to the detector6as detector gas. The flow path14constitutes a third flow path for guiding hydrogen gas generated by the gas generator10to the upstream side of the separation column4as carrier gas. The AFC20is for controlling each of a total flow rate of hydrogen gas introduced into the injector2, inlet pressure of the separation column4, a split vent flow rate, and a purge vent flow rate during analysis. A flow rate obtained by subtracting a split vent flow rate and a purge vent flow rate from a total flow rate of hydrogen gas introduced into the injector2is a flow rate of hydrogen gas introduced as carrier gas into the detector6via the separation column4. That is, the AFC20realizes a carrier gas flow rate adjustor for adjusting a flow rate of hydrogen gas introduced into the detector6as carrier gas. An APC22is for adjusting a flow rate of hydrogen gas flowing through the flow path16and a flow rate of oxygen gas flowing through the flow path18. That is, the APC22realizes a detector gas flow rate adjustor for adjusting the flow rates of hydrogen gas and oxygen gas introduced into the detector6as detector gas. Note that the detector gas flow rate adjustor does not need to be realized by a flow controller such as one of the APC22, and the flow rate of hydrogen gas flowing through the flow path16and the flow rate of oxygen gas flowing through the flow path18may be adjusted by flow controllers different from each other. Here, a structure of the detector6will be described by taking an FID as an example. In a case where the detector6is an FID, as shown inFIG.2, a nozzle102for generating a hydrogen flame is provided in the internal space of a cell100of the detector6. A pipe104forming an outlet portion of the separation column4passes through the inside of the nozzle102. A pipe108forming a part of the flow path16is fluidly connected to the nozzle102, and hydrogen gas generated by the gas generator10is introduced between an outer peripheral surface of the pipe104and an inner surface of the nozzle102as detector gas. The hydrogen gas introduced into the nozzle102as detector gas merges with carrier gas from the separation column4in a tip portion of the nozzle102and is ejected from the tip of the nozzle102. A pipe110forming a part of the flow path18passes through the internal space of the cell100, and oxygen gas generated by the gas generator10is introduced into the internal space of the cell100as detector gas. The oxygen gas introduced into the internal space of the cell100is used as supporting gas for burning the hydrogen gas ejected from the tip of the nozzle102, so that a hydrogen flame is formed at the tip of the nozzle102. Note that the pipe110may communicate with the inside of the nozzle2so that the hydrogen gas and the oxygen gas are mixed inside the nozzle2. Returning toFIG.1, description of the analysis system is continued. The control device12has a function of controlling operation of the gas generator10, the AFC20, and the APC22, and is realized by an electronic circuit including at least a central processing unit (CPU) and a data storage device. The control device12includes a controller24and an information storage area26. The controller24is a function realized by a CPU executing a program, and the information storage area26is a function realized by a part of a storage area of a data storage device. The controller24is configured not only to operate the gas generator10so that a necessary amount of hydrogen gas and oxygen gas for analysis is obtained, but also to control operation of the AFC20and APC22so that a ratio of a flow rate of hydrogen gas introduced into the detector6and a flow rate of oxygen gas introduced into the detector6is made suitable for the detector6. The ratio of flow rates of hydrogen gas to oxygen gas required for normal operation of the detector6differs depending on the type of the detector6(FID, FPD, or the like). The information storage area26stores information as to the ratio of flow rates of hydrogen gas and oxygen gas suitable for various detectors. Note that, as another embodiment, the detector6itself may hold information as to the ratio of hydrogen gas and oxygen gas suitable for the detector6. An example of operation of the gas chromatography analysis system realized by the controller24will be described with reference toFIG.1and the flowchart ofFIG.3. First, the controller24recognizes of what type the detector6is based on information input by the user or based on information read from the detector6, and obtains a ratio of flow rates of hydrogen gas and oxygen gas suitable for the type of the detector6based on the information stored in the information storage area26(Step101). Then, the controller24calculates a flow rate of each of hydrogen gas and oxygen gas required to make a ratio of a total flow rate of hydrogen gas introduced into the detector6as carrier gas via the separation column4and hydrogen gas introduced into the detector6as detector gas via the flow path16and a flow rate of oxygen gas introduced into the detector6through the flow path18to the ratio obtained from the information of the information storage area26(Step102). Based on a calculation result, the controller24operates the gas generator10so that a required amount of hydrogen gas and oxygen gas is generated (Step103), and controls operation of the AFC20and the APC22so that a ratio of flow rates of hydrogen gas and oxygen gas introduced into the detector6becomes a ratio suitable for the detector6(Step104). In a state where flow rates of hydrogen gas and oxygen gas introduced into the detector6are controlled by the above operation, a sample is injected into the injector2and the gas chromatography analysis is started. The sample injected into the injector2becomes sample gas, which is introduced into the separation column4by carrier gas so that components included in the sample are separated from each other (Step105). The components included in the sample separated by the separation column4are introduced into the detector6together with the carrier gas and detected (Step106). Note that, in the above embodiment, hydrogen gas generated by the gas generator10is used as the carrier gas. However, a carrier gas supply source may be provided separately from the gas generator10. In that case, gas other than hydrogen gas such as helium gas and nitrogen gas can be used as the carrier gas. When gas other than hydrogen gas is used as the carrier gas, the controller24controls the APC22so that the ratio of a flow rate of hydrogen gas introduced into the detector6as detector gas and a flow rate of oxygen gas is suitable for the detector6. The embodiment described above is merely an example of embodiments of the gas chromatography analysis method and system according to the present invention. The embodiments of the gas chromatography analysis method and system according to the present invention are as described below. An embodiment of the gas chromatography analysis method according to the present invention includes separating components included in a sample gas by introducing the sample gas into a separation column using carrier gas, and detecting the components in the sample gas that has passed through the separation column and has been introduce into a detector. The detecting includes taking out hydrogen gas and oxygen gas generated by electrolysis of water in a separated manner, and supplying the taken-out hydrogen gas and oxygen gas individually to the detector as detector gas with each flow rate of taken-out hydrogen gas and oxygen gas being independently controlled. According to the specific aspect of the analysis method according to the above embodiment, the detecting includes adjusting a ratio of hydrogen gas and oxygen gas introduced into the detector to a suitable ratio for the detector. Such an embodiment makes it possible to use various types of detectors in gas chromatography analysis using hydrogen gas generated by electrolysis of water. In the above specific aspect, the separating may include supplying hydrogen gas produced by electrolysis of water as the carrier gas to upstream of the separation column, and the detecting may include controlling a flow rate of each of hydrogen gas and oxygen gas so that a ratio of a total flow rate of hydrogen gas introduced into the detector as the detector gas and hydrogen gas introduced into the detector as the carrier gas and a flow rate of oxygen gas introduced into the detector becomes the suitable ratio for the detector. By such an embodiment, in a case where gas other than hydrogen gas and oxygen gas is not used as make-up gas, all gases used in the gas chromatography analysis are generated by electrolysis of water, and gas chromatography analysis not using a gas cylinder can be realized. Make-up gas can be eliminated by making the shape of the detector suitable for a flow rate during use of a capillary column and suppressing the spread of peaks in a chromatogram. Then, since the ratio of the total flow rate of hydrogen gas introduced into the detector and the flow rate of oxygen gas introduced into the detector is controlled to be suitable for the detector, various types of detectors can be used in the gas chromatography analysis not using a gas cylinder. An embodiment of the gas chromatography analysis system according to the present invention includes a separation column for separating components included in a sample gas, a detector fluidly connected to downstream of the separation column, and being for detecting the components separated by the separation column, a gas generator configured to generate hydrogen gas and oxygen gas by electrolysis of water and to individually take out generated hydrogen gas and oxygen gas, a first flow path for guiding hydrogen gas, as detector gas, generated by the gas generator to the detector, a second flow path for guiding oxygen gas, as detector gas, generated by the gas generator to the detector, and a detector gas flow rate adjustor that adjusts each of a flow rate of hydrogen gas introduced into the detector through the first flow path and a flow rate of oxygen gas introduced into the detector through the second flow path. A first aspect of the analysis system according to the above embodiment further includes a controller configured to adjust a ratio of a flow rate of hydrogen gas introduced into the detector and a flow rate of oxygen gas introduced into the detector to a suitable ratio for the detector by controlling the detector gas flow rate adjustor. By such an embodiment, various types of detectors can be used in the gas chromatography analysis system in which hydrogen gas and oxygen gas are generated by electrolysis of water and used as detector gas. The first aspect may further include a third flow path for guiding hydrogen gas, as carrier gas, generated by the gas generator to an upstream side of the separation column, and a carrier gas flow rate adjustor that adjusts a flow rate of hydrogen gas introduced into the detector as the carrier gas, and the controller may be configured to adjust a ratio of a total flow rate of hydrogen gas introduced as the detector gas into the detector and hydrogen gas introduced as the carrier gas into the detector and a flow rate of oxygen gas introduced into the detector to a suitable ratio for the detector by controlling the detector gas flow rate adjustor and the carrier gas flow rate adjustor. By such an embodiment, in a case where gas other than hydrogen gas and oxygen gas is not used as make-up gas, all gases used in the analysis are generated by the gas generation unit, and a gas chromatography analysis system not using a gas cylinder can be realized. Make-up gas can be eliminated by making the shape of the detector suitable for a flow rate during use of a capillary column and suppressing the spread of peaks in a chromatogram. Then, since the ratio of the total flow rate of hydrogen gas introduced into the detector and the flow rate of oxygen gas introduced into the detector is controlled to be suitable for the detector, various types of detectors can be used in the gas chromatography analysis system not using a gas cylinder. Further, in the first aspect, an information storage area that stores information as to a correlation between detector types and ratios of flow rates of hydrogen gas and oxygen gas suitable for each detector types may be included. In that case, the controller may be configured to recognize a type of the detector based on information input by the user or information obtained from the detector, to obtain a ratio of hydrogen gas and oxygen gas suitable for the type of the detector from the information storage area, and to use the obtained ratio of hydrogen gas and oxygen gas as the suitable ratio for the detector. In this manner, the analysis system automatically obtains a hydrogen gas flow rate and an oxygen gas flow rate suitable for the detector without the user setting the flow rates of hydrogen gas and oxygen gas. The separation column in the present invention may be a capillary column. DESCRIPTION OF REFERENCE SIGNS 2Injector4Separation column6Detector8Column oven10Gas generator12Control device13,14,16,18Flow path20AFC22APC24Controller26Information storage area | 16,879 |
11860137 | DETAILED DESCRIPTION Specific embodiments of the disclosure will now be described in detail with reference to the accompanying figures. Like elements in the various figures are denoted by like reference numerals for consistency. In the following detailed description of embodiments of the disclosure, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to one of ordinary skill in the art that the disclosure may be practiced without these specific details. In other instances, well-known features have not been described in detail to avoid unnecessarily complicating the description. Throughout the application, ordinal numbers (e.g., first, second, third, etc.) may be used as an adjective for an element (i.e., any noun in the application). The use of ordinal numbers is not to imply or create any particular ordering of the elements nor to limit any element to being only a single element unless expressly disclosed, such as using the terms “before”, “after”, “single”, and other such terminology. Rather, the use of ordinal numbers is to distinguish between the elements. By way of an example, a first element is distinct from a second element, and the first element may encompass more than one element and succeed (or precede) the second element in an ordering of elements. Embodiments of the invention provide a method and a system for detecting the presence of hydrocarbons in drilling cuttings through the addition of an artificial chemical standard to the drilling fluids. More specifically, the artificial chemical standard is used to accurately measure the amount of drilling fluid contaminates on the recovered drilling cuttings, also referred to as rock chips, from the borehole. The contamination signal from the drilling fluid hydrocarbons is then subtracted from the contamination signal of the contaminated rock chips to reveal an initial amount of natural hydrocarbons in the sample prior to contamination. Each of these aspects will be described in further detail below. During drilling, geologists utilize drilling cuttings to extract crucial information in order to identify different geological layers and their potential for containing hydrocarbons. However, drilling cuttings that are recovered from wells drilled with oil-based fluids material are often artificially contaminated with hydrocarbons. Such oil-based fluids, when mixed with drilling cuttings in the borehole, will result in overprinting the natural hydrocarbon signal in drilling cuttings. This is typically identified using pyrolysis and gas chromatography-mass spectrometry (GC-MS) techniques. Turning now toFIGS.1A-1D,FIGS.1A-1Ddepict a close similarity in composition between oil-based drilling fluids and natural hydrocarbons in crude oil and rock extracts. Specifically,FIGS.1A and1Billustrate a comparison between the composition of drilling fluids (FIG.1A) and light crude oil (FIG.1B) using a pyrolysis flame ionization detector. InFIGS.1A and1B, curve (100) represents flame ionization detector (FID) values over time, and curve (102) represent values obtained from an oven that controls the temperature over time. Referring toFIGS.1C and1D, a comparison between the composition of drilling fluids (FIG.1C) and natural hydrocarbons from rock extracts (FIG.1D) using total ion chromatogram obtained from GC-MS analysis is depicted. The labeled peaks depict the abundance of straight-chain hydrocarbons ranging in carbon number from C10to C24. Oil-based drilling fluids contain a series of n-alkane hydrocarbons at the diesel range, with carbon number and a molecular weight ranging from normal decane (n-C10) to tetracosane (n-C24). Carbon number is the total number of carbon atoms contained in a hydrocarbon or other chemical's molecule. A hydrocarbon is any of a class of organic chemicals made up of only the elements carbon (C) and hydrogen (H). Hydrocarbon type refers to the type of chemical bonds between the carbon atoms and other parts of the molecule. Hydrocarbon type includes monoaromatic (substituted benzene), naphthalene, fluorene, anthracene, olefin, iso-olefin (alkene or alkyne), olefino-naphthene, mono-naphthene (cycloalkane), decalin, indane, indene, tetralin, paraffin, isoparaffin (alkane), nitrogen, sulfur and oxygen (NSO) compounds, asphaltenes, alcohol, ether, ester, ketone, and aldehyde. Alkanes contain only single bonds, alkenes contain a carbon-carbon double bond, alkynes contain a carbon-carbon triple bond, and aromatics contain a benzene ring. The composition of oil-based drilling fluids appears in the pyrolysis instrument as one peak (refer toFIG.1A), which is also similar to the same signal of light crude oil (refer toFIG.1B). Therefore, pyrolysis analysis is considered unreliable for rock samples contaminated with drilling fluid. In addition, the GC-MS signal of an oil-based mud (FIG.1C) is closely similar to the natural hydrocarbon signal obtained from rock extracts (FIG.1D), which can negatively impact a number of geochemical parameters. Described herein is a method for detecting the presence of natural hydrocarbons in drilling cuttings through the addition of a synthetic chemical standard, having a known concentration, to the drilling fluids. The addition of the synthetic chemical standard must be introduced prior to circulating the drilling fluid through the borehole, such that, the drilling mud at the mud pit contains the drilling fluids with the added synthetic chemical compound. FIG.2illustrates an exemplary drilling well site. In general, well sites may be configured in a myriad of ways. Therefore, the drilling well site depicted inFIG.2is not intended to be limiting with respect to the particular configuration of the drilling equipment. The drilling well site is depicted as being on land. In other examples, the well site may be offshore, and drilling may be carried out with or without use of a marine riser. A drilling operation at the well site may include drilling a wellbore into a subsurface including various formations. In one or more embodiments, the well site may include a drilling cuttings collector (200) and a mud tank with a shale shaker (202) at the top. A drill string may be suspended in the wellbore by a derrick (204) with a swivel (206) and a kelly (208) extending down from the top of the derrick (204) and attached to a rotary house (210), which rotates the drill string, and a standpipe (212). Furthermore, in one or more embodiments, the drilling site may comprise a drilling mud pump (214). Mixed mud (216) is collected proximate the rig site surface (218) at which the drill pipe (220) extends down the borehole (222). The borehole (222) can penetrate a number of geological formations, including an overburden layer (224), a tight-sealing shale layer (226), and a hydrocarbon-bearing reservoir layer (228). The drilling site may further include a drilling bit (230) to cut into the subsurface rock. While penetrating the reservoir layer (228), the drilling bit (230) reaches a deepest reservoir zone (232) of the wellbore at which drill cuttings are recovered and circulated to the surface. In one or more embodiments, recovered drilling cuttings are transferred to a natural hydrocarbon detection unit (234). The hydrocarbon detection unit (234) may include an organic solvent extractor (236), which produces the rock extracts. Solvent extraction is the separation and/or concentration of components of a solution by distribution between two immiscible liquid phases. Solvent extraction provides the ability to separate mixtures into components according to their chemical type. The rock extracts may then be analyzed on a gas-chromatograph equipped with a mass-spectrometer (238) (GC-MS), and processing can be performed at a data output, display, and processing module (240). The processing module (240) may be implemented in hardware (i.e., circuitry), software, or any combination thereof. In one embodiment, the GC-MS is a triple quadrupole mass spectrometer, or GC-MS-QQQ. The GC-MS-QQQ is a tandem mass spectrometer consisting of two quadrupole mass analyzers in series with a radio frequency (RF)-only quadrupole between them to act as a cell for collision-induced dissociation. The GC-MS-QQQ allows for increased sensitivity and specificity, resulting in lower detection and quantitation limits. Referring toFIG.3,FIG.3is a flowchart depicting the workflows for establishing the detection of natural hydrocarbon in drilling cuttings contaminated with oil-based drilling fluids. The flowchart illustrates steps for tagging and calibration of oil-based drilling fluids with a synthetic chemical standard according to embodiments of this disclosure. Additionally, the flowchart illustrates the process for detecting natural hydrocarbons in rock cuttings contaminated with tagged oil-based drilling fluid described herein. In one or more embodiments, the method is divided into two main stages. The first stage is directed to tagging and calibration of the oil-based drilling fluids, while the second stage is directed to detection of natural hydrocarbon in the contaminated drill cuttings. A first step (300) of the first stage is addition of an internal synthetic chemical standard to the oil-based drilling fluid (302) at a known concentration. This addition of a synthetic chemical standard is referred to as chemical tagging. A non-limiting example of a synthetic chemical standard is 1-pentadecene, but any suitable chemical standard having the same chemical properties can be utilized. The synthetic chemical standard allows for accurate concentration quantification of all hydrocarbon compounds present in the oil-based drilling fluids, as will be described in detail below. In a second step304, the mixture of the drilling fluid and internal synthetic chemical standard is analyzed using a gas chromatography-mass spectrometry (GC-MS) analytical approach, resulting in a peak area of n-alkanes C10+(306) and a peak area of internal standard (308). In a third step (310), the concentration of each individual hydrocarbon compound in parts per million (PPM) is determined as follows: HCconcentration=HCaSTa×STc×F,(1) where HCa denotes the hydrocarbon peak area (306) from the instrument signal, STa denotes the peak area of added internal chemical standard (308), STc denotes the concentration of the internal chemical standard, and F denotes the instrument response factor. The instrument response factor is a ratio of an analyte of interest signal response and the standard internal concentration. In some embodiments, the value for the instrument response factor can range from 0.3 to 4 based on many factors related to the instrument operation condition. After determining the concentration of each hydrocarbon compound in the drilling fluids, a calibration relationship for all n-alkanes is established in a fourth step (312). The calibration relationship is a ratio factor for each compound relative to the internal synthetic chemical standard, as shown in the table of mass chromatography data below. TABLE 1AnalysisOutput(Recovered(MeasuredInput (Drilling fluidsdrilling cuttingsnaturalwith internal standard)from borehole)hydrocarbons)CompoundConcentrationRatioConcentrationConcentrationLabel *(ppm)Factor(ppm)(ppm)n-C105669.570.225799.60130.03n-C1116247.830.6416438.78190.95n-C1226865.221.0527112.26247.04n-C1329421.741.1529703.04281.30n-C1429126.091.1429484.76258.67Std25560.871.0023215.87Nonpresentn-C1527213.041.0627691.21478.17n-C1623010.870.9023439.65428.78n-C1718121.740.7118505.57383.83n-C1812404.350.4912721.73317.38n-C197742.390.307987.07244.68n-C203985.870.164165.86179.99n-C211955.430.082077.77122.34n-C221020.650.041120.0399.38n-C23494.570.02570.2175.64n-C24247.280.01297.3650.08n-C25104.350.00145.0340.68* n-C10.25denote normal alkane hydrocarbons ranging in total carbon number from 10 to 25.Std denotes 1-pentadecene internal standard which is added to the drilling fluids In the second stage, the detection of natural hydrocarbon in contaminated drilling cuttings is achieved as illustrated in the flowchart inFIG.3. The drilling fluid used is already mixed with the synthetic chemical standard in the first stage, resulting in tagged oil-based drilling fluid. In a first step of the second stage (314), the tagged oil-based drilling fluid is circulated in the borehole while drilling via a mud pump. A mud pump, or mud dripping pump, is a reciprocating piston or plunger pump designed to circulate drilling fluid under high pressure down the drill string and back up and out the drilling bit. Referring back toFIG.2, when the drilling bit (230) reaches the reservoir zone (232), the drilling cuttings from the reservoir layer (228) start to circulate to the surface and are retrieved from the shale shaker (202). This action corresponds to a second step of the second stage (316), which is recovery of rock cuttings mixed with tagged oil-based drilling fluid. In one or more embodiments, samples are then transferred to the natural hydrocarbon detection unit (refer to (234) inFIG.2), at which drilling cuttings are first subjected to intensive organic solvent extraction under preset pressure and temperature settings. Non-limiting examples of organic solvents which can be extracted include hexane, iso-octane, dichloromethane, carbon disulfide, and methanol. The organic solvent extraction, in a third step of the second stage (318), extracts all hydrocarbons present in the drilling cuttings sample in a fluid phase. The hydrocarbons removed include artificial hydrocarbons from the tagged oil-based drilling fluid and natural hydrocarbons from the oil-bearing reservoir rock, which together produce a fluid extract. In a fourth step of the second stage (320), the fluid extract is analyzed via gas chromatography-mass spectrometry (GC-MS) to measure the concentration of all hydrocarbons present in the drilling cuttings. The output of the instrument (i.e., a peak area of n-alkanes C10+(322) and a peak area of internal standard (324)) is then processed to calculate the concentration of all hydrocarbons (i.e., n-alkanes in ppm) in a fifth step of the second stage (326). Subsequently, the established calibration ratio factor from the fourth step (312) in the first stage is used to calculate the exact amount of hydrocarbons present in the drilling cuttings that result from drilling fluids (refer to Table 1 above for examples). The hydrocarbon signals from the tagged oil-based drilling fluid are then subtracted from the total hydrocarbon signal measured from the drilling cuttings fluid extracts in a hydrocarbon signal removal from oil-based drilling fluid step (328). Specifically, the subtraction process uses the known concentration of hydrocarbon in the drilling fluids prior to circulating them in the borehole and the hydrocarbon concentrations measured in the recovered drilling cuttings in order to derive the natural hydrocarbon signal. Therefore, the natural hydrocarbon signal is the initial drilling fluid hydrocarbon concentration subtracted from the hydrocarbon concentration of cuttings with drilling fluid. Non-limiting examples of these values are shown in Table 1 above. Finally, the method described herein results in a quantitative measurement of natural hydrocarbons (330). One or more embodiments may be implemented on a computing system. Any combination of mobile, desktop, server, router, switch, embedded device, or other types of hardware may be used.FIG.4illustrates an exemplary computing system (400). The computing system (400) may be one or more mobile devices (e.g., laptop computer, smart phone, personal digital assistant, tablet computer, or other mobile device), desktop computers, servers, blades in a server chassis, or any other type of computing device or devices that includes at least the minimum processing power, memory, and input and output device(s) to perform one or more embodiments disclosed herein. For example, as shown inFIG.4the computing system (400) may include one or more computer processor(s), or a processing module (240), associated memory (404) (e.g., random access memory (RAM), cache memory, flash memory), one or more storage device(s) (406) (e.g., a hard disk, an optical drive such as a compact disk (CD) drive or digital versatile disk (DVD) drive, a flash memory stick), and numerous other elements and functionalities. The computer processor(s) (240) may be an integrated circuit for processing instructions. For example, the computer processor(s) may be one or more cores, or micro-cores of a processor. The computing system (400) may also include one or more input device(s) (408), such as a camera, imager, touchscreen, keyboard, mouse, microphone, touchpad, electronic pen, or any other type of input device. Further, the computing system (400) may include one or more output device(s) (410), such as a screen (e.g., a liquid crystal display (LCD), a plasma display, touchscreen, cathode ray tube (CRT) monitor, or other display device), a printer, external storage, or any other output device. One or more of the output device(s) may be the same or different from the input device(s). The computing system (400) may be connected to a network (412) (e.g., a local area network (LAN), a wide area network (WAN) such as the Internet, mobile network, or any other type of network) via a network interface connection (not shown). The input and output device(s) may be locally or remotely (e.g., via the network (412)) connected to the computer processor(s) (240), memory (404), and storage device(s) (406). Many different types of computing systems exist, and the aforementioned input and output device(s) (408), (410) may take other forms. Further, one or more elements of the computing system (400) may be located at a remote location and be connected to the other elements over a network (412). Further, one or more embodiments may be implemented on a distributed system having a plurality of nodes, where each portion of the embodiment may be located on a different node within the distributed system. In one embodiment, the node corresponds to a distinct computing device. In other embodiments, the node may correspond to a computer processor with associated physical memory. In yet other embodiments, the node may correspond to a computer processor or micro-core of a computer processor with shared memory and/or resources. Software instructions in the form of computer readable program code to perform embodiments of the disclosure may be stored, in whole or in part, temporarily or permanently, on a non-transitory computer readable medium such as a CD, DVD, storage device, a diskette, a tape, flash memory, physical memory, or any other computer readable storage medium. Specifically, the software instructions may correspond to computer readable program code that, when executed by a processor(s), is configured to perform one or more embodiments of the disclosure. The invention described herein has multiple advantages over existing techniques. For instance, unlike prior techniques, the present invention is utilized to detect natural hydrocarbons in drilling cuttings, not crude oil samples. This aspect is significant because it enables flagging of potential oil and gas-bearing rock beds during drilling prior to any further testing. In addition, unique tagging compounds, that are similar in properties to natural hydrocarbons, are used in the present invention to tag oil-based drilling fluids. Unlike existing tracers, such as C16to C20alkenes, the invention described herein introduces 1-pentadecene of known concentration to the drilling fluids. This tagging will fixate the drilling fluid compounds concentration and can be used to measure the presence of natural hydrocarbons in drilling cuttings, prior to producing crude oil altogether. Furthermore, an ultra-sensitive analytical approach is utilized to detect hydrocarbons using GC-MS-QQQ technology, which is capable of detecting hydrocarbons at the femtogram level. GC-MS-QQQ analytical systems measure the mass of each hydrocarbon compound, thereby increasing the sensitivity and selectivity of the detected hydrocarbons compared to traditional GCxGC-FID analytical approaches. In summary, the method according to embodiments of this disclosure adds a synthetic chemical standard to oil-based drilling fluid with known concentration to quantitatively remove the hydrocarbon overprint resulting from oil-based drilling fluid in order to reveal the natural hydrocarbon abundance in drilling cuttings. The method can also be applied for detection of hydrocarbon-bearing layers while drilling in real-time, reducing the need for costly downhole well-testing for hydrocarbons, and maximizing the extracted geological information from drill cuttings. Although only a few example embodiments have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the example embodiments without materially departing from this invention. Accordingly, all such modifications are intended to be included within the scope of this disclosure as defined in the following claims. In the claims, means-plus-function clauses are intended to cover the structures described herein as performing the recited function and not only structural equivalents, but also equivalent structures. Thus, although a nail and a screw may not be structural equivalents in that a nail employs a cylindrical surface to secure wooden parts together, whereas a screw employs a helical surface, in the environment of fastening wooden parts, a nail and a screw may be equivalent structures. It is the express intention of the applicant not to invoke 35 U.S.C. § 112(f) for any limitations of any of the claims herein, except for those in which the claim expressly uses the words ‘means for’ together with an associated function. | 21,963 |
11860138 | DESCRIPTION OF EMBODIMENTS Hereinafter, modes for carrying out the present invention will be described with reference to the drawings. In the embodiments given below, “lipid” refers to an organism-derived substance containing a fatty acid or a hydrocarbon chain. First Embodiment The analysis method of the present embodiment involves detecting ions corresponding to cholesteryl ester and cholesteryl ester peroxide in a sample by liquid chromatography/mass spectrometry (LC/MS), and analyzing a degree of oxidation of the sample based on measurement data obtained by this detection. The sample is a liquid or a solid containing lipids and is not particularly limited as long as the sample can be prepared as a sample for analysis to be introduced to a liquid chromatograph. The sample is preferably a body fluid, such as blood, obtained from a biological body such as a human from the viewpoint that the analysis method of the present embodiment is suitably used for biological or medical purposes. Alternatively, from a similar viewpoint, the sample is preferably a sample containing a solid, such as a tissue (e.g., an organ), cells, or exosomes, obtained from a biological body such as a human. Hereinafter, analysis using blood obtained by blood collection from a human as the sample will be described as an example. A method for pretreating the sample is not particularly limited and can be any method by which analytes among molecules constituting lipids (hereinafter, referred to as lipid molecules) contained in the sample are separable and detectable by LC/MS. Such analyte lipid molecules are not particularly limited and can include cholesteryl ester and cholesteryl ester peroxide as well as cholesteryl ester hydroxide, phospholipid having only one acyl group (hereinafter, referred to as lysophospholipid), phospholipid having a plurality of acyl groups, triglycerol, and the like. The analyte lysophospholipid can include lysophosphatidylcholine. The analyte phospholipid can include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and the like. It should be noted that molecules other than the lipid molecules may be included in the analytes. For example, cholesterol or a cholesterol derivative such as cholesterol sulfate can be used as an analyte. Preferred examples of the pretreatment method include a method of extracting the lipid molecules, etc. with methanol. In this method, cryopreserved whole blood is weighed. Then, methanol is added thereto in an amount of a liquid such as several tens to several hundreds of times the volume of the blood, and the mixture is stirred to extract the lipid molecules into the methanol phase. After centrifugation, the supernatant is collected. The pretreatment method can be similarly performed using refrigerated blood or a sample other than blood. This permits rapid pretreatment of the sample and also facilitates automation. The sample thus pretreated is introduced to a liquid chromatograph of an analytical device. It should be noted that, when the sample collected from a biological body contains a solid, a liquid such as a buffer solution is appropriately added thereto, and a tissue is disrupted and homogenized, for example. Then, liquid components can be subjected to pretreatment. FIG.1is a conceptual diagram showing the configuration of an analytical device related to the analysis method of the present embodiment. The analytical device1includes a measurement part100and an information processing part40. The measurement part100includes a liquid chromatograph10and a mass spectrometer20. It should be noted that the analytical device1may be integrated with a pretreatment part which performs pretreatment and includes a dispensing device and a centrifuge. The liquid chromatograph10includes mobile phase containers11aand11b, liquid feed pumps12aand12b, a sample introduction part13, and an analytical column14. The mass spectrometer20includes an ionization chamber21having an ionization part211, a first vacuum chamber22ahaving an ion lens221, a capillary212which introduces ions from the ionization chamber21to the first vacuum chamber22a, a second vacuum chamber22bhaving an ion guide222, and a third vacuum chamber22c. The third vacuum chamber22cincludes a first mass separation part23, a collision cell24, a second mass separation part25, and a detection part30. The collision cell24includes an aion guide240and a CID gas introduction port241. The information processing part40includes an input part41, a communication part42, a memory part43, an output part44, and a control part50. The control part50includes a device control part51, an analysis part52, and an output control part53. The analysis part52includes a chromatograph preparation part521, a degree-of-oxidation analysis part522, and a normalization part523. The liquid chromatograph (LC)10exploits the distinctive affinity of each component in the sample for a mobile phase and for a stationary phase of the analytical column14to separate the component and elute such components at different retention times. The liquid chromatograph10is not limited by its type as long as the analyte lipid molecules can be separated with the desired accuracy such that the lipid molecules can be separated and detected by the mass spectrometer20. For example, a nano LC, a micro LC, a high-performance liquid chromatograph (HPLC) or an ultrahigh-performance liquid chromatograph (UHPLC) can be used as the liquid chromatograph10. The liquid chromatography has higher quantification accuracy than that of infusion analysis because the suppression of ionization, etc. is reduced. The mobile phase containers11aand11bincludes containers capable of storing a liquid, such as vials or bottles, and store mobile phases differing in composition from each other. The mobile phases stored in the mobile phase containers11aand11bare referred to as mobile phase A and mobile phase B, respectively. The mobile phase A and the mobile phase B are not particularly limited by their composition as long as the analyte lipid molecules can be separated with the desired accuracy. For example, an aqueous ammonium formate solution can be used as the mobile phase A, and a liquid containing acetonitrile and isopropanol at a predetermined volume ratio such as 1:1 can be used as the mobile phase B. The liquid feed pumps12aand12bfeed the mobile phase A and the mobile phase B, respectively, at predetermined flow rates. The mobile phase A and the mobile phase B sent from the liquid feed pumps12aand12b, respectively, are mixed midway through a flow channel and introduced to the sample introduction part13. The liquid feed pumps12aand12bchange the flow rates of the mobile phase A and the mobile phase B, respectively, and thereby change the composition of the mobile phase to be introduced to the analytical column14depending on time. Data that indicates the composition of a mobile phase at each time from a point in time corresponding to the start of analysis such as the introduction of the sample is referred to as gradient data. The liquid feed pumps12aand12bare controlled by device control part51mentioned later based on the gradient data so that a mobile phase having the set composition is introduced to the analytical column14. Time-dependent change in the composition of the mobile phase is not particularly limited as long as the analyte lipid molecules can be separated with the desired accuracy. The sample introduction part13includes a sample introduction device such as an autosampler and introduces a pretreated sample S to the mobile phase (arrow A1). The sample S introduced by the sample introduction part13appropriately passes through a guard column (not shown) and is introduced to the analytical column14. The analytical column14has a stationary phase and exploits the distinctive affinity of each lipid molecule for the mobile phase and for the stationary phase to elute the analyte lipid molecules in the introduced sample S at different retention times. The analytical column14is not particularly limited by its type as long as the lipid molecules can be separated with the desired accuracy. A reverse-phase column is preferred from the viewpoint of easy handling and easy ionization in mass spectrometry. The stationary phase of the analytical column14is preferably, for example, silane bound with C8 or C18 linear hydrocarbon supported by a support such as a silica gel. An elution sample containing the lipid molecules eluted from the analytical column14is introduced to the ionization part21of the mass spectrometer20. An eluate from the analytical column14is preferably input to the mass spectrometer20by online control without the need of an operation, such as dispensing, by a user of the analytical device1(hereinafter, simply referred to as “user”). The mass spectrometer20performs tandem mass spectrometry on the elution sample introduced from the analytical column14to detect the analyte lipid molecules. The route of sample-derived ions Si derived from the sample S, obtained by the ionization of the elution sample is schematically shown by arrow A2of an alternate long and short dash line. The ionization part21of the mass spectrometer20ionizes the introduced elution sample. The ionization method is not particularly limited as long as the lipid molecules are ionized to an extent that the analyte lipid molecules are detected with the desired accuracy. In the case of performing LC/MS/MS as in the present embodiment, electrospray ionization (ESI) is preferred. In the following, embodiments with ESI will be described. A sample-derived ion Si obtained by the ionization of the elution sample exiting from the ion source211moves depending on difference in pressure between the ionization chamber21and the first vacuum chamber22a, passes through the capillary212, and enters into the first vacuum chamber22a. A degree of vacuum is the highest in the third vacuum chamber22c, followed by that of the second vacuum chamber22band the first vacuum chamber22ain this order, In the third vacuum chamber22c, air is exhausted to high vacuum of, for example, 10-2 Pa or less through a vacuum pump (not shown). The sample-derived ions Si entering into the first vacuum chamber22apass through the ion lens221and are introduced to the second vacuum chamber22b. The sample-derived ions Si entering into the second vacuum chamber22bpass through the ion guide222and are introduced to the third vacuum chamber22c. The sample-derived ions Si introduced to the third vacuum chamber22care emitted to the first mass separation part23. The sample-derived ions Si, which pass through the ion lens221, the ion guide222, etc. before entering into the first mass separation part23are converged by electromagnetic action. The first mass separation part23includes a quadrupole mass filter. The sample-derived ions Si having set m/z selectively pass as precursor ions through the quadrupole mass filter by electromagnetic action based on voltage applied to the quadrupole mass filter, and exit therefrom towards the collision cell24. The first mass separation part23permits selective passage of precursor ions of the ionized analyte lipid molecules included in the sample-derived ions Si. The collision cell24dissociates the ionized analyte lipid molecules through collision induced dissociation (CID) to generate fragment ions, while controlling the movement of the sample-derived ions Si by the ion guide240. For CID, a gas containing argon, nitrogen, or the like (hereinafter, referred to as CID gas) against which the sample-derived ions Si are allowed to collide is introduced from the CID gas introduction port241(arrow A3) so as to attain a predetermined pressure within the collision cell. The sample-derived ions Si including the generated fragment ions exit therefrom toward the second mass separation part25. It should be noted that the dissociation method is not limited to CID as long as the fragment ions are detectable. The second mass separation part25includes a quadrupole mass filter. The sample-derived ions Si having set m/z selectively pass as product ions through the quadrupole mass filter by electromagnetic action based on voltage applied to the quadrupole mass filter, and exit therefrom towards the detection part30. The second mass separation part25permits selective passage of fragment ions of the analyte lipid molecules included in the sample-derived ions Si. The detection part30has an ion detector such as a secondary electron multiplier or a photomultiplier and detects the entering sample-derived ions Si including the fragment ions of the analyte lipid molecules. The detection mode may be any of a positive ion mode which detects positive ions and a negative ion mode which detects negative ions. Detection signals obtained by detecting the sample-derived ions Si are A/D-converted by an A/D converter (not shown), and the resulting digital signals are input as measurement data to the control part50of the information processing part40(arrow A4). The information processing part40has an information processing device such as a computer and not only appropriately serves as an interface with a user but performs processing, such as communication, memory, and arithmetic operation, on various pieces of data. The information processing part40serves as a processing device that performs the processing of control of the measurement part100, analysis, and display. It should be noted that the information processing part40may be configured as one device integrated with the liquid chromatograph10and/or the mass spectrometer20. A portion of data that is used in the analysis method of the present embodiment may be stored in a remote server or the like, and a portion of arithmetic processing that is performed in the analysis method may be performed in a remote server or the like. The operation of each part of the measurement part100may be controlled by the information processing part40or may be controlled by a device constituting each part. The input part41of the information processing part40is configured to comprise an input device such as a mouse, a keyboard, various buttons and/or a touch panel. The input part41accepts, from a user, for example, information necessary for processing by the control part50, such as m/z values of the sample-derived ions Si to be detected. The communication part42of the information processing part40is configured to comprise a communication device capable of communicating through wireless connection or wire-line connection via a network such as Internet. The communication part42appropriately sends and receives necessary data so as to receive data necessary for measurement by the measurement part100and to send data processed by the control part50, such as analysis results from the analysis part52. The memory part43of the information processing part40has a non-volatile memory medium. The memory part43memorizes, for example, measurement data output from the measurement part100, and a program for the control part50to execute processing. The output part44of the information processing part40is controlled by the output control part53and configured to comprise a display device such as a liquid-crystal monitor and/or a printer. The output part44outputs information on measurement by the measurement part100, analysis results from the analysis part52, etc. by display on the display device or printing on a printing medium. The control part50of the information processing part40is configured to comprise a processor such as CPU. The control part50performs various processes, such as control of the measurement part100and analysis of the measurement data output from the measurement part100, by executing a program memorized in the memory part43or the like. The device control part51of the processing part50controls the measurement operation of the measurement part100based on analysis conditions or the like set according to, for example, input mediated by the input part41. The analysis part52performs analysis such as analysis of the degree of oxidation of the sample S or quantification of the analyte lipid molecules based on the measurement data output from the measurement part100. The chromatogram preparation part521of the analysis part52prepares mass chromatogram data corresponding to a mass chromatogram. In the mass chromatogram data a retention time corresponds to detection intensity corresponding to a fragment ion of each analyte lipid molecule at the retention time. The mass chromatogram data prepared by the chromatogram preparation part521is memorized in the memory part43. The degree-of-oxidation analysis part522of the analysis part52analyzes the degree of oxidation of the sample S based on a ratio between the intensity of a detected fragment ion of cholesteryl ester (hereinafter, referred to as a non-peroxide ion) and the intensity of a detected fragment ion of cholesteryl ester peroxide (hereinafter, referred to as a peroxide ion). Values of peak intensities or peak areas (i.e., integrated intensity at a peak) corresponding to the peaks of the non-peroxide ion and the peroxide ion are preferably used as these intensities, though values used as these intensities are not particularly limited. For example, any statistical values or the like that indicate the amplitudes of detection signals corresponding to the non-peroxide ion and the peroxide ion can be used. In this context, the peak intensity refers to the maximum intensity at a peak. The degree-of-oxidation analysis part522preferably calculates statistical values such as peak intensities and peak areas by appropriately performing noise reduction processing such as smoothing or background removal. The cholesteryl ester and the cholesteryl ester peroxide for use in the analysis of the degree of oxidation after mass spectrometry by the mass spectrometer20are not particularly limited and preferably have an acyl group with a carbon number of 18, more preferably an acyl group with a carbon number of 18 having two or 3 carbon-carbon double bonds. The degree-of-oxidation analysis part522preferably analyzes the degree of oxidation based on a ratio between the intensity of the non-peroxide ion and the intensity of the peroxide ion, as to cholesteryl esters having acyl groups having the same carbon number and the same number of carbon-carbon double bonds. This achieves more accurate analysis of the degree of oxidation because molecules having the same configuration are analyzed for their degrees of oxidation by quantifying the non-peroxide and the peroxide. It should be noted that the degree of oxidation of the sample S may be analyzed based on a ratio between the sum of intensities of a plurality of detected fragment ions corresponding to a plurality of cholesteryl esters, respectively, and the sum of intensities of a plurality of detected fragment ions corresponding to a plurality of cholesteryl ester peroxides, respectively, as the cholesteryl ester and the cholesteryl ester peroxide for use in the analysis of the degree of oxidation. The degree-of-oxidation analysis part522calculates a ratio by dividing the intensity of the peroxide ion by the intensity of the non-peroxide ion (hereinafter, referred to as a degree-of-oxidation ratio), as an index for the degree of oxidation (hereinafter, referred to as a degree-of-oxidation index) of the sample S. A larger value of the degree-of-oxidation index is interpreted as a higher degree of oxidation of the sample S. It should be noted that the degree-of-oxidation index may not be the value itself of the degree-of-oxidation ratio as long as the degree-of-oxidation index is based on the degree-of-oxidation ratio. Alternatively, a ratio obtained by dividing the intensity of the non-peroxide ion by the intensity of the peroxide ion may be used as the degree-of-oxidation ratio. In this case, a larger degree-of-oxidation index is interpreted as a lower degree of oxidation of the sample S. Thus, the degree-of-oxidation index is appropriately set in coordination with the degree of oxidation of the sample S. The degree-of-oxidation analysis part522evaluates the degree of oxidation of the sample S based on the degree-of-oxidation index and a predetermined threshold (hereinafter, referred to as a degree-of-oxidation threshold). The degree-of-oxidation threshold is not particularly limited and can be appropriately set to an arbitrary value. For example, provided that two degree-of-oxidation thresholds are 0.5 and 2.0, the degree-of-oxidation analysis part522can evaluate the sample S as having high quality when the degree-of-oxidation index is less than 0.5, as having moderate quality when the degree-of-oxidation index is 0.5 or more and less than 2.0, and as having low quality when the degree-of-oxidation index is 2.0 or more. The number of degree-of-oxidation thresholds is not particularly limited and may be one or may be three or more. The value of the degree-of-oxidation threshold is appropriately preset from data, a theory, etc. that indicates the relationship between the degree of oxidation and the quality of the sample, and memorized in the memory part43or the like. The degree-of-oxidation index calculated by the degree-of-oxidation analysis part522, or information that indicates the evaluated sample quality described above (e.g., “low”, “moderate” or “high” quality) is memorized in the memory part43. The normalization part523of the analysis part52calculates a normalized intensity by dividing the intensity corresponding to the fragment ion of each analyte lipid molecule by a normalization factor (hereinafter, referred to as a normalized intensity). The normalization part523employs, as the normalization factor, an intensity of a detected ion (hereinafter, referred to as a non-variation ion), such as a fragment ion, corresponding to a substance given below (hereinafter, referred to as a non-variation substance) having a small amount of variation in long-term storage of a blood sample in Examples mentioned later. In this case as well, statistical values such as peak intensities or peak areas can be appropriately used as such intensities. The non-variation substance is preferably at least one substance selected from the group consisting of cholesteryl ester having an acyl group having a carbon number of 18 and 1 carbon-carbon double bond, cholesteryl ester having an acyl group having a carbon number of 20 and 5 carbon-carbon double bonds, lysophosphatidylcholine having an acyl group having a carbon number of 20 and 5 carbon-carbon double bonds, phosphatidylcholine having two acyl groups having a total carbon number of 32 and a total of 1 carbon-carbon double bond, phosphatidylcholine having two acyl groups having a total carbon number of 34 and a total of 1 carbon-carbon double bond, phosphatidylcholine having two acyl groups having a total carbon number of 36 and a total of 2 carbon-carbon double bonds, phosphatidylcholine having two acyl groups having a total carbon number of 38 and a total of 4 carbon-carbon double bonds, phosphatidylethanolamine having two acyl groups having a total carbon number of 34 and a total of 2 carbon-carbon double bonds, phosphatidylethanolamine having two acyl groups having a total carbon number of 36 and a total of 2 carbon-carbon double bonds and phosphatidylethanolamine having two acyl groups having a total carbon number of 38 and a total of 4 carbon-carbon double bonds. This permits quantitative comparison even when different samples have different lipid concentrations, because a sample-derived ion Si corresponding to each component of the sample S is quantified with reference to a molecule having less variation during storage of the sample S. In the non-variation substance, the phosphatidylcholine having two acyl groups having a total carbon number of 32 and a total of 1 carbon-carbon double bond is preferably phosphatidylcholine containing an acyl group having a carbon number of 16 and 0 carbon-carbon double bonds and an acyl group having a carbon number of 16 and 1 carbon-carbon double bond. In the non-variation substance, the phosphatidylcholine having two acyl groups having a total carbon number of 34 and a total of 1 carbon-carbon double bond is preferably phosphatidylcholine containing an acyl group having a carbon number of 16 and 0 carbon-carbon double bonds and an acyl group having a carbon number of 18 and 1 carbon-carbon double bond. In the non-variation substance, the phosphatidylcholine having two acyl groups having a total carbon number of 36 and a total of 2 carbon-carbon double bonds is preferably phosphatidylcholine having two acyl groups each having a carbon number of 18. In the non-variation substance, the phosphatidylcholine having two acyl groups having a total carbon number of 38 and a total of 4 carbon-carbon double bonds is preferably phosphatidylcholine containing an acyl group having a carbon number of 18 and 0 carbon-carbon double bonds and an acyl group having a carbon number of 20 and 4 carbon-carbon double bonds. In the non-variation substance, the phosphatidylethanolamine having two acyl groups having a total carbon number of 34 and a total of 2 carbon-carbon double bonds is preferably phosphatidylethanolamine containing an acyl group having a carbon number of 16 and 0 carbon-carbon double bonds and an acyl group having a carbon number of 18 and 2 carbon-carbon double bonds. In the non-variation substance, the phosphatidylethanolamine having two acyl groups having a total carbon number of 36 and a total of 2 carbon-carbon double bonds is preferably phosphatidylethanolamine containing two acyl groups each having a carbon number of 18. In the non-variation substance, the phosphatidylethanolamine having two acyl groups having a total carbon number of 38 and a total of 4 carbon-carbon double bonds is preferably phosphatidylethanolamine containing an acyl group having a carbon number of 18 and 0 carbon-carbon double bonds and an acyl group having a carbon number of 20 and 4 carbon-carbon double bonds. The normalization part523preferably normalizes the intensity of the sample-derived ion Si corresponding to each cholesteryl ester in the sample S detected by mass spectrometry in the mass spectrometer20, using the sum of intensities of non-variation ions corresponding to cholesteryl esters included in the non-variation substance. This permits quantitative comparison even when different samples have different total concentrations of cholesteryl esters. The normalization part523preferably normalizes the intensity of the sample-derived ion Si corresponding to each lysophospholipid in the sample S detected by mass spectrometry in the mass spectrometer20, using the intensity of a non-variation ion corresponding to lysophosphatidylcholine included in the non-variation substance. This permits quantitative comparison even when different samples have different concentrations of lysophospholipid such as lysophosphatidylcholine. The normalization part523preferably normalizes the intensity of the sample-derived ion Si corresponding to each phospholipid containing a plurality of acyl groups in the sample S detected by mass spectrometry in the mass spectrometer20, using the sum of intensities of non-variation ions corresponding to phosphatidylcholines and phosphatidylethanolamines included in the non-variation substance. This permits quantitative comparison even when different samples have different concentrations of phospholipids including a plurality of phospholipids such as phosphatidylcholines and phosphatidylethanolamines. It should be noted that a method for normalization using the detection intensity of the non-variation substance by the normalization part523is not particularly limited. The normalization may be performed, for example, using the sum of intensities of non-variation ions corresponding to all the non-variation substances. This permits quantitative comparison even when different samples have different total lipid concentrations. The non-variation substance is preset, and data on analysis conditions, such as a retention time and two m/z values for detection as a precursor ion and a product ion (hereinafter, the combination of these two m/z values is referred to as a transition), for the mass spectrometry of the non-variation substance is memorized in the memory part43or the like. The normalization part523calculates a detection intensity corresponding to the non-variation substance, with reference to this data, from the mass chromatogram data prepared by the chromatogram preparation part521. If data on the non-variation substance is not memorized in the memory part43or the like for a reason such as an unset non-variation substance or the mass spectrometer20does not detect a non-variation ion corresponding to the non-variation substance, the normalization part523performs normalization without using the intensity of the non-variation ion. In this case, the normalization part523calculates the sum of intensities of detected ions, such as fragment ions, corresponding to phospholipids among the sample-derived ions Si, and normalizes the respective intensities of the detected sample-derived ions Si using this sum to calculate normalized intensities (hereinafter, in this case as well, referred to as normalized intensities). In this context, the phospholipids preferably include lysophospholipid, and phospholipid containing a plurality of acyl groups. This achieves normalization more stably reflecting lipid concentrations even when the phospholipid containing a plurality of acyl groups is converted to lysophospholipid by the elimination of fatty acid during storage of the sample S or the lysophospholipid is no longer lysophospholipid through the binding of fatty acid, because the value of the normalization factor does not vary. It should be noted that when normalization using the non-variation substance is possible, data normalized using the sum of intensities of ions corresponding to phospholipids may also be prepared and used for analysis. In the method of searching for and selecting the non-variation substance as described above, when mass spectrometry is performed at least twice at a predetermined interval of time on the same sample S, a substance corresponding to an ion whose variation in intensity normalized using the sum of intensities of ions corresponding to phospholipids is equal to or less than a predetermined percentage can be selected as the non-variation substance. The predetermined interval of time is appropriately set to 1 month or longer, 1 year or longer, or the like. The predetermined percentage is appropriately set to 20% or less, 15% or less, 10% or less, or the like. The analysis part52analyzes the measurement data obtained by the mass spectrometry of the sample S based on the degree-of-oxidation index calculated by the degree-of-oxidation analysis part522, or information that indicates the evaluated quality of the sample S. For example, when the evaluated quality of the sample S is low, the analysis part52can avoid conducting a portion of analysis because of low reliability. The output control part53prepares an output image involving, for example, information on measurement conditions for the measurement part100and/or analysis results from the analysis part52, etc., and allows the output part44to output this image. The output control part53prepares an output image involving the degree-of-oxidation index, or information that indicates the degree of oxidation of the sample S based on the degree-of-oxidation index, and allows the output part44to output this image. Each ofFIGS.2to4is a flow chart showing the flow of the analysis method of the present embodiment.FIG.2is a flow chart showing the flow of mass spectrometry for searching for and setting a non-variation substance (hereinafter, referred to as non-variation substance search mass spectrometry). Each ofFIGS.3and4is a flow chart showing the flow of an analysis method of conducting analysis using a normalization factor based on the set non-variation substance. Provided that the non-variation substance is known, each step of the flow chart ofFIG.2may not be performed. It should be noted that the mass spectrometry ofFIG.2through the mass spectrometry ofFIG.4may be performed with different analytical devices. In step S1001(FIG.2), blood is obtained from a biological body by a medical worker or the like. After the completion of step S1001, step S1003is started. In step S1003, a portion of the obtained blood is stored by a medical worker, an analyst or a user or the like, and another portion of the blood is pretreated to prepare sample S. After the completion of step S1003, step S1005is started. In step S1005, the sample introduction part13introduces the sample S to the liquid chromatograph10where the sample S is subjected to liquid chromatography. After the completion of step S1005, step S1007is started. In step S1007, the mass spectrometer performs the mass spectrometry (non-variation substance search mass spectrometry) of the sample S subjected to liquid chromatography to detect respective sample-derived ions Si corresponding to components of the sample S. After the completion of step S1007, step S1009is started. In step S1009, the normalization part523calculates a sum of intensities of ions corresponding to phospholipids among the sample-derived ions Si. After the completion of step S1009, step S1011is started. In step S1011, respective intensities of the detected sample-derived ions Si are normalized using the sum calculated in the step S1009to calculate respective normalized intensities of the sample-derived ions Si. After the completion of step S1011, step S1013is started. In step S1013, the blood stored in the step S1003is pretreated by a medical worker, an analyst or a user or the like after a lapse of a predetermined period to prepare sample S. After the completion of step S1013, step S1015is started. In step S1015, the analytical device1performs liquid chromatography and non-variation substance search mass spectrometry on the sample S prepared in the step S1015in the same way as in the steps S1005to S1011, and calculates normalized intensities of sample-derived ions Si. After the completion of step S1015, step S1017is started. In step S1017, the analysis part52selects a substance having variation equal to or less than a predetermined percentage as a non-variation substance by comparing the normalized intensities calculated in the step S1011with the normalized intensities calculated in the step S1015, and the memory part43memorizes a retention time of the non-variation substance and a transition for the mass spectrometry of the non-variation substance. After the completion of step S1017, the processing of searching for the non-variation substance is completed, and step S2001is started to conduct analysis using a normalization factor based on the non-variation substance. In step S2001(FIG.3), blood is obtained from a biological body by a medical worker or the like. After the completion of step S2001, step S2003is started. The biological body may be an individual different from the individual from which the blood is obtained in the step S1001, or may be the same individual. In step S2003, the obtained blood is pretreated by a medical worker, an analyst or a user or the like to prepare sample S. After the completion of step S2003, step S2005is started. In step S2005, the sample introduction part13introduces the sample S to the liquid chromatograph10where the sample S is subjected to liquid chromatography. After the completion of step S2005, step S2007is started. In step S2007, the mass spectrometer performs the mass spectrometry of the sample S subjected to liquid chromatography to detect respective sample-derived ions Si corresponding to components of the sample S, including cholesteryl ester and cholesteryl ester peroxide as well as a non-variation substance. After the completion of step S2007, step S2009is started. In step S2009, the degree-of-oxidation analysis part522calculates a degree-of-oxidation index that indicates the degree of oxidation of the sample S based on a degree-of-oxidation ratio obtained by dividing any one of the intensity of the detected ion corresponding to cholesteryl ester and the intensity of the detected ion corresponding to cholesteryl ester peroxide by the other. After the completion of step S2009, step S2011is started. In step S2011, the normalization part523calculates respective normalized intensities of components of the sample S using a normalization factor based on the non-variation substance. FIG.4is a flow chart showing the flow of the step S2011. In step S2011-1, the normalization part523calculates a sum of intensities corresponding to cholesteryl esters included in the non-variation substance. After the completion of step S2011-1, step S2011-2is started. In step S2011-2, the normalization part523normalizes an intensity of the detected sample-derived ion Si corresponding to each cholesteryl ester using the sum calculated in the step S2011-1to calculate a normalized intensity of the sample-derived ion Si corresponding to each cholesteryl ester. After the completion of step S2011-2, step S2011-3is started. In step S2011-3, the normalization part523calculates an intensity corresponding to lysophosphatidylcholine included in the non-variation substance. After the completion of step S2011-3, step S2011-4is started. In step S2011-4, the normalization part523normalizes an intensity of the detected sample-derived ion Si corresponding to each lysophospholipid using the intensity calculated in the step S2011-3to calculate a normalized intensity of the sample-derived ion Si corresponding to each lysophospholipid. After the completion of step S2011-4, step S2011-5is started. In step S2011-5, the normalization part523calculates a sum of intensities corresponding to phosphatidylcholines and phosphatidylethanolamines included in the non-variation substance. After the completion of step S2011-5, step S2011-6is started. In step S2011-6, the normalization part523normalizes an intensity of the detected sample-derived ion Si corresponding to each phospholipid containing a plurality of acyl groups using the sum calculated in the step S2011-5to calculate a normalized intensity of the sample-derived ion Si corresponding to each phospholipid mentioned above. After the completion of step S2011-6, step S2013is started. It should be noted that the order of normalization of the intensities of the sample-derived ions Si as to the cholesteryl ester, the lysophospholipid and the phospholipid mentioned above is not particularly limited. Referring back toFIG.3, in step S2013, the analysis part52analyzes the sample S using the degree-of-oxidation index or information on the degree of oxidation of the sample S based on the degree-of-oxidation index, and the normalized intensities calculated in the step S2011. After the completion of step S2013, step S2015is started. In step S2015, the output part44displays information obtained by the analysis conducted in the step S2013. After the completion of step S2015, the processing is completed. The aforementioned embodiment produces the following working effects.(1) The analysis method of the present embodiment comprises: subjecting a sample S to liquid chromatography; performing mass spectrometry of the sample S subjected to liquid chromatography to detect a non-peroxide ion corresponding to cholesteryl ester and a peroxide ion corresponding to cholesteryl ester peroxide; and analyzing a degree of oxidation of the sample S based on a ratio between an intensity of the detected non-peroxide ion and an intensity of the detected peroxide ion. This achieves quantitative evaluation of the degree of oxidation of the sample S using mass spectrometry to evaluate the quality of stored sample S.(2) In the analysis method of the present embodiment and the analytical device1, the degree-of-oxidation analysis part522can calculate a degree-of-oxidation index that indicates the degree of oxidation of the sample S based on a degree-of-oxidation ratio obtained by dividing any one of the intensity of the non-peroxide ion and the intensity of the peroxide ion by the other. This achieves quantitative comparison of the degree of oxidation of the sample S using the index.(3) In the analysis method of the present embodiment and the analytical device1, the output part44outputs the degree-of-oxidation index, or information that indicates the degree of oxidation of the sample S based on the degree-of-oxidation index. This can easily inform a user or the like of the degree of oxidation of the sample S based on the quantitative value.(4) In the analysis method of the present embodiment and the analytical device1, the analysis part52analyzes the measurement data obtained by the mass spectrometry of the sample S based on the degree-of-oxidation index. This achieves more precise analysis of the measurement data based on the quality, such as the degree of oxidation, of the sample S.(5) In the analysis method of the present embodiment and the analytical device1, the normalization part523normalizes intensities of respective sample-derived ions Si corresponding to components of the sample S detected in mass spectrometry using an intensity of an ion corresponding to a preset non-variation substance. This achieves quantitative comparison of analyte lipid molecules even when different samples S have different lipid concentrations.(6) The analysis method of the present embodiment comprises, when non-variation substance search mass spectrometry is performed at least twice at a predetermined interval of time on the same sample S, detecting a non-variation ion whose variation in normalized intensity is equal to or less than a predetermined percentage by subsequent mass spectrometry, and normalizing respective intensities of sample-derived ions Si detected by this mass spectrometry using an intensity of the above non-variation ion. This achieves quantitative comparison of the amounts of analyte lipid molecules even when different samples S have different lipid concentrations, based on a non-variation substance obtained by actual mass spectrometry.(7) In the analysis method of the present embodiment, the sample S is a sample stored in the state of blood. This allows many findings to be gained by the analysis of lipid molecules in rapidly and conveniently collectable blood containing diverse components.(8) The analytical device of the present embodiment comprises: a sample introduction part13which introduces sample S; a liquid chromatograph10which separates the sample S; a mass spectrometry part (mass spectrometer20) which performs mass spectrometry of the sample S separated in the liquid chromatograph10to detect a non-peroxide ion and a peroxide ion; and a degree-of-oxidation analysis part522which analyzes a degree of oxidation of the sample S based on a ratio between an intensity of the detected non-peroxide ion and an intensity of the detected peroxide ion. This achieves quantitative evaluation of the degree of oxidation of the sample S using mass spectrometry to evaluate the quality of stored sample S. Modifications as given below are also included in the scope of the present invention and may be combined with the aforementioned embodiment. In the following Variations, the same reference signs will be used to designate sites exhibiting the same or similar structures or functions as in the aforementioned embodiment, so that the description will be omitted as appropriate. Variation 1 In the aforementioned embodiment, the mass spectrometer20is a tandem mass spectrometer which performs mass-separation through two quadrupole mass filters. The configuration of the mass spectrometer20, the type of the mass spectrometer20, and the method for mass spectrometry are not particularly limited as long as sample-derived ions Si corresponding to analyte lipid molecules in the sample S are detectable with the desired accuracy. For example, the sample S may be ionized by probe electrospray ionization which performs ionization by applying high voltage to a probe attached to the sample S. For the details of the probe electrospray ionization, see International Publication No. WO 2010/047399. Variation 2 In the aforementioned embodiment, the degree-of-oxidation index is calculated based on a ratio between an intensity of a detected ion corresponding to cholesteryl ester and an intensity of a detected ion corresponding to cholesteryl ester peroxide. However, the degree-of-oxidation index may be calculated based on a ratio between an intensity of a detected ion corresponding to cholesteryl ester and an intensity of a detected ion corresponding to cholesteryl ester hydroxide or cholesteryl sulfate. Since marked increase in the amounts of the cholesteryl ester hydroxide and the cholesteryl sulfate is observed in an oxidized sample, these can be properly used for the degree-of-oxidation index. Variation 3 In the aforementioned embodiment, when a normalization factor is calculated from the sum of intensities of a plurality of detected ions, this sum may exclude an intensity of a detected ion, such as a fragment ion, corresponding to a molecule having an acyl group having a carbon number of 20 and 4 carbon-carbon double bonds. This molecule preferably has an acyl group corresponding to arachidonic acid. The arachidonic acid is cleaved from phospholipids by an enzyme (PLA2) when infection, trauma, or the like occurs in a biological body such as a human. Thus, there is a possibility that the amount of arachidonic acid in phospholipids is decreased due to various diseases including diseases having chronic inflammation, such as allergic diseases (e.g., atopic dermatitis and asthma) and diabetes mellitus. Thus, the normalization factor excluding an intensity of an ion corresponding to a molecule containing arachidonic acid or an acyl group corresponding to arachidonic acid (hereinafter, referred to as “arachidonic acid, etc.”) allows analyte lipid molecules to be analyzed precisely using the more stable normalization factor. For example, when the normalization part523calculates a normalization factor from the sum of intensities of ions corresponding to phospholipids including lysophospholipid, the phospholipids can exclude phospholipid containing arachidonic acid, etc. In another example, the normalization part523can perform normalization using an intensity of an ion corresponding to at least one member selected from the group consisting of non-variation substances containing no arachidonic acid, etc., i.e., cholesteryl ester having an acyl group having a carbon number of 18 and 1 carbon-carbon double bond, cholesteryl ester having an acyl group having a carbon number of 20 and 5 carbon-carbon double bonds, lysophosphatidylcholine having an acyl group having a carbon number of 20 and 5 carbon-carbon double bonds, phosphatidylcholine having two acyl groups having a total carbon number of 32 and a total of 1 carbon-carbon double bond, phosphatidylcholine having two acyl groups having a total carbon number of 34 and a total of 1 carbon-carbon double bond, phosphatidylcholine having two acyl groups having a total carbon number of 36 and a total of 2 carbon-carbon double bonds, phosphatidylethanolamine having two acyl groups having a total carbon number of 34 and a total of 2 carbon-carbon double bonds and phosphatidylethanolamine having two acyl groups having a total carbon number of 36 and a total of 2 carbon-carbon double bonds, among the aforementioned non-variation substances. The analysis part52may determine whether the sample S is derived from a healthy person or derived from a human having a disease such as chronic inflammation, using the intensity of the detected ion, such as a fragment ion, corresponding to the molecule containing arachidonic acid, etc. The output control part53may allow the output part44to output information on the determination. The analysis part52performs this determination based on a preliminarily obtained threshold based on a detection intensity for a sample obtained from a healthy person and a detection intensity for a sample obtained from a human having a disease such as chronic inflammation. A value appropriately normalized with the aforementioned normalization factor can be used as the intensity of the ion corresponding to the molecule containing arachidonic acid, etc. In the analysis method of the present Variation, the sample-derived ions Si include a lipid molecule containing a fatty acid or acyl group having a carbon number of and 4 carbon-carbon double bonds, and the analysis part52determines whether or not the sample S has been obtained from a healthy individual based on a normalized intensity of an ion corresponding to the lipid molecule. This achieves analysis of a health condition of the biological body from which the sample S is derived, and also achieves quality evaluation of the sample S from the viewpoint of the health condition of the biological body at the time of collection. The present invention is not limited by the contents of the embodiments described above. Other possible aspects or embodiments are also included in the scope of the present invention without departing from the technical idea of the present invention. EXAMPLES In Examples given below, experimental results of subjecting a portion of blood collected from a human to mass spectrometry in March 2017, refrigerating another portion thereof, and subjecting this portion to mass spectrometry in March 2018 to analyze variation in components in the blood, and experimental results of analyzing the constitution of an acyl group of each compound in May 2017 will be described. It should be noted that the present invention is not limited by numerical values and conditions shown in Examples given below. Mass spectrometry performed in March 2017 and March 2018 Pretreatment 500 μL of methanol was added to 5 μL of whole blood, and the mixture was stirred for several minutes to extract lipid components, followed by centrifugation. 1 μL of the supernatant was collected and used as a sample to be introduced to a liquid chromatograph-mass spectrometer. Conditions for Liquid Chromatography The sample was separated by liquid chromatography under the following conditions.System: LCMS-8060 (Shimadzu Corp.)Analytical column: Kinetex C8 (Phenomenex Inc.) (inside diameter: 2.1 mm, length: 150 mm, particle size: 2.6 μm)Injection volume: 3 μLColumn temperature: 45° C.Mobile phase:(A) a 20 mM aqueous ammonium formate solution(B) a solution of acetonitrile and isopropanol mixed at a volume ratio of 1:1 Flow rate: 0.3 mL/min Gradient Program: Time (min)Concentration (%) of mobile phase B0201202402592.5 The concentration of mobile phase B was nonlinearly changed against time from 2 minutes through 25 minutes in order to increase difference in elution time among components at elution times of diacylphospholipids. Conditions for Mass Spectrometry The elution sample eluted in the liquid chromatography was detected by tandem mass spectrometry with connection directly to an elution port.System: LCMS-8060 (Shimadzu Corp.)Ionization method: electrospray ionization, positive ion mode/negative ion modeMeasurement mode: multiple reaction monitoring (MRM)Temperature:Desolvation Line (DL) temperature: 250° C.Heat block temperature: 400° C.Interface temperature (which corresponds to the temperature of heating gas around an ionization part):—Interface voltage (voltage applied to the entrance of DL from the tip of a capillary): 1.0 kVGas flow rate:Nebulizer gas flow rate: 2.0 L/minDriving gas flow rate: 10.0 L/minHeating gas flow rate: 10.0 L/min Tables A and B given below show the retention time and a transition of each compound contained in the elution sample, a peak intensity at each transition, and a normalized intensity obtained by dividing the peak intensity by the sum of intensities of peaks of detected phospholipids including lysophospholipid. Table A is based on the mass spectrometry performed in March 2017, and Table B is based on the mass spectrometry performed in March 2018. Table B further shows the rate of increase in the normalized intensity in the mass spectrometry performed in March 2018 with respect to the normalized intensity in the mass spectrometry performed in March 2017. In the compound names, CE refers to cholesteryl ester, Chol refers to cholesterol, Chol_sulfate refers to cholesterol sulfate, LPC refers to lysophosphatidylcholine, LPE refers to lysophosphatidylethanolamine, PC refers to phosphatidylcholine, PE refers to phosphatidylethanolamine, and PS refers to phosphatidylserine. In the description of x:y in the compound names, x represents the total carbon number in acyl groups contained in each compound, and y represents the total number of carbon-carbon double bonds in the acyl groups contained in each compound. CE18:2-OH is cholesteryl ester hydroxide having an acyl group having a carbon number of 18 and 2 carbon-carbon double bonds. CE18:2-OOH is cholesteryl ester peroxide having an acyl group having a carbon number of 18 and 2 carbon-carbon double bonds. CE18:3-OOH is cholesteryl ester peroxide having an acyl group having a carbon number of 18 and 3 carbon-carbon double bonds. In the description of the transition, m/z of a precursor ion is shown on the left side of “>”, and m/z of a product ion is shown on the right side of “>”. TABLE 1Table A: Measurement Conditions and Measurement Results for MassSpectrometry of Each Compound (March 2017)CompoundRetentionPeakNormalizedIDNameTimeTransitionIntensityIntensity1PS(40:6)13.404836.55 > 651.501397810.18221122CE 18:322.624664.70 > 369.404926330.64217053CE 22:523.263716.70 > 369.40363620.04739964PE(38:6)14.329764.50 > 623.50466530.06081445PE(38:5)14.816766.55 > 625.50382820.04990246PC(34:4)13.484754.55 > 184.10398730.05197637CE 22:423.967718.70 > 369.4091700.01195358PC(34:2)14.583758.55 > 184.101862803724.282539PC(36:5)13.76780.55 > 184.105098010.664549810PC(34:3)13.852756.55 > 184.102760840.359888611CE 20:422.961690.70 > 369.4038068094.962356112CE 18:223.566666.70 > 369.401189555915.50642513LPC(18:2)7.416520.35 > 184.1011280471.470462814PE(36:4)14.64740.50 > 599.501265360.164945715CE 22:622.276714.70 > 369.404932680.642998216PC(38:6)14.156806.55 > 184.1017416342.270302517PS(40:5)13.649838.55 > 653.55103030.013430418PE(36:3)14.929742.55 > 601.50520590.067861419LPE(18:2)7.53478.30 > 337.2524020.003131120PC(36:4)14.465782.55 > 184.10819941310.68832421Chol13.402369.30 > 161.10403090.052544722PS(38:5)13.049810.55 > 625.5017950.002339923CE 20:521.968688.70 > 369.402654140.3459797 TABLE 2Table A (Sequel): Measurement Conditions and Measurement Resultsfor Mass Spectrometry of Each Compound (March 2017)CompoundRetentionPeakNormalizedIDNameTimeTransitionIntensityIntensity24PE(36:2)15.728744.55 > 603.55912000.118883525PE(38:4)15.614768.55 > 627.551131020.147433826PC(38:5)14.645808.60 > 184.104287110.558845127PS(38:4)13.668812.55 > 627.50621150.080969828PC(32:1)14.39732.55 > 184.106693380.872513829PC(36:3)14.86784.60 > 184.1034365224.479669430PC(40:5)15.445836.60 > 184.102340490.30509431PE(34:2)14.745716.50 > 575.50929300.121138732PC(38:4)15.468810.60 > 184.1035142684.58101533LPC(20:5)6.771542.30 > 184.10193830.025266734PC(34:1)15.396760.60 > 184.10914011311.91457135PC(36:2)15.612786.60 > 184.10810831610.56957436CE 18:124.703668.70 > 369.408385241.093055837PC(44:12)18.543878.55 > 184.10261390.034073438PC(32:2)13.597730.55 > 184.101302520.169789739PC(30:1)14.169704.50 > 184.1029317183.821633540LPC(20:3)7.789546.35 > 184.101092470.142408641LPC(16:0)7.86496.35 > 184.1076230539.937011242PC(40:7)14.321832.60 > 184.10408810.053290343PC(38:3)15.888812.60 > 184.1012667011.651204844LPC(18:1)8.144522.35 > 184.109745861.270419145PC(40:6)15.133834.60 > 184.104772200.622078946CE 14:023.361614.70 > 369.4021480.0028 TABLE 3Table A (Sequel): Measurement Conditions and Measurement Resultsfor Mass Spectrometry of Each Compound (March 2017)CompoundRetentionPeakNormalizedIDNameTimeTransitionIntensityIntensity47PC(36:1)16.451788.60 > 184.1012421201.619162348PC(28:1)13.161676.50 > 184.101439460.187640449LPC(20:4)7.376544.35 > 184.102748790.358317850PE(34:1)15.533718.55 > 577.501195170.155796151PC(32:0)15.188734.55 > 184.109408661.226463552CE 16:123.455640.70 > 369.40889180.115908853PC(38:2)17.496814.65 > 184.103877570.505459654PC(40:4)16.197838.65 > 184.101192710.155475455LPC(22:6)7.254568.35 > 184.10350390.04567556PE(36:1)16.52746.55 > 605.55382700.049886857CE 16:024.567642.70 > 369.40995970.129829458LPE(18:1)8.275480.30 > 339.3025860.00337159LPC(18:0)8.961524.35 > 184.1024510053.195001360LPE(22:6)7.234526.30 > 385.255350.000697461PC(30:0)14.576706.55 > 184.10891420.116200862PC(38:1)18.699816.65 > 184.102163750.282055163PC(30:2)13.311702.50 > 184.102183980.284692264LPE(20:4)7.35502.30 > 361.2510230.001333565LPE(16:0)7.984454.30 > 313.2524680.003217266Chol_sulfate9.466465.30 > 97.0034330.004475167CE18:2—OH20.149682.80 > 369.4022890.002983868CE18:2—OOH18.32698.80 > 369.4036740.004789269CE18:3—OOH18.076696.80 > 369.4024330.0031715 TABLE 4Table B: Measurement Conditions and Measurement Results forMass Spectrometry of Each Compound (March 2018)CompoundRetentionPeakNormalizedRate ofIDNameTimeTransitionIntensityIntensityincrease1PS(40:6)12.848836.55 > 651.50655820.045372550.252CE 18:321.329664.70 > 369.403141770.217361630.343CE 22:521.613716.70 > 369.40286560.01982550.424PE(38:6)13.72764.50 > 623.50404770.028003790.465PE(38:5)14.175766.55 > 625.50363520.025149930.506PC(34:4)13.074754.55 > 184.10405430.028049450.547CE 22:422.53718.70 > 369.4095300.006593280.558PC(34:2)14.202758.55 > 184.101968892713.62167590.569PC(36:5)13.344780.55 > 184.105389310.372856450.5610PC(34:3)13.453756.55 > 184.103037700.21016160.5811CE 20:421.631690.70 > 369.4043489773.008815830.6112CE 18:222.172666.70 > 369.40137707389.527209380.6113LPC(18:2)7.205520.35 > 184.1013471250.932001030.6314PE(36:4)14.01740.50 > 599.501515850.104873250.6415CE 22:621.013714.70 > 369.406444350.445848810.6916PC(38:6)13.739806.55 > 184.1023085461.597154860.7017PS(40:5)12.968838.55 > 653.55139920.009680290.7218PE(36:3)14.275742.55 > 601.50719720.049793430.7319LPE(18:2)7.286478.30 > 337.2533730.002333590.7520PC(36:4)14.046782.55 > 184.10117238808.111101920.7621Chol14.22369.30 > 161.10577880.039980310.7622PS(38:5)12.332810.55 > 625.5025820.001786340.7623CE 20:5—688.70 > 369.404242820.293537170.85 TABLE 5Table B (Sequel): Measurement Conditions and Measurement Results forMass Spectrometry of Each Compound (March 2018)CompoundRetentionPeakNormalizedRate ofIDNameTimeTransitionIntensityIntensityincrease24PE(36:2)15.035744.55 > 603.551496890.103561510.8725PE(38:4)14.932768.55 > 627.551937600.134051790.9126PC(38:5)14.055808.60 > 184.107352220.50865930.9127PS(38:4)12.999812.55 > 627.501071860.074156040.9228PC(32:1)13.994732.55 > 184.1012139620.839872940.9629PC(36:3)14.468784.60 > 184.1064746964.479482831.0030PC(40:5)15.053836.60 > 184.104575590.316559681.0431PE(34:2)14.104716.50 > 575.501836700.127071081.0532PC(38:4)15.057810.60 > 184.1071416964.940943121.0833LPC(20:5)6.585542.30 > 184.10394890.027320251.0834PC(34:1)15.018760.60 > 184.101929338013.34801891.1235PC(36:2)15.242786.60 > 184.101732368111.98529351.1336CE 18:123.196668.70 > 369.4017995561.245012931.1437PC(44:12)18.281878.55 > 184.10578590.040029431.1738PC(32:2)13.891730.55 > 184.102906710.201099131.1839PC(30:1)13.709704.50 > 184.1065791804.551769511.1940LPC(20:3)7.562546.35 > 184.102503730.173219181.2241LPC(16:0)7.636496.35 > 184.101775655512.2847751.2442PC(40:7)13.9832.60 > 184.10954240.066018571.2443PC(38:3)15.514812.60 > 184.1030322172.097822661.2744LPC(18:1)7.899522.35 > 184.1023651111.636289041.2945PC(40:6)14.72834.60 > 184.1012077760.835593191.3446CE 14:021.972614.70 > 369.4055900.003867411.38 TABLE 6Table B (Sequel): Measurement Conditions and Measurement Results forMass Spectrometry of Each Compound (March 2018)CompoundRetentionPeakNormalizedRate ofIDNameTimeTransitionIntensityIntensityincrease47PC(36:1)16.108788.60 > 184.1034050262.355748521.4548PC(28:1)12.716676.50 > 184.104091180.283046041.5149LPC(20:4)7.166544.35 > 184.108061520.557731831.5650PE(34:1)14.853718.55 > 577.503510460.242869251.5651PC(32:0)14.83734.55 > 184.1028298811.957837611.6052CE 16:122.071640.70 > 369.402715190.187848931.6253PC(38:2)17.097814.65 > 184.1012281170.849665991.6854PC(40:4)15.816838.65 > 184.103828580.26487821.7055LPC(22:6)7.051568.35 > 184.101259600.087144731.9156PE(36:1)15.796746.55 > 605.551421090.098317331.9757CE 16:023.076642.70 > 369.403861820.267177892.0658LPE(18:1)7.983480.30 > 339.30109170.007552872.2459LPC(18:0)8.681524.35 > 184.10107563947.441752052.3360LPE(22:6)7.136526.30 > 385.2526050.001802252.5861PC(30:0)13.796706.55 > 184.104386350.303467212.6162PC(38:1)18.379816.65 > 184.1011065300.76554672.7163PC(30:2)12.854702.50 > 184.1012272770.849084852.9864LPE(20:4)7.253502.30 > 361.2576920.005321673.9965LPE(16:0)7.717454.30 > 313.25240400.016631945.1766Chol_sulfate9.07465.30 > 97.00507910.035139477.8567CE18:2-OH19.607682.80 > 369.402715680.1878828362.9768CE18:2-OOH17.439698.80 > 369.4012569890.86964093181.5869CE18:3-OOH17.171696.80 > 369.4013473310.93214355293.91 The aforementioned sum of the peak intensities corresponding to the phospholipids including lysophospholipid excluded the intensity of PC(34:2) (ID8) which was saturated. FIGS.5and6show mass chromatograms obtained by mass spectrometry in March 2017 and March 2018, respectively, on some analyte molecules. In the mass chromatograms ofFIGS.5and6, a mass chromatogram Mi is shown which is an enlargement of peaks around a retention time of 17.5 minutes. In these mass chromatograms, peaks corresponding to cholesteryl ester (CE), cholesteryl ester peroxide (CE-OOH), phospholipids (PLs), and lysophospholipids (LPLs) were observed. As shown by ID2, ID12, ID68 and ID69 in Table B, the amount of cholesteryl ester peroxide with respect to the amount of cholesteryl ester in the sample was largely increased by storage for approximately 1 year. As for cholesterol sulfate and cholesteryl ester hydroxide, increase in their amounts by storage for approximately 1 year was also observed (ID66 and ID67). As shown in Table B, compounds corresponding to ID #23 to 36 were each considered as a compound having less variation even by refrigeration for 1 year or longer, because the percentage of variation in the normalized intensity in the mass spectrometry performed in March 2018 with respect to the normalized intensity in the mass spectrometry performed in March 2017 was 15% or less. FIGS.7(A) and7(B)show enlarged peaks corresponding to (a) cholesteryl ester (18:1) (ID36) and (b) cholesteryl ester (20:5) (ID23) in the mass chromatograms obtained by mass spectrometry in March 2017 and March 2018, respectively. FIGS.8(A) and8(B)show enlarged peaks corresponding to lysophosphatidylcholine (20:5) (ID33) in the mass chromatograms obtained by mass spectrometry in March 2017 and March 2018, respectively. FIGS.9and10show enlarged peaks corresponding to (a) phosphatidylcholine (32:1) (ID28), (b) phosphatidylcholine (34:1) (ID34), (c) phosphatidylcholine (36:2) (ID35) and phosphatidylcholine (38:4) (ID32) in the mass chromatograms obtained by mass spectrometry in March 2017 and March 2018, respectively. FIGS.11and12show enlarged peaks corresponding to (a) phosphatidylethanolamine (34:2) (ID31), (b) phosphatidylethanolamine (36:2) (ID24) and (c) phosphatidylethanolamine (38:4) (ID25) in the mass chromatograms obtained by mass spectrometry in March 2017 and March 2018, respectively. Mass spectrometry performed in May 2018 LC/MS was performed on the refrigerated sample described above under the following analysis conditions. Conditions for Liquid ChromatographySystem: LCMS-8060 (Shimadzu Corp.)Analytical column: Kinetex C8 (Phenomenex Inc.) (inside diameter: 2.1 mm, length: 150 mm, particle size: 2.6 μm)Column temperature: 50° C.Mobile phase:(A) a 20 mM aqueous ammonium formate solution(B) a solution of acetonitrile and isopropanol mixed at a volume ratio of 1:1Flow rate: 0.4 mL/minGradient program: Time (min)Concentration (%) of mobile phase B0250.5251401492.5 The concentration of mobile phase B was nonlinearly changed against time from 1 minute through 14 minutes in order to increase difference in elution time among components at elution times of diacylphospholipids. Conditions for mass spectrometry were the same as those for the aforementioned mass spectrometry performed in March 2017 and March 2018. Transitions were set such that the carbon number and the number of carbon-carbon double bonds in acyl groups were distinguishable for analysis among some compounds having a plurality of acyl groups among compounds of ID23 to ID36 in Table B. In the description below, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) having both of an acyl group having a carbon number x1 and y1 carbon-carbon double bonds and an acyl group having a carbon number of x2 and y2 carbon-carbon double bonds are referred to as PC(x1:y1-x2:y2) and PE(x1:y1-x2:y2), respectively. FIG.13is a diagram showing a mass chromatogram corresponding to the whole PC(32:1) and two mass chromatograms from different transitions corresponding to PC(16:0-16:1). The peaks of PC(16:0-16:1) were observed at a retention time corresponding to the peak of the whole PC(32:1). FIG.14is a diagram showing a mass chromatogram corresponding to the whole PC(34:1), two mass chromatograms from different transitions corresponding to PC(16:0-18:1), and two mass chromatograms from different transitions corresponding to PC(16:1-18:0). The peaks of PC(16:0-18:1) were observed at a retention time corresponding to the peak of the whole PC(34:1). FIG.15is a diagram showing a mass chromatogram corresponding to the whole PC(36:2), two mass chromatograms from different transitions corresponding to PC(18:0-18:2), and a mass chromatogram corresponding to PC(18:1-18:1). The peaks of PC(18:0-18:2) and PC(18:1-18:1) were observed at a retention time corresponding to the peak of the whole PC(36:2). FIG.16is a diagram showing a mass chromatogram corresponding to the whole PC(38:4), two mass chromatograms from different transitions corresponding to PC(18:0-20:4), two mass chromatograms from different transitions corresponding to PC(18:1-20:3), and two mass chromatograms from different transitions corresponding to PC(18:2-20:2). The peaks of PC(18:0-20:4) were observed at a retention time corresponding to the peak of the whole PC(38:4). FIG.17is a diagram showing a mass chromatogram corresponding to the whole PE(34:2), two mass chromatograms from different transitions corresponding to PE(16:0-18:2), and two mass chromatograms from different transitions corresponding to PE(16:1-18:1). The peaks of PE(16:0-18:2) were observed at a retention time corresponding to the peak of the whole PE(34:2). FIG.18is a diagram showing a mass chromatogram corresponding to the whole PE(36:2), two mass chromatograms from different transitions corresponding to PE(18:0-18:2), and a mass chromatogram corresponding to PE(18:1-18:1). The peaks of PE(18:0-18:2) and PE(18:1-18:1) were observed at a retention time corresponding to the peak of the whole PE(36:2). FIG.19is a diagram showing a mass chromatogram corresponding to the whole PE(38:4), two mass chromatograms from different transitions corresponding to PE(18:0-20:4), and two mass chromatograms from different transitions corresponding to PE(18:1-20:3). The peaks of PE(18:0-20:4) were observed at a retention time corresponding to the peak of the whole PE(38:4). The disclosure of the following priority application is herein incorporated by reference: Japanese Patent Application No. 2018-109023 (filed on Jun. 6, 2018). REFERENCE SIGNS LIST 1. . . Analytical device,10. . . Liquid chromatograph,14. . . Analytical column, . . . Mass spectrometer,21. . . Ionization chamber,23. . . First mass separation part,24. . . Collision cell,25. . . Second mass separation part,30. . . Detection part,40. . . Information processing part,43. . . Memory part,44. . . Output part,50. . . Control part,52. . . Analysis part,100. . . Measurement part,521. . . Chromatogram preparation part,522. . . Degree-of-oxidation analysis part,523. . . Normalization part, S . . . Sample, Si . . . Sample-derived ion. | 68,629 |
11860139 | The present invention relates to a system and method for analysing the composition of a quenched flow reaction liquid.FIG.1shows a quenched flow reactor5in fluid communication with a HPLC apparatus8. The quenched flow reactor comprises a first reagent release mechanism1, a second reagent release mechanism2and a quenching reagent release mechanism3. All are shown as a syringe, but other release mechanisms are envisaged. The valve10is used to fill the syringe from a reservoir and to allow external delivery of reagents from a separate flow release mechanism. In use, the first reagent and the second reagent are released and mixed in a reaction area11which includes the pathway extension valve4. Preferably the reaction area11comprises a mixer, such as a t-format mixer or a berger ball mixer. The pathway extension valve4comprises three loops9which may or may not form part of the fluid pathway, depending on the position of the valve. It will be appreciated that other lengths and numbers of option loops are included in the present invention. The liquid then flows to the quenching area12, where it mixes with the quenching reagent released from the quenching reagent release mechanism3to form a quenched flow reaction liquid. The quenching area12preferably comprises a mixer, such as a t-format mixer or a berger ball mixer. The quenched flow reaction liquid is then transferred, preferably piped into a bypass valve6. A first proportion of the quenched flow reaction liquid is then transferred, preferably piped out of the system to waste7or to a container7. This allows the liquid to flow through the quenched flow reactor at a fast rate, such as about 0.2 to about 30 ml/s, preferably about 0.5 to about 20 ml/s while the first reagent and the second reagent are mixing and the reaction is taking place. It will be appreciate that the fast flow rates are required to mix the first reagent and second reagent effectively, and that the flow rates may be reduced, or even stopped to give the desired reaction time, prior to pushing the reaction liquid into the quenching area. A second proportion of the quenched flow reaction liquid is directed into the HPLC injection valve13, and in particular through the HPLC injection valve loop14. The HPLC apparatus8comprises a HPLC pump17which pumps solvent to the HPLC injection valve13through the solvent line16. The HPLC injection valve loop14has two positions. In a first position, the HPLC injection valve loop14is connected to waste15or to a container15. This allows the HPLC injection valve loop14to be loaded with the desired first part of the second proportion of the quenched flow liquid and some of the quenched flow reaction liquid to be removed from the system. Once the desired first part of the second proportion of the quenched flow reaction liquid is loaded into the HPLC injection valve loop14, the HPLC injection valve13is moved to a second position, in line with the solvent line16of the HPLC apparatus to load the selected quenched flow reaction liquid onto the column. The HPLC apparatus8may comprise a digestion column, such as a pepsin column. Further, the HPLC analyte resulting from the HPLC analysis may be further piped into an analysis apparatus, preferably a mass spectrometer (not shown). FIG.2shows a pathway extension valve4in a first position, whereby the fluid pathway through the pathway extension valve is from the inlet23, directly to the outlet24. The additional passageway extensions25,26and27do not form part of the passageway at the first position. FIG.3shows a pathway extension valve4in a second position, whereby the fluid pathway through the pathway extension valve is from the inlet23, through a first extension passageway25and then to the outlet24. The additional passageway extensions26and27do not form part of the passageway at the second position. FIG.4shows a pathway extension valve4in a third position, whereby the fluid pathway through the pathway extension valve is from the inlet23, through a first passageway extension25, through a second passageway extension26and then to the outlet24. The additional passageway extension27does not form part of the passageway at the third position. FIG.5shows a pathway extension valve4in a fourth position, whereby the fluid pathway through the pathway extension valve is from the inlet23, through a first passageway extension25, through a second passageway extension26, through a third passageway extension27and then to the outlet24. There are no unused passageway extensions in the fourth position. It will be appreciated that each passageway extension is shown as a loop. Each loop may be the same length, or a different length to the other loops present. Further, the length of the passageway through the pathway extension valve can be selected from at least 2 predetermined lengths, preferably from 2 to about 10 predetermined lengths, preferably from about 3 to about 8 predetermined lengths, most preferably from 4 to 6 predetermined lengths. Further, the pathway extension valve may be arranged such that the liquid can flow through a first passageway extension, or a second passageway extension, or a third passageway extension, or a fourth passageway extension, or a fifth passageway extension, or a sixth passageway extension, or a seven passageway extension, or an eight passageway extension, or a ninth passageway extension or a tenth passageway extension, or any combination thereof where each length may be different. It will be appreciated that there may be any number of different passageway extensions in the pathway extension valve, such as at least one, preferably about 2 to about 10, preferably about 3 to about 8, preferably about 4 to about 6. FIGS.6a-8bshow an example of the pathway extension valve. It will be appreciated that other arrangements are possible, such as a plug type valve with passageway extensions along the radius of the plug and the passageway path diagonally or right angled drilled through the middle of the plug to a common port. FIG.6ashows a rotor31of a pathway extension valve. The rotor31comprises a rotor sealing surface34. The rotor sealing surface34comprises three passageway extensions36which may optionally be included in the passageway through the pathway extension valve. The sealing surface further comprises part of the passageway33. FIG.6bshows a cross-section view of the rotor31. One of the passageway extensions36and part of the passageway33are each shown as an indent in the sealing surface34. FIG.7ashows a stator32of the pathway extension valve. The stator comprises a stator sealing surface40. The stator sealing surface40comprises a plurality of threaded fluid tube sealing ports38shown arranged around the outer portion of the stator sealing surface40. The stator sealing surface40has one common threaded fluid tube sealing port39shown in the centre of the sealing surface. The common threaded fluid tube sealing port39is either the inlet or the outlet. One of the plurality of threaded fluid tube sealing ports38is the other of the inlet or the outlet. The arrangement will depend on how the tubing is connected. The stator has a stator sealing surface40. Adjusting the valve, such as by turning the valve will determine which of the passageway extensions are included in the passageway through the pathway extension valve. FIG.7bshows a cross-sectional view of the stator32. The stator sealing surface40has threaded fluid tube sealing ports38through the surface. The common threaded fluid tube sealing port39is shown in the centre of the stator sealing surface40. FIG.8ashows the rotor31in engagement with the stator32. The threaded fluid tube sealing ports38and the common threaded fluid tube sealing port39are each engaged with a passageway extension36or a part of the passageway33. The rotor sealing surface engages with the stator sealing surface40. FIG.8bshows a cross-sectional view of the rotor31in engagement with the stator32. Part of the passageway33lines up with the common threaded fluid tube sealing port39. Each of the threaded fluid tube sealing ports38engage with a passageway extension36. The rotor sealing surface34and the stator sealing surface40are in engagement. Adjusting the valve by turning will move the position of the passageway extensions36to move them to form part of the passageway or remove them from the passageway, thus allowing the length of the passageway through the pathway extension valve to be adjusted, and thus the length of the reaction area fluid pathway to be changed. It will be appreciated that the pathway extension valve may have a different number, type and arrangement of sealing ports and tubing. FIG.9shows a prereaction system41that can be optionally incorporated into the quenched flow reactor5ofFIG.1. The prereaction system41comprises a first precursor release mechanism42, a second precursor release mechanism43. All are shown as a syringe, but other release mechanisms are envisaged. The valve10is used to fill the syringe from a reservoir and to allow external delivery of reagents from a separate flow release mechanism. In use, the first precursor and the second precursor are released and mixed in a prereaction area44, to form the first reagent. The prereaction area44, may preferably comprise a pathway extension valve as described herein (not shown). The first reagent flows to the reaction area11, where it mixes with the second reagent released from the second reagent release mechanism2(not shown). The system and method for analysing the composition of a quenched flow reaction liquid then continues to proceed as described above with reference toFIG.1. | 9,653 |
11860140 | DETAILED DESCRIPTION Currently, the quantitative determination of low levels of the PFAS analytes in water sources often requires extraction of the PFAS analytes from the water. This requirement is typically due to the limitations of the analytical methods employed for sample analysis. When analyte concentrations are too low to be quantitated by established analytical techniques, extraction thereof serves to provide a more concentrated sample than the originally collected unconcentrated water sample. These extraction steps are often time-consuming, costly, and inherently introduce the possibility of errors in the analysis along with an increase in possible sample contamination. In some cases, up to one liter of water from a contaminated water source must be extracted to provide 1 mL of an aqueous sample after evaporation of extracting solvent and subsequent aqueous dissolution of the isolated extract. Embodiments of the present invention recognize that extraction steps contribute to increased costs and errors in the qualitative and quantitative analysis of PFAS analytes in water samples. Embodiments of the present invention provide a method and LC/MS/MS system for the determination of concentrations and amounts of low levels of PFAS analytes in unconcentrated as well as concentrated samples. Thus, extraction techniques may be avoided in the analysis of PFAS analytes in unconcentrated samples, such as finished drinking water, ground water, raw source water, and water at an intermediate stage of treatment between raw source water and finished drinking water. As described herein, “PFAS analyte” indicates a poly- or perfluorinated alkyl carboxylic or sulfonic acid and/or the corresponding conjugate bases. It will be readily understood by one having ordinary skill in the art that the relative quantity of the acid and conjugate base will be dependent on the pH of the sample and/or standard that contains the PFAS analyte as well as the pKa(H2O) of the PFAS acid component within the given sample and/or standard solution. The PFAS analytes, as acids and/or the corresponding conjugate bases, that are detected in a solution or an unconcentrated sample include without limitation: i) perfluorobutanoic acid (C3F7CO2H) and/or the conjugate base thereof, i.e., perfluorobutanoate (C3F7CO2-); ii) perfluorobutanesulfonic acid (C4F9SO3H) and/or the conjugate base thereof, i.e., perfluorobutane sulfonate (C4F9SO3-); iii) perfluoropentanesulfonic acid (C5F11SO3H) and/or the conjugate base thereof, i.e., perfluoropentane sulfonate (C5F11SO3-); iv) perfluorohexanoic acid (C5F11CO2H) and/or the conjugate base thereof, i.e., perfluorohexanoate (C5F11CO2-); v) perfluorohexanesulfonic acid (C6F13SO3H) and/or the conjugate base thereof, i.e., perfluorohexane sulfonate (C6F13SO3-); vi) perfluoroheptanoic acid (C6F13CO2H) and/or the conjugate base thereof, i.e., perfluoroheptanoate (C6F13CO2-); vii) perfluoroheptanesulfonic acid (C7F15SO3H) and/or the conjugate base thereof, i.e., perfluoroheptane sulfonate (C7F15SO3-); viii) perfluorooctanoic acid (C7F15CO2H) and/or the conjugate base thereof, i.e., perfluorooctanoate (C7F15CO2-); ix) perfluorooctanesulfonic acid (C8F17SO3H) and/or the conjugate base thereof, i.e., perfluorooctane sulfonate (C8F17SO3-); x) perfluorononanoic acid (C8F17CO2H) and/or the conjugate base thereof, i.e., perfluorononanoate (C8F17CO2-); and xi) perfluorodecanoic acid (C9F19CO2H) and/or the conjugate base thereof, i.e., perfluorodecanoate (C9F19CO2-). The method and system encompasses the concentration determination of, in a solution or an unconcentrated sample, all known isomers of PNAS analytes with the general formula CnF(2n+1)—X as described herein. PFAS analyte acronyms used throughout this description are as shown in Table 1. TABLE 1Acronym Definitions.Analyte Name (Acid/Conjugate Base)AcronymPerfluorobutanoic acid/PerfluorobutanoatePFBAPerfluorobutanesulfonic acid/Perfluorobutane sulfonatePFBSPerfluorodecanoic acid/PerfluorodecanoatePFDAPerfluoroheptanoic acid/PerfluoroheptanoatePFHpAPerfluoroheptanesulfonic acid/PerfloroheptanesulfonatePFHpSPerfluorohexanoic acid/PerfluorohexanoatePFHxAPerfluorohexanesulfonic acid/PerflorohexanesulfonatePFHxSPerfluorononanoic acid/PerfluorononanoatePFNAPerfluoropentanesulfonic acid/PerfloropentanesulfonatePFPeSPerfluorooctanoic acid/PerfluorooctanoatePFOAPerfluorooctanesulfonic acid/PerflorooctanesulfonatePFOS Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, and use of the methods and systems disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the methods and systems specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present invention. The terms “substantially”, “approximately”, “about”, “relatively,” or other such similar terms that may be used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to +10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%. The ranges disclosed herein include increments governed by significant figures as recited in the ranges. For example, a temperature range of approximately 120° C. to approximately 125° C. indicates three significant figures, hence approximately 120 to approximately 121° C., approximately 121 to approximately 122° C., approximately 122 to approximately 123° C., approximately 123 to approximately 124° C., and approximately 124 to approximately 125° C. are thereby included as range subsets. In various embodiments, unconcentrated samples are analyzed for detection and quantitation of PFAS analytes. As used herein, “unconcentrated sample” typically refers to an aqueous sample collected from a water source such as, but not limited to, finished drinking water, ground water, raw source water, and water at an intermediate stage of treatment between raw source water and finished drinking water. The sample may also be collected from an effluent from processes that utilize one or more PFAS analytes, such as from a factory that produces PFAS-containing products. The unconcentrated sample is not concentrated by any deliberate or substantial evaporation of the solvent, i.e., water. Further, the unconcentrated sample is not concentrated by, for example, extraction into an organic solvent to subsequently make a non-aqueous or aqueous solution of PFAS analyte(s) that have higher concentrations than the originally collected sample. An unconcentrated sample also includes a sample that is diluted with respect to the originally collected sample. The diluent may be water or a water-miscible solvent such as, but not limited to, an alcohol (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol sec-butanol, iso-butyl alcohol, tert-butyl alcohol, diols such as ethylene glycol, triols such as glycerol, etc.), acetonitrile, etc. In some embodiments, unconcentrated samples also contain added chemicals, such as ammonium chloride, buffers, etc., for purposes of dechlorination, sample preservation, pH adjustment, etc. Unconcentrated samples include such water samples which are not diluted or concentrated such that they may be directly injected from the source, with or without minimal processing, into the system for analysis. The term “minimal processing” includes the addition of preservatives, buffers, etc. in order to modulate sample stability, pH, etc. In various embodiments, concentrated samples are analyzed for detection and quantitation of PFAS analyte(s) at extremely low levels. As used herein, “concentrated samples” include samples obtained via one or more of the following steps: i) the extraction of PFAS analyte(s) from a first volume of water (typically an aqueous sample obtained directly from a water source) into second volume of a water-immiscible solvent, wherein the second volume of a water-immiscible solvent is less than or substantially the same; ii) partial or complete evaporation of the water-immiscible solvent to concentrate the PFAS analyte(s) contained therein; and iii) re-dissolving the PFAS analyte(s) into a third volume of water with or without the concomitant introduction of preservatives, buffers, and/or dechlorination agents, wherein the third volume of water is of a lesser volume than the first volume of water. In some embodiments of the present invention, concentrated and unconcentrated samples of PFAS analyte(s) include samples collected and prepared from soil and plants, as described elsewhere (e.g., see Huset and Barry, “Quantitative determination of perfluoroalkyl substances (PFAS) in soil, water, and home garden produce”, MethodsX 5 (2018) 697-704). In some embodiments, concentrated and unconcentrated samples of PFAS analyte(s) include samples collected from urine and blood. As used herein, the term “PFAS analyte solution,” “PFAS analyte(s) in a solution,” “a solution containing PFAS analyte(s),” and the like, includes a homogeneous solution of PFAS analyte(s), which includes concentrated and unconcentrated PFAS analyte samples as well as standards, etc. As is well-known in the art, for any analyte to be injected onto an LC/MS/MS system, it must be in a homogeneous solution of a solvent suitable for injection onto an LC column. It will be understood that within a known volume of an analyte solution that has a known concentration, the amount of analyte is also known and readily calculated. For example, 75 microliters (∝L or ∝l) of a PFAS analyte solution that has a concentration of 0.010 micrograms per liter (0.010 ∝g/L or ∝g/l) contains 7.5×10−7∝g of the PFAS analyte according to the equation: (0.010 ∝g/L)×(75 ∝L)×(10-6 L/∝L)=7.5×10−7∝g. Thus, 75 ∝L of a 0.0020 ∝g/L PFAS analyte solution contains 1.5×10−7∝g of the PFAS analyte, 75 ∝L of a 0.25 ∝g/L PFAS analyte solution contains 1.9×10−5∝g of the PFAS analyte and 75 ∝L of a 0.070 ∝g/L PFAS analyte solution contains 5.3×10−6∝g of the PFAS analyte. Throughout this description and claims, it will be understood that any known volume of an analyte solution with a known concentration of said analyte may be expressed in terms of a known mass of said analyte. Herein, analyte concentration may be expressed as parts per trillion (ppt) according to the relationship 1 ng/L=1 ppt. Thus, 0.010 μg/L may be expressed as 10 ppt, 0.0020 μg/L may be expressed as 2 ppt, 0.070 μg/L may be expressed as 70 ppt, and 0.25 μg/L may be expressed as 250 ppt. Because the relationship between ppt and μg/L is defined above, it is now established that a known volume containing a known ppt of an analyte may also be expressed in terms of a known mass of said analyte. Embodiments of the present invention provide a method and system to determine the concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples within a range of approximately 0.0020 μg/L to approximately 0.25 μg/L based on approximately a 75 μL injection volume using ESI conditions on an LC/MS/MS instrument as described infra. It will be readily apparent to one skilled in the art that, since the sensitivity of the method and system described herein is dependent on injection volume, injection volumes greater than approximately 75 μL will produce quantitation of solutions having a concentration of PFAS analyte of formula CnF(2n+1)—X in solutions that is lower than 0.0020 ∝g/L. In some embodiments, a concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples is determined within a range of approximately 0.0020 ∝g/L to approximately 0.24 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.23 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.22 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.21 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.20 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.19 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.18 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.17 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.16 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.15 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.14 cc g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.13 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.12 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.11 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.10 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.090 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.080 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.070 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.060 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.050 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.040 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.030 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.020 ∝g/L. In some embodiments, the range is approximately 0.0020 ∝g/L to approximately 0.010 ∝g/L. Embodiments of the present invention provide a method and system to determine a concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples within a range of approximately 0.010 μg/L to approximately 0.25 μg/L based on a 75 μL injection volume using ESI conditions on an LC/MS/MS instrument as described infra. In some embodiments, a concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples is determined within a range of approximately 0.010 μg/L to approximately 0.24 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.23 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.22 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.21 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.20 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.19 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.18 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.17 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.16 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.15 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.14 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.13 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.12 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.11 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.10 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.090 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.080 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.070 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.060 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.050 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.040 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.030 μg/L. In some embodiments, the range is approximately 0.010 μg/L to approximately 0.020 μg/L. Embodiments of the present invention provide a method and system to determine a concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples within a range of approximately 0.070 μg/L to approximately 0.25 μg/L based on a 75 μL injection volume using ESI conditions on an LC/MS/MS instrument as described infra. In some embodiments, a concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples is determined within a range of approximately 0.070 μg/L to approximately 0.24 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.23 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.22 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.21 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.20 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.19 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.18 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.17 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.16 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.15 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.14 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.13 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.12 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.11 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.10 μg/L. In some embodiments, the range is approximately 0.070 μg/L to approximately 0.090 μg/L. In some embodiments, the range is approximately 0.070 μg/to approximately 0.080 μg/L. Embodiments of the present invention provide a method and system to determine a concentration of a PFAS analyte of formula CnF(2n+1)—X in solutions such as unconcentrated samples within a range of approximately 2.0 ppt to approximately 250 ppt based on a 75 μL injection volume using ESI conditions on an LC/MS/MS instrument as described infra. As explained supra, wherein 1 ng/L=1 ppt (i.e. 1.0 μg/L=1.0×103 ppt), the embodiments described supra of determinable μg/L concentration ranges of PFAS analytes apply when expressed as ppt. It will be readily understood by a person having ordinary skill in the art that virtually any concentration of a PFAS analyte of formula CnF(2n+1)—X above 0.250 μg/L (250.0 ppt) within a solution is determinable by the method and system described herein via the use of well-known dilution techniques. In fact, such techniques are exemplified by the preparation of analyte standards as described infra. Embodiments of the present invention provide a method and system to determine an amount of a PFAS analyte of formula CnF(2n+1)—X that is injected from a solution such as unconcentrated sample onto an LC/MS/MS instrument based on a known injected volume of determined concentration. The individual amount of a PFAS analyte that are determinable per injection range from approximately 1.5×10-7 μg to approximately 1.9×10-5 μg using ESI conditions as described infra. In some embodiments, the amount of a PFAS analyte determinable per injection is within a range of approximately 1.5×10-7 μg to approximately 1.8×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 1.7×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 1.6×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 1.5×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μs to approximately 1.4×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×μg to approximately 1.3×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 fig to approximately 1.2×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μs to approximately 1.1×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 9.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 9.0×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μs to approximately 8.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 7.5×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 fig to approximately 6.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 6.0×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 5.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 4.5×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 3.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 3.0×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 2.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 1.5×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 1.5×10-7 μg to approximately 7.5×10-7 μg. Embodiments of the present invention provide a method and system to determine an amount of a PFAS analyte of formula CnF(2n+1)—X that is injected from a solution such as unconcentrated sample onto an LC/MS/MS instrument based on a known injected volume of determined concentration. The individual amount of a PFAS analyte that are determinable per injection range from approximately 7.5×10-7 μg to approximately 1.9×10-5 μg using ESI conditions as described infra. In some embodiments, the amount of a PFAS analyte determinable per injection is within a range of approximately 7.5×10-7 μg to approximately 1.8×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7)μg to approximately 1.7×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.6×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.5×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.4×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.3×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.2×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.1×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 9.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 9.0×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 8.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 7.5×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 6.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 6.0×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 5.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 4.5×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 3.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 3.0×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 2.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 7.5×10-7 μg to approximately 1.5×10-6 μg. Embodiments of the present invention provide a method and system to determine an amount of a PFAS analyte of formula CnF(2n+1)—X that is injected from a solution such as unconcentrated sample onto an LC/MS/MS instrument based on a known injected volume of determined concentration. The individual amount of a PFAS analyte that are determinable per injection range from approximately 5.3×10-6 μg to approximately 1.9×10-5 μg using ESI conditions as described infra. In some embodiments, the amount of a PFAS analyte determinable per injection is within a range of approximately 5.3×10-6 μg to approximately 1.8×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 1.7×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 1.6×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 1.5×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 1.4×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 1.3×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 1.2×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 fig to approximately 1.1×10-5 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 9.8×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 lag to approximately 9.0×10-6FIG.1nsome embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 8.3×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 7.5×10-6 μg. In some embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 6.8×10-6FIG.1nsome embodiments, the determinable amount is within a range of approximately 5.3×10-6 μg to approximately 6.0×10-6 μg. Embodiments of the present invention utilize ESI on an LC/MS/MS system to determine PFAS analyte concentration and amount. ESI is an ionization technique used in mass spectrometry to produce ions using an electrospray in which a high voltage is applied to a liquid to create an aerosol that is ionized. FIG.1depicts a block diagram of components100of an LC/MS/MS system used to determine a concentration and/or amount of a PFAS analyte in samples in accordance with an exemplary embodiment of the present invention. It should be appreciated thatFIG.1provides only an illustration of one implementation and does not imply any limitations with regard to other systems in which embodiments of the present invention may be implemented. Other modifications to the depicted system may be made without departing from the scope of the present invention. LC/MS/MS system100includes injector110, LC column115, ESI ionizer component120, triple quadrupole mass spectrometer (TQMS) component125, ion detector130, and mass spectrum read-out software135. TQMS125includes two quadrupole mass analyzers in series (125Q1and125Q3) with a non-mass-resolving quadrupole (125Q2) between them to act as a cell for collision-induced dissociation. All three quadrupole mass analyzers consist of four cylindrical rods (for reasons of simplicity they are schematically represented by the labeled parallel bars inFIG.1). The four cylindrical bars are set parallel to each other. For125Q1and125Q3, each opposing rod pair is connected together electrically and a radio frequency (RF) voltage with a DC offset voltage is applied between one pair of rods and the other. Ions travel down the quadrupole between the rods. Only ions of a certain mass-to-charge ratio will reach detector130for a given ratio of voltages. Other ions have unstable trajectories and will collide with the rods. This permits selection of an ion with a particular m/z or allows the operator to scan for a range of m/z-values by continuously varying the applied voltage. Quadrapole125Q2is an RF-only quadrupole (non-mass filtering) for collision induced dissociation of selected parent ion(s) from125Q1. Subsequent fragments are passed through to125Q3where they may be filtered or fully scanned. In an embodiment, an aliquot of PFAS analyte sample105is injected into injector110and the injection liquid101is resolved into various PFAS analytes by LC column115using, for example, the column, conditions, and gradient example shown and described for Table 6. After eluting through LC column115, the PFAS analyte-containing eluent102is subjected to ESI120. Conditions for ionization of PFAS analytes using ESI techniques as depicted by ESI120will be detailed and described infra in embodiments of the present invention. After ionization of the PFAS-containing eluent by ESI120, the ion(s)103are passed through the first quadrupole mass analyzer,125Q1, which serves as a filter for selecting desired PFAS analyte ions104. The second quadrupole mass analyzer,125Q2, allows for collision of selected ions104to produce one or more children ions106that then pass through the third quadrupole mass analyzer,125Q3. Quadrupole mass analyzer125Q3provides a scan of the entire m/z range of the product ion(s)106, providing output107for fragments106. Quantification of selected ion104can then be deduced from the ion fragmentation output107received by ion detector130and processed by mass spectrum read-out software135. Embodiments of the present invention employ ESI settings on an LC/MS/MS system such as the system described above that include: i) an ion polarity setting to cause the formation of negative or positive ions; ii) a probe gas temperature setting for controlling the temperature of an inert drying gas (typically nitrogen) that is used to promote the removal of solvent from aerosol particles in spray ionization; iii) a gas flow setting for controlling the volume per unit time that the drying gas is dispersed; iv) a nebulizer setting for controlling the pressure utilized for the mass spectrometer nebulizer, which delivers a fine mist using the specified pressure; v) a sheath gas heater setting for controlling a temperature setting for heating a sheath gas, which is an inert gas (typically nitrogen) introduced through a tube that is coaxial with the electrospray emitter to pneumatically assist the formation of the sprayed droplets; vi) a sheath gas flow setting for controlling a volume per unit time that the sheath gas is dispersed; vii) a capillary voltage setting for controlling a voltage applied to the tip of a metal capillary relative to the surrounding source-sampling cone or heated capillary, which creates a strong electric field causes the dispersion of the sample solution into an aerosol of highly charged electrospray droplets; and viii) a V charging setting for controlling a charging electrode within the instrument. A non-limiting example of ESI settings used on an AGILENT 6495 mass spectrometer for analyzing concentrations and amounts of PFAS analytes in solutions such as unconcentrated samples is shown in Table 2 below. TABLE 2Example of ESI settings used in an embodimentof the present invention.PolarityNegative ionESI ConditionsGas Temp (° C.)120Gas Flow (1/min)11Nebulizer (psi)20Sheath Gas Heater400Sheath Gas Flow8Capillary (V)1500V Charging0Ion Funnel ParametersHigh Pressure RF110Low Pressure RF80 For Table 2 above, the “Ion Funnel Parameters” refers to settings for an ion funnel, which is used to focus a beam of ions using a series of stacked ring electrodes with decreasing inner diameter. A combined radio frequency (RF) and fixed electrical potential is applied to the grids. In various embodiments of the present invention, concentrations and amounts of PFAS analytes in solutions such as unconcentrated samples are analyzed using ESI conditions include a probe gas temperature setting (“Gas Temp (° C.)”) of approximately 120° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 150° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 145° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 140° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 135° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 130° C. In some embodiments, the probe gas temperature setting is approximately 120° C. to approximately 125° C. In some embodiments, the probe gas temperature setting is approximately 120° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 125° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 165° C. In some embodiments, ESI conditions include a probe probe gas temperature setting of approximately 125° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 150° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 145° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 140° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 135° C. In some embodiments, the probe gas temperature setting is approximately 125° C. to approximately 130° C. In some embodiments, the probe gas temperature setting is approximately 125° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 130° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 150° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 145° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 140° C. In some embodiments, the probe gas temperature setting is approximately 130° C. to approximately 135° C. In some embodiments, the probe gas temperature setting is approximately 130° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 135° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 150° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 145° C. In some embodiments, the probe gas temperature setting is approximately 135° C. to approximately 140° C. In some embodiments, the probe gas temperature setting is approximately 135° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 140° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 150° C. In some embodiments, the probe gas temperature setting is approximately 140° C. to approximately 145° C. In some embodiments, the probe gas temperature setting is approximately 140° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 145° C. to approximately 150° C. In some embodiments, the probe gas temperature setting is approximately 145° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 150° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 150° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 150° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 150° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 150° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 150° C. to approximately 155° C. In some embodiments, the probe gas temperature setting is approximately 150° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 155° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 155° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 155° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 155° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 155° C. to approximately 160° C. In some embodiments, the probe gas temperature setting is approximately 155° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 160° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 160° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 160° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 160° C. to approximately 165° C. In some embodiments, the probe gas temperature setting is approximately 160° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 165° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 165° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 165° C. to approximately 170° C. In some embodiments, the probe gas temperature setting is approximately 165° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 170° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 170° C. to approximately 175° C. In some embodiments, the probe gas temperature setting is approximately 170° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 175° C. to approximately 180° C. In some embodiments, the probe gas temperature setting is approximately 175° C. In some embodiments, ESI conditions include a probe gas temperature setting of approximately 180° C. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 180° C. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFBS as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 180° C. In some embodiments when the PFAS analyte solution includes PFBS as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFHpA as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 180° C. In some embodiments when the PFAS analyte solution includes PFHpA as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFHxA as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 180° C. In some embodiments when the PFAS analyte solution includes PFHxA as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFHxS as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 180° C. In some embodiments when the PFAS analyte solution includes PFHxS as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFPeS as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 180° C. In some embodiments when the PFAS analyte solution includes PFPeS as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFDA as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 160° C. In some embodiments when the PFAS analyte solution includes PFDA as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFHpS as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 160° C. In some embodiments when the PFAS analyte solution includes PFHpS as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFOS as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 160° C. In some embodiments when the PFAS analyte solution includes PFOS as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFNA as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 140° C. In some embodiments when the PFAS analyte solution includes PFNA as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFNS as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 160° C. In some embodiments when the PFAS analyte solution includes PFNS as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments when the PFAS analyte solution contains PFOA as analyte, the probe gas temperature setting ranges from approximately 120° C. to approximately 160° C. In some embodiments when the PFAS analyte solution includes PFOA as analyte, the probe gas temperature setting is approximately 120° C. In some embodiments, the ESI probe gas temperature setting is set on an AGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, the ESI gas temperature setting is set on an AGILENT 6495 mass spectrometer. In various embodiments, ESI conditions include a sheath gas heater setting (“Sheath Gas Heater”) of approximately 250° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 280° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 275° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 270° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 265° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 260° C. In some embodiments, the sheath gas heater setting is approximately 250° C. to approximately 255° C. In some embodiments, the sheath gas heater setting is approximately 250° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 255° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 280° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 275° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 270° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 265° C. In some embodiments, the sheath gas heater setting is approximately 255° C. to approximately 260° C. In some embodiments, the sheath gas heater setting is approximately 255° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 260° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 280° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 275° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 270° C. In some embodiments, the sheath gas heater setting is approximately 260° C. to approximately 265° C. In some embodiments, the sheath gas heater setting is approximately 260° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 265° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 280° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 275° C. In some embodiments, the sheath gas heater setting is approximately 265° C. to approximately 270° C. In some embodiments, the sheath gas heater setting is approximately 265° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 270° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 280° C. In some embodiments, the sheath gas heater setting is approximately 270° C. to approximately 275° C. In some embodiments, the sheath gas heater setting is approximately 270° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 275° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 275° C. to approximately 280° C. In some embodiments, the sheath gas heater setting is approximately 275° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 280° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 280° C. to approximately 285° C. In some embodiments, the sheath gas heater setting is approximately 280° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 285° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 285° C. to approximately 290° C. In some embodiments, the sheath gas heater setting is approximately 285° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 290° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 290° C. to approximately 295° C. In some embodiments, the sheath gas heater setting is approximately 290° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 295° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 295° C. to approximately 300° C. In some embodiments, the sheath gas heater setting is approximately 295° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 300° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 300° C. to approximately 305° C. In some embodiments, the sheath gas heater setting is approximately 300° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 305° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 305° C. to approximately 310° C. In some embodiments, the sheath gas heater setting is approximately 305° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 310° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 310° C. to approximately 315° C. In some embodiments, the sheath gas heater setting is approximately 310° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 315° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 315° C. to approximately 320° C. In some embodiments, the sheath gas heater setting is approximately 315° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 320° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 320° C. to approximately 325° C. In some embodiments, the sheath gas heater setting is approximately 320° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 325° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 325° C. to approximately 330° C. In some embodiments, the sheath gas heater setting is approximately 325° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 330° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 330° C. to approximately 335° C. In some embodiments, the sheath gas heater setting is approximately 330° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 335° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 335° C. to approximately 340° C. In some embodiments, the sheath gas heater setting is approximately 335° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 340° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 340° C. to approximately 345° C. In some embodiments, the sheath gas heater setting is approximately 340° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 345° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 345° C. to approximately 350° C. In some embodiments, the sheath gas heater setting is approximately 345° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 350° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 350° C. to approximately 355° C. In some embodiments, the sheath gas heater setting is approximately 350° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 355° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 355° C. to approximately 360° C. In some embodiments, the sheath gas heater setting is approximately 355° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 360° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 360° C. to approximately 365° C. In some embodiments, the sheath gas heater setting is approximately 360° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 365° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 365° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 365° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 365° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 365° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 365° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 365° C. to approximately 370° C. In some embodiments, the sheath gas heater setting is approximately 365° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 370° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 370° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 370° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 370° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 370° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 370° C. to approximately 375° C. In some embodiments, the sheath gas heater setting is approximately 370° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 375° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 375° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 375° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 375° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 375° C. to approximately 380° C. In some embodiments, the sheath gas heater setting is approximately 375° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 380° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 380° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 380° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 380° C. to approximately 385° C. In some embodiments, the sheath gas heater setting is approximately 380° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 385° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 385° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 385° C. to approximately 390° C. In some embodiments, the sheath gas heater setting is approximately 385° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 390° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 390° C. to approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 390° C. In various embodiments, ESI conditions include a sheath gas heater setting of approximately 395° C. to approximately 400° C. In some embodiments, the sheath gas heater setting is approximately 395° C. In some embodiments, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the sheath gas heater setting ranges from approximately 250° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFBS as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFBS as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFHpA as analyte, the sheath gas heater setting ranges from approximately 275° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFHpA as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFHxA as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFHxA as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFHxS as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFHxS as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFPeS as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFPeS as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFDA as analyte, the sheath gas heater setting ranges from approximately 275° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFDA as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFHpS as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFHpS as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFOS as analyte, the sheath gas heater setting ranges from approximately 350° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFOS as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFNA as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFNA as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFNS as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFNS as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments when the PFAS analyte solution contains PFOA as analyte, the sheath gas heater setting ranges from approximately 300° C. to approximately 400° C. In some embodiments when the PFAS analyte solution includes PFOA as analyte, the sheath gas heater setting is approximately 400° C. In some embodiments, the ESI sheath gas heater setting is set on an AGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, the ESI sheath gas heater setting is set on an AGILENT 6495 mass spectrometer. In various embodiments, ESI conditions include a sheath gas flow (“Sheath Gas Flow”) of approximately 8 L/min to approximately 12 L/min. In some embodiments, the sheath gas flow is approximately 8 L/min to approximately 11 L/min. In some embodiments, the sheath gas flow is approximately 8 L/min to approximately 10 L/min. In some embodiments, the sheath gas flow is approximately 8 L/min to approximately 9 L/min. In some embodiments, the sheath gas flow is approximately 8 L/min. In various embodiments, ESI conditions include a sheath gas flow of approximately 9 L/min to approximately 12 L/min. In some embodiments, the sheath gas flow is approximately 9 L/min to approximately 11 L/min. In some embodiments, the sheath gas flow is approximately 9 L/min to approximately 10 L/min. In some embodiments, the sheath gas flow is approximately 9 L/min. In various embodiments, ESI conditions include a sheath gas flow of approximately 10 L/min to approximately 12 L/min. In some embodiments, the sheath gas flow is approximately 10 L/min to approximately 11 L/min. In some embodiments, the sheath gas flow is approximately 10 L/min. In various embodiments, ESI conditions include a sheath gas flow of approximately 11 L/min to approximately 12 L/min. In some embodiments, the sheath gas flow is approximately 11 L/min. In some embodiments, the sheath gas flow is approximately 12 L/min. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFBS as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution contains PFBS as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFHpA as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 10 L/min. In some embodiments when the PFAS analyte solution contains PFHpA as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFHxA as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 10 L/min. In some embodiments when the PFAS analyte solution contains PFHxA as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFHxS as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution contains PFHxS as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFPeS as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution contains PFPeS as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFDA as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 9 L/min. In some embodiments when the PFAS analyte solution contains PFDA as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFHpS as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution contains PFHpS as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFOS as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 9 L/min. In some embodiments when the PFAS analyte solution contains PFOS as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFNA as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 9 L/min. In some embodiments when the PFAS analyte solution contains PFNA as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFNS as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution contains PFNS as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments when the PFAS analyte solution contains PFOA as analyte, the sheath gas flow setting ranges from approximately 8 L/min to approximately 10 L/min. In some embodiments when the PFAS analyte solution contains PFOA as analyte, the sheath gas flow setting is approximately 8 L/min. In some embodiments, the ESI sheath gas flow setting is set on an AGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, the ESI sheath gas flow setting is set on an AGILENT 6495 mass spectrometer. In various embodiments of the present invention, concentrations and amounts of PFAS analytes in solutions such as unconcentrated samples are analyzed using ESI conditions that include a capillary voltage setting (“Capillary (V)”) of approximately 1500 V to approximately 4500 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 4400 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 4300 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 4200 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 4100 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 4000 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3900 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3800 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3700 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3600 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3500 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3400 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3300 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3200 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3100 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 3000 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2900 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2800 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2700 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2600 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2500 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2400 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2300 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2200 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2100 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 2000 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 1900 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 1800 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 1700 V. In some embodiments, the capillary voltage setting is approximately 1500 V to approximately 1600 V. In some embodiments, the capillary voltage setting is approximately 1500 V. In various embodiments of the present invention, concentrations and amounts of PFAS analytes in solutions such as unconcentrated samples are analyzed using ESI conditions include a capillary voltage setting of approximately 4500 V. In some embodiments, the capillary voltage setting is approximately 4400 V. In some embodiments, the capillary voltage setting is approximately 4300 V. In some embodiments, the capillary voltage setting is approximately 4200 V. In some embodiments, the capillary voltage setting is approximately 4100 V. In some embodiments, the capillary voltage setting is approximately 4000 V. In some embodiments, the capillary voltage setting is approximately 3900 V. In some embodiments, the capillary voltage setting is approximately 3800 V. In some embodiments, the capillary voltage setting is approximately 3700 V. In some embodiments, the capillary voltage setting is approximately 3600 V. In some embodiments, the capillary voltage setting is approximately 3500 V. In some embodiments, the capillary voltage setting is approximately 3400 V. In some embodiments, the capillary voltage setting is approximately 3300 V. In some embodiments, the capillary voltage setting is approximately 3200 V. In some embodiments; the capillary voltage setting is approximately 3100 V. In some embodiments, the capillary voltage setting is approximately 3000 V. In some embodiments, the capillary voltage setting is approximately 2900 V. In some embodiments, the capillary voltage setting is approximately 2800 V. In some embodiments, the capillary voltage setting is approximately 2700 V. In some embodiments, the capillary voltage setting is approximately 2600 V. In some embodiments, the capillary voltage setting is approximately 2500 V. In some embodiments, the capillary voltage setting is approximately 2400 V. In some embodiments, the capillary voltage setting is approximately 2300 V. In some embodiments, the capillary voltage setting is approximately 2200 V. In some embodiments, the capillary voltage setting is approximately 2100 V. In some embodiments, the capillary voltage setting is approximately 2000 V. In some embodiments, the capillary voltage setting is approximately 1900 V. In some embodiments, the capillary voltage setting is approximately 1800 V. In some embodiments, the capillary voltage setting is approximately 1700 V. In some embodiments, the capillary voltage setting is approximately 1600 V. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 2000 V. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFBS as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 2500 V. In some embodiments when the PFAS analyte solution includes PFBS as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFHpA as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 3000 V. In some embodiments when the PFAS analyte solution includes PFHpA as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFHxA as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 3000 V. In some embodiments when the PFAS analyte solution includes PFHxA as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFHxS as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 3000 V. In some embodiments when the PFAS analyte solution includes PFHxS as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFPeS as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 2500 V. In some embodiments when the PFAS analyte solution includes PFPeS as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFDA as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 4500 V. In some embodiments when the PFAS analyte solution includes PFDA as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFHpS as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 3000 V. In some embodiments when the PFAS analyte solution includes PFHpS as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFOS as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 2500 V. In some embodiments when the PFAS analyte solution includes PFOS as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFNA as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 4000 V. In some embodiments when the PFAS analyte solution includes PFNA as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFNS as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 4000 V. In some embodiments when the PFAS analyte solution includes PFNS as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments when the PFAS analyte solution includes PFOA as analyte, the capillary voltage setting ranges from approximately 1500 V to approximately 3000 V. In some embodiments when the PFAS analyte solution includes PFOA as analyte, the capillary voltage setting is approximately 1500 V. In some embodiments, the ESI capillary voltage setting is set on an AGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, the ESI capillary voltage setting is set on an AGILENT 6495 mass spectrometer. In various embodiments, ESI conditions include a gas flow setting (“Gas Flow (1/min)”) of approximately 11 L/min to approximately 20 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 19 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 18 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 17 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 16 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 15 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 14 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 13 L/min. In some embodiments, the gas flow setting is approximately 11 L/min to approximately 12 L/min. In some embodiments, the gas flow setting is approximately 11 L/min. In various embodiments, ESI conditions include a gas flow setting of approximately 20 L/min. In some embodiments, the gas flow setting is approximately 19 L/min. In some embodiments, the gas flow setting is approximately 18 L/min. In some embodiments, the gas flow setting is approximately 17 L/min. In some embodiments, the gas flow setting is approximately 16 L/min. In some embodiments, the gas flow setting is approximately 15 L/min. In some embodiments, the gas flow setting is approximately 14 L/min. In some embodiments, the gas flow setting is approximately 13 L/min. In some embodiments, the gas flow setting is approximately 12 L/min. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFBA as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFBS as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFBS as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFHpA as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFHpA as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFHxA as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFHxA as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFHxS as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFHxS as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFPeS as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFPeS as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFDA as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFDA as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFHpS as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFHpS as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFOS as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 12 L/min. In some embodiments when the PFAS analyte solution includes PFOS as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFNA as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFNA as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFNS as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFNS as analyte, the gas flow setting is approximately 11 L/min. In some embodiments when the PFAS analyte solution contains PFOA as analyte, the gas flow setting ranges from approximately 11 L/min to approximately 20 L/min. In some embodiments when the PFAS analyte solution includes PFOA as analyte, the gas flow setting is approximately 11 L/min. In some embodiments, the ESI gas flow setting is set on an AGILENT 6490 or 6495 mass spectrometer. In exemplary embodiments, the ESI gas flow setting is set on an AGILENT 6495 mass spectrometer. An example of minimum detection levels (MDL), minimum reporting levels (RL or MRL), and reporting ranges for the ESI settings shown above is shown in Table 3. TABLE 3Minimum Detection Levels (MDL), Minimum ReportingLevels (RL or MRL), and Reporting Ranges for anExemplary Embodiment of the Present Invention.MDLRL (MRL)Reporting RangeAcronym[μg/L][μg/L][μg/L]PFBA0.00250.0100.010-0.25PFBS0.00410.0100.010-0.25PFDA0.00440.0100.010-0.25PFHpA0.00550.0100.010-0.25PFHpS0.00870.0100.010-0.25PFHxA0.00170.0100.010-0.25PFHxS0.00570.0100.010-0.25PFNA0.00520.0100.010-0.25PFPeS0.00450.0100.010-0.25PFOA0.000770.00200.0020-0.25PFOS0.000950.00200.0020-0.25 MDLs (Method Detection Limits) are statistical values used to determine RLs/MRLs as described infra. RLs/MRLs (Reporting Limits or Minimum Reporting Levels) are practical and routinely achievable values of analyte concentration given ESI parameters such as described supra. The determination of RLs/MRLs is described infra. FIG.2illustrates processes, generally designated200, for validating a method to determine concentrations and amounts of PFAS analytes in unconcentrated samples and consequently determining concentrations and amounts of PFAS analytes in unconcentrated samples, in accordance with an exemplary embodiment of the present invention. In step205, working standards of PFAS analytes are prepared. Working standards include standards to calibrate and verify the calibration of the LC/MS/MS system both initially and ongoing (described in more detail infra) as well as quality control standards analyzed in an analytical run such as laboratory fortified blanks, laboratory fortified matrix standards, laboratory fortified matrix duplicates, etc. Laboratory fortified blanks are an aliquot of preserved reagent water to which known quantities of the method analytes are added in the laboratory. Laboratory fortified blanks are analyzed like samples. The laboratory fortified blanks are used to determine whether a method can make accurate and precise measurements. Laboratory fortified matrix standards are aliquots of environmental samples to which known quantities of the method analytes are added in the laboratory. Laboratory fortified matrix standards are analyzed like samples. The laboratory fortified matrix standards are used to determine whether a sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix are determined in a separate aliquot and the measured values in the laboratory fortified matrix standards are corrected for background concentrations. Laboratory fortified matrix duplicates are a second aliquot of the environmental sample used to prepare the laboratory fortified matrix standards. They are fortified, processed, and analyzed identically to the laboratory fortified matrix standards. Laboratory fortified matrix duplicates are used instead of a laboratory duplicate to assess method precision when the occurrence of target analytes is low. In some embodiments, a quality control sample is a solution of method analytes obtained from a source external to the laboratory and different from the source of calibration standards. The quality control sample is used to verify the accuracy of the calibration standards. Preserved reagent water is deionized water (resistance of 18.2 megaohms or greater) wherein a preservative has been added. In one embodiment, preserved reagent includes the addition of approximately 200 mg of ammonium chloride per liter of deionized water. As used herein, “fortified” indicates that the sample, standard, blank, etc. has the one or more target PFAS analytes added to the solution. In other words, the sample, standard, blank, etc. that is “fortified” has had the target analyte added in a specified amount to the fortified sample, standard, blank, etc. Because the inventive method and system concerns PFAS detection at ppt levels, Teflon products are fastidiously excluded. If PFAS contamination is unavoidable from the LC/MS/MS system, the detection of such contamination is not integrated or included in the determination of concentration and/or amount of the PFAS in question. The use of blanks allows detection of such contamination and correction is taken based on such detection. Blank subtraction is not a valid or acceptable correction for contamination. A continuing calibration verification standard (CCV) is a calibration standard containing a specified concentration of method analytes, which is analyzed at specified periods to verify the accuracy of the existing calibration for said analytes. For some embodiments of the present invention, there is no substantially significant difference between a laboratory fortified blank and a continuing calibration verification standard. A laboratory fortified matrix standard is an aliquot of an environmental sample to which known quantities of method analytes are added in the laboratory. The laboratory fortified matrix standard is analyzed like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix should preferably be determined in a separate aliquot and the measured values in the laboratory fortified matrix standard corrected for background concentrations. In various embodiments, a laboratory fortified matrix duplicate standard is a second aliquot of an environmental sample used to prepare the laboratory fortified matrix standard. The laboratory fortified matrix duplicate standard is fortified, processed, and analyzed in the same way as the laboratory fortified matrix standard. The laboratory fortified matrix standard duplicate is used instead of a laboratory duplicate to assess method precision when the occurrence of target analytes is low. Method blanks are aliquots of preserved reagent water that are treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, etc. In various embodiments, the method blanks are used to determine if method analytes or other interferences are present in the laboratory environment, the reagents, or the apparatus. An internal standard is a pure compound added equally and in a known amount to all standard solutions and samples. They are used to measure the relative response of the method analyte. In some embodiments, the internal standard includes isotopically labeled analogues (e.g., 13C) of method analyte. In some embodiments, an analysis batch is analyzed on the LC/MS/MS system. An analysis batch is a set of up to 20 field samples (not including quality control samples such as method blanks, continuing calibration verification standards, laboratory fortified matrix standards and laboratory fortified matrix duplicate standards) that are analyzed on the same instrument during a 24-hour period that begins and ends with the analysis of the appropriate continuing calibration verification standard. In some embodiments, an additional continuing calibration verification standard is analyzed after analysis of 10 field samples. Standards for initial calibration, ongoing calibration verification, and quality control samples, etc. are prepared by adding appropriate volumes of primary dilution standard solutions to preserved reagent water or sample. Primary dilution standard (PDS) solutions are solutions of one or more method analytes prepared in the laboratory from stock standard solutions and diluted as needed to prepare calibration solutions and other required analyte solutions. Stock standard solutions are concentrated solutions containing one or more method analytes prepared in the laboratory using assayed reference materials or purchased as certified from a reputable commercial source. For example, standards or other solutions of desired concentrations may be prepared from primary dilution standard solutions or a stock standard solutions if the desired concentrations are more dilute than the primary dilution standard solutions or a stock standard solutions. Analogously, serial dilution of, e.g., calibration standards of a given concentration, provide calibration standards lower and lower in concentration than the given concentration with every dilution. Table 4 shows an example for the preparation of stock standard and primary dilution standards. TABLE 4Stock Standards (SS) and Primary Dilution Standards (PDS) Example Preparation.Stock Standard Custom Mix-(SS)Primary dilution Standards (PDS)WeightVolumeConc.5Volume ofConc.Final VolumeStandardAnalyte(g)3(mL)4(mg/mL)SS used (∝L)(∝g/mL)(mL)6IDPFOS*10.053850.001.002.500.1025.00PDS 1PFHxS*10.05481.002.50PFBAPurchased as0.050050.00PFBS*solution of indicated0.044257.00PFDAconcentration.0.050050.00PFHpA0.050050.00PFHpS**0.047652.0PFHxA0.050050.00PFNA0.050050.00PFPeS**0.046953.00PFOA0.050050.00PFOS*10.032330.001.002.500.1025.00PDS 2PFHxS*10.03291.00PFBA0.03001.00PFBS1.00PFDA1.00PFHxA1.00PFNA1.00PFHxA1.00PFOA***2Analytes purchased0.041460.4PFHpS**as solution of0.047652.00PFPeS**indicated0.046953.00concentration.13C-Analytes purchased0.0500015.000.0075100.00ISPFHxAas solution of13C-indicatedPFDAconcentration.13C-PFOA13C-PFOS**0.047815.70*Potassium salt**Sodium salt***Ammonium salt1Technical grade quantitative standards containing branch and linear isomers2Technical grade qualitative standard containing branch and linear isomers3Analyte compound purchased neat from vendor.4Weighed analyte dissolved with indicated volume of methanol.5After dilution with methanol.6After dilution with methanol. Table 5 shows an example for the preparation of working standards. TABLE 5Example Preparation of Working Standards (WS).Volume ofWS FinalWS FinalPDS/ICSVolumeSolventConcentrationWS NameUsed (∝L)(mL)Used(∝g/L)ICS 6/CCV HL250/PDS1100.00Preserved0.25ICS 5/CCVML100/PDS1100.00reagent0.10ICS 1/CCV LLa100/ICS 55.00water0.0020ICS 2/CCV LLb500/ICS 55.000.010ICS 31250/ICS 55.000.025ICS 42500/ICS 55.000.050MDL a50/ICS 55.000.0010MDL b250/ICS 55.000.0050LFB ML2500/ICS 55.000.050(for DOC)QCS50.0/PDS2100.000.050LFM/LFMD50.0/PDS1100.00Sample0.050100.0 ∝L of IS is added to 5.00 mL of each WS resulting in a concentration of 0.15 ∝g/L.ICS 1 is only used for PFOS and PFOA.The RL for PFOS and PFOA is 0.0020 ∝g/L, the RL for all other compounds is 0.010 ∝g/L In step210, acceptable settings are determined for ESI mass spectrometer conditions of individual PFAS analytes and mixtures thereof as described supra. An example of ESI mass spectrometer conditions is shown in Table 2 (supra). In step215, a chromatography gradient is developed that allows the analysis of PFAS mixtures analyzed in the present invention. An example of a chromatography gradient suitable for separation and analysis of PFAS mixtures is shown in Table 6. TABLE 6Example of an LC Gradient for PFAS Analysis of PFAS.LC Gradient Program for PFAS Analysis% 5 mM AmmoniumTime (min)Acetate% Methanol0851518515359555955.185156.68515 In step220the chromatographic gradient developed in step215is combined with the mass spectroscopy settings determined in step210. Table 7 shows an example of triple quadrapole MS/MS method conditions for PFAS analysis after combination with the Table 6 LC gradient. The chromatographic gradient and mass spectroscopy conditions must be optimized to allow an appropriate number of scans across the peak. To produce good, reproducible peak shape and recoveries, a minimum of 10 scans across the peak is required. TABLE 7Example of LC/MS/MS Method Conditions for PFAS Analysis.Triple Quadrupole MS/MS Method ConditionsRetentionPrecursorProductCollisionCellInt Std UsedScanTimeIonIonMS1FragEnergyAccelerationforAnalyteType(min)(m/z)(m/z)aMS2Voltage(ev)b(V)QuantitationPFBAPrimary3.97212.99168.9Unit38054PFHxA 13CPFBSPrimary5.529298.9998.8Unit380371PFOS 13CPFBSQualifier5.529298.9980Unit380411PFDAPrimary6.068512.99468.9Unit38093PFDA 13CPFDAQualifier6.068512.99218.9Unit380173PFDAInternal6.068514.99470.4Unit38093NA13CStandardPFHpAPrimary5.815362.99318.8Unit38051PFHxA 13CPFHpAQualifier5.815362.99168.9Unit380171PFHpSPrimary5.905448.9998.7Unit380451PFOS 13CPFHpSQualifier5.905448.9980.1Unit380451PFHxAPrimary5.686312.99268.9Unit38052PFHxA 13CPFHxAQualifier5.686312.99119Unit380212PFHxAInternal5.686314.99270.1Unit38052NA13CStandardPFHxSPrimary5.805398.9998.9Unit380412PFOS 13CPFHxSQualifier5.805398.9979.9Unit380552PFNAPrimary5.997462.99419.1Unit38054PFHxA 13CPFNAQualifier5.997462.99218.9Unit380174PFOAPrimary5.915412.99368.9Unit38044PFOA 13CPFOAQualifier5.915412.99168.8Unit380174PFOAInternal5.848414.99370Unit38054NA13CStandardPFOSPrimary5.987498.9998.8Unit380452PFOS 13CPFOSQualifier5.987498.9979.9Unit380552PFOSInternal5.987502.9979.8Unit380522NA13CStandardPFPeSPrimary5.694348.9998.8Unit380412PFHxA 13CPFPeSQualifier5.694348.9979.9Unit380412 For Table 7, the precursor ion is the deprotonated molecule ([M-H]—) of the target analyte. In MS/MS, the precursor ion is mass selected and fragmented by collisionally activated dissociation to produce distinctive product ions of smaller m/z. The product ion is one of the fragment ions produced in MS/MS by the collisionally activated dissociation of the precursor ion. In the examples shown in Tables 6 and 7 the following instrumentation is used:i) AGILENT LC/MS/MS System (Column: Analytical column ZORBAX ECLIPSE PLUS C18, 2.1×50 mm, 1.8 um);ii) AGILENT 1290 INFINITY Autosampler;iii) AGILENT 1290 Binary Pump;iv) AGILENT 1290 TCC Column Compartment; andv) AGILENT 6495 Mass Spectrometer. In the examples shown in Tables 6 and 7 the data software used is AGILENT MASS HUNTER. In step225, a practical range of detection is determined using calibration standards prepared as described supra and method validation samples. In these steps, quality control (QC) includes a demonstration of capability (DOC) requirement, a determination of the method detection limit (MDL), and confirmation of the minimum reporting limit (MRL). In various embodiments, an initial demonstration of capability (IDC) is performed prior to analyzing any field samples and any time major method modifications are made. The following steps are exemplary: i) Generate an acceptable instrument calibration and demonstrate a low system background by analyzing an acceptable method blank. The mass spectrometer is calibrated according to the manufacturer's recommendations. Prior to the analysis of samples, the instrument's performance is optimized, and an instrument calibration curve is generated. The instrument is calibrated using standards at several concentrations. They are analyzed with every analytical run. A calibration curve is generated for each analyte by plotting the responses against known concentrations. In some embodiments, linear and/or quadratic regression models are used. Both weighted and unweighted models are used. In various embodiments, a calibration curve regression model and a range of calibration levels is used for all routine sample analysis. The initial calibration is verified by analyzing various concentrations of CCV ((low level) LL, (medium level) ML, (high level) HL) prior to sample analysis and after every 10 samples (see Table 5 supra). ii) Analyze a method blank to demonstrate low background contamination. iii) Demonstrate method precision and accuracy by analyzing 4 replicates of a laboratory fortified method blank. iv) Establish the method detection limit (MDL) by analyzing seven replicates of a laboratory fortified blank fortified at less than the concentration of the reporting limits (RL) over a period of three days. The determination of the MDL is described in more detail infra. Table 8 illustrates an example of a laboratory analytical run sequence for this method, with QC parameters frequency, concentrations and acceptance criteria. TABLE 8Method Analysis Sequence with QC Frequency and Acceptance Criteria.SampleQC and Instrument CalibrationAnal #NameQCs, ICSs, CCVs Acceptance CriteriaFrequency1ICS 11. Instrument Calibration isAnalyzed with every analytical2ICS 2updated and recalculated againstrun.3ICS 3the newly generated calibration4ICS 4curve.5ICS 52. Each analyte in each6ICS 6calibration point, except for theconcentrations ≤RL, mustcalculate to be ±30% of the truevalue.3. Each analyte incalibration points atconcentrations ≤RL mustcalculate to be ±50% of the truevalue.4. ICS 1 is only used forPFOS and PFOA.7QCS1. Recovery for targetAnalyzed after instrumentanalytes must be ±30%calibrationof the true value8MB1. Must be free fromAnalyzed with each batch of up tocontamination that could prevent20 samples processed as a groupthe determination of any targetwithin a work shift.analyte.Concentration of target analytesmust be ≤1/3 RL.9CCV LLa1. Recovery for PFOA andAnalyzed at the beginning of anPFOS must be ± 50% of the trueanalytical batch of 20 samplesvalueprocessed as a group within a10CCV LLb1. Recovery for targetwork shift.analytes must be ±50% of thetrue value.11Sample 1*12LFM1. LFM/D: Recovery forAnalyzed with each batch13LFMDtarget analytes should be ±40%of up to 20 samples14Sample 2*of the true value for all analytesprocessed as a group15Sample 3*except for PFOA and PFOSwithin a work shift.16Sample 4*should be ± 30% of the true17Sample 5*value f; precision as RPD should18Sample 6*be ≤30%.19Sample 7*20Sample 8*21Sample 9*22Sample 10*23CCV ML1. Recovery for targetAnalyzed with each analyticalanalytes must be ±30%batch of up to 20 samples after theof the true value.first 10 samples.24Sample 11*25Sample 1*26Sample 13*27Sample 14*28Sample 15*29Sample 16*30Sample 17*31Sample 18*32Sample 19*33Sample 20*34CCV HL1. Recovery for targetAnalyzed with each analyticalanalytes must be ±30%batch of up to 20 samples after theof the true value.second 10 samples.Internal Standard Response Relative Percent Deviation (ISRPD) must not exceed ±50% for each analyte except PFDA.ISRPD for PFDA must not exceed ±60%*If sample contains a method analyte(s) at or above the MRL, analyze an associated FB.If a method analyte(s) found in the field sample is present in the associated FB at a concentration greater than 1/3MRL, then the sample results are invalid. An example of a demonstration of capability (DOC) study including the demonstration of laboratory precision and accuracy are presented in Table 9 using 75 ∝L injection volumes: TABLE 9Example of a DOC Study for PFAS.Data File17181920AccuracyMethod'sPrecisionMethod'sAmountAmount Recoveredas MeanAccuracyStandardasPrecisionAnalyteAddedDEMODEMODEMODEMOMeanRecoveryLimitsDeviationRSD *LimitsName[μg/L][μg/L][μg/L][μg/L][μg/L][μg/L][%][%][μg/L][%][%]PFBA0.0500.05240.04820.04780.04770.04998.070.0-130.00.00234.6<20.0PFBS0.0500.04130.04520.04300.04340.04386.570.0-130.00.00163.6<20.0PFHxA0.0500.04830.04950.04300.04540.04793.170.0-130.00.00296.3<20.0PFPeS0.0500.05220.04720.04760.05060.04998.870.0-130.00.00244.9<20.0PFHpA0.0500.05420.05180.04680.05040.051101.670.0-130.00.00316.1<20.0PFHxS0.0500.04840.05040.05080.04820.04998.970.0-130.00.00132.7<20.0PFHpS0.0500.05220.06100.05840.05650.057114.070.0-130.00.00376.5<20.0PFOA0.0500.04870.05420.04890.05020.050101.070.0-130.00.00265.1<20.0PFOS0.0500.04910.05170.05460.05380.052104.670.0-130.00.00254.7<20.0PFNA0.0500.06160.05160.05170.05470.055109.870.0-130.00.00478.5<20.0PFDA0.0500.04900.05390.04880.05350.051102.670.0-130.00.00285.4<20.0 Determination of MDL MDL (Method Detection Limits) are the minimum concentration of a substance that can be reported with 99% confidence that the measured concentration is distinguishable from Method Blank results. An example of a procedure for determining MDL is as follows: First, an estimate is made of an initial MDL using one or more of: i) a mean determined concentration plus three times the standard deviation of a set of MB; ii) a concentration value that corresponds to an instrument signal/noise in the range of 3 to 5; iii) a concentration equivalent of three times the standard deviation of replicate instrumental measurements of spiked blanks; iv) a region of the calibration where there is a significant change in sensitivity, such as a break in the slope of the calibration; v) an instrumental limitation; and vi) a previously determined MDL. Second, an initial MDL determination is made by selecting a spiking level, typically 2-10 times the estimated method detection limit from above, but less than the value of the laboratory established RL and less than or equal to a regulatory authority reported required detection limit (RDL), if one exists. Once the spiking level is determined, a minimum of seven laboratory standards in reagent water (containing all method preservatives, if applicable) are made at the selected spiking level concentration and they are processed through all steps of the method. Generally, the standards used for the MDL are prepared in at least three batches on three separate calendar dates and analyzed on three separate calendar dates. Preparation and analysis may be performed on the same day. In general, statistical outlier removal procedures are not used to remove data for the initial MDL determination since the total number of observations is small and the purpose of the MDL procedure is to capture routine method variability. However, documented instances of gross failures (e.g., instrument malfunctions, mislabeled samples, cracked vials) may be excluded from the calculations, provided that at least seven spiked samples and seven method blanks are available. After the method is run, the spiking level is evaluated. If any result for any individual analyte from the spiked samples does not meet a qualitative method identification criterion or does not provide a numerical result greater than zero, then the method is repeated with spiked samples at a higher concentration. The method MDL is the greater of either an MDL based on spiked samples (MDLs) or an MDL based on method blanks (MDLb). The MDL is calculated as shown below: First, a mean of the measured concentration values X is calculated as shown below: X=∑XinWhere:i=from 1 to n;n=the number of data points; andXi=the measured concentration value of an individual laboratory standard. Second, a mean percent recovery (R) is calculated as shown below: R=XT×100%Where:X=mean of the measured concentration values; andT=true concentration used. Third, a standard deviation (Ss) is calculated as shown below: Ss=∑(Xi-X)2n-1Where:i=from 1 to nn=the number of data points;Xi=the measured concentration value of an individual laboratory standard; andX=mean of the measured concentration values. The MDLs is then calculated as shown below: MDLs=t(n−1,1−a=0.99)*SsWhere:t(n−1, 1−α=0.99)=the Student's t-value appropriate for a single-tailed 99thpercentile t statistic and a standard deviation estimate withn-1 degrees of freedom (see Table 10 below); andSs=standard deviation of the replicate spiked sample analyses. For the MLDb, one of the following criterion is applied:i) If none of the method blanks give numerical results for an individual analyte, the MDLb does not apply and the MDLs is used. A numerical result includes both positive and negative results, including results below a current MDL, but not results of “ND” (not detected) commonly observed when a peak is not present in chromatographic analysis;ii) If some (but not all) of the method blanks for an individual analyte give numerical results, set the MDLb equal to the highest method blank result; oriii) If all of the method blanks for an individual analyte give numerical results, then the MDLb is calculated as shown below: First, a mean of the measured concentration values X is calculated as shown below: X=∑XinWhere:i=from 1 to n;n=the number of data points; andXi=the measured concentration value of an individual MB. Second, a standard deviation (Sb) is calculated as shown below: Sb=∑(Xi-X)2n-1Where:i=from 1 to nn=the number of data points;Xi=the measured concentration value of an individualMB; andX=mean of the measured MB concentration values. Third, the MDLb is then calculated as shown below: MDLb=X+t(n−1,1—α=0.99)*SbWhere:X=mean of the MB results (zero is used in place of the mean if the mean is negative);t(n−1, 1−α=0.99)=the Student's t-value appropriate for a single-tailed 99thpercentile t statistic and a standard deviation estimate withn-1 degrees of freedom (see Table 8 below); andSb=standard deviation of the MB analyses. TABLE 10Student's Single-Tailed 99thPercentile t Statistic Values.DegreesDegreesDegreesReplicateofStudent'sReplicatesofStudent'sReplicatesofStudent'sNumberFreedomt- ValueNumberFreedomt-ValueNumberFreedomt-Valuenn − 1t(n−1, 0.99)nn − 1t(n−1, 0.99)nn − 1t(n−1, 0.99)763.14341402.42375742.378872.99842412.42176752.377982.89643422.41877762.3761092.82144432.41678772.37611102.76445442.41479782.37512112.71846452.41280792.37413122.68147462.41081802.37414132.65048472.40882812.37315142.62449482.40783822.37316152.60250492.40584832.37217162.58351502.40385842.37218172.56752512.40286852.37119182.55253522.40087862.37020192.53954532.39988872.37021202.52855542.39789882.36922212.51856552.39690892.36923222.50857562.39591902.36824232.50058572.39492912.36825242.49259582.39293922.36826252.48560592.39194932.36727262.47961602.39095942.36728272.47362612.38996952.36629282.46763622.38897962.36630292.46264632.38798972.36531302.45765642.38699982.36532312.45366652.385100992.36533322.44967662.3841011002.36434332.44568672.383∞∞2.32635342.44169682.38236352.43870692.38237362.43471702.38138372.43172712.38039382.42973722.37940392.42674732.379 In general, an MDL verification is performed each time an MDL study is performed and on an annual basis. In one scenario, if an MDL value is greater than or equal to the concentration used for the MDL study, the concentration used for MDL study will be the MDL verification concentration. In another scenario, if an MDL value is less than the concentration used for MDL study, a laboratory standard is prepared and analyzed in reagent water (with preservatives if applicable), wherein the, laboratory standard prepared has an analyte concentration: i) greater than or equal to the MDL value; ii) no more than 2-3 times the MDL value; iii) less than the concentration used for the MDL study and the RL; and iv) less than or equal to the RDL if applicable. Determination of RL/MRL The RL (or MRL) is established for each method/analyte using its calculated MDL value. The RL/MRL is set at a value of 1 to 5 times the MDL value and then this set RL/MRL value is confirmed by processing and analyzing seven replicates of laboratory fortified blanks (LFB) that are fortified with analyte at or below the set RL/MRL concentration. The LFB also include all method-specified dechlorination agents (e.g., ammonium chloride) and preservatives, which are included in typical sample preparation. First, the results of the analytical run are used to determine the mean concentrations of the LFB and their standard deviations. Second, the Half Range for the prediction interval of results (HRPIR) are calculated using the following equation: HRPIR=3.963X SWhere:S=standard deviation of the seven LFB concentration measurements; and3.963 is the factor specific to seven replicates. An upper and lower limit of the Prediction Interval of Result (PIR=Mean±HRPIR) provides confirmation of the set RL/MRL if it meets two criteria: The Upper PIR Limit (PIRUL) must be≤150% recovery and The Lower PIR Limit (PIRLL) must be ≥50% recovery for the RL/MRL to be confirmed. The calculations for PIR LL and PIRLLare as follows: PIRUL=Mean+HPPIRFortifiedConcentration×100≤150%PIRLL=Mean-HPPIRFortifiedConcentration×100≥50% In step230, samples from various sources of water are collected and analyzed using the above described method. For example, samples are collected in 250 mL polypropylene bottles. In some cases, the 250 mL bottles are pre-charged with approximately 50 mg of ammonium chloride. In an example for the analysis of PFAS in tap water, the water tap is allowed to run freely until the water temperature has stabilized, and the flow is reduced to permit bottle filling without splashing. The bottle is filled to the neck, taking care not to flush out the ammonium chloride, if present. The bottle is then capped and agitated to dissolve the ammonium chloride, if present, and placed in a cooler with frozen gel packs. In some cases, the samples received at the laboratory on the collection day are transported in coolers with frozen gel packs and their temperature is maintained between 1° C. and 10° C. for the first 48 hours. In other cases, the samples that will not be received at the laboratory on the day of collection are maintained at a temperature range between approximately 1° C. to 6° C. until analysis is initiated at a receiving laboratory. Typically, a maximum holding time from collection to analysis is 14 days. Samples are typically prepared for analysis by removing from refrigeration and allowing the samples to equilibrate to ambient temperature. In most cases, the samples are checked for dechlorination efficiency by testing with free chlorine strips to ensure that the free chlorine level is <0.1 mg/L. Samples, standards, and QCs are next loaded into 2 mL autosampler vials. In some cases, the samples and QCs are spiked with 10 μL of an internal standard as described supra. The samples are then analyzed by injection alongside the standards and QCs into an LC/MS/MS with ESI using the conditions described supra. After the initial calibration is confirmed valid with the QCS and CCV, analyzing field and QC samples is typically begun at the frequency outlined in Table 8 (supra). The instrument's MASS HUNTER software is used in the calibration procedure. MASS HUNTER analytical software uses peak areas and the internal standard technique to calculate concentrations of the method analytes. Data may be fit with either a linear or quadratic regression with weighting if necessary. The calibration curve for PFOS and PFOA should be forced through the origin. The percent recovery calculation for CCV, LFB, QCS and LFM is performed using the following formula: P=A-BT×100%Where:P=percent recovery;A=measured concentration of analyte after spiking;B=measured background concentration of analyte; andT-true concentration of the spike. Relative percent difference for the fortified matrix duplicate is calculated using the following formula: RPD=❘"\[LeftBracketingBar]"LMF-LFMD❘"\[RightBracketingBar]"LFM+LFMD2×100%Where:RPD=relative percent difference;LFM=measured concentration of analyte in the fortified sample; andLFMD=measured concentration of analyte in the fortified sample duplicate. Internal Standard Response Relative Percent Deviation (ISRPD) is calculated as follows: ISRPD=ISResponseintheSample-AverageISResponseintheInitialCalibrationAverageISResponseintheInitialCalibration×100 It should be appreciated that all combinations of the foregoing embodiments and additional embodiments discussed in greater detail herein (provided such embodiments are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein. Although the invention has been described by reference to specific examples, it should be understood that numerous changes may be made within the spirit and scope of the inventive concepts described. Accordingly, it is intended that the disclosure not be limited to the described examples, but that it have the full scope defined by the language of the following claims. | 152,718 |
11860141 | DETAILED DESCRIPTION OF THE INVENTION The cerebral creatine deficiency syndromes (CCDS), inborn errors of creatine metabolism, include the two creatine biosynthesis disorders (guanidinoacetate methyltransferase [GAMT] deficiency and L-arginine:glycine amidinotransferase [AGAT or GATM] deficiency), and the X-linked creatine transporter [SLC6A8] deficiency. Intellectual disability and seizures are common to all three CCDS. The majority of individuals with GAMT deficiency have a behavior disorder that can include autistic behaviors and self-mutilation; a significant proportion have pyramidal/extrapyramidal findings. Onset is between the ages of three months and three years. The phenotype of SLC6A8 deficiency in affected males ranges from mild intellectual disability and speech delay to severe intellectual disability, seizures, and behavior disorder, with age at diagnosis ranging from two to 66 years. Females heterozygous for SLC6A8 deficiency may have learning and behavior problems. Biochemical diagnosis of CCDS relies on the measurement of guanidinoacetate (GAA), creatine, and creatinine in urine and plasma. In certain embodiments, the methods provided herein are for detecting or determining the amount of guanidinoacetate (GAA), creatine, and creatinine comprising (a) purifying GAA, creatine, and creatinine in the sample; (b) ionizing GAA, creatine, and creatinine in the sample; and (c) detecting or determining the amount of the GAA, creatine, and creatinine ion(s) by mass spectrometry; wherein the amount of the GAA, creatine, and creatinine ion(s) is related to the amount of GAA, creatine, and creatinine in the sample. In certain embodiments, the methods provided herein are for detecting or determining the amount of guanidinoacetate (GAA) comprising (a) purifying GAA in the sample; (b) ionizing GAA in the sample; and (c) detecting or determining the amount of the GAA ion(s) by mass spectrometry; wherein the amount of the GAA ion(s) is related to the amount of GAA in the sample. In certain embodiments, the methods provided herein are for detecting or determining the amount of creatine comprising (a) purifying GAA, creatine, and creatinine in the sample; (b) ionizing creatine in the sample; and (c) detecting or determining the amount of the creatine ion(s) by mass spectrometry; wherein the amount of the creatine ion(s) is related to the amount of creatine in the sample. In certain embodiments, the methods provided herein are for detecting or determining the amount of creatinine comprising (a) purifying creatinine in the sample; (b) ionizing creatinine in the sample; and (c) detecting or determining the amount of the creatinine ion(s) by mass spectrometry; wherein the amount of the creatinine ion(s) is related to the amount of creatinine in the sample. In some embodiments, guanidinoacetate (GAA), creatine, and creatinine are underivatized prior to mass spectrometry. In some embodiments, the sample is urine or serum or plasma. In a preferred embodiment, the sample is urine. In some embodiments, the sample is whole blood. In some embodiments, the sample is saliva. In some embodiments, purifying provided herein comprises liquid chromatography. In some embodiments, the liquid chromatography comprises high performance liquid chromatography (HPLC). In some embodiments, purifying provided herein comprises solid phase extraction (SPE). In some embodiments, the ionization comprises electrospray ionization (ESI). In some embodiments, the ionization comprises ionizing in positive mode. In some embodiments, the ionization comprises ionizing in negative mode. In some embodiments, methods provided herein further comprise adding an internal standard. In some embodiments, the internal standard is isotopically labeled. In certain embodiments, the limit of quantitation of the methods is less than or equal to 1 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.9 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.8 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.7 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.6 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.5 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.4 mg/L. In some embodiments, the limit of quantitation of the methods is less than or equal to 0.3 mg/L. In certain embodiments, the limit of detection of the methods is less than or equal to 1 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.9 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.8 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.7 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.6 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.5 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.4 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.3 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.2 mg/L. In some embodiments, the limit of detection of the methods is less than or equal to 0.1 mg/L. In some embodiments, the methods may include adding an agent to the sample in an amount sufficient to deproteinate the sample. Suitable test samples include any test sample that may contain the analyte of interest. In some preferred embodiments, a sample is a biological sample; that is, a sample obtained from any biological source, such as an animal, a cell culture, an organ culture, etc. In certain preferred embodiments samples are obtained from a mammalian animal, such as a dog, cat, horse, etc. Particularly preferred mammalian animals are primates, most preferably male or female humans. Particularly preferred samples include blood, plasma, serum, hair, muscle, urine, saliva, tear, cerebrospinal fluid, or other tissue sample. Such samples may be obtained, for example, from a patient; that is, a living person, male or female, presenting oneself in a clinical setting for diagnosis, prognosis, or treatment of a disease or condition. The test sample is preferably obtained from a patient, for example, blood serum. Sample Preparation for Mass Spectrometry Methods that may be used to enrich in analyte relative to other components in the sample (e.g. protein) include for example, filtration, centrifugation, thin layer chromatography (TLC), electrophoresis including capillary electrophoresis, affinity separations including immunoaffinity separations, extraction methods including ethyl acetate extraction and methanol extraction, and the use of chaotropic agents or any combination of the above or the like. Protein precipitation is one preferred method of preparing a test sample. Such protein purification methods are well known in the art, for example, Polson et al.,Journal of Chromatography B785:263-275 (2003), describes protein precipitation techniques suitable for use in the methods. Protein precipitation may be used to remove most of the protein from the sample leaving analyte in the supernatant. The samples may be centrifuged to separate the liquid supernatant from the precipitated proteins. The resultant supernatant may then be applied to liquid chromatography and subsequent mass spectrometry analysis. In certain embodiments, the use of protein precipitation such as for example, acetonitrile protein precipitation, obviates the need for high turbulence liquid chromatography (HTLC) or other on-line extraction prior to HPLC and mass spectrometry. Accordingly in such embodiments, the method involves (1) performing a protein precipitation of the sample of interest; and (2) loading the supernatant directly onto the HPLC-mass spectrometer without using on-line extraction or high turbulence liquid chromatography (HTLC). In some preferred embodiments, HPLC, alone or in combination with one or more purification methods, may be used to purify analyte prior to mass spectrometry. In such embodiments samples may be extracted using an HPLC extraction cartridge which captures the analyte, then eluted and chromatographed on a second HPLC column or onto an analytical HPLC column prior to ionization. Because the steps involved in these chromatography procedures can be linked in an automated fashion, the requirement for operator involvement during the purification of the analyte can be minimized. This feature can result in savings of time and costs, and eliminate the opportunity for operator error. It is believed that turbulent flow, such as that provided by HTLC columns and methods, may enhance the rate of mass transfer, improving separation characteristics. HTLC columns separate components by means of high chromatographic flow rates through a packed column containing rigid particles. By employing high flow rates (e.g., 3-5 mL/min), turbulent flow occurs in the column that causes nearly complete interaction between the stationary phase and the analyte(s) of interest. An advantage of using HTLC columns is that the macromolecular build-up associated with biological fluid matrices is avoided since the high molecular weight species are not retained under the turbulent flow conditions. HTLC methods that combine multiple separations in one procedure lessen the need for lengthy sample preparation and operate at a significantly greater speed. Such methods also achieve a separation performance superior to laminar flow (HPLC) chromatography. HTLC allows for direct injection of biological samples (plasma, urine, etc.). Direct injection is difficult to achieve in traditional forms of chromatography because denatured proteins and other biological debris quickly block the separation columns. HTLC also allows for very low sample volume of less than 1 mL, preferably less than 0.5 mL, preferably less than 0.2 mL, preferably 0.1 mL. Examples of HTLC applied to sample preparation prior to analysis by mass spectrometry have been described elsewhere. See, e.g., Zimmer et al.,J. Chromatogr. A854:23-35 (1999); see also, U.S. Pat. Nos. 5,968,367; 5,919,368; 5,795,469; and 5,772,874. In certain embodiments of the method, samples are subjected to protein precipitation as described above prior to loading on the HTLC column; in alternative preferred embodiments, the samples may be loaded directly onto the HTLC without being subjected to protein precipitation. The HTLC extraction column is preferably a large particle column. In various embodiments, one of more steps of the methods may be performed in an on-line, automated fashion. For example, in one embodiment, steps (i)-(v) are performed in an on-line, automated fashion. In another, the steps of ionization and detection are performed on-line following steps (i)-(v). Liquid chromatography (LC) including high-performance liquid chromatography (HPLC) relies on relatively slow, laminar flow technology. Traditional HPLC analysis relies on column packings in which laminar flow of the sample through the column is the basis for separation of the analyte of interest from the sample. The skilled artisan will understand that separation in such columns is a diffusional process. HPLC has been successfully applied to the separation of compounds in biological samples but a significant amount of sample preparation is required prior to the separation and subsequent analysis with a mass spectrometer (MS), making this technique labor intensive. In addition, most HPLC systems do not utilize the mass spectrometer to its fullest potential, allowing only one HPLC system to be connected to a single MS instrument, resulting in lengthy time requirements for performing a large number of assays. Various methods have been described for using HPLC for sample clean-up prior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring22:608-12 (2000); and Salm et al.,Clin. Therapeutics22 Supl. B:B71-B85 (2000). One of skill in the art may select HPLC instruments and columns that are suitable for use with analyte. The chromatographic column typically includes a medium (i.e., a packing material) to facilitate separation of chemical moieties (i.e., fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups, preferably C-18 bonded groups. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an effluent that includes the fractionated sample. In one embodiment, the sample (or pre-purified sample) is applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes may be selected for eluting the analyte(s) of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (i.e. mixed) mode. During chromatography, the separation of materials is effected by variables such as choice of eluent (also known as a “mobile phase”), elution mode, gradient conditions, temperature, etc. In certain embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. Such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample. In one preferred embodiment, the HTLC may be followed by HPLC on a hydrophobic column chromatographic system. In certain preferred embodiments, a TurboFlow Cyclone P® polymer-based column from Cohesive Technologies (60 m particle size, 50×1.0 mm column dimensions, 100 Å pore size) is used. In related preferred embodiments, a Synergi Polar-RP® ether-linked phenyl, analytical column from Phenomenex Inc (4 μm particle size, 150×2.0 mm column dimensions, 80 Å pore size) with hydrophilic endcapping is used. In certain preferred embodiments, HTLC and HPLC are performed using HPLC Grade Ultra Pure Water and 100% methanol as the mobile phases. By careful selection of valves and connector plumbing, two or more chromatography columns may be connected as needed such that material is passed from one to the next without the need for any manual steps. In preferred embodiments, the selection of valves and plumbing is controlled by a computer pre-programmed to perform the necessary steps. Most preferably, the chromatography system is also connected in such an on-line fashion to the detector system, e.g., an MS system. Thus, an operator may place a tray of samples in an autosampler, and the remaining operations are performed under computer control, resulting in purification and analysis of all samples selected. In certain preferred embodiments, analyte or fragments thereof in a sample may be purified prior to ionization. In particularly preferred embodiments the chromatography is not gas chromatography. Detection and Quantitation by Mass Spectrometry In various embodiments, analyte or fragments thereof may be ionized by any method known to the skilled artisan. Mass spectrometry is performed using a mass spectrometer, which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electron ionization, chemical ionization, electrospray ionization (ESI), photon ionization, atmospheric pressure chemical ionization (APCI), photoionization, atmospheric pressure photoionization (APPI), fast atom bombardment (FAB), liquid secondary ionization (LSI), matrix assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, surface enhanced laser desorption ionization (SELDI), inductively coupled plasma (ICP) and particle beam ionization. The skilled artisan will understand that the choice of ionization method may be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc. In preferred embodiments, analyte or a fragment thereof is ionized by heated electrospray ionization (HESI) in positive or negative mode. In alternative embodiments, analyte or a fragment thereof is ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in positive or negative mode. After the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio. Suitable analyzers for determining mass-to-charge ratios include quadrupole analyzers, ion traps analyzers, and time-of-flight analyzers. The ions may be detected using several detection modes. For example, selected ions may be detected i.e., using a selective ion monitoring mode (SIM), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM). Preferably, the mass-to-charge ratio is determined using a quadrupole analyzer. For example, in a “quadrupole” or “quadrupole ion trap” instrument, ions in an oscillating radio frequency field experience a force proportional to the DC potential applied between electrodes, the amplitude of the RF signal, and the mass/charge ratio. The voltage and amplitude may be selected so that only ions having a particular mass/charge ratio travel the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments may act as both a “mass filter” and as a “mass detector” for the ions injected into the instrument. One may enhance the resolution of the MS technique by employing “tandem mass spectrometry,” or “MS/MS”. In this technique, a precursor ion (also called a parent ion) generated from a molecule of interest can be filtered in an MS instrument, and the precursor ion is subsequently fragmented to yield one or more fragment ions (also called daughter ions or product ions) that are then analyzed in a second MS procedure. By careful selection of precursor ions, only ions produced by certain analytes are passed to the fragmentation chamber, where collisions with atoms of an inert gas produce the fragment ions. Because both the precursor and fragment ions are produced in a reproducible fashion under a given set of ionization/fragmentation conditions, the MS/MS technique may provide an extremely powerful analytical tool. For example, the combination of filtration/fragmentation may be used to eliminate interfering substances, and may be particularly useful in complex samples, such as biological samples. The mass spectrometer typically provides the user with an ion scan; that is, the relative abundance of each ion with a particular mass/charge over a given range (e.g., 100 to 1000 amu). The results of an analyte assay, that is, a mass spectrum, may be related to the amount of the analyte in the original sample by numerous methods known in the art. For example, given that sampling and analysis parameters are carefully controlled, the relative abundance of a given ion may be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, molecular standards may be run with the samples, and a standard curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion may be converted into an absolute amount of the original molecule. In certain preferred embodiments, an internal standard is used to generate a standard curve for calculating the quantity of analyte. Methods of generating and using such standard curves are well known in the art and one of ordinary skill is capable of selecting an appropriate internal standard. For example, an isotope of analyte may be used as an internal standard. Numerous other methods for relating the amount of an ion to the amount of the original molecule will be well known to those of ordinary skill in the art. One or more steps of the methods may be performed using automated machines. In certain embodiments, one or more purification steps are performed on-line, and more preferably all of the purification and mass spectrometry steps may be performed in an on-line fashion. In certain embodiments, such as MS/MS, where precursor ions are isolated for further fragmentation, collision activation dissociation is often used to generate the fragment ions for further detection. In CAD, precursor ions gain energy through collisions with an inert gas, and subsequently fragment by a process referred to as “unimolecular decomposition”. Sufficient energy must be deposited in the precursor ion so that certain bonds within the ion can be broken due to increased vibrational energy. In particularly preferred embodiments, analyte is detected and/or quantified using MS/MS as follows. The samples are subjected to liquid chromatography, preferably HPLC, the flow of liquid solvent from the chromatographic column enters the heated nebulizer interface of an MS/MS analyzer and the solvent/analyte mixture is converted to vapor in the heated tubing of the interface. The analyte is ionized by the selected ionizer. The ions, e.g. precursor ions, pass through the orifice of the instrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e., “precursor” and “fragment” ions) based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ions are fragmented. The first quadrupole of the mass spectrometer (Q1) selects for molecules with the mass to charge ratios of analyte. Precursor ions with the correct mass/charge ratios of analyte are allowed to pass into the collision chamber (Q2), while unwanted ions with any other mass/charge ratio collide with the sides of the quadrupole and are eliminated. Precursor ions entering Q2 collide with neutral argon gas molecules and fragment. This process is called collision activated dissociation (CAD). The fragment ions generated are passed into quadrupole 3 (Q3), where the fragment ions of analyte are selected while other ions are eliminated. The methods may involve MS/MS performed in either positive or negative ion mode. Using standard methods well known in the art, one of ordinary skill is capable of identifying one or more fragment ions of a particular precursor ion of analyte that may be used for selection in quadrupole 3 (Q3). If the precursor ion of analyte includes an alcohol or amine group, fragment ions are commonly formed that represent dehydration or deamination of the precursor ion, respectfully. In the case of precursor ions that include an alcohol group, such fragment ions formed by dehydration are caused by a loss of one or more water molecules from the precursor ion (i.e., where the difference in mass to charge ratio between the precursor ion and fragment ion is about 18 for the loss of one water molecule, or about 36 for the loss of two water molecules, etc.). In the case of precursor ions that include an amine group, such fragment ions formed by deamination are caused by a loss of one or more ammonia molecules (i.e. where the difference in mass to charge ratio between the precursor ion and fragment ion is about 17 for the loss of one ammonia molecule, or about 34 for the loss of two ammonia molecules, etc.). Likewise, precursor ions that include one or more alcohol and amine groups commonly form fragment ions that represent the loss of one or more water molecules and/or one or more ammonia molecules (i.e., where the difference in mass to charge ratio between the precursor ion and fragment ion is about 35 for the loss of one water molecule and the loss of one ammonia molecule). Generally, the fragment ions that represent dehydrations or deaminations of the precursor ion are not specific fragment ions for a particular analyte. Accordingly, in preferred embodiments of the invention, MS/MS is performed such that at least one fragment ion of analyte is detected that does not represent only a loss of one or more water molecules and/or a loss of one or more ammonia molecules from the precursor ion. As ions collide with the detector they produce a pulse of electrons that are converted to a digital signal. The acquired data is relayed to a computer, which plots counts of the ions collected versus time. The resulting mass chromatograms are similar to chromatograms generated in traditional HPLC methods. The areas under the peaks corresponding to particular ions, or the amplitude of such peaks, are measured and the area or amplitude is correlated to the amount of the analyte of interest. In certain embodiments, the area under the curves, or amplitude of the peaks, for fragment ion(s) and/or precursor ions are measured to determine the amount of analyte. As described above, the relative abundance of a given ion may be converted into an absolute amount of the original analyte, using calibration standard curves based on peaks of one or more ions of an internal molecular standard. The following examples serve to illustrate the invention. These examples are in no way intended to limit the scope of the methods. EXAMPLES Example 1: Detection and Quantitation of GAA, Creatine, and Creatinine by Mass Spectrometry Samples were prepared by diluting urine 1:50 fold with ultrapure water. A minimum sample volume of 100 μL was used. Following dilution, samples were spiked with an internal standard mixture (deuterium-labeled creatinine, C13-labeled guanidinoacetate) and mixed. The diluted sample mix was injected onto an Agilent 1200 Series HPLC system using a reverse-phase column (Reversed Phase BDS 250×4.6 mm). HPLC mobile phases: 0.1% formic acid/acetonitrile. Analysis was performed by positive electrospray ionization using an Agilent 6410 triple quadrupole mass spectrometer. The run time was 10 minutes. The calibration curves showed consistency in reproducibility and linearity. The method provides linear results over a range of 0.4-2500 mg/L for guanidinoacetate and creatine and 0.8-5000 mg/L for creatinine. The lower limits of quantitation were 0.3 mg/L for guanidinoacetate and creatinine and 0.4 mg/L for creatine. Inter-assay coefficients of variation were 8.1 to 4.7% at 25-500 mg/L for guanidinoacetate, 9.9 to 6.1% at 25-500 mg/L for creatine, and 1.8 to 4.1% at 120-2300 mg/L for creatinine. TABLE 1Limit of quantitation and spiked recovery studiesAnalyteLOQ (mg/L)Mean Recovery (%)*Guanidinoacetate0.397Creatine0.4101Creatinine0.3102 TABLE 2Guanidinoacetate Intra- and Inter-assay Precision and AccuracyLowMediumHighIntra-assayMean (mg/L)26.3105.0511.0CV (%)1.20.90.6Accuracy (%)105.0105.0102.2N101010Inter-assayMean (mg/L)25.7102.8497.5CV (%)8.15.64.7Accuracy (%)102.8102.899.5N313131 Theoretical concentration of spiked material: 25, 100, and 500 mg/L for low, medium and high, respectively. TABLE 3Creatine Intra- and Inter-assay Precision and AccuracyLowMediumHighIntra-assayMean (mg/L)27.5107.0509.8CV (%)1.31.30.9Accuracy (%)110.0107.0102N101010Inter-assayMean (mg/L)25.6105.0525.0CV (%)9.94.46.1Accuracy (%)102.4105.0105.0N313131 Theoretical concentration of spiked material: 25, 100, and 500 mg/L for low, medium and high, respectively. TABLE 4Creatinine Intra- and Inter-assay Precision and AccuracyLowMediumHighIntra-assayMean (mg/L)120.5741.22318.1CV (%)2.12.21.2Accuracy (%)96.698.892.7N101010Inter-assayMean (mg/L)119.9731.42295.2CV (%)1.82.04.1Accuracy (%)95.997.591.8N313131 Theoretical concentration of spiked material: 125, 750, and 2500 mg/L for low, medium and high, respectively. Precision/Sensitivity/Range Intra Assay PrecisionGAA:Low Control: 1.2%Mid Control: 0.9%High Control: 0.6%Creatine:Low Control: 1.3%Mid Control: 1.3%High Control: 0.9%CreatinineLow Control: 2.1%Mid Control: 2.2%High Control: 1.2%Inter Assay PrecisionGAA:Low Control: 8.1%Mid Control: 5.6%High Control: 4.7%Creatine:Low Control: 9.9%Mid Control: 4.4%High Control: 5.6%CreatinineLow Control: 1.8%Mid Control: 2.0%High Control: 4.1%Recovery StudyGAA: SD < Tea/4 for all samplesCreatine: SD < Tea/4 for all samplesCreatinine SD < Tea/4 for all samplesAnalytical SensitivityGAA: 2.84 nmol/mL(Limit of Detection)Creatine: 0.22 nmol/mLCreatinine: 1.65 nmol/mLAnalytical SensitivityGAA: 7.50 nmol/mL(Limit of Quantitation)Creatine: 1.15 nmol/mLCreatinine: 2.34 nmol/mLLinearityGAA: 3 - 21348 nmol/mLCreatine: 3 - 19065 nmol/mLCreatinine: 7 - 44202 nmol/mLAnalytical MeasurementGAA: 3 - 21348 nmol/mLRange (AMR)Creatine: 3 - 19065 nmol/mLCreatinine: 7 - 44202 nmol/mLClinical ReportableGAA: 3 - 21348 nmol/mLRange (CRR)Creatine: 3 - 19065 nmol/mLCreatinine: 7 - 44202 nmol/mL Measurements of these three analytes in urine allow for the biochemical diagnosis of CCDS. The ability to measure all three analytes directly in the urine, with a very simple method of sample preparation, is an improvement over previous methods involving derivatization and indirect calculations. The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. The methods illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the invention embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the methods. This includes the generic description of the methods with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the methods are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. | 32,347 |
11860142 | DESCRIPTION OF EMBODIMENTS First Embodiment Apparatus Configuration FIG.1is a schematic diagram illustrating a configuration of a liquid chromatograph mass spectrometer according to a first embodiment. The liquid chromatograph mass spectrometer includes solution tanks1ato1c, a mass spectrometry unit2, liquid feed pumps3ato3c, a separation column4, a selector valve5(first valve), a first flow path11, and a second flow path12, pressure gauges13ato13c, a sampler14, and a controller100. The solution tanks1aand1bcontain a solution to be a mobile phase. As the solution to be the mobile phase, a solution generally used in liquid chromatography can be used depending on a sample. For example, water, an aqueous solution of salts, an organic solvent such as methanol, acetonitrile or hexane may be used alone, or can be mixed and used. The solution tanks1aand1bare connected to the first flow path11via the liquid feed pumps3aand3b(first liquid feed pump), respectively. The solution tank1ccontains a solution to be fed to the second flow path12, and is connected to the second flow path12via the liquid feed pump3c(second liquid feed pump). The solution contained in the solution tank1cmay be the same as or different from the solution contained in the solution tanks1aand1b. The liquid feed pumps3ato3cpressurize and feed the solutions in the solution tanks1ato1c, respectively. The pressure gauges13aand13bare connected to the liquid feed pumps3aand3bthat feed the solutions to the first flow path11, respectively, and the pressure gauge13cis connected to the liquid feed pumps3cthat feed the solution to the second flow path12. The pressure gauges13ato13cmeasure the pressure of the solution flowing through the flow path, respectively. It is preferable to use the pressure gauges13ato13ccapable of measuring a pressure in a range that can be pressurized by the liquid feed pumps3ato3c, typically a pressure of about 0 to 200 MPa. If the dead volume of the pressure gauges13ato13cis large, the time required for the pressure of the solution to stabilize is long. Therefore, it is preferable to reduce the dead volume of the pressure gauges13ato13c, typically 10 μL or less. The first flow path11and the second flow path12are composed of, for example, pipes. The sampler14is connected to the first flow path11, and the separation column4is connected to the downstream side of the sampler14. As the sampler14, for example, an autosampler, a manual injector, or the like can be used. The sample is introduced into the first flow path11by the sampler14. The second flow path12is a flow path that does not pass through the separation column4, and no sample is introduced into the second flow path12. Since the first flow path11has the separation column4having a small conductance on the flow path, it is preferable to use the liquid feed pumps3aand3bcapable of feeding the liquid, typically, at a pressure of 0.1 to 100 MPa in order to obtain a sufficient flow rate for analysis. On the other hand, since the second flow path12has a higher conductance than the first flow path11, a liquid feed pump having a lower pressure upper limit than the liquid feed pumps3aand3bfeeding the liquid to the first flow path11as the liquid feed pump3cfeeding the liquid to the second flow path12. The first flow path11and the second flow path12are connected to the selector valve5, and by switching the selector valve5, one of the first flow path11and the second flow path12is connected to the mass spectrometry unit2. Details of the selector valve5will be described later. Hereinafter, in the liquid chromatograph mass spectrometer of this embodiment, the area from the solution tanks1aand1bto immediately before the mass spectrometry unit2may be referred to as a “liquid chromatograph (LC)”. Although not illustrated, the mass spectrometry unit2has components such as an ion source, a vacuum chamber, and an ion detector, which are provided by a general mass spectrometer (MS). The mass spectrometry unit2ionizes the sample introduced from the first flow path11with the ion source, introduces ions into the vacuum chamber, separates ions for each mass-to-charge ratio (m/z), and detects the ionic strength by the ion detector. The ion detector outputs a detection signal of the ionic strength to the controller100. Alternatively, the ion detector may output the ion current as a detection signal to the controller100. Examples of the ion source include an electrospray ionization ion source, an atmospheric pressure chemical ionization ion source, and an atmospheric pressure photoionization ion source. In either ionization method, the solution containing the sample is sprayed into the vacuum chamber through a capillary of an ion source probe. An inner diameter of an appropriate capillary depends on the flow rate of the liquid chromatograph (LC), and it is preferable to use a thinner capillary as the flow rate decreases. The inner diameter of a typical capillary is about 30 μm to 150 μm. When a capillary with an inner diameter larger than 150 μm is used, the ionization efficiency is lowered and the sample distribution is widened by diffusion while the solution flows through the capillary, so that the LC separability may be deteriorated. Since the capillary has a small inner diameter and may be heated depending on the ionization conditions, it is easily clogged due to the contamination of foreign substances and the accumulation of impurities. The controller100is a computer terminal such as a personal computer, and is configured to control the operation of the entire liquid chromatograph mass spectrometer. Although not illustrated, the controller100includes a data processor that processes the detection signal (measured value) of the ion detector of the mass spectrometry unit2, a storage that stores various data, an input unit for a user to input an instruction to the liquid chromatograph mass spectrometer, and a display for displaying the results of mass spectrometry and various GUI screens, and the like. FIGS.2A and2Bare schematic diagrams illustrating a connection example of the flow paths by the selector valve5.FIG.2Aillustrates a state in which the first flow path11is connected to the mass spectrometry unit2, andFIG.2Billustrates a state that the second flow path12is connected to the mass spectrometry unit2. The controller100drives the selector valve5to switch the flow path connected to the mass spectrometry unit2. In this way, either one of the first flow path11and the second flow path12is introduced into the mass spectrometry unit2. As illustrated inFIGS.2A and2B, the flow path not connected to the mass spectrometry unit2is connected to a waste liquid tank15, which prevents the solution from flowing out to the outside. Operations FIG.3is a flowchart illustrating the operation of the liquid chromatograph mass spectrometer according to the first embodiment. The operation when an autosampler is used as the sampler14will be described below. First, the user prepares a sample in advance and introduces it into the sampler14. By adding an internal standard substance having a known concentration to the sample, the influence of adsorption on the flow path and instability of ionization can be eliminated, so that the signal strength of the sample can be accurately quantified. After introducing the sample into the sampler14, the user inputs an operation start instruction from the input unit of the mass spectrometry unit2. Upon receiving the operation start instruction, the controller100operates the liquid chromatograph mass spectrometer in Step S1to start normal measurement. That is, the controller100confirms that the first flow path11is connected to the mass spectrometry unit2by the selector valve5and drives the liquid feed pumps3aand3bto introduce the solution from the solution tanks1aand1binto the first flow path11. The pressure gauges13aand13boutput the pressure values of the liquid feed pumps3aand3bto the controller100. Further, the controller100drives the sampler14to introduce the sample into the first flow path11, and causes the mass spectrometry unit2to analyze the sample. The ion detector of the mass spectrometry unit2outputs the signal strength of the sample and the signal strength of the internal standard substance to the controller100. The output signal of the ion detector may be an ionic strength or an ion current. The data processor of the controller100obtains a chromatogram based on the output signal of the ion detector. The display of the controller100may receive a chromatogram from the data processor and display the chromatogram. In Step S2, the controller100compares the signal strength of the internal standard substance or the output values of the pressure gauges13aand13bwith a predetermined threshold value stored in the storage. In this way, the controller100determines whether there is an abnormality in the measurement. The predetermined threshold value is, for example, a value set based on the measured value obtained by the past measurement or the variation thereof, and the upper limit of the range of the values obtained by the normal measurement can be set as the threshold value. When the controller100determines that there is no abnormality (No), the controller100returns to Step S1and continues the normal measurement. If the controller100detects an abnormality (Yes), there is a high possibility that clogging has occurred in the LC or the mass spectrometry unit2, so the process proceeds to Step S3to identify the location where the clogging has occurred. In Step S3, the controller100switches the selector valve5, connects the second flow path12to the mass spectrometry unit2, and drives the liquid feed pump3cto feed the solution from the solution tank1cto the mass spectrometry unit2at a constant flow rate. Further, the controller100receives the pressure value of the liquid feed pump3cfrom the pressure gauge13c. In Step S4, the controller100compares the output value of the pressure gauge13cwith a predetermined threshold value stored in the storage. The predetermined threshold value to be compared with the output value of the pressure gauge13cis set based on, for example, the pressure in the absence of clogging. If the clogging occurs between the selector valve5and the outlet of the ion source capillary, especially in the ion source capillary, the pressure measured by the pressure gauge13crises. Therefore, it can be determined whether there is clogging between the selector valve5and the capillary outlet of the ion source depending on whether the pressure measured by the pressure gauge13cis equal to or higher than the threshold value. When the pressure measured by the pressure gauge13cis less than the threshold value (No), it can be assumed that there is no clogging between the selector valve5and the capillary outlet of the ion source, and the clogging occurs on the LC side, that is, the separation column4. After that, the process proceeds to Step S5, and the user replaces the separation column4. When the controller100detects that the separation column4has been replaced, the controller100switches the selector valve5to connect the first flow path11to the mass spectrometry unit2, returns to Step S1, and returns to the normal measurement. If the pressure measured by the pressure gauge13cis equal to or higher than the threshold value (Yes), it can be seen that there is clogging between the selector valve5and the capillary outlet of the ion source. At this time, the controller100displays, for example, a message requesting maintenance for clearing the clogging of the ion source on the display, and stops the measurement operation. After that, the process proceeds to Step S6, and the user performs a restoration process for clearing the clogging. As the restoration process, for example, the type or the flow rate of the solution in the solution tank1cis changed. Since the second flow path12does not have a part having a small conductance such as the separation column4on the flow path, pressure can be directly applied to the clogged part by increasing the flow rate, and the clogging can be efficiently washed away. Further, by using a solvent that easily dissolves impurities (salts and polymers) that cause clogging as the solution to be introduced into the second flow path12, the clogging can be efficiently removed. Specifically, as the solution to be introduced into the second flow path12, an organic solvent such as pure water is suitable for dissolving salts and isopropanol is suitable for dissolving polymers. In this step, the controller100determines whether the output value of the pressure gauge13cis less than a predetermined threshold value. The controller100determines that the clogging has disappeared when the value of the pressure gauge13cis less than the threshold value, switches the selector valve5, connects the first flow path11to the mass spectrometry unit2, returns to Step S1, and can return to normal measurement. It is also possible to add an internal standard substance having a known concentration to the solution flowing through the second flow path12in Step S6. In this case, by monitoring the signal strength of the internal standard substance in addition to the pressure value of the solution, it is possible to determine whether the state of the flow path has returned to normal. Technical Effect As described above, when the liquid chromatograph mass spectrometer according to this embodiment detects an abnormality, the liquid chromatograph mass spectrometer switches from the first flow path11to the second flow path that does not pass through the separation column4, measures the pressure of the second flow path12. When the pressure of the second flow path12is equal to or higher than the predetermined threshold value, it is possible to specify the location where the clogging has occurred as an ion source. Therefore, according to this embodiment, the location where the clogging has occurred can be automatically identified, and the recovering from the clogging can be achieved in a short time. Second Embodiment Apparatus Configuration FIG.4is a schematic diagram illustrating a configuration of a liquid chromatograph mass spectrometer according to a second embodiment. As illustrated inFIG.4, the liquid chromatograph mass spectrometer according to this embodiment is different from the first embodiment in that the first flow path11is branched into three, and each of the three separation columns4passes through the branched first flow path11, and there are included selector valves and7(second valves) on the upstream side and the downstream side of the three separation columns4, respectively. The characteristics of the plurality of separation columns4may be similar or different from each other. The controller100controls the drive of the selector valves6and7to select the separation column4to be used for measurement. The number of the separation columns4is not limited to three. Other configurations and operations are the same as those in the first embodiment, and thus the description thereof will be omitted. Technical Effect Since the liquid chromatograph mass spectrometer according to this embodiment has the plurality of separation columns4, the characteristics of the plurality of separation columns4can be made different, and the separation column4can be selected according to the characteristics of the sample to be measured. Alternatively, analysis can be performed in parallel using a plurality of separation columns4having similar characteristics, and the sample can be introduced into the mass spectrometry unit2only for a time near the peak, which has an advantage that the throughput can be improved. The liquid chromatograph mass spectrometer of this embodiment includes the plurality of separation columns4, which increases the number of selector valves and complicates the flow path. In addition, the selector valves6and7may be clogged, or carryover may occur due to the sample remaining in the selector valves6and7. Here, in the liquid chromatograph sorting apparatus described in PTL 1, even if a plurality of separation columns are provided as in this embodiment and the pressure of the liquid feed pump is measured to detect clogging, it is not possible to specify whether the location where the clogging has occurred is the selector valve, the selector column, or the ion source. Therefore, it is troublesome because it is necessary for the user to specify the clogged location. On the other hand, according to this embodiment, when an abnormality is detected, the first flow path11is switched to the second flow path12that does not pass through the separation column4, and the pressure of the second flow path12is measured. When the pressure of the second flow path12is equal to or higher than the predetermined threshold value, it can be specified that the ion source is clogged, and when the pressure of the second flow path12is lower than the predetermined threshold value, it can be specified that any of the separation column4and the selector valves6and7is clogged. Therefore, according to this embodiment, the location where the clogging has occurred can be automatically identified, and the recovering from the clogging can be achieved in a short time. Third Embodiment Apparatus Configuration FIG.5is a schematic diagram illustrating a configuration of a liquid chromatograph mass spectrometer according to a third embodiment. As illustrated inFIG.5, in this embodiment, at least one of the plurality of separation columns4provided in the liquid chromatograph mass spectrometer according to the second embodiment is a hollow dummy column10, and the flow path can be switched by the selector valves6and7(second valves) provided in the upstream side and the downstream side of the separation column4and the dummy column10respectively. In this embodiment, among the flow paths between the selector valve6and the selector valve7, the flow path that passes through the separation column4is referred to as the first flow path11, and the flow path that passes through the dummy column10is referred to as the second flow path12. In other words, the dummy column10is provided in parallel with the plurality of separation columns4, and the second flow path12is a flow path passing through the dummy column10. The dummy column10is a hollow column without a filler inside. Therefore, the dummy column10has a larger conductance than the separation column4. The controller100connects any of the plurality of first flow paths11and the second flow path12to the mass spectrometry unit2by switching the selector valves6and7. Since other apparatus configurations are the same as those of the first embodiment and the second embodiment, the description thereof will be omitted. Operations The operation of the liquid chromatograph mass spectrometer according to this embodiment will be described. In this embodiment, during the normal measurement (Step S1illustrated inFIG.3), liquid is fed from the solution tank1aby the liquid feed pump3a(first liquid feed pump). If it is determined to be abnormal during the normal measurement (Yes in Step S2illustrated inFIG.3), the controller100switches the selector valves6and7(Step S3) to connect the second flow path12passing through the dummy column10to the mass spectrometry unit2. Then, the controller100feeds the solution from the solution tank1bby the liquid feed pump3b(second liquid feed pump), and measures the pressure of the second flow path12by the pressure gauge13b(Step S4). Since other operations are the same as the operations in the first embodiment, detailed description thereof will be omitted. Technical Effect As described above, according to this embodiment, the second flow path12is configured to pass through the dummy column10provided in parallel with the separation column4of the first flow path11instead of the piping as in the first embodiment. Therefore, it is possible to simplify the configuration of the apparatus without the need of any special flow path, and reduce the manufacturing cost. Further, since the dummy column10can be removed like the separation column4, it can be easily replaced with another column. Furthermore, since it is possible to determine whether there is clogging between the liquid feed pump3band the capillary outlet of the ion source, it is possible to automatically specify whether the clogging occurs on the separation column4side or the ion source side, and it can be recovered from the clogging in a short time. Fourth Embodiment Apparatus Configuration A liquid chromatograph mass spectrometer according to a fourth embodiment will be described.FIG.6is a schematic diagram illustrating a configuration of a mass spectrometry unit according to the fourth embodiment. As illustrated inFIG.6, the liquid chromatograph mass spectrometer of this embodiment is different from the first embodiment in that the mass spectrometry unit2has two or more ion source probes9, and a selector valve8(third valve) for switching between a plurality of ion source probes are included. Switching of the selector valve8is controlled by the controller100. Although not illustrated, the ion source probe9has a capillary, and the outlet of the capillary is arranged toward a vacuum chamber16. Since other apparatus configurations are the same as those in the first embodiment, description thereof will be omitted. Operations The operation of the liquid chromatograph mass spectrometer according to this embodiment will be described. The operation of this embodiment is almost the same as that of the first embodiment, but is different from the first embodiment in that, at the time of normal measurement (Step S1in the first embodiment), one of the plurality of ion source probes9is used, and when it is determined that the ion source is clogged (Yes in Step S4), the controller100switches the selector valve8in Step S5to connect another ion source probe to the mass spectrometry unit2. In this way, when it is determined that the ion source is clogged, it is possible to recover from the clogging more reliably and in a short time by switching the ion source probe9. Technical Effect As described above, in this embodiment, as in the first embodiment, the liquid chromatograph mass spectrometer is configured to switch from the first flow path11to the second flow path12that does not pass through the separation column4when an abnormality is detected so as to measure the pressure of the second flow path12. As a result, according to this embodiment, the location where the clogging has occurred can be automatically identified, and the recovering from the clogging can be achieved in a short time. Further, this embodiment has a configuration in which the flow path is switched to another ion source probe9when it is determined that there is clogging between the selector valve5and the capillary outlet of the ion source. As a result, it is possible to recover from the clogging more reliably and in a short time. MODIFICATIONS The present disclosure is not limited to the examples described above, but includes various modifications. For example, the above embodiments have been described in detail for easy understanding of the present disclosure, and the invention does not necessarily have all the configurations described. In addition, some of certain embodiment can be replaced with the configuration of the other embodiment. Further, it is possible to add the configuration of one embodiment to the configuration of another embodiment. It is also possible to add, delete, or replace a part of the configuration of another embodiment with respect to a part of the configuration of each embodiment. REFERENCE SIGNS LIST 1a-1csolution tank2mass spectrometry unit3a-3cliquid feed pump4separation column5-8selector valve9ion source probe10dummy column11first flow path12second flow path13a-13cpressure gauge14sampler15waste liquid tank16vacuum chamber | 23,989 |
11860143 | DETAILED DESCRIPTION Reference will now be made in detail to various embodiments of the present invention(s), examples of which are illustrated in the accompanying drawings and described below. While the invention(s) will be described in conjunction with exemplary embodiments, it will be understood that the present description is not intended to limit the invention(s) to those exemplary embodiments. On the contrary, the invention(s) is/are intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents, and other embodiments, which may be included within the spirit and scope of the invention(s) as defined by the appended claims. Chromatography is a separation technique where analytes within a sample mixture are separated while going through a chromatography column based on the analytes' distinct affinity for a stationary phase versus a mobile phase. In ion chromatography (IC), the separation is specific to ions. Following separation, analytes may be detected by conductivity detectors due to the electrical properties of ions. This inherently presents a challenge as the separated analytes are enveloped by a sea of eluent, which eluent is also conductive, whereby conductive detection of the eluting analyte can be impossible. This challenge may be resolved by utilizing a suppressor between the separation column and the conductivity detector that removes background conductivity of the eluent by turning the eluent into water, which effectively enhances the signal of the analyte. The mechanisms are slightly different for anion and cation analyses. In the case of an Anion suppressor, sodium or potassium ions are respectively removed from the eluent flowing through the suppressor of sodium hydroxide or potassium hydroxide and the remaining hydroxide ions combine with hydronium ions to form water, which has a very low conductivity and thus lowers the background signal of the eluent. The counter cations of the analytes are replaced with hydronium, thus changing the analytes from a salt form to an acid form therefore enhancing their signals. In the case of a Cation suppressor, metasulfunate and sulfunate are respectively removed from the eluent flowing through the suppressor of metasulfonic acid and sulfuric acid and the remaining hydronium ions combine with hydroxide ions to form water, which again lowers the background signal of the eluent. Similarly, the counter anions of the analytes are replaced with hydroxide, thus transforming the analytes from a salt form to their base form therefore enhancing their signals. Over time, suppressors have evolved from single column devices that needed several regeneration cycles (such as those described in U.S. Pat. Nos. 3,897,213, 3,920,397, 3,925,019, 3,926,559, and 5,597,734), to continuously regenerated in-line devices (such as those described in U.S. Pat. No. 4,474,664), to more recent electrolytically regenerating devices (such as those shown in U.S. Pat. Nos. 4,459,357, 4,403,039, 4,999,098 and 5,248,426), the entire content of which patents is incorporated herein for all purposes by this reference. Generally, a voltage is applied to a suppressor to effect an ion exchange between eluent and regenerant channels. The amount of voltage needed to sufficiently suppress the eluent is generally dependent on the eluent flow rate and concentration. In accordance with various aspects of the present invention, the methods and systems herein allow for self-regulation of suppressors by determining the state of the suppressor to distinguish whether there is insufficient current, optimal current, or too much current being provided to the suppressor. The state of the suppressor may be determined based upon the impedance of the suppressor, in which capacitance may indicate an unsuppressed state, resistance may indicate a suppressed state, and resistance with thermal effects may indicate an over-suppressed state. Turning now to the drawings, wherein like components are designated by like reference numerals throughout the various figures, attention is directed toFIG.1which illustrates an exemplary chromatography (IC) system30in accordance with various aspects of the present invention. The IC system generally includes an eluent source32, a sample injection valve33, an ion-chromatography column35, a suppressor37, and a conductivity detector39. In accordance with various aspects of the present invention, the system also includes a power supply40and a control unit42, which are configured to monitor and adjust the voltage applied to the suppressor in order to improve or optimize the performance of the suppressor. The control unit generally includes one or more processors and memory, with the processor(s) being configured to run software for performing various steps. The power supply may be a dedicated power supply providing the electric potential to the suppressor, which configuration may be particularly well suited for retrofitting existing IC systems. The power supply and the control unit may be discrete components, or they may be integrated into a power-supply module44that may be integrally provided in a new IC system or separately provided to retrofit an existing IC system. Generally, a sample is introduced into an eluent through sample injection valve33and the resulting solution flows to and through column35, which is packed with a chromatographic separation medium to separate analytes within the sample from one another. The solution leaving column35is directed downstream to suppressor37, which suppresses the conductivity of the eluent but not the ionic conductivity of the separated analytes. Typically, suppressor37includes a primary eluent or liquid-sample channel46through which sample containing an ionic species flows, and a regenerant or ion-receiving channel47through which a regenerant flows. One will appreciate that such suppressors are particularly well suited for IC suppression, however, such suppressors may be used for sample pre-treatment and other uses. As such, the primary channel may suppress an eluent with an ionic species, or alternatively, may simply pretreat a liquid including an ionic species. The suppressed eluent is then directed downstream to a detection means such as a conductivity detector39for detecting the resolved ionic species. In the conductivity detector, the presence of ionic species produces an electrical signal proportional to the amount of ionic material, thus permitting detection of the concentration of separated ionic species. The conductivity detector may be operably connected to a computer, processing device, data acquisition system, or other suitable means for acquiring and/or processing the data. After passing through conductivity detector39, the eluent may be directed to ion-receiving channel47of suppressor37, thus providing a source of water to the suppressor37, in manner similar to that described in U.S. Pat. No. 5,352,360, the entire content of which is incorporated herein for all purposes by this reference. The suppressed eluent may then be directed to waste. To prevent eluent in conductivity detector39from out-gassing, the system may include a back pressure coil49downstream from the conductivity detector through which eluent flows before flowing to the ion-receiving channel of the suppressor. The back pressure coil may help prevent gases generated during suppression, from out-gassing and thus prevent the formation of bubbles in the conductivity detector, thus reducing noise and improving accuracy of the detector. As noted above, the suppressor includes liquid-sample channel46through which sample flows containing an ionic species, and ion-receiving channel47through which a regenerant flows. An ion-exchange membrane51between the channels and is configured to substantially block bulk liquid flow between the liquid-sample and ion-receiving channels while allowing passage of ions of one charge, positive or negative, between the channels. The suppressor is provided with a first electrode53in electrical communication with liquid-sample channel46, and a second electrode54in electrical communication ion-receiving channel47. The electrodes may be in the form of flat plates or other structure that can be mounted or embedded in the respective channels. The electrodes may be formed of highly conductive materials that are inert to the solutions passed through the suppressor. Platinum is a preferred material for this purpose, however, one will appreciate that other suitable materials may be utilized. An electrical potential is applied between the electrodes from the power supply. Power supply40is configured to apply an electric potential to suppressor37via first and second electrodes53,54. An external power supply may be utilized, such as a N6774A power supply in conjunction with an N6705C power analyzer, both by Keysight Technologies of Colorado Springs, CO. One will appreciate that other suitable power supply devices may be utilized, either incorporated within one or more components of system30, or provided externally to the system. The power supply is configured to provide an operating or offset voltage VOSto the suppressor. The power supply is also configured to provide an applied voltage waveform VAto the suppressor in addition to the offset voltage VOS, the purpose of which will become apparent as described below. In various embodiments, the power-supply module and/or control unit may utilize engineering software for measurement, hardware control and data insights. A suitable engineering software is the LabVIEW system engineering software by National Instruments of Austin, TX. One will appreciate that such software may be provided with a standalone computing device, incorporated into firmware of the power-supply module and/or control unit, or incorporated into other firmware or software of the IC system. One will appreciate that a wide variety of power supplies and control units may be utilized in accordance with various aspects of the present invention. In order to determine the state of the suppressor, characteristics are identified to distinguish the suppressor in its three main phases: (1) unsuppressed; (2) suppressed; and (3) over-suppressed. Unfortunately, these states are difficult to assess using a conductivity detector because conductivity signals are high in the unsuppressed state, often resulting in negative peaks in the presence of analyte. Conductivity signals are generally acceptable in the suppressed state (e.g., often near or less than 1.0 μS/cm) with positive peaks identifying analyte. And conductivity signals in the over-suppressed state are not easily distinguishable from those of the suppressed state, namely because conductivity signals in the over-suppressed state are higher than but generally within the same magnitude as those of the suppressed state, with similarly positive peaks. Accordingly, the systems and methods described herein do not rely on conductivity signals. Instead, and in accordance with various aspects of the present invention, the systems and methods described herein rely on a measured current signal of the suppressor itself. And the measured current signal of the suppressor may be used to distinguish the primary operational states of the suppressor based on the impedance of the suppressor. Voltage and current within a system are generally related by impedance, in accordance with Ohm's law: V=Z*IEq. (1) where V is voltage, Z is impedance, and I is current. Impedance is the obstruction to a current at a given applied voltage. And the three forms of impedance are resistance, capacitance, and inductance. In the case of pure resistance, the voltage signal and the current signal are in phase and generally proportional to one another. In the case of capacitance, the voltage signal lags the current signal. And in inductance, the voltage signal leads the current signal. One way to illustrate such impedance is by applying a square voltage signal and observing the response of the current signal. For example,FIG.3illustrates various forms of impedance. The resistance form of impedance has a square current signal i(t) that closely resembles the input square voltage signal V(t)—both the voltage and current waveforms are in phase and proportional. The capacitance form of impedance has a square current signal i(t) that is lagging as compared to the input square voltage signal V(t)—here the current signal is the derivative of the voltage signal. And the inductance form of impedance has a current signal i(t) that is leading as compared to the input square voltage signal V(t)—here the current signal is the integral of the voltage signal. Thus, when an offset voltage VOSis applied to the suppressor to operate the suppressor, a relatively small applied voltage waveform VAmay be applied to the suppressor in addition to the offset voltage VOSin order to monitor suppressor's current responses to the applied voltage. When the applied voltage waveform VAis applied in an oscillating square waveform having a frequency F and amplitude A, the combined offset and applied voltage waveform may be represented as shown inFIG.4. And when the current of the suppressor is measured, the measured current response resulting from the offset and applied voltages provides an indication as to the impedance and corresponding operational state of the suppressor. As shown inFIG.5, when the suppressor is not suppressed it is highly capacitive, and the measured current response exhibits a diminishing waveform, as shown in the leftmost measured current response. As capacitance decreases when the suppressor approaches suppression, the measured current response exhibits a less diminishing waveform, as shown in the second-from-left measured current response. When the suppressor is properly suppressed, the resulting waveform is substantially a square waveform. As shown inFIG.5, the middle measured current response largely approximates the applied voltage waveform shown inFIG.4. In the suppressed state the impedance is overwhelmingly driven by resistance due to the fact that the eluent is mainly water. Thus, the measured current response is substantially in phase and proportional to the applied voltage waveform shown inFIG.4. In the over-suppressed state, there may be additional thermal effects as excessive voltage that may cause higher current through the suppressor. Since there is more than enough current for the suppression process, the excess current may be translated into heat which is reflected in the upward motion of the observed measured current signal. It can be seen in the second-from-right measured current response that the waveform is increasing slightly, and the rightmost measured current response is increasingly increasing. With an exemplary system described above, an exemplary method for self-regulating a suppressor of an IC system in accordance with various aspects of the present invention can now be described. With reference toFIG.2, the power supply may be set to provide an oscillating offset voltage VOSto the suppressor. And the power supply may be activated to provide an applied voltage waveform VAto the suppressor in addition to the offset voltage VOS. An ion chromatography run may then be commenced on the IC system in which eluent flows through the suppressor. During the ion chromatography run, a current of the suppressor is cyclically measured responsive to the offset and applied voltages VOSand VA, and a suppressor state of the suppressor is determined based upon the measured current waveform. Diminishing current corresponding to the upper voltage square waveform may indicate an unsuppressed state of the eluent flowing through the suppressor, which may be due to electrical capacitance and resistance within the suppressor. Substantially constant current may indicate a suppressed state, which may be due to substantially constant electrical resistance within the suppressor. And increasing current corresponding to the upper voltage square waveform may indicate an over-suppressed state, which may be due to increased electrical resistance and thermal effects within the suppressor. The offset voltage VOSsupplied to the suppressor may be adjusted based upon the suppressor state. Offset voltage VOSmay be increased for an unsuppressed state. Offset voltage VOSmay be maintained for a suppressed state. And offset voltage VOSmay be decreased for an over-suppressed state. Such adjustments may vary the offset voltage VOSover time in response to varied concentration of eluent flowing through the suppressor over time. The voltage waveform may have a voltage amplitude A and a voltage frequency F, and the applied voltage waveform VAmay be a waveform voltage having a positive pulse width and a negative pulse width. In various embodiments, an applied square-waveform voltage is utilized to provide readily identifiable positive and negative pulse widths, from which the resulting current response signals would provide readily identifiable current slopes. A current slope of a positive pulse width (SP) less than a first predetermined threshold may indicate an unsuppressed state, a substantially neutral current slope of the positive pulse width (SP) greater than the first predetermined threshold and less than a second predetermined threshold may indicate a suppressed state, and a current slope of the positive pulse width (SP) greater than the second predetermined threshold may indicate an over-suppressed state. The applied voltage VAmay have a square waveform voltage having a positive pulse width and a negative pulse width. The applied voltage VAmay be an oscillating voltage having period T, wherein the measuring, determining, and adjusting steps are performed for each period T. The measured current response has a positive pulse width slope SP(e.g., slope SPinFIG.7) and a negative pulse width slope SN(e.g., slope S N inFIG.7). In accordance with various aspects of the present invention, (a) a slope Spof less than approximately 0.1 mA/s indicates an unsuppressed state, (b) a slope Spof approximately 0.1 mA/s to 0.3 mA/s indicates a suppressed state, and (c) a slope Spgreater than approximately 0.3 mA/s indicates an over-suppressed state. And (a) a slope SNgreater than approximately −0.05 mA/s indicates an unsuppressed state, and (b) a slope SNless than approximately −0.05 indicates a suppressed or over-suppressed state. An adjusting step may adjust the offset voltage VOSby an adjusted voltage ΔV each period T. In various embodiments, the adjusted voltage ΔV is less than the applied voltage VA. And in some embodiments the adjusted voltage ΔV is less than 10% of the Amplitude A. For example, the adjusted voltage ΔV may be approximately mV. One will appreciate that a ΔV of approximately 0.01 to 10% of the Amplitude A may efficiently adjust the voltage while avoiding overcorrection. In various embodiments, the power supply and control unit may be configured in various ways to apply offset voltages VOSand applied voltage waveforms VAto the suppressor, and to measure the current of the suppressor response to the offset and applied voltages VOS, VA. For example, the power supply and control unit may be configured to execute a repeat-cycle program similar to that illustrated inFIG.6. Such a program allows the self-adjustment of the offset voltage provided to the suppressor based on the current response of the suppressor, thereby creating a self-regulating feedback loop. The program may generally run as follows:A) The power supply is turned on and parameters are set;B) A voltage frequency and cycle time are set (e.g., 0.1 Hz with a cycle time of 10 seconds) allowing a single square wave within that cycle (e.g., an upper part of the resulting current wave occurs in the first 5 seconds and the lower part occurs in the last 5 seconds);C) After each cycle, the upper slope (or Sp) of the current wave is calculated using the current and time information from 0.01 seconds to 4.99 seconds, and the same is done for the lower slope (or S N) from 5.01 seconds to 9.99 seconds (e.g., in mA/s), wherein the slope may be calculated by fitting the data to the best linear function with the least error and extracting the slope value of that linear function;D) After each cycle, the offset voltage VOSis increased or decreased based on the slope values, wherein preset threshold values for the upper (or Sp) and lower slopes (or SN) are set to increase or decrease the offset voltage accordingly. The above is but one example of how the power supply and control unit may be configured to operate the suppressor. One will appreciate that various protocols and parameters may be utilized to apply predetermined operating voltages (i.e., offset voltage VOS) and observable applied voltage waveforms (i.e., applied voltage VA) to the suppressor and measure the resulting current of the suppressor in order to self-regulate the suppressor. For example, either the upper or the lower slope (e.g., slopes SPand SNinFIG.7) could be used individually or together to assess operational states of the suppressor. In one exemplary experimental method in accordance with various aspects of the present invention, the following parameters were used for applying a square waveform voltage to a Dionex™ AERS™ 500 4 mm suppressor: cycle time of 10 seconds; frequency of 0.1 Hz; amplitude of 100 mV; and a delta voltage ΔV of +/−5 mV (i.e., the amount by which the offset voltage is changed each cycle time every 10 seconds). The current response is shown inFIG.7, and it can be seen that diminishing measured current response signals indicate unsuppressed states of the suppressor, negligible slopes indicate suppressed states, and increasing wave signals indicate over-suppressed states. Based upon such behavior, upper and lower slope criteria of the measured current response signals may be defined to distinguish each suppressor state. For example, the following table sets forth slope criteria that may be utilized to distinguish each suppressor state. Slope (mA/s) CriteriaUpper (or SP)Lower (or SN)Unsuppressed<0.100>−0.050Suppressed0.100-0.300<−0.050Over-Suppressed>0.300<−0.050 With reference toFIG.7, the unsuppressed state may be identified when the upper slope (e.g., SPinFIG.7) is below approximately 0.100 mA/s, the suppressed state when the upper slope is approximately 0.100 to 0.300 mA/s, and the over-suppressed state when the upper slope is above approximately 0.300 mA/s. The unsuppressed state may also be identified when the lower slope (e.g., S N) is above approximately −0.050 mA/s. For the above conditions, the power supply may regulate voltage to the suppressor as follows:A) A square voltage of 0.1 Hz with an oscillating amplitude of 100 mV and given offset voltage is applied to the suppressor;B) The upper and lower slopes of the resulting current signal are calculated;C) If the lower slope is greater than −0.050 mA/s, the status of the suppressor is automatically classified as unsuppressed, and the offset voltage is increased by a predetermined ΔV of 5 my;D) If the lower slope is less than −0.050 mA/s, the upper slope is evaluated and based on the range where the upper slope falls, and the offset voltage is adjusted accordingly;E) When the unsuppressed state is determined, the offset voltage is increased by 5 my;F) When the suppressed state is determined, the offset voltage stays the same;G) When the over-suppressed state is determined, the offset voltage is decreased by 5 my; andH) The cycle is repeated every 10 seconds. One will appreciate that the upper slope alone (e.g., SPinFIG.7) may be capable of identifying the unsuppressed, suppressed, and over-suppressed states of the suppressor. One will also appreciate that the lower slope alone (e.g., SNinFIG.7) may be capable of identifying whether or not the suppressor is unsuppressed. And one will also appreciate that both slopes may be utilized together to identify the three suppressor states. In accordance with various aspects of the present invention, it is possible to self-regulate a suppressor by ensuring that enough power is delivered to the suppressor to fully suppress the eluent and increase the accuracy of conductive detection results. And it is possible to prevent over suppression by inadvertently overpowering the suppressor, which may significantly increase suppressor lifetime. The systems and methods described herein may provide simpler equipment configurations as desired voltages for the suppressor are determined without any feedback from the conductivity detector. In fact, the systems and methods described herein may provide desired voltages to the suppressor without knowledge of eluent concentrations and/or flow rates through the suppressor. In the case of gradients or changing concentrations, the systems and methods described herein allow for automatically voltage changes to the suppressor. For example, as eluent concentration through the suppressor increases, a constant voltage to the suppressor would result in under-suppression of the greater eluent concentration. However, the present systems and methods allow for identifying such an unsuppressed state and automatically take corrective action. As the present methods and systems rely solely on a measured current response of the suppressor, a power supply and/or control unit may be readily retrofit to existing IC systems. For convenience in explanation and accurate definition in the appended claims, the terms “upper” and “lower” and similar terms are used to describe features of the exemplary embodiments with reference to the positions of such features as displayed in the figures. The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and their practical application, to thereby enable others skilled in the art to make and utilize various exemplary embodiments of the present invention, as well as various alternatives and modifications thereof. It is intended that the scope of the invention be defined by the Claims appended hereto and their equivalents. | 26,528 |
11860144 | DETAILED DESCRIPTION The measuring cell1according to the present disclosure can be used for carrying out various chemical analyses, such as titration, digestion and/or pre-reaction before titration. InFIG.1, the liquid3to be analyzed is placed in the vessel2and provided with a stir bar11, which mixes the liquid by means of the magnetic stirrer10arranged below the vessel2. There is no limitation with regard to the shape and size of the vessel2; especially, small, standardized vessels can be used. The heating wire4, which heats the liquid inside the vessel in a uniform and controlled manner, is at least partially guided around an outer wall of the vessel2. By way of example, the heating wire is guided around the vessel between the bottom of the vessel and the upper edge of the liquid. The optional elastic molded jacket13, which fixes the heating wire4in its position, is additionally shown. The heating wire4is shown in detail inFIG.2. A thermal fuse14or a second temperature sensor15can additionally be arranged in the molded jacket; this determines and/or monitors a second temperature in the area of the heating wire4and by means of which it can be established if a maximum permissible value of the second temperature is exceeded. The vessel2is closed by means of a cover6, which closes the vessel2in an essentially gas-tight manner, so that no or only little liquid3in the form of vapor escapes from the vessel2during heating. A series of ducts7a,b,c,dare introduced into the cover6and serve to accommodate further elements. A first analysis sensor8, which determines and/or monitors at least one chemical and/or physical variable of the liquid3of the vessel2, is accommodated in a first duct7a. For example, the first analysis sensor8can be a pH, conductivity, redox electrode or a photometric transceiver system. A second duct7bis provided for a liquid line9. By means of the liquid line9, liquid is added to the vessel2, for example titrated, or removed from the vessel2. A plurality of liquid lines for various liquids may also be used. A first temperature sensor5, which determines and/or monitors a first temperature of the liquid3, is accommodated by way of example in a third duct7cof the cover6. Alternatively, the first temperature sensor can be integrated into the first analysis sensor8, such as a redox electrode, which additionally determines and/or monitors the first temperature of the liquid3. For example, the first temperature sensor5regulates the heating of the vessel2by the heating wire4. Optionally, a fourth duct7dis provided in the cover6, which duct serves to vent the vessel2and to which duct a tube18is attached, for example. For the thermal insulation of the vessel2and of the heating wire4, a housing16can optionally at least partially surround the vessel2and the heating wire4. The housing16can, for example, be connectable to the cover6and/or the magnetic stirrer10, as indicated inFIG.1by the screws19, so that a compact, stable measuring cell1is produced. With that said, other possibilities for connecting the housing16and the vessel2and/or the cover6are not ruled out. In order to rapidly cool the measuring cell1again after a heating process and prepare it for the next chemical analysis, a blower17is optionally arranged in the vicinity of the vessel2. The blower17cools the vessel2by directing an air flow, which is indicated by arrows, toward the vessel2. The blower17can also be used, for example, during a heating process, in order to cool the region between the cover6and an upper edge of the liquid3of the vessel2and thus promote condensation and recirculation of the evaporated liquid3. FIG.2shows the heating wire schematically in detail. By way of example, the heating wire4is wound around the outer wall of the vessel2as a compact package. By way of example, the heating wire4is surrounded by an insulating jacket12and a molded jacket13. The molded jacket13can be, for example, a shrink tube. In order to avoid local temperature peaks at the heating wire4, the heating wire4can be designed such that the resistance of the heating wire4increases with increasing temperature. | 4,140 |
11860145 | It should be understood that the above-referenced drawings are not necessarily to scale, presenting a somewhat simplified representation of various preferred features illustrative of the basic principles of the disclosure. The specific design features of the present disclosure, including, for example, specific dimensions, orientations, locations, and shapes, will be determined in part by the particular intended application and use environment. DETAILED DESCRIPTION Wetstock management can involve the usage of fuel data sensors to monitor the fuel stock at a fuel storage facility, evaluating measurement data to detect anomalies affecting the fuel stock, and performing corrective actions as necessary. The sensors can measure fuel data characterizing myriad possible aspects of the fuel storage facility. For example, fuel losses due to leaks, theft, delivery shortages, or the like can be detected and damage to storage equipment can be identified. By automating these processes in a “smart” manner applying, for instance, artificial intelligence and/or machine learning techniques, as described below, wetstock management can be performed more efficiently, economically, and safely. Embodiments of methods and systems for automated wetstock management are discussed herein below. FIG.1illustrates one embodiment of an exemplary automated wetstock management system. The automated wetstock management system (“wetstock system”)100can provide an automated, alert-driven wetstock management service using automated processing, artificial intelligence, and/or machine learning technologies to dynamically create workflows for the purpose of resolving issues arising in a given fuel storage facility. In some embodiments, operation of the wetstock system100can be carried out by a remote, cloud-based wetstock management server (not shown) configured to perform one or more operations described below. The wetstock system100can collect electronic fuel data characterizing one or more aspects of the fuel storage facility including, but not limited to, fuel stored in the facility, storage equipment (e.g., tanks), monitoring equipment, etc., from multiple sources such as, for example, automatic tank gauges (ATGs), point of sale devices, forecourt controllers, back office systems, fuel dispensers, and the like, as well as manually submitted fuel data (e.g., through a wetstock management computer application, website, etc.). The wetstock system100can process the collected data automatically through various algorithms, machine learning, and/or artificial intelligence to create alerts and/or exceptions. Additionally, the wetstock system100can apply risk categorization to a dynamically created diagnostic workflow. The wetstock system100can evaluate incoming data when an exception is raised, validate the exception to filter out any invalid exceptions, categorize the exception based on risk, identify the most likely fault, as well as the probability of said fault, and notify a user (e.g., fuel storage facility operator or manager, fuel merchant, etc.) in a bespoke manner. In addition to exception-specific faults, the wetstock system100can utilize the individual exceptions to generate a consolidated risk-versus-probability model and solution. Finally, resolved issues can be tracked and logged by the wetstock system100. The data can used as training data in a machine learning context to enable the wetstock system100to learn from previous diagnoses and more effectively diagnose similar situations in the future. According to some embodiments, as shown inFIG.1, the wetstock system100can be configured to embody a supporting component110and an analytics component120, each of which composed of multiple individual elements. However, the wetstock system100is not limited solely to such configuration. The supporting component110of the wetstock system100can include the components necessary to enable an analytics model, implemented by the analytics component120to function and be serviced. The analytics component120of the wetstock system100can include the components according to which collected fuel data is processed and analyzed. It is understood that the supporting and analytics components110and120are not limited solely to the configurations shown inFIG.1and described below, but can be re-configured and/or re-arranged in any suitable manner, as would be understood by a person of ordinary skill in the art, consistent with the scope of the present claims defined herein. Operationally, the wetstock system100can execute individual units of the supporting and analytics components110and120in a particular order, such as the order depicted inFIG.1. However, the ordering of units shown inFIG.1is provided merely for demonstration purposes, and operation of the wetstock system100is not limited solely thereto. Thus, units of the supporting and analytics components110and120, respectively, can be executed in any suitable order, as would be understood by a person of ordinary skill in the art, consistent with the scope of the present claims defined herein. Referring now toFIG.1, the supporting component110can initialize the wetstock system100by executing units that support or enable operation of the analytics component120whereby the collected fuel data is automatically processed and analyzed. Firstly, for example, an onboarding unit111can be executed whereby features required to initialize organizations, sites, wetstock details, and the like are carried out. Similarly, a user and system management unit112can be performed whereby features required to initialize one or more users (e.g., fuel storage facility operator or manager, fuel merchant, etc.) of the wetstock system100are carried out. For example, one or more registered users of the wetstock system100can be loaded, preferences of the one or more users can be imported, user permissions can be set, and so forth. In addition, features required to initialize the wetstock system100itself can be executed. For example, security settings, site groups, and the like associated with the wetstock system100can be initialized. The user and system management unit112can initialize user and system settings using operation data stored in a local or remote memory (not shown), depending on the configuration, characterizing one or more aspects of previous operations of the wetstock system100. In cases where said operation data does not exist, the user and system management unit112can initialize user and system settings according to a default configuration. Once the supporting component110has initialized the wetstock system100by executing units that support or enable operation of the analytics component120, units of the analytics component120can be executed. Firstly, for example, a data processing unit121, which encompasses both the import and export of data, can be performed. In detail, the data processing unit121can begin by collecting fuel data characterizing one or more aspects of a fuel storage facility from a wide variety of devices such as sensors or other measurement tools. These devices can include, for example, ATGs, fuel leak detection sensors, magnetostrictive probes, point of sale devices, forecourt controllers, back office systems, fuel dispensers, and so on. Also, users of the wetstock system100can manually submit data to be processed. All inputted data can be combined and exported for automated evaluation using predefined algorithms (122) of the wetstock system100. An algorithms unit122can then be executed whereby the fuel data collected in the data processing unit121is inputted to one or more predefined models and/or rules of the wetstock system100. Algorithms of the wetstock system100can include any models and/or rules for processing the collected fuel data to generate one or more exceptions (123) when said one or more exceptions exist. The algorithms can be used to evaluate the collected input data for myriad purposes such as analyzing fuel loss, flow rates, delivery yields, etc., in order to alert the user of any issues occurring in the fuel storage facility. Such algorithms can encompass, but are not limited to, anomaly detection (e.g., detecting the presence of a value outside of a calculated or pre-configured normal range for a value during a given time slice), trend analysis (e.g., detecting the tendency of a value to move toward a range considered to be unacceptable), cross-value correlation (e.g., detecting the tendency of a value to change based on values of another variable or external events), and so on. To these ends, the input data can be analyzed in various ways such as calculating a maximum or minimum value over a given time slice, calculating an average, mean, or median value over a given time slice, calculating a standard deviation over a given time slice, and so on. For example, an average value in a given time slice (e.g., week, month, quarter, year, etc.) can be compared with a corresponding average value associated with a past time slice to detect anomalies. In some embodiments, multiple algorithms can be combined to create new algorithms. Output data generated by execution of these algorithms can be used to identify exceptions, escalate risk, and/or apply to further algorithms. Next, an exceptions and services unit123can be executed whereby exceptions generated through the processing of collected fuel data via the algorithms described above can be delivered to the user. For the purpose of the present disclosure, an exception can refer to any data outputted via the algorithms unit122having a value which is outside of a predefined normal, or safe, range or threshold. For example, a fuel leak can cause a sudden decrease in fuel tank level. If an ATG detects that said level is less than a predefined minimum tank level threshold, an exception indicative of a fuel leak can be present. A wide range of services can be provided to the user based on the generated exceptions including, for example, predictive maintenance of impending faulty equipment, regulatory report generation and delivery to the appropriate standards bodies, authority notification when product theft is detected, predictive delivery of product based on trends, vendor notification of incorrect delivery of product (e.g., insufficient delivery, incorrect product, etc.), automated shutdown of fuel pumps due to detected issues (e.g., leaks, mechanical pump issues, etc.), and so on. Next, a risk escalation unit124can be executed whereby a risk category can be assigned to an exception generated through the algorithms unit122and exceptions and services unit123based on a variety of factors. The risk assignment can be used to determine whether or not to escalate the exception, as well as the extent to which the exception is escalated. Moreover, risk categorization can allow users to assign rules to a particular risk category that is specific to their needs. In some cases, as anomalies in the input data are detected and exceptions are generated in the manner described above, a machine learning-based system can examine the actions taken to address an anomalous situation, such as a fuel leak, as it occurs in real-time. Thus, when the exception re-occurs, the response time can be compared against both configured service level agreements and past resolutions to determine whether the correct resources are being applied and the appropriate attention is being given to the fuel leak. Further, as new anomalies in the fuel data are detected, and exceptions are generated which can exacerbate the situation, machine learning techniques can use the past resolutions as training data to change the resources assigned or invoke automated reactions to a new higher or lower risk. Examples of these reactions could be shutting down devices, notifying the authorities, notifying more experienced personnel, and so on. Next, a workflow unit125can be executed whereby a workflow including a series of steps for assisting the user to resolve an identified operational issue can be generated in real-time based on an identified exception and the risk category assigned thereto. The workflow can provide end-to-end support for the user to resolve an operational issue in the most appropriate and efficient manner, taking into account on-site equipment, depending on the threat and seriousness of the issue. For example, when the operational issue is a fuel leak, the workflow can include steps intended to correct or prevent exacerbation of the fuel leak. The workflow can be provided to the user in a manner determined according to the generated exception and the level of risk assigned thereto. In some embodiments, a device (e.g., a computing device such as a computer, mobile device, tablet device, etc.) coupled to a wetstock management server (not shown) responsible for performing elements of the analytics component120can display a visual characterization of the workflow, via a display unit of the device, enabling the user to read and follow the displayed workflow steps. Next, a notifications unit126can be executed whereby a notification or alert, each of which is used interchangeably herein, characterizing the operational issue can be generated and sent to the user through a variety of possible communication channels or mechanisms. The notifications can be generated to allow for specific messages and channels of communications to be used depending on the type of alert. Multiple different users can be notified at a time which can vary based on the time of day. Also, the notification can be created and transmitted in a manner determined based on the assigned risk category, such that users are alerted only to on-site equipment issue when certain rules and/or risks are breached. Upon resolution of the operational issue, e.g., a detected fuel leak has been eliminated, a data reassessment unit127, a resolve and learning unit128, and a tools unit113can be executed, thereby completing the analytics component120and the supporting component110for the particular exception. The assigned risk category can be de-escalated in response to issue resolution, but the fuel data can still be collected and monitored to ensure the exception no longer occurs. Moreover, the wetstock system100can maintain records for continual improvement thereof, such as validation of the workflow and training of models. In this regard, machine learning techniques can be applied to train rules, thresholds, and/or settings, using available information (e.g., collected fuel data, generated exception, workflow, notifications, etc.) as input. As a result, the workflows and notifications provided through the wetstock system100in response to exceptions can improve throughout the operational lifespan of the system. As an illustrative example, it is assumed that a fuel storage facility is equipped with a tank overfill alarm that activates when an ATG coupled to a fuel tank detects a particular stock level volume. A fuel data collection device (not shown), such as an Internet of Things (IoT) device, located on-site can collect the ATG data and transmit the collected data to a remotely located wetstock management server (not shown) configured to perform operations of the wetstock system100. Particularly, the wetstock management server can execute the aforementioned units of the analytics components120including the data processing unit121to collect the ATG data from the fuel data collection device in conjunction with fuel data measured by other on-site devices and/or manually inputted data, the algorithms unit122to process the collected data according to one or more predefined rules and/or models, the exceptions and services unit123to determine whether an exception, e.g., a fuel tank level outside of a safe range, exists, the risk escalation unit124to assign a detected exception a risk category, and the workflow unit125to generate a workflow providing end-to-end support for the user to resolve the issue causing the exception. The wetstock management server can further execute the notifications unit126to determine a notifying action dependent upon user-specific and tank-specific settings. In some cases, multiple risk categories each of which corresponding to one or more predefined exception criteria can be created. Each risk category can also correspond to one or more channels of electronic communication through which a notification is to be delivered, such as an automated phone call, short message service (SMS) message (text message), e-mail, push notification to wetstock management application, and so on. As the risk category increases in urgency, more communication channels can be selected for transmission of the notification. A risk category among the plurality of possible risk categories can be assigned to the exception based upon the exception criteria(s) associated with each risk category. For illustration, an example set of risk categories and corresponding criteria and communication channel is provided below in Table 1. TABLE 1Risk CategoryCommunicationTypeException CriteriaChannelRisk Category 1Nominal capacity has beenPhone call; SMS; E-equaled or exceededmail; Push notificationRisk Category 2Safe working capacity (SWC)SMS; Push notificationhas been breached by morethan 100 litersRisk Category 3SWC has been breached byPush notificationless than 100 litersRisk Category 4SWC has not been breachedNo alert Based on the risk category of the generated exception, a notification describing the operational issue can be generated and electronically transmitted via the corresponding electronic communication channel(s). The notification can include any available data characterizing the nature of the issue. As the risk of the operational issue, e.g., the severity of the fuel leak, increases, so too does the number of communication channels through which the notification is transmitted. The day and time of the detected operational issue can determine the user or users that are notified. The notified user(s) can then log on to an application of the wetstock system100and review the workflow generated by the wetstock management server for the exception that will guide them through resolving the issue (e.g., fuel leak). The workflow can include recommendations such as checking particular locations, e.g., interceptors, forecourt sensors, etc., for signs of fuel spills, contacting relevant authorities or response teams, and so on. After the issue has been resolved, the wetstock system100can maintain records for learning purposes so that in the event of a future stock reading at the same height, the risk of such issue is already known and can be more efficiently addressed. The wetstock system100can implement a wetstock management computer application with a user interface through which the user can interact with the wetstock system100by viewing workflows, receiving notifications (e.g., push notifications), and the like. In this regard,FIG.2is an exemplary user interface implemented by the wetstock system100. The user interface200can include a variety of interactive elements intended to inform the user of information characterizing one or more aspects of the fuel storage facility provided by the wetstock system100. For example, the user interface200can include a test site selection section210in which the user can select a test site of the fuel storage facility (e.g., Test Site1), as well as a particular tank (e.g., Tank1) of the selected site. The test site selection section210can also include selectable elements (e.g., buttons, drop-down menus, test input bars, etc.) enabling the user to quickly select a current site, receive confidence predictions (described below), and/or reset all input data collected by the wetstock system100. Additionally, the user interface200can include a fuel data section220displaying information based on collected fuel data characterizing one or more aspects of the fuel storage facility. For example, the fuel data section220can include status indicators of sensors, alarms, and so forth within the fuel storage facility. Moreover, the fuel data section220can include visual indicators of both active and inactive exceptions as determined automatically by the wetstock system100based upon the collected fuel data. As shown inFIG.2, the fuel data objects221can indicate inactive exceptions, while the fuel data objects222can indicate active exceptions. As such, the wetstock system100is not limited to recognizing only a single exception at a time, but can recognize multiple exceptions under certain circumstances. In some embodiments, the wetstock system100can combine the multiple exceptions as indicated by fuel data objects221and222in order to predict the most likely cause of the exceptions. In this regard, the user interface200can include a confidence prediction section230in which one or more possible faults are provided in order of probability calculated using wetstock system100analytics described above. As shown inFIG.2, for example, a line issue can be predicted as the most likely fault or cause of the current exceptions. Based upon the predicted most likely fault, the wetstock system100can generate a workflow in the manner described above, which can be displayed for the user through the user interface200. In some embodiments, the user can select a particular predicted cause of the exceptions, and the workflow can be generated based upon the selected cause. FIG.3is a flowchart illustrating an exemplary, simplified procedure implemented by the wetstock system100. The procedure300can start at step305, and continue to step310, where, as described in greater detail below, the wetstock system100can perform automated wetstock management to enable resolution of exceptions identified during operation of a fuel storage facility. At step305, one or more sensors of a plurality of sensors (e.g., ATGs, fuel leak detection sensors, magnetostrictive probes, point of sale devices, forecourt controllers, back office systems, fuel dispensers, etc.) disposed in the fuel storage facility can sense fuel data of the fuel storage facility. The fuel data can include any type of measurement data characterizing one or more aspects of the fuel storage facility including, for example, fuel tank levels, water content, leak detection, flow readings, equipment status, and so on. At step310, a wetstock management server communicatively coupled to the plurality of sensors can collect the acquired fuel data, via data processing unit121, and process the fuel data to detect whether the fuel data satisfies an exception indicative of an operational issue of the fuel storage facility based on one or more predefined rules or models stored in the wetstock management server, via algorithms unit122and exceptions and services unit123. The wetstock management server can be a remote, i.e., cloud-based, server located outside of the fuel storage facility. In some embodiments, the measured fuel data can be collected by a fuel data collection device (not shown), such as an IoT device, located on-site, and the fuel data collection device can transmit the collected data to the wetstock management server for processing. Upon detecting that the collected fuel data satisfies an exception, the procedure300can proceed toward one or more outputs including generating and displaying a workflow (steps315through325) and identifying a risk category and transmitting an alert via select communication channels (steps330through340). In some embodiments, only one of the outputs can be carried out. In other embodiments, both outputs, or any combination thereof, can be carried out. At step315, the operational issue of the fuel storage facility can be identified based on the exception. For instance, if the exception derives from a sudden decrease in a fuel tank level, the operational issue can be identified as a fuel loss or leak. At step320, a workflow can be generated, via workflow unit125, for assisting a user of the fuel storage facility to resolve the operational issue identified in step315. The workflow can include a series of steps providing end-to-end support for the user to resolve the operational issue in the most appropriate and efficient manner. The workflow can be generated dynamically, that is, in real-time, taking into account on-site equipment and the seriousness of the issue. At step325, a device communicatively coupled to the wetstock management server can display a visual characterization of the workflow, such as a listing of the workflow steps. The device, e.g., a computer, a mobile device, a tablet, or the like, can include a display unit configured to display the visual characterization of the workflow. In some embodiments, the user can interact with the device by, for example, indicating through the device that a workflow step has been completed, that additional assistance is necessary, or the like. Meanwhile, at step330, a risk category among a plurality of predefined risk categories can be assigned to the exception, via risk escalation unit124. Identifying the risk category can be carried out based on one or more exception criteria associated with each of the plurality of predefined risk categories. For example, as shown in Table 1, criteria relating to an amount by which fuel exceeds a predefined maximum limit can correspond to each risk category. The exception detected in step310can be compared with the exception criteria to assign the appropriate risk category to the exception. At step335, one or more electronic communication channels among a plurality of predefined electronic communication channels can be selected for transmission of an alert characterizing the operational issue to the user. Referring again to Table 1, each of the predefined risk categories can correspond to a particular set of electronic communication channels. Thus, the one or more electronic communication channels can be selected based on the assigned risk category. At step340, an alert or notification characterizing the operational issue can be electronically transmitted to the user, via notifications unit126, using the one or more electronic communication channels selected in step335. The number of users receiving the alert can depend upon the degree of urgency associated with the assigned risk category as well as the date and time at which the exception is detected. Also, the alert can be created and transmitted such that users are alerted only to on-site equipment issues when certain rules and/or risks are breached. The procedure300can continue throughout operation of the fuel storage facility. The techniques by which the steps of procedure300may be performed, as well as ancillary procedures and exception criteria, are described in detail above. It should be noted that the steps shown inFIG.3are merely examples for illustration, and certain other steps may be included or excluded as desired. Further, while a particular order of the steps is shown, this ordering is merely illustrative, and any suitable arrangement of the steps may be utilized without departing from the scope of the embodiments herein. Even further, the illustrated steps may be modified in any suitable manner in accordance with the scope of the present claims. Accordingly, the automated wetstock management system as discussed herein can combine all known alerts and data points, site equipment, and infrastructure details into a model to provide a user with the most likely on-site fault based on both risk, likelihood, real-life probability, and the equipment on-site. By applying artificial intelligence and machine learning techniques to wetstock management procedures, wetstock management can be performed more efficiently, thereby saving costs and improving safety. It should be understood that terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “includes,” or variations thereof, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. The term “coupled” denotes a physical relationship between two components whereby the components are either directly connected to one another or indirectly connected via one or more intermediary components. Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about,” “approximately,” and “substantially,” are not to be limited to the precise value specified. In at least some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Here and throughout the specification and claims, range limitations may be combined and/or interchanged, such ranges are identified and include all the sub-ranges contained therein unless context or language indicates otherwise. Additionally, it is understood that one or more of the above methods, or aspects thereof, may be executed by at least one control unit. The term “control unit” may refer to a hardware device that includes a memory and a processor. The memory is configured to store program instructions, and the processor is specifically programmed to execute the program instructions to perform one or more processes which are described above. The control unit may control operation of units, modules, parts, devices, or the like, as described herein. Moreover, it is understood that the above methods may be executed by an apparatus, such as a wetstock management server, comprising the control unit in conjunction with one or more other components, as would be appreciated by a person of ordinary skill in the art. The foregoing description has been directed to embodiments of the present disclosure. It will be apparent, however, that other variations and modifications may be made to the described embodiments, with the attainment of some or all of their advantages. Accordingly, this description is to be taken only by way of example and not to otherwise limit the scope of the embodiments herein. Therefore, it is the object of the appended claims to cover all such variations and modifications as come within the true spirit and scope of the embodiments herein. | 30,935 |
11860146 | DETAILED DESCRIPTION In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, to avoid unnecessarily obscuring aspects of the present teachings. Unless specifically stated otherwise, and as may be apparent from the following description and claims, it should be appreciated that throughout the specification descriptions utilizing terms such as “processing,” “computing,” “calculating,” “determining,” or the like, refer to the action and/or processes of a computer or computing system, or similar electronic computing device, that manipulate and/or transform data represented as physical, such as electronic, quantities within the computing system's registers and/or memories into other data similarly represented as physical quantities within the computing system's memories, registers or other such information storage, transmission or display devices. As used herein, a soil “nutrient” measurement refers to a measurement of ions in the soil, soil pH and soil moisture. Ion measurement can include nitrogen, nitrate, potassium, phosphate, chloride, and the like. The present disclosure generally relates to a soil nutrient measurement platform that includes a soil nutrient measurement device having a housing with a plurality of elements extending to an exterior of the housing. These elements include a metal plug of a reference electrode, one or more sensing electrodes, a moisture sensor and one or more optional water outlets. Automation circuitry may be included inside the housing to receive signals from the reference electrode, the one or more sensors, and the moisture sensors, and determine levels of certain nutrients and/or soil pH, as described in greater detail below. Referring toFIG.1, a reference electrode100is shown. The reference electrode100can include a metal plug102that extends beyond a sealed bottom108thereof. A reference metal, such as a titanium nitride (TiN) plate104may extend from inside the reference electrode100to extend out a top end thereof. A buffer solution106may be present inside the reference electrode100, providing liquid communication between the metal plug102and the titanium nitride plate104. Referring also toFIG.2, the reference electrode100may be electrically connected to a voltage source, such as power supply149as part of automation circuitry148. A sensing surface110can be connected to a transducer112. The transducer may be a bipolar junction transistor (BJT), a field effect transistor (FET), or the like. The change in the surface potential of the sensing surface110due to binding of target ions causes the signal at the transducer112to change. The reference electrode100applies a voltage to the medium in which it is inserted, such as the soil. This can be used to determine a desired measurement, such a pH, nutrient content, or the like. While the Figures illustrate a single transducer block112, it should be understood that where there are multiple sensing surfaces110, each sensing surface110may be connected to its own transducer112. Referring now toFIGS.3and4, a soil nutrient measurement device130can include a housing132having a plug surface134penetrating from the housing132at a bottom surface133thereof. The plug surface134is the bottom portion of the metal plug102of the reference electrode100as shown inFIGS.1and2. Further, one or more sensing surfaces136can penetrate through the bottom surface133of the housing132. The one or more sensing surfaces136may be similar or differently shaped surfaces, including planar surfaces, rod-shaped protrusions, or the like. Finally, a moisture sensor138can penetrate through the bottom surface133of the housing132. The one or more sensing surfaces136may include four sensing surfaces136, as shown inFIG.3. Each sensing surface136may be configured to make a different measurement. For example, the sensing surfaces136ofFIG.3may be configured to measure one or more of pH, nitrogen levels, phosphorus levels, potassium levels, chloride levels, temperature, and the like, for example. The sensing electrodes can be formed from an appropriate material that specifically binds the target ion to make the various measurements. For example, the sensing electrodes for pH may be constructed out of or coated with titanium nitride. The housing132may be a tubular material, such as a PVC pipe, metal tube, or the like. The side of the housing132may vary, depending on the particular application, and may be from about 1-inch to over 6-inches. The housing132may be hollow or may be filled with a shock absorbing material or fluid. While the Figures show a round housing, it should be understood that the housing may be configured in various shapes, sizes, and configurations based on the type of soil/terrain. Referring toFIG.4, an automation circuitry148may interconnect the one or more sensing surfaces136, the moisture sensor138and the reference electrode100. The automation circuitry148can receive signals from these components and determine the appropriate measurements, such as pH, nutrient levels, moisture levels, temperature, or the like. WhileFIG.4shows the one or more sensing surfaces136simply connected to the automation circuitry148, it should be understood that each of the one or more sensing surfaces136are connected to a transducer112within the automation circuitry148. Further, whileFIG.4shows the reference electrode100simply connected to the automation circuitry148, it should be understood that the reference electrode is connected to the power supply149, as illustrated inFIG.2. The automation circuitry148can further include a wireless transceiver140for sending and receiving data, a processor142for executing program code stored in a memory144, such program code configured at least for determining the appropriate measurements from the signals received from the various sensors. The memory144may further be used to store the data from the sensors or the processed data of the appropriate measurements determined from the signals from the sensors. The automation circuitry148can also include a GPS locating device147, permitting measurements to be linked with a specific location the measurement was performed. Finally, the automation circuitry148can include the transducer112, or the set of transducers, as discussed above. In some embodiments, an external notification device146can be provided on the exterior of the housing. The notification device146can provide a visual or audio notification to the user of a particular nutrient level or moisture level. For example, the notification device146can light green to illustrate proper fertilizer application (e.g., nutrient levels within a predetermined range), red for too low fertilizer application, yellow for low moisture levels, or the like. In other embodiments, a user may have a portable electronic device to receive data, in real time, from the wireless transceiver140. In other embodiments, the user may make measurements and analyze those at a later time. Referring now toFIGS.5and6, the soil nutrient measurement device130ofFIGS.3and4can be configured with a water reservoir160that can provide water to soil when the moisture sensor138detects a moisture level below which the sensing surfaces136may make an accurate measurement. One or more water outlets152of the soil nutrient measurement device150can be provided at the bottom surface133of the housing132to expel water from the water reservoir160when needed to make an accurate measurement. A valve162may be controlled by the automation circuitry148to control the flow of water from the water reservoir160to the water outlets152. Referring now toFIG.7, a soil nutrient measurement device170can be configured in a similar manner to those described above, except that the sensing surfaces136A,136B, the moisture sensors138A,138B, and the metal plug134A,134B, may be disposed at sides of a housing172of the measurement device170. A first set of sensing surfaces136A, moisture sensors138A and a metal plug134A can be disposed at a lower end of the housing172. A second set of sensing surfaces136B, moisture sensors138B and a metal plug134B can be disposed at an upper end of the housing172. Thus, the measurement device170can provide measurements at more than one depth. While two depths for making measurements are shown inFIG.7, additional sets of sensing surfaces, moisture sensors and the metal plug can be provided at various locations along a longitudinal length of the measurement device170, or at the bottom surface of the measurement device170, to provide data at more than two depths, thereby providing different soil parameters at different depths at a same topographical location. A lower transducer112A, or set of lower transducers where there are multiple lower sensing surfaces, and an upper transducer112B, or a set of upper transducers where there are multiple upper sensing surfaces can be provided to receive signals from the lower and upper sensing surfaces and metal plugs of the reference electrodes to make the appropriate measurements, as discussed above. While not the automation circuitry148does not identify specific components for clarity, the automation circuitry148can be similar to that described above with reference toFIG.4described above. Referring toFIGS.7and8, the bottom surface174of the housing172may lack any sensors protruding therethrough. In one embodiment, the bottom surface174may include a soil penetrating shaped tip182, such as a pointed tip, as shown inFIG.8. Such a tip182can help the measurement device180penetrate the soil to achieve the appropriate depths for making multiple measurements at multiple depths. Referring now toFIG.9, a soil nutrient measurement device190can be similar to that of the measurement device170ofFIG.7, with the addition of a water reservoir192that can feed water to a plurality of water outlets194. The operation of the water reservoir192is similar to that described above with respect toFIG.6. Similar to that described with reference toFIGS.2and4, with reference toFIGS.6through9, the one or more sensing surfaces136are shown to simply connect to the transducers112A,112B within the automation circuitry148. It should be understood that each of the one or more sensing surfaces136are connected to a transducer within the automation circuitry148. Further, while the reference electrode100and the moisture sensors138are shown simply connected to the automation circuitry148, it should be understood that the reference electrode is connected to the power supply149, as illustrated inFIG.2. The circuitry for operating the sensor, which includes the transducer112and the reference electrode, is included in the automation circuitry148. Referring toFIG.10, the soil nutrient measuring device130,150,170,190can be attached to a tractor1000. As the tractor1000tills a field, the tractor1000can be intermittently stopped and the device130,150,170,190can be lowered into the ploughed soil. Soil moisture can be measured and, if the moisture is too low, then water can be injected to wet the soil. After a wait of a fixed amount of time, nutrients (nitrate, pH and other ions) can be measured at that location. Based on the nutrient reading, fertilizer can be applied to the field in an appropriate amount, thus avoiding over or under fertilization. Referring toFIG.11, the soil nutrient measuring device130,150,170,190can be attached to a drone1100. The drone1100can be used to lower the device130,150,170,190into a field to measure soil nutrients. As discussed above, GPS technology may be used to track the location of the measurements so that a user can determine specific locations that may require fertilization. CONCLUSION The descriptions of the various embodiments of the present teachings have been presented for purposes of illustration but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein. While the foregoing has described what are considered to be the best state and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings. The components, steps, features, objects, benefits and advantages that have been discussed herein are merely illustrative. None of them, nor the discussions relating to them, are intended to limit the scope of protection. While various advantages have been discussed herein, it will be understood that not all embodiments necessarily include all advantages. Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain. Numerous other embodiments are also contemplated. These include embodiments that have fewer, additional, and/or different components, steps, features, objects, benefits and advantages. These also include embodiments in which the components and/or steps are arranged and/or ordered differently. While the foregoing has been described in conjunction with exemplary embodiments, it is understood that the term “exemplary” is merely meant as an example, rather than the best or optimal. Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims. It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms “comprises,” “comprising,” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element. The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments have more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter. | 16,657 |
11860147 | DETAILED DESCRIPTION A device for determining the real evapotranspiration of a vegetated surface10of a soil12, comprises a sensor14for measuring the humidity of the soil12, a porous evaporator16, a tank18of liquid water underlying the evaporator16to which it is connected by a suction tube20, and a control CPU22. The tank18kept in depression to allow the water to rise through the tube20towards the evaporator16, is provided with a water level meter24, the measured values whereby are transmitted to the CPU22. The evaporator16has a composite structure comprising a lower plate28of ceramic material enclosed by a waterproofed ceramic capsule30, an upper layer32of fabric exposed to the atmosphere and having a color and albedo corresponding to those of the vegetated surface, and an intermediate layer34having resistance to the water vapor flow which depends on the temperature parameter. Preferably, the upper layer32is Green Canvas acrylic fabric produced by Sunbrella Fabrics (1831 N. Park Avenue Glen Raven, NC, USA), green Erin color and albedo of about 0.2. Advantageously, the intermediate layer34is of a fabric which incorporates shape memory polymeric material which varies its geometry following the variation of a thermal parameter such as temperature. Preferably, such shape memory polymeric material is poly-NiPAAm/chitosan microgel. In the intermediate layer34a resistor36is embedded which is part of an electrical circuit (not shown in the diagram ofFIG.1) and whose heat generation is controlled by the CPU22on the basis of the temperature values of the intermediate layer34detected by a sensor, such as a thermocouple38. A process for determining the real evapotranspiration of the vegetated soil surface by using the aforementioned device installed therein is now described. Liquid water previously fed into the tank18is suctioned through the tube20into the evaporator16. Crossing the latter, the water vaporizes and finally passes into the atmosphere exiting from the upper layer32. Thanks to the level measurements26performed by the meter24, the CPU22is able to calculate the decrease in the quantity of water present in the tank18, which corresponds to the vaporized quantity. The CPU22is also able to control the resistance, or rather its inverse, i.e. the permeance, to the vapor flow of the intermediate layer34of the evaporator16depending on the humidity value of the soil12measured by the sensor14, in a manner such that the calculated flow of vaporized water, or the quantity of vapor flowing in the unit of time through the surface unit, substantially corresponds to the flow of evapotranspiration which actually takes place through the vegetated surface10of the soil12and which is therefore estimated by the device in a suitably accurate manner. In particular, the more the soil12is wet, the more the resistance to the vapor flow of the evaporator16is decreased, while the more the soil12is dry the more the resistance to the vapor flow of the evaporator16is increased. For this purpose, the fact that the resistance/permeance to the water vapor flow of the intermediate layer34of the evaporator16depends on its temperature is exploited. Therefore, the CPU22controls the latter parameter, so as to cause the evaporator16to assume the desired value of resistance/permeance to the vapor flow at a given rate of humidity of the soil12. Specifically, the temperature is controlled by the CPU22by regulating the generation of heat by the resistor36based on the temperature values detected by the thermocouple38, according to well-known principles of system thermal regulation. For example, if the detected temperature is lower than the desired one, heat generation is increased by increasing the intensity of electric current flowing through the resistor and/or the resistance of the latter, or vice versa, so as to compensate for the deviation of the temperature parameter from the desired value. Of course, the principle of the invention being unchanged, the details of construction and the embodiments may widely vary with respect to what has been described purely by way of example, without thereby departing from the scope of the invention as defined in the appended claims. In particular, any “thermally-driven” fabric could be used as constitutive of the intermediate layer of the evaporator, in which the temperature induced through a thermo-resistor causes the permeance to vary due to the vapor flow as it exceeds the so-called activation temperature of the constitutive shape-memory polymers. Also porous membranes based on nano-polyurethane fibers sensitive to thermal stimuli belong to this category of fabrics, which are inserted through electrospinning and laid therein (Charly Azra et al., 2015: Mondal et al., 2006). Such polymers in particular have a permeability to water vapor variable in a representative domain of the main agricultural crops, and in particular <1000 g*m−2*d−1, which corresponds to a stomatal resistance of about 100 s*m−1. It is also possible to use as constitutive materials of the intermediate layer of the evaporator polymers which can be activated through low-power electrical stimuli, thus avoiding the generation of heat and the consequent alteration of the heat balance in the area of the device affected by the water exchange process: in fact, one thermal stimulus, different from the environmental one, modifies the water state and therefore the natural vapor concentration gradient of the system. Such a stimulus is for example represented by a voltage difference which does not cause the material to heat up, but causes its porosity to vary in combination with the thickness of the material itself. A combined variation of porosity and thickness results in a variation of the tortuosity, much more effective than the variation of porosity alone in order to influence the resistance to the water vapor flow. Furthermore, the controlled creation of a gap between the porous plate and the intermediate layer of the evaporator allows controlling the global permeance of the device also through this way. For this purpose, graphene actuators or actuators based on polythiophene gels capable of converting an electrical stimulus into mechanical energy can be used, so as to generate a gap between the porous plate and the intermediate layer of the evaporator, varying the resistance to the water vapor flow. | 6,394 |
11860148 | DETAILED DESCRIPTION OF THE DRAWINGS While the concepts of the present disclosure are susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that there is no intent to limit the concepts of the present disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives consistent with the present disclosure and the appended claims. FIG.1shows an illustrative arrangement of an electrical transformer10including a gas analysis system12for determining characteristics of dissolved gases within fluid of the transformer10. The transformer10illustratively includes a housing14defining an interior16and electrical windings18arranged within the interior16of the housing14. The electrical windings18illustratively comprise windings of electrical wiring forming a series of turns about limbs of the transformer10to produce electromagnetic effect when current is passed through the wiring. The transformer10is illustratively embodied as a high-voltage, three-phase, core type transformer, but in some embodiments, may include any manner of electromagnetic device including but not limited to shell type and/or single or multi-phase. In the illustrative embodiment as shown inFIG.1, the housing14contains fluid20for cooling and/or electrically insulating the components of the transformer10, such as the electrical windings18. As mentioned above, dissolved gases can develop within the fluid20as a result of operable use of the fluid20for cooling and/or insulation (for example, by fluid breakdown and/or faulty operational issues of the transformer10, generally, including leaks in the housing14) and/or the fluid20may carry gases generated from the degradation of other insulation materials in the transformer, such as paper. The gas analysis system12is illustratively arranged to extract dissolved gases from the fluid20for analysis. Referring toFIG.1, the gas analysis system12illustratively includes an extraction probe22for extracting gas from the fluid20. The extraction probe22is illustratively arranged in contact with the fluid20and is formed of a gas-permeable material to permit permeation of dissolved gases from the fluid20. The extraction probe22is illustratively embodied as a conduit defining an interior passage for receiving and communicating gas. The gas-permeable material illustratively permits dissolved gases to permeate into the interior passage while inhibiting ingress of liquids, for example, dielectric oils. Suitable gas-permeable materials may include one or more fluoropolymers. In the illustrative embodiment, the extraction probe22is formed as an extraction coil having coil loops in contact with the fluid20. In some embodiments, the extraction probe22may include any suitable shapes and/or forms. As shown inFIG.1, the gas analysis system12illustratively includes a gas analysis module24for conducting analysis of gas. The gas analysis module24is illustratively fluidly connected with the extraction probe22and forms a gas circuit for circulation of gas between the extraction probe22and the gas analysis module24. An exemplary housing25of the gas analysis module24is shown inFIG.1. The gas analysis module24illustratively includes a gas cell26for receiving gas extracted from the fluid20. The gas cell26illustratively includes a cell body28defining a cavity30through which gas is passed for analysis. In the illustrative embodiment, the gas analysis system12conducts optical analysis of gas to determine gas characteristics. In the illustrative embodiment, portions of the gas circuit other than the extraction probe22, including the cavity30, are hermetically sealed to ambient air such that only the extraction probe22is arranged to allow permeation of gases into and out of the gas circuit, thereby allowing gas exchange with the transformer fluid20. As shown inFIG.1, the gas analysis module24illustratively includes a gas analysis device32for conducting analysis of gas within the gas cell26. In the illustrative embodiment, the gas analysis device32is an optical device embodied as a light spectroscopy device, namely a Fourier transform infrared (FTIR) spectrometer. In some embodiments, the gas analysis module24may perform any manner of gas analysis techniques and may include any suitable configuration and/or components to perform such techniques, for example, but without limitation, ultra violet light spectroscopy, Raman spectroscopy, photoacoustic spectroscopy, tunable diode laser absorption spectroscopy (TDLAS). The gas analysis device32illustratively performs light spectrum analysis of gas within the gas cell26. In some embodiments, the gas cell26may use optical path length enhancement techniques such as multi-pass cells or resonant cavities. Multi-pass cells may include White cell, Herriot cell, folded path cells, and/or other multi-pass cells. Resonant cavities may include Fabry-Perot cavities, cavities designed for cavity ring-down spectroscopy, integrated cavity output spectroscopy (ICOS), off-axis integrated cavity output spectroscopy (OA-ICOS), and/or other optical path length enhancement techniques. As shown inFIG.1, the gas analysis device32illustratively includes a light source34and detectors36,38for receiving light from the light source34. As discussed in additional detail below, the light source34illustratively generates infrared (IR) light for propagation through gas for observation of the light absorption characteristics of the gas. The light directed through the gas is received by the detectors36,38. The detectors36,38are illustratively embodied as photodetectors that receive light propagated through gas (but that has not been absorbed by the gas) and that generate an electrical signal indicating the light received. The detectors36,38are illustratively embodied as analog detectors that generate an analog signal that is converted to a digital signal by an analog-to-digital converter. In some embodiments, the detectors36,38may include any suitable arrangement of signal generation for gas analysis. The gas analysis device32illustratively determines characteristics of the gas based on the light received by the detectors36,38. In the illustrative embodiments, the gas analysis module24can determine characteristics of dissolved gas within the fluid20by analysis of gas extracted by the gas analysis system12from the transformer10. Relevant characteristics of dissolved gases within the fluid20of the transformer10include the presence and/or identification of such gases and their dissolved concentrations within the fluid20. A non-exhaustive list of gases of interest within the fluid20may include, for example, oxygen (O2), nitrogen (N2), hydrogen (H2), carbon dioxide (CO2), and/or hydrocarbons (e.g., methane, ethane, acetylene, and/or ethylene), among other gases. The gas analysis device32may also monitor water vapor (H2O) extracted from the moisture dissolved in transformer oil20. Referring now toFIG.2, the transformer10is shown in partial cross-section for descriptive purposes. The housing14of the transformer10illustratively includes a sampling portal40defining a portion of the interior16containing fluid20as part of the housing14. The sampling portal40illustratively includes pipe extension42connected with a wall43of the transformer10and a shroud44secured with the pipe extension42. The extraction probe22is illustratively mounted within the shroud44in contact with fluid20. The extraction probe22is illustratively mounted within a fluid chamber46defined by the shroud44as a part of the interior16. The chamber46illustratively contains fluid20as part of the housing14and fluidly communicating through the pipe extension42. In the illustrative embodiment, the pipe extension42illustratively includes a valve48disposed fluidly between the wall43and the chamber46to permit isolation of the extraction probe22, but in some embodiments, the valve48may be excluded. In some embodiments, the extraction probe22may be arranged inside of the wall43. The gas analysis system12illustratively includes an extraction module50as shown inFIGS.2-4. The extraction module50illustratively provides a packaging platform for mounting the extraction probe22within the housing14as shown inFIG.2. Referring toFIGS.3and4, the extraction module50illustratively includes a mounting frame52and the extraction probe22secured with the mounting frame52. In the illustrative embodiment, a pump54is mounted to the mounting frame52and is fluidly connected with the extraction probe22to provide a motive pressure source for circulation of gas within the gas circuit. Control valves and/or other flow distribution devices for operation of the gas circuit may be mounted to the mounting frame52. As shown inFIGS.3and4, the mounting frame52illustratively includes an engagement wall56and a probe arm58extending from the engagement wall56. The engagement wall56illustratively includes a face60that forms at least a portion of fluid boundary of the chamber46. The engagement wall56illustratively supports the probe arm58for extension within the chamber46for contact with fluid20. In the illustrative embodiment as shown inFIGS.3and4, the probe arm58includes a spool62having the extraction probe22(embodied as an extraction coil) looped around the spool62. In the illustrative embodiment, the extraction coil is looped around the spool62to form a number of coil turns having a successively stacked arrangement for exposure to fluid20. Increasing the number of coils may improve the effective exchange surface between oil and gas phase and may reduce the response time of the measurement. Gas circulated through the extraction probe22portion of the gas circuit illustratively flows successively through each of the coil turns and out for circulation to the gas analysis device32. In the illustrative embodiment, the extraction probe22is fluidly connected with the pump54for communication of extracted gas through the gas circuit. As best shown inFIG.4, the spool62is illustratively cantilevered from the engagement wall56and provides structure for arranging the extraction probe22for contact with fluid20. In some embodiments, the extraction probe22may be secured to the mounting frame52in any suitable manner and/or arrangement. The spool62is illustratively formed as a structural frame defining an annular spool bed61for receiving the extraction probe22wrapped thereon and defining openings63extending through the spool bed61to permit fluid20to contact interior portions of the extraction probe22to increase the effective exchange surface between oil and gas phase. The spool62is illustratively shaped as a hollow cylinder to permit fluid20therein. The spool62illustratively includes a strut65bridging radially across the spool bed61to provide structural support and defining openings67to permit circulation of fluid20through the spool62. Returning briefly toFIG.1, as previously mentioned, the extraction probe22and the gas analysis module24are fluidly connected to define a gas circuit for circulation of gas therebetween. In the illustrative embodiment, the extraction probe22and the gas analysis module24are fluidly connected by transport conduit64including conduit segments66,68. The segment66is illustratively embodied as a supply segment for providing gas from the extraction probe22to the gas analysis module24and the segment68is embodied as a return segment for providing gas from the gas analysis module24to the extraction probe22. In the illustrative embodiment, the pump54is arranged fluidly along the supply segment66and provides a lower pressure level at the output of the extraction probe22(relative to the pressure of the gas cell26), which may assist with extraction of dissolved gases. In some embodiments, the gas circuit may be formed substantially or entirely by the extraction probe22and gas analysis module24being fluidly connected with each other by direct connection and/or with little or no transport conduit64. In some embodiments, the extraction probe22and gas analysis module24may be partly or wholly combined into a common module and/or arranged within a common housing for compact arrangement. The gas circuit illustratively provides a circulation loop for communication of gas between the extraction probe22and the gas analysis module24. In the illustrative embodiment, the gas circuit encourages the gas extracted from the fluid20to reach and maintain equilibrium with dissolved gases within the fluid20. Such passive extraction and non-destructive analysis can avoid practical challenges with active sampling, such as fluid leaks, contamination, and waste materials, among others. Passive extraction does not rely on a precise determination of the extraction rate of the gas and thus reduces the need for factory calibration of each analyzer extraction rate. As mentioned above, the pump54illustratively assists circulation of the gas through the gas circuit and may assist extraction, but in some embodiments, circulation of the gas through the gas circuit may be provided by any suitable device(s), including but not limited to redundant pumps arrangements or arrangements without a pump such as convective and/or diffusive transport. Referring now toFIG.5, a diagrammatic illustration of the gas analysis module24is shown. As mentioned above, the gas analysis module24illustratively includes the gas analysis device32arranged for conducting analysis of gas within the gas cell26. The light source34of the gas analysis device32illustratively includes a light generation source70. In the illustrative embodiment, the light generation source70includes an interferometer for modulating mid-IR light, for example, with a wavelength within a range of about 1 microns to about 50 microns (in some illustrative embodiments), about 2.5 microns to about 25 microns (in other illustrative embodiments), and about 2.5 to about 16 microns (in still other illustrative embodiments). The light generation source70also illustratively includes at least one light generator72for generating the mid-IR light and may include various relays, filters, and/or other conditioning devices (collectively indicated as74) for providing suitable light for gas analysis. A non-limiting example of a suitable light generator72may include a glow bar (globar). The light source34illustratively includes a relay mirror76arranged to receive a beam of light78from the light generation source70and a beam splitter80arranged to receive the beam78from the relay mirror76. As shown inFIG.5, the gas analysis device32illustratively includes two optical channels as explained herein. The beam splitter80illustratively divides the beam78into two beams of light82,84for spectrum analysis. The beam splitter80is illustratively embodied to have a beam-splitting ratio of 50:50 (50/50 splitter) dividing the beam78evenly into the two beams82,84, but in some embodiments, the beam splitter80may have other suitable beam-splitting ratios. In some embodiments, any suitable arrangement of relays, filters, splitters, and/or other conditioning devices may be employed to propagate light accordingly for gas analysis. Beams82,84propagate through respective defined spaces for collection by detectors36,38. In the illustrative embodiment as shown inFIG.4, analysis of the beams82,84propagated through respective defined spaces can determine characteristics of the gas extracted from the fluid20. The beam82illustratively propagates through the gas cell26for reception by detector36. The beam82illustratively enters the gas cell26through a window86, propagates through the cavity30for interaction with gas therein, and exits the gas cell26through another window88. Light from the beam82exiting the gas cell26is received by the detector36for analysis. The gas within the cavity30affects the beam82in a manner such that the affected light received by detector36can indicate characteristics of the gas within the cavity30. As explained below, the detector36can generate a signal related to the absorption spectrum of the gas within the cavity30based on the light received from beam82. In the illustrative embodiment, the gas within the cavity30absorbs energy from the beam82in the form of electromagnetic radiation. The remaining energy of beam82passes through the gas and is received by the detector36to generate a signal related to an absorption spectrum in the illustrative embodiment. The absorption spectrum of the relevant gas can include the fraction of incident radiation absorbed by the gas sample (in this instance, the gas within the cavity30) over a range of wavelengths and/or frequencies of propagated light. By analysis of the light received by the detector36(for example, but without limitation, the wavelength and/or frequency thereof), the characteristics of the gas within the cavity30can be reliably determined. Moreover, characteristics of the dissolved gases within fluid20can be determined based on the characteristics of the gas within the cavity30. In some embodiments, other analytical techniques and/or equipment may be used to determine gas characteristics. In some embodiments, additional gas analysis devices may be included in the gas cell to detect certain gases, such as hydrogen (H2), oxygen (O2), and/or nitrogen (N2), and some of those additional gas analysis devices may use non-optical measurement principles that do not require gas interaction with light, such as resistive, capacitive, and/or thermo-conductive sensors, by way of example. Accurate determination of the characteristics of gas within the cavity30(and ultimately the dissolved gases within fluid20) should account for contaminants and/or artifacts. Common sources of artifacts includes constituents within the air contained in the gas analysis module24and/or constituents within the air in the vicinity of the transformer10that may enter the gas analysis module24. For example, ambient air within the gas analysis module24can reduce the light received by the detector36even though it cannot enter into the cavity30. Accordingly, reference information regarding the ambient environment can be useful in interpreting the light received by the detector36. In the present disclosure, the terms “air” and “ambient air” are not intended to limit the gas constituents which can be considered, but may include any gas constituent, including constituents of the same species as the dissolved gases of interest in the fluid20. By considering such reference information of ambient air, the characteristics of the gas within the cavity30(and by correspondence, the characteristics of the dissolved gases within the fluid20) can be accurately determined by correction and/or calibration of the light received by the detector36(absorption spectrum). Such corrective approaches can reduce the need for purging, scrubbing, desiccants, relay adjustment, and/or other resource-laden or mechanically demanding techniques to achieve accurate results. As shown inFIG.5, the beam84(split from the beam82) illustratively propagates through a reference space90to provide characteristics of ambient air as reference information. The reference space90illustratively contains ambient gas (illustratively embodied as ambient air) which affects the beam84in a manner such that the affected light received by detector38can indicate characteristics of the ambient gas. The characteristics of the ambient gas can be used in interpreting the light received by detector36. Analysis of the light received by the detector36in combination with the light received by the detector38can allow determination of characteristics of the gas within the cavity30(and, hence, the characteristics of the dissolved gases within the fluid20) by reducing artifacts from the light absorbed by the ambient gas. Reduction of artifacts from the light absorbed by the ambient gas is illustratively achieved by consideration of the corresponding absorption spectra perceived by detectors36,38. In some embodiments, reference information may be obtained by any suitable technique and/or equipment. In the illustrative embodiment as shown inFIG.5, the beam splitter80effectively provides a reference source point92for propagation of light through the defined spaces30,90. The reference source point92is illustratively represented as a single point on the beam splitter80for descriptive purposes. As shown inFIG.5, a propagation distance diis illustratively defined between the reference source point92and each of the detectors36,38. A first propagation distance, referred to as a cell distance dcell, is illustratively defined between the reference source point92and the detector36. The cell distance dcellillustratively corresponds to the propagation of the beam82. A second propagation distance, referred to as a reference distance dRef, is illustratively defined between the reference source point92and the detector38. The reference distance dRefillustratively corresponds to the propagation of the beam84. A third propagation distance, referred to as the cell body distance L, is illustratively defined between the first window86and the second window88delimiting the cavity30of the gas cell26. In the illustrative embodiment, the distance (span) resulting from the subtraction of the cell body distance L from the cell distance dcell(e.g., the span may include the sum of the distance between the reference source point92and the cavity30, S1, and the distance between the detector36and the cavity30, S2, as indicated inFIG.5, either or both of which may contain ambient gas) is substantially equal to the reference distance dRefin such a way that the propagation distances in ambient air between the reference source point92and each of the detectors36,38are substantially equal. In other embodiments, however, the propagation distances in ambient air between the reference source point92and each of the detectors36,38may be different from each other and a correlation can be applied to equate their corresponding absorption spectra. In some embodiments, the cell distance dcellmay be substantially equal to the sum of the reference distance dRefand the cell body distance L. In some embodiments, the propagation distances between the reference source point92and each of the detectors36,38may be substantially equal. In some embodiments, the propagation distances may be different from each other and a correlation can be applied to equate their corresponding absorption spectra. In the illustrative embodiment, the light source34provides the beam of light78for division into beams82,84for respective propagation through each of the cavity30and reference space90. Thus, the light source34illustratively provides each of beams82,84simultaneously from the same source for use in two optical channels; one channel analyzing light propagated through the gas cell26, and another channel analyzing light propagated through the reference space90. The dual channel arrangement using the same source of light can promote uniformity between the spectral characteristics of the channels and decrease adjustable parameters (e.g., moving optics, pressure/temperature modulation of gas samples) and/or the use of commodities (e.g., purge gas, desiccants, scrubbers) in obtaining reliable readings. Devices, systems, and methods of the present disclosure can be advantageous for remote operation where commodities and/or maintenance availability is of concern. Moreover, arrangements of the present disclosure can account for unexpected and/or unknown contaminants, even without identifying the exact contaminant. In some embodiments, the reference information of the ambient gas may not identify one or more of the substances in the gas analysis module24and/or located between the light generator72and detectors36,38. However, the reference information of the unidentified substance can still be considered in accurately determining the characteristics of the gas within the cavity30. Referring now toFIG.6, an illustrative embodiment of the gas cell26is shown. The gas cell26illustratively includes a housing94, which is shown partially cutaway (and semi-transparent) to reveal a cell body96that defines the cavity30therein (the cell body96being an illustrative embodiment of the cell body28ofFIG.1). The cell body96illustratively includes openings98penetrating through the cell body96on opposite ends100,102to connect with the cavity30. Each opening98is enclosed by a respective one of the windows86,88. The cell body96illustratively includes gas ports104,106that each penetrate through the housing94and fluidly connect with the cavity30to form a portion of the gas circuit to communicate gas with the extraction probe22. The gas port104is illustratively embodied as an inlet port (relative to the gas cell26) for receiving gas from the extraction probe22and the gas port106is embodied as an outlet port for sending gas to the extraction probe22. The cell body96illustratively includes pressure and temperature sensor ports108for insertion of pressure and temperature sensors122,124(shown inFIG.1) to monitor the conditions within the cavity30. A cell heater110including electrical leads111is illustratively connected with the cell body96within the housing94to provide temperature control of the cavity30. Referring toFIG.7, an illustrative flow diagram is shown. A process200for determining characteristics of gases is described relative to boxes202-208. In box202, dissolved gases are illustratively extracted from fluid20of the transformer10. In the illustrative embodiment, the dissolved gases are extracted by permeation into the extraction probe22to enter the gas circuit. The process illustratively proceeds from box202to box204. In box204, extracted gas illustratively enters a detection field. In the illustrative embodiment, the extracted gas enters the detection field as it circulates through the gas cell26and light is propagated through the extracted gas for reception by the detector36. In embodiments in which reference information is used for correction, in box206, the characteristics of ambient gases are detected. In the illustrative embodiment, the second channel of the gas analysis module24propagates light through the reference space90and the ambient gas therein for reception by the detector38. The process proceeds from box204to box208. In box208, gas within the detection field circulates out of the detection field. In the illustrative embodiment, gas within the gas cell26is circulated through the gas circuit to return to the extraction probe22. The circulation of the gas within the gas circuit promotes non-destructive testing and enables equilibrium between gas in the gas circuit and dissolved gas within the fluid20. Returning briefly toFIG.1, operation of the gas analysis system12and the various methods and functions described herein is illustratively governed by a control system112. The control system112illustratively includes a processor114, memory device116, and communications circuitry118in communication with each other. The memory device116stores instructions for execution by the processor114to conduct operations of the gas analysis system12. In the illustrative embodiment, the instructions include at least one algorithm for conducting the disclosed operations, but in some embodiments, the instructions may include any of look up tables, charts, and/or other reference material. The communications circuitry118illustratively includes various circuitry arranged to send and receive communication signals between the control system112and various components as directed by the processor114. It will be appreciated that the communications circuitry118also allows the control system112to communicate with other devices, including remote devices, and along various communications networks, such that the gas analysis system12(as well as the transformer10) can be connected to and form part of the Internet of Things. As a result, various components of the gas analysis system12may be sensed and/or controlled remotely across existing network infrastructure. The control system112is illustratively arranged in communication with the gas analysis module24and the pump54through communication links120to communicate signals to govern their operation. Communication links120illustratively include hardwired connections, but in some embodiments may include any of hardwired and wireless connections, and/or combinations thereof. In the illustrative embodiment, the control system112is in communication with each of the light source34, the detectors36,38, gas cell temperature and pressure sensors122,124through individual links120, but in some embodiments, the control system112may be in communication with components of the gas analysis module24by one or more shared links120. The control system112illustratively performs spectrum analysis of the light received by the detectors36,38and determines the characteristics of the gas within the cavity30and the corresponding characteristics of the dissolved gas within the fluid20. As shown inFIG.1, the transformer10illustratively includes a pump126arranged to circulate fluid20within the housing14. Circulating the fluid20can assist in providing uniform distribution of dissolved gases and can assist in extracted gases reaching accurate equilibrium faster than with stagnant fluid conditions. In the illustrative embodiment, the pump126is a thermal pump circulating the fluid20by convective movement. In other embodiments, any suitable device for circulating the fluid20may be used, including, for example, a displacement pump and/or an agitator. In the illustrative embodiment, the control system112is in communication with the pump126to govern operation of the pump126. In the illustrative embodiment, the control system112is embodied to govern operations of all components of the gas analysis system12. In some embodiments, the control system112may govern operation of other systems of the transformer10. In some embodiments, the control system112may include multiple processors, memory devices, and/or communications circuitry that may have any suitable arrangement including but not limited to dedicated and partly or wholly shared arrangements. In some embodiments, another control system112may be dedicated to govern operation of the gas analysis module24and the remainder of the gas analysis system12may be governed by the control system112. As mentioned above, the extraction probe22may include a suitable permeable material, for example, fluoropolymers. Suitable gas-permeable materials may include, for example, but without limitation, amorphous fluoroplastics. such as Teflon® AF and/or Chemours® AF, as marketed by Professional Plastics, Inc. (under affiliation and/or with permission from DuPont®), with typical properties as shown in the table below: Typical Properties of Teflon ® AFOptical ClarityClear: >95%Upper Use Temperature, ° C.285 (545)(° F.)Thermal Stability, ° C. (° F.)360 (680)Thermal Expansion (linear),80ppm/° C.Water AbsoptionNoWeatherabilityOutstandingFlame Resistant LOI, %95Tensile Modulus, Mpa (psi)950-2150(137, 786-311, 832)Creep ResistanceGoodSolubilitySelected solventsResistence to Chemical AttackExcellentSurface-Free EnergyLowRefractive Index1.29-1.31Dielectric Constant1.89-1.93 Non-limiting examples may include Teflon® AF 1600 and/or Teflon® AF 2400 (and/or Chemours® AF 1600 and/or AF 2400) having typical properties as described within the table below: Typical Property Data for Teflon ® AF Amorphous FluoroplasticsASTMGradePropertyMethodUnit16002400ElectricalDielectricD1501.931.90ConstantDissipationD1500.0001-0.00020.0001-0.0003FactorDielectricD149kV/0.1 mm2.11.9StrengthOpticalOpticalD1003%>95>95TransmissionRefractive IndexD5421.311.29ABBE Number92113MechanicalYield StrengthMPa23° C. (73° F.)27.4 ± 1.026.4 ± 1.9150° C. (302° F.)6.7 ± 5.98.7 ± 4.0220° c.(428° F.ITensile StrengthD638MPa23° C. (73° F.)26.9 ± 1.526.4 ± 1.9150° C. (302° F.)7.7 ± 6.1220° c. (428° F.I4.2 ± 1.8Elongation atD638%Break23° C. (73° F.)17.1 ± 5.07.9 ± 2.3150° C. (302° F.)89.3 ± 13.1220° c. (428° F.I8.4 ± 4.1Tensile ModulusD638GPa1.61.5Flexural ModulusD790GPa23° C. (73° F.)1.8 ± 0.11.6 ± 0.1150° C. (302° F.)1.0 ± 0.10.7 ± 0.1220° c. (428° F.IHardnessRockwellD78523° C. (73° F.)10397.5DurometerD1706Shore D777523° C. (73° F.)150° C.(302° F.I7065220° c.(428° F.IImpact StrengthNotched IzodN——DeflectionD648° C. (° F.)Temperature(66 psi)156 (313)200 (392)(264 psi)154 (309174 (345)ChemicalContact AngleD570Degrees104105with WaterCritical SurfaceDynes/cm15.715.6EnergyTaber Abrasioncc/2000 cycles0.1070.2Chemical ResistanceWater Absorption%<0.01<0.01Gas PermeablityH20Barrer1142402602Barrer340990N2Barrer130490CO2Barrer2800OtherT9D3418° C. (°Fl160 (320) ± 5240 (464) ± 10Specific GravityD7921.781.67Melt ViscosityD3835Pa s2657 at 250° C.540 at 350° C.100 s−1100 s−1VolumeE831ppm/° C.260301Coefficient ofThermal Expansion In some embodiments, any suitable materials for gas-permeable, liquid-resistant extraction of dissolved gases from transformer fluid may be applied. The present disclosure includes devices, systems, and methods for oil and gas management for dissolved gas analyzers for use in transformer monitoring. The devices, systems, and methods of the present disclosure may include detecting dissolved gases in insulating oil of electrical equipment using gas equilibrium theory. Equilibrium can be achieved relative to the solubility of a gas in a transformer fluid20, such as mineral oils, ester-based oils, or other insulation fluids, at a given temperature and for a given partial vapor pressure of a gas. Gas solubility can be described with quantities such as Ostwald coefficients of gas solubility that are specific to the type of fluid and to each gas constituent and may have temperature dependency. Gas solubility coefficients can be used to relate the partial pressure of gas in the gas cell with the concentration of dissolved gas in oil. The extracted gases being in equilibrium with the dissolved gases in oil may provide more accurate readings without requiring precise knowledge of extraction rates. In some embodiments, the extraction probe22of the present disclosure may comprise at least one ring of highly gas permeable tubing that is not permeable to liquid. In some embodiments, the extraction probe22may be connected to a closed-circulation system. The closed-circulation system may include one or more pumps for gas circulation and a gas cell, for example, gas cell26, for analytical measurement of the gas. The present disclosure includes devices, systems, and methods adapted to monitor the health of a transformer by measuring dissolved gases within insulating oil of the transformer. For example, the concentration of specific gases can give indications of specific aspects of the operation of the transformer. Direct oil sampling and analysis of dissolved gases contained in transformer oil use active extraction of the gases and active measurement technics that consume the gases through the analyses. They are often implemented by circulating and/or conditioning oil samples outside the transformer in an oil circuit and may present a risk of oil leakage in case of breakage of the oil circuit. By contrast, embodiments of the devices, systems, and methods of the present disclosure permit online measurement with high accuracy and without active extraction. In some of the disclosed embodiments, oil containing the dissolved gases is circulating around highly permeable material tube within a fluid chamber46communicating fluidly with the transformer10through the pipe extension42. In some embodiments, the oil circulation around the permeable tube may be generated by pump, propeller and/or other mechanical systems and/or using thermally induced convection. Gases contained in oil can pass through permeable material to reach the gas phase loop. The permeable material properties can assist in obtaining equilibrium between gases in the liquid and gases in the gas phase loop. The gas loop may include a gas cell with optical inlet and outlet allowing examination of the gases by optical analyzer. Devices, systems, and methods of the present disclosure may include highly permeable fluoropolymer tubing, such as Teflon AF family of amorphous fluoroplastics, by way of example. Highly gas permeable material can promote gas equilibrium and can improve measurement response time. The tubing may be rolled to form one or more turns of a coil. Devices, systems, and methods of the present disclosure may include circulation of the transformer fluid (e.g., oil) around this coil. A structural ring may support the tubing. According to the present disclosure, the fluoropolymer tubing may be connected to a gas circulating loop. The gas circulating loop may include one or more pumps to enhance reliability. In some embodiments, stainless steel tubing may transport gas to a gas cell for analysis. In some embodiments, a spectrometer may perform analysis of the gases. In some embodiments, in-oil sensors may be used for H2and/or H2O measurement. Devices, systems, and methods of the present disclosure may include passive extraction of dissolved gases and measurement, in lieu of active principles for gas separation and measurement. In some embodiments, the present disclosure may include transport of extracted gases without a carrier medium (e.g., a carrier gas). In some embodiments, a lower pressure may be formed within the extraction probe22, relative to the pressure within the gas cell26to assist with extraction of dissolved gases. Devices, systems, and methods of the present disclosure can be used in transformer monitoring and/or specifically in monitoring of dissolved gases analysis in transformer fluid such as oil. For gas phase analysis, gases can be extracted from the transformer oil. Measurement of the gases can require a complex system for analysis, and in some embodiments, the gas sample can be transported to a gas analyzer. The devices, systems, and methods of the present disclosure can be helpful in avoiding transporting the transformer oil itself to an analyzer, which can present a risk of oil leakage in case of tubing breakage. Use of passive measurement and passive extraction of the gases can simplify the calibration and installation of gas analysis systems. Use of high porosity and/or highly permeability material can help to reach equilibrium between gases in oil and gases in the sample gas phase. Using gases equilibrium, without requiring new gases to be sampled, can reduce risk of contamination of the oil. Use of a lower pressure (relative to the pressure within the gas cell) in the gas sampling probe can reduce the response time of the systems. The use of multiple transport pumps can help to reduce risk of failure. In some embodiments, measurement of H2may be conducted in gas phase to reduce the cost. In some embodiments, measurement of O2, H2, and/or N2may be performed optically and/or with non-optical sensors. In some embodiments, O2can be measured by paramagnetic analyzer. In some embodiments, gas leak detection may be performed by monitoring the presence of CO2or H2O with the gas cell, whether by direct and/or indirect sampling. The present disclosed devices, systems, and methods may involve advanced analyses and identification of interferent and outlier. The present disclosure includes devices, systems, and methods for dual channel optical gas analyzers for compensation of ambient air constituents. Spectrometers can be used to measure light absorption spectra of gases. When gases of interest in a sample under observation are also present in ambient air (e.g., air either in the analyzer and/or around the sampling system) or when other gases in ambient air might interfere with the measurement of the gases of interest, spectrometers often must be purged, for example, with a purified gas to determine the contribution due to the absorption of only the gases of interest in the gas sample. The present disclosure includes devices, systems, and methods to reduce and/or remove the need for conditioning of the air in the analyzer or around the sampling system. The present disclosure includes spectrometers with two measurement channels. One channel can receive light propagating through ambient air and through a sampling gas cell. The transmitted light is then detected by a photodetector which generates an electrical signal that is digitized using an analog to digital converter. Another channel receives light propagating through ambient air only. Unlike in the first channel, the light of this second channel is not propagating through the sampling gas cell. The gas absorption contribution to the transmitted light in this second channel is related to ambient air constituents. The transmitted light of this second channel is detected by a second photodetector which generates an electrical signal that is digitized using a second analog to digital converter. Devices, systems, and methods within the present disclosure may include light sources that split the light (e.g., by beam splitter, light divider, and/or any other suitable light splitting technique), a gas cell that may contain one or many gases of interest, components to insert gases into the gas cell, a first detector measuring the light transmitted through the gas cell and through ambient air, a second detector measuring the light transmitted only through ambient air, a processor to determine the concentration of one or more gases present in the sampling gas cell from the first channel signal, and remove interferences and/or contribution of gases in ambient air of the first channel based on the ambient air signal recorded from the second channel. In some embodiments, a light source may be modulated by an interferometer. The light source may be divided in two different beams by a 50/50 Wedged ZnSe Beamsplitter. One of the beams may propagate through the gas cell and may reach the gas cell detector. The other beam may be directed towards a reference detector, to sense the ambient air composition only. The propagation distance in ambient air can be adjusted for both beams. The adjustment can be performed in a manner such that both the light transmitted by the gas cell and reaching the first detector and the light reaching the reference detector of the second channel propagate through similar distances in ambient air. In some embodiments, it may be assumed that ambient air composition in the instrument is homogeneous, and the light absorption due to the gases from ambient air should be the proportional to the gases concentration as well as to the respective propagation distance of both channels. In some embodiments, the gas cell may be a closed container with one inlet and one outlet to fluidly connect to form a gas circulation loop. The light from the interferometer can enter the gas cell from one side and exit through the other side to the gas cell detector. The gas cell can be temperature controlled by a cartridge heater. The pressure and temperature of the gases in the gas cell can be measured and used as input parameters to the calculation of gases concentrations. The present disclosure includes devices, systems, and methods in which the need for a purging system can be reduced and/or removed. Reducing and/or removing the need for a purging system can be an advantage when an analyzer is located in remote areas and purging systems are not available and/or are costly to install and operate. Concentration of gases in the gas cell that may also be present ambient air can be determined without purge, scrubber, desiccant and/or analyzer sealing. Other ambient air gases which have absorption signatures that may interfere with the determination of the gases concentration in the gas cell may also be compensated without purge, scrubber, desiccant and/or sealing. With the teachings of the present disclosure, the gases in ambient air can be measured simultaneously with the gases in the gas cell if desired, as opposed to calibration methods where only one channel can be used. Single channel calibration may perform reference background measurement taken apart from and/or without the gases of interest in the gas cell. The devices, systems, and methods of the present disclosure can provide an advantage when ambient air composition varies over time. The devices, systems, and methods of the present disclosure can include calibration for spectral intensity of the source, and calibration for the spectral characteristics of optical components that are common to both the first and second channels. The present disclosure can be used in the field of transformer monitoring by analysis of dissolved gases. The teachings of the present disclosure are generally applicable to other fields where optical methods require purge, scrubber, desiccant and/or sealing in order to calibrate, remove, and/or correct for ambient air constituents. The devices, systems, and methods of the present disclosure can provide an alternative to systems taking reference measurements using only one detector, by removing the gases of interests from the gas cell and/or bypassing the gas cell. Measuring low concentration gases by spectroscopy with accuracy can be challenging, particularly when the same gases or other interfering gases are present in ambient air, either in the analyzer or around the sampling system. Concentration of these gases in ambient air and/or the relative propagation distance of the light in ambient air could be non-negligible compared to the concentration of the gases in the gas cell and the propagation distance in the gas cell. Furthermore, the concentration of these gases in ambient air may vary with time, and unexpected gases can appear in ambient air in some sites. Pressure and temperature of the ambient air may differ from the pressure and temperature of the gas sample in the gas cell. To remove the contribution of ambient air gases, analyzers are often purged with purified gases (by way of example, the MB3000 spectrometer marketed by ABB Inc., includes a purging option). Purging can require bottles of purified gases, like Nitrogen, and/or a purified gas generator. Purge air is often dried to remove humidity, which can be a significant interferent in some instances, and CO2is often removed as well with a scrubber. In other systems where a purge is not possible and/or desirable, desiccants and/or scrubbers are used to remove humidity and/or other gases, but must be replaced or regenerated after some time. Other exemplary techniques can include moving relay mirrors to the gas cell in and out of the first channel in order to bypass the gas cell and direct the light to the detector to take background measurement. The relay optics can be designed such that the propagating distance in air with and without the relay optics is the same. Still other exemplary techniques can include using a scrubber to remove the gas component of interest from the gas cell after measuring the gas sample with the gas component of interest and inferring its concentration by the comparison of those two alternate measurements. Still other techniques may vary the pressure and/or the temperature of the gas sample to discriminate its composition over ambient air composition. In cases where the purge gas is supplied from an exhaustible source, such as a bottle, the exhaustible source will need to be refilled and/or changed at periodic maintenance intervals. Purge generators can be costly equipment that can require maintenance as well. Scrubbers and desiccants also require maintenance. Thus, the purge-based systems can increase the cost of operating spectrometers. As mentioned above, the present disclosure can include reducing and/or removing the need for a purging system, desiccants, scrubbers and/or instrument sealing. Accordingly, the devices, systems, and methods of the present disclosure can reduce installation and/or maintenance costs related to the spectrometer, and can enable solutions for remote sites where purging systems are not available and/or maintenance cannot be performed frequently due to cost and/or safety issues. In some embodiments, the devices, systems, and methods of the present disclosure do not require moving optics and/or sample gas pressure modulation, and the ambient air constituents can be measured simultaneously with the gas cell constituents. Since spectrometers using certain teachings of the present disclosure can measure spectra of ambient air, they may also detect and/or compensate for unexpected gases present in the ambient air, as opposed to scrubbers that are designed for specific constituents. Devices, systems, and methods of the present disclosure may be used to detect other defects around the transformer, for example but without limitation, detection of insulation gas leaks, such as SF6. By measuring and removing ambient air absorption, the devices, systems, and methods of the present disclosure can reduce sensitivity to ambient air compositions. The composition of the air inside the optical analyzer and/or around the sampling system may not need to be controlled by use of purge system, desiccants, scrubbers and/or instrument sealing. In some embodiments, the devices, systems, and methods of the present disclosure may use factory calibration to characterize the difference of light propagating distances in air between first and second channels. In some embodiments, the devices, systems, and methods of the present disclosure may use factory calibration of the system to measure the spectral response of the first and second detectors as well as spectral response of optical components not common to first and second channel. In some embodiments, the devices, systems, and methods of the present disclosure may use factory calibration to characterize the spectral response and/or instrument line shape of the first and second channels in order to improve the compensation of air constituents in the first channel using the second channel. Factory calibration may include purge of the analyzer. The present disclosure include techniques developed to adjust the position of system components (mirrors, lenses, detectors, etc.) to minimize the difference of light propagating distance in air between first and second channel. In some embodiments, one or more algorithms may be used to compensate for the ambient constituents of the first channel using the second channel signal. While certain illustrative embodiments have been described in detail in the figures and the foregoing description, such an illustration and description is to be considered as exemplary and not restrictive in character, it being understood that only illustrative embodiments have been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. There are a plurality of advantages of the present disclosure arising from the various features of the methods, systems, and articles described herein. It will be noted that alternative embodiments of the methods, systems, and articles of the present disclosure may not include all of the features described yet still benefit from at least some of the advantages of such features. Those of ordinary skill in the art may readily devise their own implementations of the methods, systems, and articles that incorporate one or more of the features of the present disclosure. | 51,673 |
11860149 | DETAILED DESCRIPTION OF THE DISCLOSURE Embodiments provided herein include systems and methods for providing dynamic real-time water-cut monitoring of fluid from an oil well or plurality of wells. These embodiments may utilize field data and machine learning methods to arrive at water-cut (WC) estimations. Because this approach provides superior results, new analytics and new advisories become feasible. Some of these embodiments provide a mechanism to benchmark multi-phase flowmeters, instantly identify malfunctioning flowmeters, and optimize flowmeter calibration frequencies and schedules. These embodiments may provide a mechanism to interpolate between the often-sparse WC measurements and to automatically determine production allocation per well in real-time or near real-time. These embodiments can be implemented in many fields that utilize pressure and temperature sensors and an estimated liquid gross rate. Unlike WC, liquid gross rate can be reliably measured through flowmeters (e.g. Venturi based, Coriolis based, etc.) or estimated through artificially intelligent systems. Further, pressure and temperature can also be reliably measured at surface through wellhead sensors and at subsurface through permanent downhole gauges or electronic stability program (ESP) sensors. The systems and methods for providing dynamic real-time water-cut monitoring of fluid from a well or plurality of wells incorporating the same will be described in more detail, below. Referring now to the drawings,FIG.1depicts a computing environment for dynamic real-time water-cut monitoring, according to embodiments described herein. As illustrated, the embodiment ofFIG.1illustrates a network coupled to a user computing device102and a remote computing device104. The network100may include any wide area network (such as the internet, cellular network, mobile data network, WiMax network, etc.), any local network (such as a local area network, Wi-Fi network, mesh network, etc.), and/or any peer-to-peer network (such as via Bluetooth, ZigBee, etc.). The user computing device102may be configured as any personal computer, laptop, mobile device, database, server, etc. for interfacing with a user and thus may include input devices and output devices for facilitating such interface. The remote computing device104may include any server, database, personal computer, tablet, mobile device, and/or other device for storing data described and/or performing the calculations described herein. As depicted inFIG.1, the remote computing device104may include a memory component140that stores data gathering logic144aand calculation logic144b. As described in more detail below, the data gathering logic144amay be configured for machine learning and/or as a neural net for causing a computing device to accumulate data, perform the calculations, assemble graphical depictions of wells, determine historical well data, pressure data, temperature data, flow data, etc. The calculation logic144bmay cause the computing device to perform one or more calculations described herein and/or provide output for display and/or upload. Also depicted inFIG.1are an upstream pressure sensor106aand a downstream pressure sensor106b(collectively referred to as “pressure sensors106”). The upstream pressure sensor106amay be disposed at an upstream portion of the pipe202(FIG.2) in a bottom hole, near the reservoir200(FIG.2), while the downstream pressure sensor106bmay be disposed at a downstream portion of the pipe202(FIG.2), near the surface. The pressure sensors106may be utilized to determine pressure readings for a well, as described in more detail below. The pressure sensors106may be configured as off the shelf sensors in some embodiments and/or as more sophisticated sensors in other embodiments. Similarly, the pressure sensors106may also represent other types of sensors, such as temperature sensors, flowmeters, etc. Similarly, while two sensors are depicted inFIG.1, any number may be utilized, depending on the sensors and desired functionality. It will be understood that whileFIG.1depicts a particular network configuration, this is merely one example. Some embodiments may be configured such that the user computing device102performs the calculations and recommendations (and thus stores the data gathering logic144aand/or the calculation logic144b) and only retrieves data from the remote computing device104. FIG.2depicts a reservoir200that may be selected for dynamic real-time water-cut monitoring, according to embodiments described herein. As illustrated, the reservoir200may be disposed below the ground and may include one or more different fluids including oil, natural gas, water, and/or other fluids. As such, when a well is created, a pipe202may be drilled into the ground to access the reservoir200. A pump204, which may be configured as a “Christmas tree” structure, a pump jack, and/or other apparatus may be coupled to the pipe for extracting fluid from the reservoir200. It will be understood that depending on the particular embodiment a plurality of wells may be utilized for a single reservoir200. As such, a plurality of different pipes202and/or pumps204may be utilized, thus increasing the complexity of dynamic water cut monitoring. FIG.3depicts a pipe202with pressure sensors106coupled thereto for dynamic real-time water-cut monitoring, according to embodiments provided herein. As illustrated, a plurality of pressure sensors106may be disposed a predetermined distance apart in order to capture pressure readings and thus calculate pressure changes as the fluid traverses the pipe202. As discussed above, while the sensors ofFIG.3may be depicted as pressure sensors106, these sensors may be configured as any type of sensors that may be utilized for performing the measurements described herein. Further, while a plurality of pressure sensors106is depicted inFIG.3, this is also merely one example. Some embodiments may utilize a single device that is configured to take measurements at a plurality of positions in the pipe202or several devices for this purpose. The embodiments depicted inFIGS.1-3may be configured to determine a pressure change from one point to another as a function of a plurality of factors, including WC. Hence, if all other factors are isolated, WC can be determined from the pressure change across two points. The learning of WC impact on pressure change between two sensors may be accomplished via machine learning of large scrutinized datasets or via other mechanisms. In general, the pressure drop across two points for incompressible fluids can be broken down to pressure losses due to potential energy (PE), kinetic energy (KE), and friction (F) as per the following equation: Δp=ΔpPE+ΔpKE+ΔpF. In many cases, pressure loss due to kinetic energy can be ignored because the diameter of the pipe202between the pressure sensors106is often constant. On the other hand, frictional pressure loss is often significant and may be accounted, especially when the distance between the pressure sensors106is large. In many current solutions, frictional pressure losses are estimated from physical models or lab-based correlations. This conventional approach limits estimate accuracy because field conditions may differ from ideal models or lab conditions. In contrast, frictional pressure loss described herein may be estimated based on a data-driven machine learning approach. Some of the parameters that correlate to frictional pressure losses are gross rate, an area of the pipe202, length in measured depth, WC, and/or fluid properties. Other parameters that can be indicative of WC and are included in machine learning, if available, are ESP parameters (e.g., Volts, Amps, horsepower, speed, motor temperature, discharge temperature, number of stages, etc.). It will be understood that in some cases when the distance between sensors is small and/or pipe diameter is large, frictional effects may be negligible. Additionally, the pressure losses due to potential energy (PE) can be estimated via the following equation: ΔpPE=(1−WC)gohTVD+WC gwhTVD, wheregorefers to a gravity of oil;hTVDrefers to total vertical depth difference between the pressure sensors 106; andgwrefers to the gravity of water. As presented above, one challenge for calculating pressure drop across two points is the calculation of frictional pressure losses and if applicable kinetic pressure losses. Embodiments described herein utilize machine learning to determine frictional and kinetic pressure losses, based on historical data associated with measured frictional and/or kinetic losses of similar systems, and/or calculated and verified frictional and/or kinetic losses. Once all pressure losses are reliably modeled, WC can be inverse determined. These embodiments receive a sufficient amount of data points from real-time data and a sufficient amount of reliable historical water-cut measurements (e.g. via separator testing, sampling, a well-calibrated meter). These points may be used as the truth model for data training. Using reliable historical WC measurements via a truth model, embodiments described herein estimate pressure losses due to potential energy using the following equation: ΔpPE=(1−WC)gohTVD+WC gwhTVD, whereWCis a fraction. Some embodiments described herein calculate the pressure losses due to friction and kinetic energy, henceforth called dynamic energy losses, as follows: ΔpDyn=ΔpF+ΔpKE=pdownstream−pupstreamΔpPE, where pdownstreamrepresents the pressure reading from the downstream pressure sensor106band pupstreamrepresents the pressure reading from the upstream pressure sensor106a. As mentioned above, pressure losses due to kinetic energy are often negligible. Accordingly, embodiments described herein may utilize machine learning to relate at least one dynamic pressure loss (or dynamic pressure losses) to parameters such as gross rate, pressure at the two gauges, temperature at the two gauges, distance between gauges in measured depth, pipe area, and fluid properties and if applicable volts, amps, horsepower, motor speed, motor temperature, discharge temperature, and number of stages. It should be understood that multi-variate nonlinear regression and/or deep learning may be used. If a match is obtained without including WC, embodiments herein may utilize the direct approach. In other words, if dynamic pressure losses are independent of WC (e.g., WC may be neglected in the dynamic pressure losses calculation), WC may be directly calculated from the equations above. Specifically, the direct approach utilizes the machine learning algorithm to estimate the dynamic pressure losses. One can estimate the ΔpPEas follows: ΔpPE=pdownstream−pupstream−ΔpF−ΔpKE. As such, in the direct approach, WC can be estimated by re-arranging the following equation: ΔpPE=(1−WC)gohTVD+WC gwhTVD. If a match is not obtained, embodiments may utilize an iterative approach or iterative process. When utilizing the iterative approach, embodiments start with an initial guess of water-cut (WCi). Using WCiand the other known parameters, embodiments estimate the dynamic pressure losses. Embodiments may then estimate ΔpPEas follows: ΔpPE=pdownstream−pupstream−ΔpF−ΔpKE. Next, embodiments may estimate WC by re-arranging the following equation: ΔpPE=(1−WC)gohTVD+WC gwhTVD. If the initial guess is within a predetermined threshold of WC (such as WCi0.001 of WC, WCi<0.01 of WC, or other predetermined threshold) the process stops, otherwise WC is used as the new guess WCi=WC and the process repeats. Accordingly, embodiments described herein provide a continuous WC estimate in real-time or near-real time, as well as provide a mechanism to benchmark multi-phase flowmeters. Embodiments may provide a mechanism to instantly identify malfunctioning flowmeters and a mechanism to optimize flowmeter calibration frequencies and schedule. Some embodiments provide a mechanism to interpolate between the often-sparse WC measurements, as well as a mechanism to automatically determine production allocation per well in real-time. Some embodiments described herein can be implemented in many fields, as these embodiments may only utilize pressure and temperature sensors and an estimated liquid gross rate. Some embodiments may be utilized to flag meters that are due for calibration. This will not only enhance the measurement quality of existing meters, but will also optimize cost by optimizing calibration frequency from periodic calibration to as-needed-basis calibration. FIG.4depicts a flowchart for dynamic real-time water-cut monitoring, according to embodiments provided herein. As illustrated in block450, a determination may be made regarding whether to use the direct approach or the iterative approach. As discussed above, this determination may be made based on whether dynamic pressure losses may be calculated without using a WC value and/or via other mechanisms, such as time needed for a solution, preference of the user, and/or for other reasons. If the direct approach is desired, WC may be calculated, as described above. If the direct approach is not desired (and/or if the indirect approach is desired), at block452, an initial guess for WCimay be determined. The guess WCimay be made from historical values for WC from machine learning algorithms based on sensor data, and/or via other mechanisms. At block454, dynamic pressure losses may be estimated. As described above, dynamic pressure losses may be estimated using the formula above, with the pressure data from the pressure sensors106. At block456, at least one potential energy pressure loss (or potential energy pressure losses) may be estimated. Again, the potential energy pressure losses may be calculated from ΔpPE=pdownstream−pupstream−ΔpFΔpKE. In block458, WC may be estimated by solving the following for WC ΔpPE=(1−WC)gohTVDWC gwhTVD. If in block460, WC−WCiis <0.001, the process may end. If not, the process may return to block454, using this iteration WC for WCi. FIG.5depicts a graph of portable testing versus new method estimation, as provided in embodiments described herein. As illustrated, there is a roughly linear relationship between new method estimation and portable testing. Specifically, new method estimation may range from about 0 to about 0.8 and portable testing may range from about 0 to about 0.8. FIG.6depicts a computing device for dynamic real-time water-cut monitoring, according to embodiments described herein. As illustrated, the remote computing device104includes a processor630, input/output hardware632, a network interface hardware634, a data storage component636(which stores production data638aand/or other data638bas described with reference toFIG.2), and a memory component140. The memory component140may be configured as volatile and/or nonvolatile memory and as such, may include random access memory (including SRAM, DRAM, and/or other types of RAM), flash memory, secure digital (SD) memory, registers, compact discs (CD), digital versatile discs (DVD) (whether local or cloud-based), and/or other types of non-transitory computer-readable medium. Depending on the particular embodiment, these non-transitory computer-readable mediums may reside within the remote computing device104and/or external to the remote computing device104. The memory component140may store operating logic642, the data gathering logic144a, and the calculation logic144b. Each of these logic components may include a plurality of different pieces of logic, each of which may be embodied as a computer program, firmware, and/or hardware, as an example. A local interface646is also included inFIG.6and may be implemented as a bus or other communication interface to facilitate communication among the components of the remote computing device104. The processor630may include any processing component operable to receive and execute instructions (such as from a data storage component636and/or the memory component140). As described above, the input/output hardware632may include and/or be configured to interface with speakers, microphones, and/or other input/output components. The network interface hardware634may include and/or be configured for communicating with any wired or wireless networking hardware, including an antenna, a modem, a LAN port, wireless fidelity (Wi-Fi) card, WiMAX card, mobile communications hardware, and/or other hardware for communicating with other networks and/or devices. From this connection, communication may be facilitated between the remote computing device104and other computing devices. The operating logic642may include an operating system and/or other software for managing components of the remote computing device104. As discussed above, the data gathering logic144amay include machine learning characteristics and/or be configured as a neural net. The data gathering logic144amay reside in the memory component140and may be configured to cause the processor630to gather data, create models based on historical data, predict future values based on the historical data, and/or perform similar functions. The calculation logic144bmay be configured to cause the processor630to perform the calculations described herein for acquiring the water-cut data, perform other calculations, and/or output results to a display device or other output device. It should be understood that while the components inFIG.6are illustrated as residing within the remote computing device104, this is merely an example. In some embodiments, one or more of the components may reside external to the remote computing device104or within other devices, such as the user computing device102depicted inFIG.1. It should also be understood that, while the remote computing device104is illustrated as a single device, this is also merely an example. In some embodiments, the data gathering logic144aand the calculation logic144bmay reside on different computing devices. As an example, one or more of the functionalities and/or components described herein may be provided by the remote computing device104and/or the user computing device102. Depending on the particular embodiment, any of these devices may have similar components as those depicted inFIG.6. To this end, any of these devices may include logic for performing the functionality described herein. Additionally, while the remote computing device104is illustrated with the data gathering logic144aand the calculation logic144bas separate logical components, this is also an example. In some embodiments, a single piece of logic may provide the described functionality. It should also be understood that while the data gathering logic144aand the calculation logic144bare described herein as the logical components, this is also an example. Other components may also be included, depending on the embodiment. As illustrated above, various embodiments for dynamic real-time water-cut monitoring are disclosed. These embodiments may be configured to provide continuous, regular, periodic, on-demand, or other type of water-cut reporting. Additionally, these embodiments do not require expensive sensors, cut off the shelf pressure sensors that are easily calibrated and maintained. Further, these embodiments can also provide ultra-fast monitoring or more robust reporting, depending on the particular desires of the system and/or user. This invention provides a continuous WC estimate at the wellhead in real-time. Some embodiments provide a mechanism to benchmark multi-phase flowmeters, instantly identify malfunctioning flowmeters, optimize flowmeter calibration frequencies and schedules, interpolate between the often-sparse WC measurements, automatically determine production allocation per well in real-time. Some embodiments may utilize the functionality described herein in many fields as it only requires pressure and temperature sensors and an estimated liquid gross rate. While particular embodiments and aspects of the present disclosure have been illustrated and described herein, various other changes and modifications can be made without departing from the spirit and scope of the disclosure. Moreover, although various aspects have been described herein, such aspects need not be utilized in combination. Accordingly, it is therefore intended that the appended claims cover all such changes and modifications that are within the scope of the embodiments shown and described herein. It should now be understood that embodiments disclosed herein include systems, methods, and non-transitory computer-readable mediums for dynamic real-time water-cut monitoring. It should also be understood that these embodiments are merely exemplary and are not intended to limit the scope of this disclosure. | 20,761 |
11860150 | DETAILED DESCRIPTION OF EMBODIMENTS The embodiments are given to better illustrate the disclosure, but the content of the disclosure is not limited to the embodiments. Therefore, it is still within the scope of protection of the disclosure for those skilled in the art to make non-essential improvements and adjustments to the embodiments according to the above summary. The disclosure provides a method for evaluating damage-healing characteristics of paving asphalt based on energetics principle, the method includes the following steps:S1, obtaining a numerical integral AHof a stored pseudo strain energy required by asphalt to compensate damage-healing to loading times, and local life compensation delta ΔN of asphalt benefiting from a damage-healing effect;S2, calculating an average stored pseudo strain energy QHof the asphalt according to the following formula: QH=AHΔN;andS3, evaluating the damage-healing characteristics of paving asphalt according to the average stored pseudo strain energy QHof the asphalt. Specifically, under test conditions of different asphalt damage states and rest periods, the pseudo strain energy evolution law of an asphalt material in a secondary loading process after a rest period of healing is analyzed, the average stored pseudo strain energy (QH) required for compensation the damage-healing under a given strain load level is calculated to serve as an energetics evaluation index for revealing the asphalt damage-healing characteristic, this index is independent of the selection of the damage state of the material and the rest periods, and only depends on the magnitude of the strain load, so that the intrinsic healing behavior characteristic of the material is reflected, and the test efficiency for evaluating the healing characteristics of different types of asphalt materials can be greatly improved. Furthermore, the method for evaluating the damage-healing characteristics of paving asphalt based on the energetics principle includes the following steps: obtaining cumulative cyclic loading times NHwhen a damage intensity reaches a damage intensity level before a rest period and cumulative cyclic loading times Nmat beginning of the rest period of the asphalt in a secondary loading process after the rest period of healing of the asphalt. Furthermore, the method for evaluating the damage-healing characteristics of paving asphalt based on energetics principle includes the following steps: obtaining the stored pseudo strain energy WSRof the asphalt, and calculating the numerical integral of the stored pseudo strain energy required by the asphalt to compensate the damage-healing to the loading times according to the following formula: AH=∫NmNHWSR. In some embodiments, the method for evaluating the damage-healing characteristics of paving asphalt based on the energetics principle further includes the following steps: obtaining a pseudo stiffness C and a pseudo strain γpRof the asphalt; and calculating the stored pseudo strain energy WSRaccording to the following formula: WSR=12×C×(γpR)2. Furthermore, the method for evaluating the asphalt damage-healing characteristics based on the energetics principle further includes the following steps: obtaining a peak shear strain γpof the asphalt in any loading period and a linear viscoelastic modulus |G*|0of the asphalt at a test temperature and a loading frequency; and calculating a pseudo strain γpRof the asphalt according to the following formula: γpR=γp×|G*|0. Furthermore, the method for evaluating the damage-healing characteristics of paving asphalt based on the energetics principle further includes the following steps: obtaining a peak shear stress τpof the asphalt in any loading period; and calculating a pseudo stiffness C of the asphalt according to the following formula: C=τpγpR. Furthermore, the method for evaluating the damage-healing characteristics of paving asphalt based on energetics principle further includes the following step: calculating the local life compensation ΔN of the asphalt benefiting from the damage-healing effect according to the following formula: ΔN=NH−Nm. It should be noted that the method for analyzing the influence of the average stored pseudo strain energy (QH) on the asphalt damage-healing characteristics in the disclosure is as follows:(1) Under a selected material damage state, a certain rest period of healing is set in a continuous loading fatigue test to complete the “fatigue-healing-fatigue” test of the asphalt material under a target strain load level.(2) Mechanical response parameters of the fatigue damage performance of the asphalt material before and after the rest period of healing are calculated and analyzed according to the formula (1) to the formula (3), and the mechanical response parameters include a pseudo stiffness and a damage intensity: C=τpγpR(1)γpR=γp×❘"\[LeftBracketingBar]"G*❘"\[RightBracketingBar]"0(2)S=∑i=1N[DMR2(rp)2(Ci-1-Ci)]α1+α(ti-1-ti)11+α(3) Where C is the pseudo stiffness of the material; τpis the peak shear stress in any loading period; γpRis the pseudo strain; γpis the peak shear strain in the loading period of the asphalt; |G*|0is the linear viscoelastic modulus of the asphalt at this temperature and loading frequency, which can be obtained by frequency sweep test; S is the damage intensity of the asphalt; i is the number of loading times selected for asphalt damage calculation; a is the material constant in the non-damage state, α=1/m, and m is the slope fitting value of the dynamic shear modulus master curve in the linear viscoelastic range of the asphalt.(3) The average stored pseudo strain energy (QH) required for compensation the damage-healing characteristics is calculated according to formulas (4) to (7) in the secondary loading process after the analysis rest periods: WSR=12×C×(γpR)2(4)QH=AHΔN(5)AH=∫NmNHWSR(6)ΔN=NH-Nm(7) Where WSRis the stored pseudo strain energy of the asphalt; AHis the numerical integral of the stored pseudo strain energy required by the asphalt to compensate the damage-healing in the secondary loading process after the rest periods; ΔN is the local life compensation of asphalt benefiting from the damage-healing effect; NHis the cumulative cyclic loading times when the damage intensity reaches the level of the damage intensity before the rest periods in the secondary loading process after the rest periods of asphalt; Nmis the cumulative cyclic loading times at the beginning of the rest periods.(4) The independence of the QHindex on the damage state and the rest periods is verified by changing the damage state and the duration of rest periods, which indicates that the QHindex has nothing to do with the damage state and the rest periods.(5) The target strain load in the “fatigue-healing-fatigue” test is further changed to verify the dependence of the QHindex on the strain load, which indicates that the QHindex only depends on the strain load. In another aspect, the disclosure provides an apparatus for evaluating the asphalt damage-healing characteristics based on energetics principle, which includes: a data obtaining module, a calculating module and an analyzing module, the data obtaining module is configured to obtain a numerical integral AHof a stored pseudo strain energy required by asphalt to compensate damage-healing to loading times and obtain local life compensation ΔN of the asphalt benefiting from a damage-healing effect; the calculating module is configured to calculate an average stored pseudo strain energy QHof the asphalt; and the analyzing module is configured to evaluate the asphalt damage-healing characteristics according to the average stored pseudo strain energy QHof the asphalt. Specifically, the above method for evaluating the asphalt damage-healing characteristics based on energetics principle may be implemented by using relevant apparatus as a carrier. For example, the apparatus including the data obtaining module, the calculating module and the analyzing module realize the data obtaining, calculating and analyzing, so as to evaluate the asphalt damage-healing characteristics. In another aspect, the disclosure provides a non-transitory computer-readable storage medium, on which computer readable instructions are stored, when the computer readable instructions are executed by a processor of a computer, the computer executes the method for evaluating the asphalt damage-healing characteristics based on energetics principle. Specifically, in addition to the above related apparatus as the carrier, the computer program can also be used as a carrier, and the computer program can be used to execute the process of data acquiring, calculating and analyzing to obtain the asphalt damage-healing characteristics. The technical schemes of the disclosure are clearly and completely described below in combination with specific embodiments. In the following embodiments, a time sweep (TS) test with continuous loading is performed on asphalt samples using a parallel plate loading mold with a diameter of 8 mm and a thickness of 2 mm of a dynamic shear rheometer to obtain the fatigue life (Nf) of the asphalt. Then the loading times corresponding to the damage states of 25% Nffailure, 50% Nffailure and 75% Nffailure are determined, and four rest periods with different durations of 1 minute, 15 minutes, 60 minutes and 180 minutes are introduced under the damage states to test the healing performance of asphalt under different damage states and rest periods, and the “fatigue-healing-fatigue” (i.e., TS based healing (TSH)) test and analysis are completed. Embodiment 1 The fatigue performance of No. 90 base asphalt from an oil source in China is tested under continuous loading at 20° C. and 10 Hz. The fatigue life (Nf) of the asphalt is 6961 under the fatigue load strain level of 2.5% TS fatigue test. Further, the TSH test analysis is completed by adding a rest periods, and an energetics index-average stored pseudo strain energy (QH) reflecting the asphalt damage-healing characteristic is calculated and obtained according to the following implementation steps:Step 1: the fatigue load strain level is kept at 2.5%, a rest period of healing of 15 minutes is set under the selected damage state of 50% Nffailure (i.e., when loading 3480 times), and the TSH test of asphalt is performed.Step 2: the mechanical response parameters: pseudo stiffness and damage intensity of the fatigue damage performance of the asphalt material before and after the rest period of healing are calculated and analyzed according to the above formulas (1) to (3). The calculation results of the parameters are shown in Table 1. TABLE 1Mechanical response parameters of fatigue performance ofasphalt materials before and after the rest period of healingMechanical responsepseudo stiffnessdamage intensityparameter(C)(S)before the healing0.8990.253after the healing1.0390.170Step 3: The average stored pseudo strain energy (QH) required to compensate the damage-healing effect is calculated according to above formulas (4) to (7) during the secondary loading after the rest periods, as shown inFIG.2, the parameter calculation results are shown in Table 2. TABLE 2Calculation results of average stored pseudo strain energy(QH) during secondary loadingMechanical response parameterNHNmΔNAHQHCalculation result35543480740.8540.012 Embodiments 2 to 12 The fatigue load strain level is kept unchanged at 2.5%, and the steps in Embodiment 1 are repeated by changing the damage state of materials (25% Nffailure, 50% Nffailure, 75% Nffailure) and the duration of rest periods (1 minute, 15 minutes, 60 minutes, 180 minutes), to calculate and verify the independence of QHindex on the damage state and the rest periods. The calculation results of the QHindex of Embodiments 1 to 12 are shown as TSH-2.5% inFIG.3. Embodiments 13 to 24 The implementation steps are the same as those in Embodiments 1 to 12, but the difference is that the fatigue load strain level is 3%, and the calculation results of QHindex are shown as TSH-3% inFIG.3. Embodiments 25 to 36 The implementation steps are the same as those in Embodiments 1 to 12, but the difference is that the fatigue load strain level is 3.5%, and the calculation results of the QHindex are shown as TSH-3.5% inFIG.3. The above results show that the average stored pseudo strain energy (QH) of asphalt material to compensate the damage-healing can well normalize the healing characteristics of asphalt material under different test conditions, and this index has nothing to do with the damage state and the rest periods, and only depends on the magnitude of the strain load. Finally, the above embodiments are only used to illustrate the technical schemes of the disclosure, but not to limit it. Although the disclosure has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical schemes of the disclosure can be modified or replaced by equivalents without departing from the purpose and scope of the technical schemes, which should be included in the scope of the claims of the disclosure. | 13,171 |
11860151 | Below is the description of a form of exemplary actuation, and as such not limiting, of the method for the extraction and the determination of microplastics in samples with organic and inorganic matrices. DESCRIPTION OF THE INVENTION In reference toFIG.1, the method for the extraction and determination of microplastics in samples with organic and inorganic matrices comprises the following fundamental stages:a) sampling and pre-treatment,b) acid digestion of the sample,c) extraction of microplastics during the liquid phase,d) identification and quantification of the microplastics. The method inherent to this invention is applied to the extraction and determination of microplastics belonging to the group comprising: polyethylene, ultra high molecular weight polyethylene (UHMWPE), polycarbonate, polyethylene terephthalate (PET) and polyvinyl chloride (PVC) in samples of organic and inorganic matrices. Said microplastics have dimensions less than 500 μm, more specifically with dimension less than 100 μm, and even more specifically with dimensions between 1 and 5 μm. In the processing of the method inherent to this invention, any sample will be implicitly considered a gram or a milliliter if not otherwise specified. Said sampling stage provides the selection of samples with a watery base or of an organic matrix comprised in the group consisting of human, plant, and animal biological tissues and liquids, pharmaceuticals, cosmetics, foods, mineral waters, waters destined for human consumption, ground waters, surface waters, sea waters, waters from pools and aquaculture installations, urban and industrial, and sewage, liquid wastes. Alternatively, said sampling stage provides the selection of airborne samples or soil samples. Conveniently, said acid digestion state or mineralization of the sample with organic or inorganic matrices provides the treatment with strong acid, in a 1:1 m/v or v/v ratio with the abovementioned sample, for a duration of at least 24 hours at a temperature of 60° C. Said strong acid is selected from the group comprising hydrochloric acid, nitric acid, and sulphuric acid. Said extraction stage of the microplastics includes the following phases:1. mix an aliquot of ultra-pure water with said sample treated with strong acid in a ratio of 1:3 m/v or v/v, to obtain a liquid mixture;2. mix a first aliquot of non-polar halogenated solvent with said liquid mixture in a ratio of 1:3 m/v or v/v, to obtain a heterogeneous liquid mixture;3. agitate said heterogeneous liquid mixture for 30 s and centrifuge it at 4000 rpm for 15 minutes to obtain two fractions: A and B, in which fraction A is watery and fraction B is organic;4. separate the watery fraction A from the organic fraction B;5. add a second aliquot of non-polar halogenated solvent to the watery fraction A to obtain a second heterogeneous mixture;6. agitate said second heterogeneous liquid mixture for 30 s and centrifuge it at 4000 rpm for 15 minutes to obtain two liquid phases: C and D, in which fraction C is watery and fraction D is organic;7. recombine the organic fractions B and D and then evaporate to dryness at a temperature of 70° C., to obtain an extract of microplastics;8. Suspend said extract of microplastics in acetonitrile with a volume between 100 and 500 microliters. Said non-polar halogenated solvent is selected from the group that includes dichloromethane (DCM) and chloroform. The agitation carried out in phases 3 and 6 of the extraction stage is conveniently done with a vortex for the duration of 30 s. Said stage of identification and quantification of the microplastics provides the transfer of said extract of the microplastics suspended in acetonitrile onto a metallic support, preferably a stub of aluminum for scanning electronic microscope (SEM Specimen Stub) with a diameter of 25 mm, taking care to distribute the extract onto the entire surface of the stub. Where the saturation of the stub surface is evident, the volume of acetonitrile is increased and more stubs are used. Then it is necessary to wait for the evaporation of the acetonitrile, taking care not to overturn the stub, preventing the particles from slipping off or modifying their dispersion. Finally, the stub is subjected to metallization with gold and inserted into the positioning chamber of the SEM samples for successive identification and quantification of the microplastics by means of electronic microscopy combined with energy dispersive X-ray, more specifically through scanning electronic microscope (SEM) combined with an Energy Dispersive X-ray Analysis (EDX). The identification and quantification of microplastics is conducted in a total reading area within the stub of 1 mm2, corresponding to a total of 228 fields at 1500 enlargement. Conveniently, the number of fields is reduced in case of an abundance of particles. The stage of identification and quantification of the microplastics calls for microanalytic acquisition phase (or the reading and recognition of the particle) and the phase of measurement and counting (or determination of the dimensions of the particle and the count). The microanalytical acquisition phase provides the verification of the chemical structure of the microplastics constituted exclusively by carbon. More specifically, it provides the determination and count of the microplastic of any dimension and form within the field of observation without discriminating position, measuring the length and width of each microplastic particle (FIG.6-10). Conveniently, every measurement of length and width of each microplastic particle is reported in the calculation sheet in electronic format created especially for the application of the method for the extraction and the determination of microplastics in samples with organic and inorganic matrices, (MICROPLAST software, of which the exemplary screens can be found inFIG.3-5). Said software automatically calculates the concentration of the microplastic particles, updating the results in real time according to the varying number and the dimensions of the microplastic particles counted and in function of the values of the other parameters, including quantity of samples analyzed, diameter of stub, etc. Moreover, the total weight of the microplastics contained in the sample is automatically estimated: the average radius of each particle; the volume, considering it as a regular sphere and, finally, the weight of the particle, considering the average density of the microplastics. Conveniently, the determination of the microplastics in samples with organic and inorganic matrices is expressed in terms of number of particles on gram (or milliliter) of sample and cubic meter of air, both in terms of micrograms of microplastics in gram (or milliliter) of sample or cubic meter of air. Conveniently, the extended uncertainty associated with the determination of the microplastics in samples with organic and inorganic matrices is expressed in confidence intervals (MIN and MAX). The evaluation of the experimental error in the measurement of concentration C of polyethylene in a massive sample is essentially due to two contributions: to the sampling statistic of N particles during the reading of the stub, assuming the casual Poisson distribution of the particles on the stub, and the width of the granulometric spectrum of the particles contained in the sample, so to the error made by adopting the average calculated only based on N particles identified as the average weight pmof the particles within the entire sample. The experimental error on the concentration (or read error) can hence be evaluated using the relation ΔC/C≈ΔNN+Δpmpm where Δpmpm corresponds to the relative error associated with the average weight pmof the N particles identified and ΔNN is the relative error associated with the N particles counted. Assuming a Poisson distribution for N particles counted, the relation ΔNN can also be expressed as 1N, while the relative error on the average weight pmof the N particles piidentified can be expressed using the standard error Σi(pm-pi)^2N(N-1)/pm. Hence, the previous relation becomes ΔC/C≈1N+Σi(pm-pi)^2N(N-1)/pmorUrelreading≈(1N)2+(Σi(pm-pi)^2N(N-1)/pm)2 Based on the following relations, it is deemed useful to consider, in order to obtain a more precise evaluation of the total uncertainties, the contribution of uncertainty deriving from the sample preparation procedure, which calls for the phases of grinding/blending, weighing, extraction, and dispersion on the stub. The resulting relation is the following: UCrel %=√{square root over ((Up)2+(UN)2+(Upr)2)} or UCrel %=√{square root over ((Ulett rel)2+(Upr)2)} where Upis the uncertainty associated with the average weight pmof the N particles identified Σi(pm-pi)^2N(N-1)/pm, UNis the uncertainty associated with the N particles counted 1N and Upr is the contribution deriving from the uncertainty of preparation. Conveniently, the software automatically calculates Upand UN. The operator can estimate the uncertainty Uprby carrying out n repeatability tests and calculating the standard deviation. The Upr, finally, must be inserted in the specific cell of the Software. In a first embodiment, the method of this invention provides the extraction of microplastics from samples with a watery base and/or of an organic matrix belonging to the group comprising of human, plant, and animal biological tissues and liquids, pharmaceuticals, cosmetics, foods, mineral waters, waters destined for human consumption, ground waters, surface waters, sea waters, waters from pools and aquaculture installations, urban and industrial, and sewage, liquid wastes. Said samples with a watery base and/or organic matrix can be found under natural conditions or previously lyophilized. The abovementioned samples with watery base and/or organic matrix are sampled according to known state-of-the-art methods. Conveniently, the above mentioned samples with watery base and/or with organic matrix are pre-treated by means of grinding and/or blending, with the duration of the pre-treatment depending on the hardness of the sample, to obtain a homogenized sample, and then subjected to weighing. An aliquot of the sample is transferred into a glass test tube—preferable with a volume of 16 ml, 100 mm tall, with a conical base, ground neck with a diameter of 16 mm and glass stopper—and an aliquot of strong acid in a 1:1 m/v or v/v ratio is added to carry out an acid digestion, or mineralization, for a duration of at least 24 h at a temperature of 60° C. In this first embodiment, the strong acid is nitric acid at 37%. The duration of the mineralization can be less than 24 h using a mineralizer in either a closed or open container. The success of the mineralization process makes it possible to obtain a somewhat transparent liquid. The abovementioned stage of acid digestion makes it possible to destroy all the organic fraction of the sample through oxidation processes and increase the solubility of the mineral salts, otherwise present in crystal form, also amorphous, which may represent a factor of confusion during the stages of identification and quantification of microplastics. Successively the mineralized sample is subjected to the liquid phase microplastics extraction stage as previously described in phases 1-8. The stages of identification and quantification of the microplastics proceeds as previously described. In a second embodiment, the method of this invention provides the extraction and the determination of airborne microplastics from air samples drawn from living and work environments, both indoors and outdoors. The sampling and pre-treatment phase of the method proposed by this invention provides the use of filtering membranes in mixed cellulose esters with a diameter of 25 mm and a porosity of at least 0.8 μm. The filter holder used is exclusively in metal (aluminum or steel) and open-faced with a cylindrical hood, also in metal, that extends between 30 mm and 50 mm in front of the filter and allows the exposure of a circular area with a diameter of at least 20 mm. The sampling stage for airborne microplastics can be conducted using two different methods: personal and environmental. The “personal” method provides the use of a sample with a good capacity to compensate load losses in order to obtain samplings even in intensely dusty conditions, making it possible to maintain, for the entire duration of the sampling, a capacity of at least 10% of the initial one. Conveniently, the filter holder is fixed in a zone near the airways with the opening facing downward. The aspiration flow is set between 1 and 5 l/min, preferably between 2 and 3 l/min. The “environmental” method provides the positioning of the filter holder at a height of 150 cm from the ground, using a tripod with the hood facing downward; the sample connected is an environmental type with a constant high aspiration flow between 18 and 25 l/min. The sampling time depends on the aspiration flow and requires a minimum volume of air aspired of no less than 3000 liters. Conveniently, it is possible to insert the filters in special fraction collectors in order to sample only the particles with diameters less than 10 micrometers, or less than 2.5 micrometers. Each sampled filter is positioned inside glass or metallic boxes; these are then positioned inside a sealed container so as to avoid generating impacts and to prevent the possibility of contamination of the filters with dust or other particles. The abovementioned filter is subjected to pre-treatment prior to the acid digestion of the sample. Another advantage is that the abovementioned filter is fragmented by suitable devices in metal and then placed fragmented in a 16 ml transparent glass test tube with a height of 100 mm, conical base, ground neck with a diameter of 16 mm and glass stopper. Then the process provides the stages of acid digestion of the sample, extraction of microplastics in liquid phase, identification and quantification of the microplastics through scanning electronic microscope coupled with Energy Dispersive X-ray Analysis (SEM-EDX), as previously described above. In a third embodiment, the method described in the present invention provides the extraction and determination of microplastics from soil samples. The abovementioned microplastics from soil samples are subjected to a pre-treatment comprising the phases of:sifting with a sieve with a 10 mm metal meshtransfer of the sifted sample to a transparent glass test tube,acid digestion with strong acid, in a ratio of 1:1 m/v or v/v with the abovementioned sample, for a duration of at least 24 hours at a temperature of 60° C.,neutralization of the sample with sodium hydroxide 1 M,vacuum filtration by means of a filtering membrane in mixed cellulose esters with a porosity of 10 μm or 100 μm. The method inherent to this invention requires that in the third embodiment, the successive phases of extraction of the microplastics follow the same methods described for the first embodiment of this invention, beginning with acid digestion. The following examples can be considered, by way of example and not of limitation, of this invention. Example 1 By way of example, below is a potential experimental application of the method for the extraction and the determination of microplastics in samples with organic and inorganic matrices for the determination of microparticles of polyethylene deriving from the wear of arthroplasty in periprosthetic tissues and in synovial fluid. Among the various types of polyethylene, the one applied in orthopedics is ultra high molecular weight polyethylene (UHMWPE with a density of 0.96 g/cm3), as it offers the best mechanical properties. The purpose of the application of the method inherent to this invention is that of determining the presence of polyethylene particles in the synovial fluid and in periprosthetic tissues of patients with hip replacements, supposing a potential release of these particles following the mechanical wear of the acetabular insert. Hence an intraoperative sample of periprosthetic tissue and a sample of synovial fluid were taken from a patient (CASE) subjected to an intervention to revise the hip replacement. In parallel, the same samples taken from a subject subjected to a primary arthroplasty hip replacement intervention were analyzed (CONTROL). Simultaneously, a test was conducted to evaluate the recovery of a biological matrix similar in structure (animal muscle) with no contamination of polyethylene, previously fortified with microparticles of polyethylene obtained through the pulverization of an acetabular insert (standard or also known as material of reference). An aliquot of 1 gram of each sample was transferred to a 16 ml transparent glass test tube with a height of 100 mm, conical base, ground neck with a diameter of 16 mm and glass stopper. A milliliter of nitric acid at 37% was then added to the samples and they were left to rest for 24 h, at a temperature of 60° C., to enable the chemical mineralization to take place as described in the method inherent to this invention. Then 3 ml of ultra-pure water and 3 ml of dichloromethane (DCM) were added to the mineralized samples. The heterogeneous mixture was then effectively agitated using a high-speed vortex mixer for approximately 30 seconds. At this point the samples were centrifuged at 4000 rpm for 15 minutes with a benchtop centrifuge with mobile recipient and angle rotor. Successively, the more watery phase was transferred to a new test tube with characteristics identical to those described above. Following a further addition of another 3 ml of dichloromethane (DCM), the solution was again agitated by means of vortex and centrifuged for another 15 min at 4000 rpm with the same centrifuge. The watery phase was eliminated and the organic phase was combined with the previous one. The extract of each sample was left to evaporate to dryness, promoting the process only by means of heating with a plate set at a temperature of 70° C. Successively, the dry residue was mixed with 100 microliters of acetonitrile. Then, after having “pipetted” multiple times using a glass Pasteur pipette in order to favor the dispersion of the particles, the suspension of each sample was transferred to a dedicated aluminum stub for scanning electronic microscope (SEM Specimen Stub) with a diameter of 25 mm, striving to distribute the solution on the entire surface of the stub. After having let the solvent evaporate, the stubs were metalized with gold and inserted into the sample positioning chamber of the SEM. More specifically, the Scanning Electronic Microscope (SEM) of Cambridge StereoScan 360 was used. The abovementioned recovery tests, conducted by repeating the extraction and analysis of a white matrix fortified with 0.2 milligrams of micronized polyethylene twice, made it possible to identify particles with diameters that varied between 2 and 25 μm. The microanalysis highlighted that the chemical structure of the particles studied is on average composed of 92.5% in carbon. The remaining percentage consists exclusively of aluminum and copper, or by elements that make up the metallic support (stub) on which the extracted particles are dispersed (FIG.2and Table 1). TABLE 1Chemical composition of the particleexamined with microanalysis in FIG. 2SpectrumIn stats.CAlCuTotalSpectrum 1Yes96.801.930.4599.18Spectrum 2Yes95.084.520.0099.60Spectrum 3Yes95.873.640.0099.61Spectrum 4Yes91.348.020.2599.30Spectrum 5Yes89.359.610.3499.40Spectrum 6Yes93.825.320.2699.33Spectrum 7Yes91.747.590.0099.75Spectrum 8Yes81.8717.370.5199.53Spectrum 9Yes96.153.240.1499.18Mean92.456.800.2299.47Std. deviation4.704.680.20Max.96.8017.370.51Min.81.871.930.00 The quantitative analyses (FIG.3andFIG.4), carried out via MICROPLAST software, have made it possible to calculate the average diameters of the particles, respectively equal to 4.52 μm and 4.37 μm. The concentrations of the two replicas were respectively equal to 268 μg/g and 218 μg/g. The average recovery calculated was equal to 121.5%. The quantitative analysis of the non-fortified matrix (FIG.5) made it possible to ascertain the low degree of contamination during the process. The concentration was equal to 2.81 μg/g. Results of the Synovial Fluid (SINOVIA) The application of the method inherent to this invention on samples of the “Case” and the “Control” highlights the significant differences between the synovial fluid relative to the “Case” and that relative to the “Control”. The successive SEM analysis of the extract of the “Case” synovial fluid highlighted the presence of polyethylene particles with diameters varying between a few micrometers (approximately 1-10 μm) and three millimeters. The particles present all the morphological characteristics similar to those of the Standard, like irregular curvature of the edges and an apparently compact structure. The results of the microanalyses highlight a chemical composition made up almost exclusively of carbon with an average percentage of 95.1%. The remaining percentage consists exclusively of aluminum and copper, or by elements that make up the metallic support (stub) on which the extracted particles are dispersed. Rare traces of trace elements like K, Na, Ca, Cl were found (FIG.6-7, Tables 2-3). TABLE 2Percentage chemical composition of the particle in FIG. 6SpectrumIn stats.CAlCuTotalSpectrum 1Yes97.061.310.3899.68Spectrum 2Yes100.000.000.00100.00Spectrum 3Yes98.360.260.2099.88Spectrum 4Yes98.440.000.0099.39Spectrum 5Yes97.990.660.2699.87Mean98.370.450.1799.76Std. deviation1.060.550.17Max.100.001.310.38Min.97.060.000.00 TABLE 3Percentage chemical composition of the particle in FIG. 7SpectrumIn stats.CAlCuTotalSpectrum 1Yes93.735.260.7199.70Spectrum 2Yes94.154.401.1199.66Spectrum 3Yes94.215.100.4799.78Mean94.034.920.7699.71Std. deviation0.260.460.32Max.94.215.261.11Min.93.734.400.47 The synovial liquid sample relative to the “Control” did not highlight the presence of polyethylene particles, demonstrating the effectiveness and efficiency of the method inherent to this invention. It must be pointed out how no polyethylene particles were found in the sample of synovial fluid of the CONTROL. The microanalysis carried out in three different points of the STUB found that aluminum is the most abundant chemical element (FIG.8and Table 4). Therefore, the invention is effective and discriminating, identifying and quantifying polyethylene only in positive samples. TABLE 4Percentage chemical composition of the CONTROL in FIG. 8SpectrumIn stats.CAlCuTotalSpectrum 1Yes18.9779.051.98100.00Spectrum 2Yes16.1983.810.00100.00Spectrum 3Yes0.00100.000.00100.00Mean11.7287.620.66100.00Std. deviation10.2410.981.14Max.18.97100.001.98Min.0.0079.050.00 Results on Tissue Sample The application of the method inherent to this invention on samples of the “CASE” and the “CONTROL” highlights the significant differences between the tissue relative to the “CASE” and that relative to the “CONTROL”. The SEM analysis following the application of the invention on the tissues of the “CASE” highlighted the presence of polyethylene particles with much smaller dimensions than those found in the “CASE” synovial fluid (from 0.8 μm to 7 μm in diameter). The particles analyzed presented all the morphological similarities of the Standard and of the microparticles found in the synovial fluid of the “CASE”, like irregular curvature of the edges and an apparently compact structure. The results of the microanalyses highlight a chemical composition made up almost exclusively of carbon with an average percentage of 80.1%. The remaining percentage consists exclusively of aluminum and copper, or by elements that make up the metallic support (stub) on which the extracted particles are dispersed. Rare traces of trace elements like K, Na, Ca, Cl were found (FIG.9and Table 5). TABLE 5Percentage chemical composition of the particles in FIG. 9SpectrumIn stats.CAlCuTotalSpectrum 1Yes90.019.150.6699.82Spectrum 2Yes93.217.150.2599.61Spectrum 3Yes89.858.151.4599.45Spectrum 4No90.898.490.7199.99Mean90.748.210.7799.72Std. deviation1.080.830.50Max.93.219.151.45Min.89.897.150.25 It is highlighted how no polyethylene particles were found in the tissue sample of the CONTROL. The microanalysis carried out in three different points of the STUB found that aluminum is the most abundant chemical element (FIG.10and Table 6). This result highlights the discriminatory effectiveness of the invention and denotes the absence of production of false positives and false negatives of the same, a fundamental characteristic in the pharmaceutical, forensic, food, medical/biomedical applications and quality control for all other types of samples for commercial or environmental use. TABLE 6Percentage chemical composition of the CONTROL in FIG. 10SpectrumIn stats.CAlCuTotalSpectrum 1Yes28.8770.260.87100.00Spectrum 2Yes31.1768.180.65100.00Spectrum 3Yes27.0871.631.29100.00Mean29.0470.020.94100.00Std. deviation2.051.740.33Max.31.1771.631.29Min.27.0868.180.65 The object of the invention is susceptible to numerous modifications and variants, all under the same concept of the inventive concept expressed in the attached claims. All parts may be replaced with other technically equivalent elements, and the materials may differ according to needs, without departing from the scope of protection of the present invention. Although the object was described with particular reference to the attached figures, the reference numbers used in the description and in the claims are used for a better understanding of the invention and do not constitute any limitation to the disclosed scope of protection. BIBLIOGRAPHY CITED Kandahari A M, Yang X, Laroche K A, Dighe A S, Pan D2, Cui Q; A review of UHMWPE wear-induced osteolysis: the role for early detection of the immune response; Bone Res. 2016 Jul. 12; 4:16014. doi: 10.1038/boneres.2016.14. eCollection 2016.Choudhury D, Ranuša M, Fleming R A, Vrbka M, Křupka I, Teeter M G, Goss J, Zou M. Mechanical wear and oxidative degradation analysis of retrieved ultra-high molecular weight polyethylene acetabular cups. J Mech Behav Biomed Mater. 2018 March; 79:314-323. doi: 10.1016/j.jmbbm.2018.01.003).Armstrong B L, Senyurt A F, Narayan V, Wang X, Alquier L, Vas G., Stir bar sorptive extraction combined with GC-MS/MS for determination of low level leachable components from implantable medical devices, J Pharm Biomed Anal. 2013 Feb. 23; 74:162-70. doi: 10.1016/j.jpba.2012.10.019. Epub 2012 Oct. 22.Pokorný D, Slouf M, Horák Z, Jahoda D, Entlicher G, Eklová S, Sosna A., Method for assessment of distribution of UHMWPE wear particles in periprosthetic tissues in total hip arthroplasty, Acta Chir Orthop Traumatol Cech. 2006 August; 73(4):243-50.Sosna A, Radonský T, Pokorný D, Veigl D, Horák Z, Jahoda D., Polyethylene disease Acta Chir Orthop Traumatol Cech. 2003; 70(1):6-16.Narayan V S., Spectroscopic and chromatographic quantification of an antioxidant-stabilized ultrahigh-molecular-weight polyethylene, Clin Orthop Relat Res. 2015 March; 473(3):952-9. doi: 10.1007/s11999-014-4108-6.Vesely F, Zolotarevova E, Spundova M, Kaftan F, Slouf M, Entlicher G., Simple colorimetric methods for determination of sub-milligram amounts of ultra-high molecular weight polyethylene wear particles, Acta Biomater. 2012 May; 8(5):1935-8. doi: 10.1016/j.actbio.2012.01.010. Epub 2012 Jan. 18.Wright S L, Kelly F J. Plastic and Human Health: A Micro Issue? Environ SciTechnol. 2017 Jun. 20; 51(12):6634-6647. doi: 10.1021/acs.est.7b00423 | 27,687 |
11860152 | DETAILED DESCRIPTION The following disclosure provides many different embodiments, or examples, for implementing different features of the invention. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. Further, spatially relative terms, such as “beneath,” “below,” “lower,” “above,” “upper” and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. The spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. The apparatus may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein may likewise be interpreted accordingly. Nanopore sequencing of a biomolecule involves passing a biomolecule through a nanopore of a nanopore device, and sensing changes in the nanopore device as the biomolecule passes through the nanopore. A nanopore sequencing device includes a sensing component, typically a nanopore device, and a sequencing component, which may be an external circuit. The external circuit controls the application of voltages across a nanopore device. The external circuit also senses responsive changes in electromechanical characteristics of the sensing component as a biomolecule passes through a nanopore of the nanopore device. Nanopore sequencing can be performed with a sensing device, e.g. a nanopore device, coupled to a remote sequencing device through electrode wires. As variations in electromechanical characteristics of a nanopore device are sensed, an analog signal associated with a change in current or voltage is created. The analog signal thus represents variations in electromechanical characteristics of a nanopore device and thereby characteristics of a biomolecule passing through a nanopore. But as electromechanical devices become smaller and smaller, e.g. on the micrometer, nanometer, or picometer scale, changes in detected currents or voltages are small, and increasingly susceptible to introduced noise. An analog signal describing variations in the electromechanical characteristics of a nanopore device, however, must be accurately conveyed to a sensing circuit in order to accurately characterize a biomolecule passing through a nanopore. Electric wires are typically used to couple a nanopore senor device to a biomolecule characterization device, which may be a biomolecule detection device for example. But coupled noise and parasitic capacitances may be produced across the electrode wires thereby distorting signal. Thus, longer electrode wires introduce more noise than shorter electrode wires, and biomolecule sequences obtained by sensing electromechanical changes may become inaccurate as a result of noise and distortion introduced by the wires. Thus, by forming sensing circuitry in a semiconductor integrated circuit layer coupled directly to a nanopore sensor, a sensed signal may be converted to a digital form by in situ formed integrated circuitry. The digital signal can then be transmitted off chip without concern for introduced noise and distortion affecting the sensed signals over long distances, so long as the noise level is low enough to distinguish binary highs (e.g., ones or zeros) to binary lows (e.g., zeroes or ones). Also, traditionally, a semiconductor nanopore is formed by e-beam ion milling. The cost and throughput of e-beam ion milling present substantial hurdles when commercializing a device, and increases the complexity of placing a nanopore device having a pre-formed nanopore such that the formed nanopore is appropriately situated. In order for a nanopore device to be properly situated, a nanopore in the nanopore device may need to be precisely placed such that the nanopore is co-axial with an orifice between two chambers of a semiconductor device. In this way, a nanopore device may function as a membrane covering an orifice separating two solution filed chambers. In this way a molecule may transit from one chamber of to another chamber through the nanopore. Such a semiconductor device may also have an integrated circuit coupled directly to the nanopore device that including pore forming circuitry such that a nanopore device need not be pre-formed, but instead may be formed in situ. Thus, a nanopore device coupled directly to a semiconductor device both minimizes noise and distortion by minimizing the length of a conducting path between a nanopore device and correspond sensing circuitry, while also allowing a nanopore to be formed in a nanopore device in place in precisely the correct location. Also, a nanopore formed in a nanopore device coupled with an integrated semiconductor circuit in accordance with this disclosure creates two measureable signal paths, as described below, which carry two signals that may be compared to increase detection accuracy. For example, as described further below, where a nanopore device is a liquid gated 2D transistor, a first current passes through the nanopore, i.e. in an solution in which the nanopore device is immersed, while a second current flows through the nanopore device itself. Each current is, in part, determined based on the size of the nanopore, and the presence of a biomolecule within the nanopore. In this way, both currents are deterministically affected by the presence of a biomolecule. As different aspects of a biomolecule pass through the nanopore, each having unique effects on the RC characteristics of the nanopore, each current varies proportionally to the changing RC characteristics. FIGS.1A-1Cillustrate various aspects of various embodiments of a microfluidic chip196.FIG.1Ais an illustration of various aspects of a biomolecule sensing device including an array180of semiconductor devices100integrated with control circuitry110A disposed in a microfluidic chip196.FIG.1Bis a cross sectional view of a semiconductor device100illustrating aspects of exemplary semiconductor device100with integrated nanopore device140. In embodiments, semiconductor device100is used as a biomolecule sensing cell in a biomolecule detection device.FIG.1Cis a top-down view illustrating aspects of exemplary semiconductor device100having an integrated nanopore device140.FIG.1Dillustrates aspects of an electric circuit of an exemplary biomolecule sensing device, e.g. device100. In embodiments, microfluidic chip196serves as a biomolecule detection device. Microfluidics device, e.g. microfluidic chip196, are device that deal with the precise control and manipulation of working fluids while such fluids are geometrically constrained to small, typically micrometer, or smaller, scale. In some applications microfluidic channels employ passive fluid control techniques, e.g. capillary forces, to control the movement of fluids, e.g. the working fluid. In other applications, microfluidic applications apply active microfluidic components, including micropumps and microvalves, e.g.195A or195B, to control the movement and direction of flow of working fluids. Other exemplary microfluidic structures include micropneumatic systems in order to handle delivery of external fluid, applied to an interface, to various aspects of a microfluidic system. Such microfluidic structures coordinate and control the movement of small volumes of working fluid (e.g., as small as nanoliters or picoleters) throughout such a system. A microfluidic chip, e.g.196, is a set of micro-channels etched or molded into a material (glass, silicon, or polymer such as PDMS, for PolyDimethylSiloxane). The micro-channels, e.g.194, forming the microfluidic chip are connected together in order to achieve the desired features (mix, pump, sort, or control the fluidic environment, e.g. a biochemical environment). Inputs and outputs, e.g.192, connecting a microfluidic chip to an external environment are created by piercing the chip to create an interface to the macro-world. Through these holes, fluids (or gases) are injected and removed from the microfluidic chip (e.g. through tubing, injectors, syringe adapters, wicks, or by other means passive or active). At the nanometer scale, microfluidics may be referred to as nanofluidics. MEMS, in its most general form, are miniaturized devices, structures, or systems that are made by employing microfabrication techniques and that integrate electrical and mechanical elements. MEMS are devices, such as nanopore layer140, formed by microfabrication techniques. MEMS is broad term for describing microscopic devices, and methods of fabricating such devices, which may range in dimensions from hundreds of micrometers down to the nanoscale where MEMS encompasses nanoelectromechanical systems (NEMS). An application of MEMS is in microsensors, e.g. semiconductor device100, which may be integrated with microfluidic systems, that are capable of detecting the presence of target substances in small (microliter or less) volumes of liquid. A characteristic of a biomolecule can be determined using a semiconductor device, such as semiconductor device100, or an array180of semiconductor devices100. One semiconductor device100, or an array180of semiconductor devices100, are disposed in a microfluidic chip196. Microfluidic chip196includes one or more micro-channels194coupling one or more inputs192to the one or more micro-channels194for carrying small volumes of working fluids to an array180of one or more semiconductor devices100. Working fluids include electrolytic solutions provided to various first chambers130A or second chambers130B of the semiconductor devices100. Working fluids may also carry one or more biomolecules from inputs192to the one or more semiconductor device100chambers130A,130B. Each semiconductor device100in an array of semiconductor devices180(which may be only a single device100) is coupled via signal carrying conductors, or interconnections,111to one or more semiconductor circuits110A (whileFIG.1Aillustrates only a single semiconductor circuit110A, it is to be understood that circuit110A may be a plurality of distinct circuits coupled respectively to a plurality of semiconductor devices in array180, or circuit110A may be disposed in a single semiconductor layer110, and comprise a plurality of subcircuits associated with each of the semiconductor devices100, or semiconductor circuit110A may be a single circuit for controlling each semiconductor device100in array180). A circuit layer110may be formed on a wafer, e.g. wafer630. In determining a characteristic of a biomolecule, e.g., sequence/order of nucleotide bases of a strand of DNA, first and second chambers130A,130B of a semiconductor device100are filled with a chemical solution, e.g. provided through microchannels194A or194B. For example, a DNA strand is provided in a chemical solution to a first chamber130A via microchannel194A. Fluid provided to a first chamber130A may pass through a micro-valve195A which controls the transfer of liquids into first chamber130A (alternatively micro-valve195A is omitted and other structures in the microfluidic chip (not illustrated) control the flow of liquids through microchannel194A. Next, a DNA strand (not particularly illustrated) is electrophoretically driven from a first chamber130A to a second chamber130B through a pore140A in a nanopore device140by a sensing device (e.g., a CMOS control circuit110A formed in semiconductor layer110). One or more characteristics of the DNA strand are detected as the DNA strand passes through the pore140A. Electrophoresis refers to causing particles to migrate through a stationary medium, like a solution, under the influence of an applied electric field. An applied electric field may be provided by immersed electrodes, e.g. electrodes150A,620. In one example, a pair of electrodes150A,620are inserted into the chemical solutions contained in respective first and second chambers130A,130B and the sensing device (e.g. circuit110A) applies a drive voltage across the electrodes150A,620. As the DNA strand passes through the pore140A, the sensing device (e.g. circuit110A) detects a drive current through the electrodes150A,620(which are electrically connected through the chemical solutions forming a circuit loop155, as illustrated inFIG.1D). The drive current detected is a function of the associated drive voltage and a corresponding change in the RC characteristics of the nanopore device140(e.g., changes in Rporeor Cpore), where different biomolecules, e.g. nucleotide bases (A, T, C, G), are known to have different deterministic effects on the RC characteristics of a nanopore device as they pass through the nanopore. For example, DNA a sequence may be identified by determining changes in resistance of a nanopore sensing changes in a drive current detected by the sensing device. A sensing device, e.g., may sense such changes in current using integrated circuitry, e.g.110A1. Embodiments of the present disclosure address the deficiencies of known approaches by coupling a nanopore biosensor device with a semiconductor integrated circuit, e.g. silicon CMOS technology, which includes sensing circuitry (e.g.110A1). Such sensing circuitry may be formed by a suitable semiconductor manufacturing process for forming integrated circuits within a semiconductor layer, e.g.110, of a semiconductor device. The semiconductor process includes forming a semiconductor device to support a nanopore device, e.g. nanopore layer140. A nanopore layer, e.g.140, is formed and disposed upon the semiconductor device, e.g.100, using known microelecromechanical systems (MEMS) techniques. In embodiments, a nanopore device140is coupled onto a semiconductor layer110, or onto an insulator layer182formed over a semiconductor layer110. Integrated circuit layer110has an integrated circuit (e.g,110A,110A) configured to sense changes in currents and voltages (e.g. iBor VB) across such a nanopore layer, e.g.140. In this way, a length of a conductor coupling a nanopore device to a sensing circuit is minimized and noise is reduced. An integrated sensing circuit110A1converts a sensed analog signal, e.g. iB, to a digital word for transmission to an external biomolecule characterization device via output198. Since all electrical components are integrated with a nanopore sensor device, e.g., a SNR is greatly improved due to the elimination of noise that incurs when long interconnect wire, or conductor, couples a sensing device with its corresponding sensing electronics. Such a biomolecule sensing device, e.g. semiconductor device100, is thus configured to detect and encode variations in sensor currents or voltages into a digital signal transmitted to a characterization device in order to detect a particular biomolecule. When a biomolecule passes through a nanopore, aspects of the biomolecule are sensed by a sensor circuit that includes either an electrolytic fluid passing through a nanopore,140A, or that includes a nanopore device140. Known semiconductor based or organic nanopore devices typically have thicknesses that are substantially larger than single biomolecule targeted for detection, and therefore have low resolution for sensing aspects of a biomolecule, e.g. a nucleotide. However, 2D transistors, such as a graphene nanoribbon, may be utilized as nanopore devices140. Such a 2D transistor may have a thickness on the order of a nucleotide, and are therefore able to resolve individual nucleotides with enhanced accuracy. Thus, a 2D transistor nanopore layer, e.g.140, is disposed upon a surface of semiconductor device100and coupled by interconnections111to an integrated circuit layer110formed below the nanopore layer. As described further below, a 2D transistor disposed on and electrically coupled to a semiconductor device may pass a current between two electrodes coupled to the nanopore layer, for example, when a voltage is applied between solutions in contact with opposing surfaces of a 2D transistor, e.g.140. Applying such a voltage may be referred to as liquid-gating such a 2D transistor. The voltage applied to solutions in chambers130A and130B act like a gate signal causing conduction along the 2D transistors length (e.g. iTflowing perpendicular to iB). The semiconductor device100and nanopore140are disposed within a solution in a fluid chamber130such that the nanopore layer separates a fluid chamber130into a portion above the nanopore layer130A and a portion below the nanopore layer130B. In this way, the upper and lower portions are separated by the nanopore device140while allowing biomolecules molecules to pass between the chambers through the nanopore140A. In this way a resulting fluid chamber130includes the portion above the nanopore layer130A and the portion below the nanopore layer130B and a relatively small portion defined by the nanopore140A itself. A fluid chamber contains a solution that suspends biomolecules. In embodiments a bias voltage (e.g., VB) is applied between the solution in the portion above the nanopore device130A and the solution in the portion below the nanopore device130B. As a biomolecule passes through the pore140A, a current (iB) passing through the nanopore140A changes as different parts of a target molecule passes through the nanopore142A. For example, different nucleotides of a DNA strand may change the resistance encountered by a current iBas it passes through the nanopore. The passing biomolecule also alters the source to drain RC characteristics of such a liquid gated 2D transistor140, thereby altering a current iTpassing through the nanopore device perpendicular to iB(i.e., through the 2D transistor). In embodiments, a device100also eliminates the need for traditional e-beam ion milling by allowing in situ, i.e. in place, nanopore140A formation. That is, an unformed nanopore device, e.g., such a 2D graphene nano-ribbon transistor may be disposed upon a semiconductor device (e.g.100) and coupled to metal contacts (e.g.150B,150C) that are integrated with portions of integrated circuit (e.g.110A,110A1). Then the nanopore device (e.g.140), or layer, may be formed by creating a nanopore (e.g.140A), or a pore, in the 2D transistor (e.g.140). Forming and disposing a nanopore layer (e.g.140) on a semiconductor device may be accomplished according to any suitable technique, the details of which are beyond the scope of this disclosure. A 2D transistor, such as140, may be a graphene nanoribbon 2D transistor. Graphene is essentially a single atomic layer of graphite that is capable of excellent conduction that may vary based on a translayer (i.e transmembrane) applied voltage (akin to a transistor with an applied gate voltage). Graphene is an allotrope of carbon, typically a dielectric, that includes tightly bonded carbon atoms. Because graphene's width is on the order of one atomic unit biomolecules passing through it may be resolved at the atomic level and so may be detected at a higher resolution. A nanopore, e.g.140A, may be formed in a graphene layer, e.g.140, by causing a forming voltage to be applied across the width of a graphene nanoribbon, e.g. perpendicular to the length of the 2D transistor. When voltage is applied in this manner, the unformed graphene nanoribbon layer initially acts like a dielectric. Such applied voltage eventually causes leakage current perpendicular to the length of the nanoribbon thereby breaking down the dielectric material of the 2D transistor in a localized area until a pore, e.g.140A, is formed in the localized area, e.g. a desired area over orifice142A, thereby allowing current to pass through the now formed nanopore device. Once formed, the size of the pore, e.g.140A, may be enlarged by appropriate voltages as discussed further below. In embodiments, a formed nanopore layer140with nanopore140A coupled to an integrated sensing circuit, e.g.,110A, formed in semiconductor layer110, is able to sense aspects of a biomolecule that passes through pore140A in the nanopore layer140. In embodiments a plurality of integrated semiconductor nanopore devices100are configured in an array180of semiconductor devices with integrated nanopores100, each including a nanopore140A and each may be electrically separated from each other semiconductor device100, or cell, with integrated nanopore. By electrically separating of each nanopore140A in an array180, each nanopore140A coupled to each semiconductor device100can be electrically formed independently, thus the size of each individual nanopore140A can be individually tailored as desired under reliable controls using electrional methods. Electrical formation of a nanopore140A may occur directly in a solution, as described herein, by first breaking down the dielectric, e.g. the carbon lattice of a graphene nanoribbon, of the nanopore layer140using relatively high voltages and then soaking the device with relatively low voltages to enlarge the nanopore to a desired size. Thus, each electrically separated nanopore140A in an array180may be addressed individually, and can individually and concurrently sense biomolecules disposed in each respective semiconductor device chamber (e.g.,130A,130B). The size of each nanopore140A can be formed individually and controllably to an intended size by integrated circuitry110A. Exemplary, non-limiting, nanopore device structures are based on 2D transistors of either graphene nano ribbon 2D transistors or MOS2 2D transistors. In any case, both the ion current (iB) and transistor source/drain current iT(e.g. a currently flowing along the length of the transistor between electrodes150B,150C, and perpendicular to iB) may be measured independently of each other by the integrated circuit110A within the formed semiconductor device circuit layer110. As a result, noise is significantly reduced by amplifying and digitizing a sense signal (e.g. iBor iT) locally without large noise interference from outside the devices or from lengthy electrodes. The particular mechanisms for forming and transferring suitable 2D transistors are known in the art and need not further be discussed. Systems and methods as described herein in various embodiments include a semiconductor device, e.g., semiconductor device100inFIG.1, with a circuit110A configured to sense a current associated with resistances associated with a biomolecule sensed by such a semiconductor device. By integrating a nanopore biosensor (e.g.140) with a semiconductor device (e.g.110), e.g. silicon CMOS technology integrated with a nanopore, the overall semiconductor device100minimizes electrical separation between the nanopore sensor140and the sensing electronics110A. This enables a higher signal to noise ratio (SNR) over known semiconductor nanopore devices by eliminating long interconnection wires from the signal path. Sensing a signal from a nanopore biosensor in accordance with this disclosure allows the sampled signal to be converted to a signal encoded with a relatively lossless signal protocol, such as standard digital signals. The example semiconductor device100includes a circuit layer110and a first chamber130A. The first chamber130A is above the circuit layer110and is defined by a chamber wall130. Disposed in the first chamber130A is a nanopore layer140, and an insulating layer160. The chamber wall130is formed, or disposed, on the circuit layer110, such as by bonding the chamber wall130to the circuit layer110with the use of an adhesive. The chamber wall130defines a first chamber cavity130A therein configured to receive a chemical solution (not particularly illustrated here, but see solution800, e.g.FIG.8T-8U). The chamber wall130may, for a non-limiting example, have a length of about 20 μm and a width of about 20 μm. The chamber wall130may be formed, for example, of a silicon cap bonded to the semiconductor device100surface. The chamber wall130is capable of retaining an ionic solution that allows conduction. The ionic solution may be, in embodiments, KCL. The upper chamber may be a cis chamber, and may have a negative voltage applied relative to a corresponding trans chamber. It will be appreciated that embodiments in accordance with this disclosure may include chamber cavities defined by chamber walls of varies sizes dictated by design considerations. Examples of materials for the chamber wall130include, but are not limited to, Si, Ge, ceramic, quartz, glass, silicon, or polymer such as PDMS, for PolyDimethylSiloxane and the like. Semiconductor device100may also include a second chamber130B formed beneath and through the circuit layer110, and may in part be defined by a cavity formed by surfaces formed within the circuit layer110, or the second chamber130B is formed by a second cap bonded to an opposite surface than the surface defining the first chamber130A. The second chamber130B is also configured to retain an ionic fluid, and may form a trans chamber and may have a positive polarity applied relative to the cis chamber). And in other embodiments, first chamber130A forms a trans chamber and the second chamber130B forms a cis chamber. In embodiments, the nanopore layer140is disposed in the first chamber cavity130A upon the circuit layer110and has first and second end portions and an intermediate portion between the first and second end portions. The nanopore layer includes a pore140athere-through formed in situ (or pre-formed). The intermediate portion of the nanopore layer140may take a variety of forms, for example in one embodiment it may have a 2D area formed of a first length of about 100 nm to about 160 nm and a second length of about 80 nm. The pore140ahas a diameter/width of, e.g., about 2.0 nm and extends through the nanopore layer perpendicular to the 2D area. In an embodiment, the nanopore layer140is ribbon-like shaped and includes a monolayer, e.g., about 1 nm thick, graphene. In an alternative embodiment, the nanopore layer140includes MoS2, MoSe2, WS2, WSe2, other suitable monolayer material, a dielectric material, such as Si3N4, an oxide-based material, such as Al2O3, or a combination thereof. The nanopore layer140may be a 2D transistor, for example a graphene nanoribbon field effect transistor, which forms a membrane and exhibits conduction along its length when a liquid-gating voltage is applied across the membrane (e.g. a voltage between a trans and cis chamber130A,130B separated by a nanopore device140layer). A first electrode layer150A is disposed in the first chamber cavity130A and is formed over the circuit layer110. The first electrode layer150A includes metal resistant to corrosion/oxidation in the chemical solution. Examples of such metal include, but are not limited to, silver, gold, platinum, other corrosion/oxidation-resistant metal, and an alloy thereof. Additional electrode layers150B,150C are disposed in the first chamber cavity130A, are formed over the circuit layer110, and are coupled to first and second end portions of the nanopore layer140, respectively. Layers150B and150C may be electrically isolated from an ionic solution within chamber130A by an insulation layer160. Electrode layers150B,150C may have a different material than the first electrode layer150B. Examples of materials for the electrode layers150B,150C include, but are not limited to, copper, aluminum, titanium, tungsten, other conductive material, and an alloy thereof. The insulating layer160covers the electrode layers150B,150C and is configured to prevent exposure of the electrode layers150B,150C to the chemical solution retained in chamber130A by chamber wall130. The insulating layer160further covers the first and second end portions of the nanopore layer140. The insulator layer160covering the first and second end portions of the nanopore layer140may also prevents biomolecules passing through the pore140A from contacting portions the nanopore layer140, which undesirably alter a resistance of the nanopore layer140. The semiconductor device100is configured to act in concert with an external device such as a biomolecule characteristic-identifying device (not particularly illustrated), e.g., during a biomolecule sequencing. During such a biomolecule sequencing, a biomolecule characteristic-identifying device may be part of a microfluidic device, e.g. microfluidic chip196, into which the semiconductor device100, or an array180, is inserted. The semiconductor device100provides one or more biomolecule characteristics based on electrical characteristics sensed by the combination of nanopore sensor device140coupled to integrated circuit layer110. As illustrated inFIGS.1B,1C, the semiconductor device100further includes a pair of conductive pads170A,170B which enable connection to external circuits. Pads170A,170B may couple exemplary circuits110,110A,110A1to output198, which provides an output to an external device. Structures depicted inFIG.1Care integrated with circuitry (aspects110A1of which are illustrate inFIG.1D) in underlying layers. Such circuitry may be formed in circuit layer110. Circuit layer110may, for example, include semiconductor device integrated circuits, e.g., portions of which are depicted inFIG.1D. Circuit portion110A1for example includes a sensing portion comprising an amplifier configured in voltage follower mode (as depicted). One of skill will appreciate that a voltage follower will convert variations in iBto a voltage, Vout, provided with sufficient current ioutto supply follow-on stages while allowing for low input currents in e.g. iB. Circuit portion110A1may include additional stages, such as an analog to digital converter320(ACD) thereby providing a digital signal to output198(e.g. in embodiments via pads170A,170B, not particularly depicted inFIG.1D). It will be appreciated that the semiconductor integrated circuit depicted by the schematic inFIG.1Dis only intended to illustrate the integration of semiconductor circuitry in layer110with nanopore device140, and additional sensing and control circuitry may also be formed in semiconductor layer110as needed. FIG.2Ais a functional block diagram illustrating an exemplary first circuit200connected to the first electrode layer150A in cavity130A and a second electrode layer620disposed in a second cavity130B opposite the nanopore layer140from cavity130A, as discussed above. The first circuit200is configured to apply a drive voltage (e.g. Vbias1) across the electrode layers150A,620for driving a biomolecule through the pore140A. In embodiments, first circuit200is further configured to also detect a drive current (idrv) through the electrode layers150A,620that is associated with both a drive voltage and corresponding RC characteristics of a nanopore layer, e.g.140. Alternatively, drive module220may alternate between a drive voltage, e.g. Vbias1, and a sensing voltage, Vbias2, thereby first driving a biomolecule by applying Vbias1and then sensing a pore current (ipore1) by applying a second bias voltage Vbias2. In embodiments Vbias2may be the same as, or different than, Vbias1. As a biomolecule passes through the nanopore140A, the RC characteristics of the nanopore device140, as experienced by a current iBpassing through a pore, e.g.140A, vary causing the current to increase and decrease in a measurable and deterministic fashion. One or more characteristics of the biomolecule may be determined by sensing changes in drive currents (idrv) detected by the first circuit200while a drive voltage is applied between terminals150A,620. As described below with respect to the manufacture of the semiconductor device100, the first circuit200further configured to form the pore140A and to measure the diameter of the pore140A after initially formed. When first disposed above a circuit layer110, a nanopore layer140is initially unformed (not pre-formed), meaning no nanopore has yet been formed. Circuit200enables in situ pore formation of a pore140A in nanopore layer140. Once a nanopore140A is formed in nanopore layer140, nanopore layer140is referred to as a formed nanopore layer. A pore may be formed in an unformed nanopore layer140by applying sufficient voltage across the nanopore layer, e.g. between first chamber130A and second chamber130B, to cause a dielectric breakdown of the nanopore layer140dielectric material (e.g. carbon in graphene form). This dielectric breakdown in an exemplary graphene 2D transistor140will cause a predictable increase in current iBthrough nanopore layer140when a pore140A is formed. As further voltage is applied, a pore140A will enlarge causing further predictable current increases as the pore140A diameter increases. As will be appreciated, once a nanopore140A is formed, the nanopore layer140RC characteristics will change in a predictable manner as a biomolecule passes through a formed pore140A in a nanopore layer140. Pore140A is dimensioned to a desired size. To form the nanopore140A, the first circuit200is configured to apply pore-forming pulses Vpulseacross the electrode layers150A,620to form the pore140A in the nanopore layer140. The first circuit200is further configured to detect a diameter of a formed pore140A by applying a pore voltage across the electrode layers150A,620. The first circuit is further configured to detecting a pore current (ipore) through the electrode layers150A,620that is associated with a pore voltage and a corresponding resistance of the nanopore140A. A diameter/width of the pore140amay be estimated using the pore currents detected by the first circuit200. In this way the dimensions of pore140acan be accurately controlled. Exemplary device100may take many forms, for example, device100includes a graphene layer140that is approximately 1-5 nm in thickness, suspended over a 1 μm wide diameter hole140A in a 40 nm thick silicon nitride (SiN) membrane182. In embodiments, SiN membrane182is suspended over an approximately 50×50 μm2aperture in a semiconductor layer coated with a 5 μm thick layer of SiO2. This exemplary device100may be inserted into a portable biometric detection device (not shown) that includes circuitry110A for applying and sensing voltages (e.g., VB) and currents (e.g., iB) through electrodes150A and620. These sensed currents or voltages may be used by circuitry110A to create a nanopore, e.g. nanopore140, as described herein or to drive a biomolecule through hole140A, or to sense variations in VBor iBas a biomolecule passes through hole140A that may interpreted by circuitry110A formed in semiconductor layer110to allow biomolecule detection. Circuitry110A may be formed within device110and suitably coupled to electrodes150A,620. In the example ofFIG.2A, the first circuit200includes a voltage generator210, a driving module220, a pore-forming module230, and a switch240controlled by a control signal CS1. The voltage generator210is connected to the electrode layer620(which is in contact with chemical solution in the second chamber130B). Voltage generator210may be responsive to input signal IN1originating in control circuitry110A. As one of skill will appreciate voltage generator210is configured to receive input control signal (IN1) which controls the generation of generate a reference voltage (Vref1). Vref1may be a constant voltage, e.g., ground, VDD, or any suitable reference voltage. Vref1may be applied to the electrode layer620. Signal IN1and reference voltage Vref1may be provided by any suitable semiconductor structure within the circuit layer110. The particulars of creating a reference voltage based on an input signal are known in the art and need not further be discussed. Signal IN1may alternatively be a control signal from external control circuitry; e.g., control circuitry in a biological characteristic-identifying device coupled to device100. IN1may be an enable signal, or characteristics of IN1may dictate characteristics of the reference voltage (Vref1), for example Vref1=A*IN1, where A is a scalar factor such that IN1may dictate the magnitude Vref1in a linear fashion. It will be appreciated that IN1may control the magnitude and duration of Vref in any suitable fashion. For example, IN1may be a digital word that encodes a magnitude (e.g. 40 mV) with a duration (e.g. 10 ns). IN1may be an enable signal that enables a predetermined Vref (e.g. 50 mV), or IN1may have dynamic qualities that determine the characteristics of Vref, for example IN1may vary dynamically according to a customized encoding as desired by the design consideration of a device100. For example IN1may vary between two voltages in a first power domain (e.g. 0v-5v), such that the variation in IN1voltage correspond to voltage changes in Vref in a second power domain (−100 mV-100 mV) as the system requires based on design considerations that are beyond the scope of this disclosure. In other embodiments IN1may be an external reference, e.g. VDD or ground or 0V, in which case Vref may likewise be VDD, or ground, or 0V from an external reference. Control signal IN1may also be a current domain signal, such that Vref varies in response to variations in current supplied by IN1. In embodiments, the driving module220, may be programmed to respond to any of Idrv, Ipore, Vbias1or Vbias2by controlling Vref using IN1. For example, Vrefmay be established to ensure, in coordination with other bias voltages, a particular direction of DC flow, e.g. a DC current flow from electrode150A to electrode620, or visa versa. Control circuitry may react to voltage variations in semiconductor device100to ensure that the direction of DC flow does not change, thereby ensuring that biomolecules flow in a particular direction through a nanopore140A. Thus, in the context of this disclosure, IN1may control the reference voltage Vref as needs dictate based on design considerations that are beyond the scope of this disclosure and according to well-known principles that need not be discussed further. The driving module220is selectively connected to the electrode layer150A and is configured to generate a bias voltage (Vbias1) applied to the electrode layer150A. The reference voltage (Vref1) at the electrode layer620and the bias voltage (Vbias1) at the electrode layer150aresult in a drive voltage across the electrode layers620,150A for driving a biomolecule through the pore140A. The driving module220is further configured to detect a drive current (Idrv) through the electrode layers620,150A that is associated with the drive voltage and a corresponding resistance of the nanopore layer140. One or more of the characteristics of the biomolecule may be determined using the drive currents (Idrv) detected by the driving module220. As explained above, as a biomolecule passes through nanorpore140, the RC characteristics of the nanopore vary with the molecule currently passing through the nanopore140, thereby causing known, deterministic, variations in Idrvthat may be simultaneously sensed by circuitry coupled to electrodes150A,620. In this way the sequence of molecules in a biomolecule may be resolved. The pore-forming module230is selectively connected to the electrode layer150A, e.g. using switch240and controlled by control signal CS1, and is a voltage generator configured to generate pore-forming pulses (Vpulse) applied to the electrode layer150afor forming the pore140a.For example, pore-forming module230may receive a reference voltage from voltage generator210and amplify the reference voltage using known principles to generate a pore forming volutage (Vpulse). The driving module220is further configured to generate a bias voltage (Vbias2) applied to the electrode layer150A. Reference voltage (Vref1) at the electrode layer620and the bias voltage (Vbias2) at the electrode layer150A together result in a pore voltage applied between electrode layers620,150a.Driving module220is further configured to detect a pore current (Ipore1) through electrode layers620,150A. Ipore1is associated with the pore voltage and a corresponding resistance of the nanopore140A, which varies in a deterministic manner as the diameter of nanopore140A increases. A diameter/width of the pore140A may be estimated using the pore currents (Ipore1) detected by the driving module220. Before a pore only very small leakage currents may pass through the dielectric membrane, e.g. graphene. Thus, when a pore140A is formed an increased flow of current iBfrom a first chamber, e.g.130A, into a second chamber, e.g.130B, through the nanopore is detected. An increase in diameter of the pore can be detected as a decrease in resistance of (or an increase in the current through) the nanopore as illustrated below. The switch240is connected to the driving module220, the pore-forming module230, and the electrode layer150a.Switch240is configured to receive a control signal (CS1) and to selectably connect either the driving module220or the pore-forming module230to electrode layer150a.CS1may be generated in semiconductor circuits formed in layer110, or may be received from an external control circuit in an attached biomolecule sensing/characterization device. FIG.2Billustrates nanopore Vpulseduring formation in a single layer of graphene, e.g. in layer140, according to various embodiments. The single layer of graphene140A sits atop an aperture142A in semiconductor and insulator layers110,182between a first chamber130A and a second chamber130B and separates two ionic solutions in each respective first and second chambers130A,130B. A first electrode150A is in the first chamber130A, and a second electrode620is in the second chamber130B. Electrically connected between the first electrode150A and the second electrode620is a circuit formed in a semiconductor circuit layer110allowing for both the measurement of current and for driving pulse formation, e.g. first circuit200. As shown inFIG.2B, before a nanopore (or defect in the graphene layer) is formed in graphene layer140, circuit200may be configured to apply Vpulse as a series of electrical pulses264of a first voltage across the first electrode150A and the second electrode620. The first voltage may be any suitable voltage for inducing nucleation in a selected nanopore layer (here, e.g., made of a single layer of graphene, but in other embodiments made of any desirable 2D transistor). For example, in the embodiments depicted, the first voltage may be about 7v. Between pulses, circuit200is configured to sense a current flowing between the first electrode150A and the second electrode620. As shown, prior to nucleation of the graphene layer, e.g.140, at time266, at approximately twenty-seconds of Vpulsepulses, current is approximately zero indicating that little or no current flows between first electrode150A and second electrode620. After time266, when nucleation of the graphene layer occurs, forming a nanopore, current begins to flow through aperture140A,142A from first electrode150A to second electrode620. In the exemplary embodiments depicted, beginning at about a time268equal to 42 s, Vpulseincludes a second series of electrical pulses270of a second voltage, here lower than the first voltage pulses264, are applied causing the defect, or nanopore, to increase in diameter over a period of approximately 40 seconds from about a diameter of 0.1 nm to a diameter of about 2.2 nm. This lengthier series of second pulses270of a lower voltage may be referred to as a voltage soaking. The second voltage may be any suitable voltage for enlarging a nanopore in a desired nanopore layer, for example in the embodiments depicted with a nanopore layer formed from a single layer of graphene, the second voltage may be about 5v. Additionally, the relative periods of time the first voltage pulses264and the second voltage pulses270are applied may be any suitable periods to achieve the desired purposes, and are not limited to the exemplary values of 7v and 5v. The relationship between current and pore diameter for a given nanopore layer may be obtained by direct measurement. FIGS.3A-3Cdepict example supporting circuitry for the first circuit200.FIG.3Ais schematic diagram illustrating an exemplary driving module220in accordance with some embodiments. These circuitry are provided by way of example and other suitable circuitry are within the scope of the present disclosure. The driving module220includes a current-to-voltage converter (IVC)310and an analog-to-digital converter (ADC)320. In this way circuitry220formed in semiconductor device layer110senses the analog value changes in iB, and converts the current signal (Idrv/Ipore1) signal first to a voltage then to a digital signal for transmission offchip to an external device. By converting the signal to digital in situ, noise and distortion introduced by transmitting analog signals off chip are avoided entirely. IVC310is configured to generate the bias voltage (Vbias1), to detect the drive current (Idrv), and to convert the drive current (Idrv) to a drive voltage (Vdrv). The ADC320is connected to the IVC310and is configured to convert the drive voltage (Vdrv), which is in an analog format, into a digital format so that the detected signal may be transmitted to the output198thereby to an external device in a digital format without concern of introduced noise and distortion masking small changes in an analog signal. One or more characteristics of the biomolecule may then be determined using the digitized drive voltages (Vdrv) provided by the ADC320. The IVC310is further configured to generate the bias voltage (Vbias2), to detect the pore current (Ipore1), and to convert the pore current (Ipore1) to a pore voltage (Vpore1). The ADC320is further configured to convert the pore voltage (Vpore1), which is in an analog format, into a digital format. A diameter/width of the pore140A may be estimated using the digitized pore voltages (Vpore1) provided by the ADC320. As illustrated inFIG.2B, nanopore diameter for a given nanopore layer may be measured based on a measured current through the nanopore. Because Vpore1is derived from Ipore1by IVC310according to a function selected by the designer, measuring Vpore1similarly provides a measurement of the nanopore. While in embodiments the nanopore diameter may be measured by directly measuring the current through the nanopore, in other embodiments it is advantageous to first convert the current to be measured to a voltage and measurement taken of the converted voltage as an alternative to measuring the current directly. In an alternative embodiment, the driving module220further includes an amplifier, e.g., a cascade amplifier, connected between the IVC310and the ADC320and configured to amplify the drive/pore voltages (Vdrv/Vpore1) provided by the IVC310prior to receipt by the ADC320. This may be advantageous where fluctuations in pore voltages or drive currents is small, and to provide sufficient driving current where input drive currents may be too small to drive follow on sensor loads. For example, an amplifier configured in voltage follower may provide a gain of 1, such that an input voltage is equal to an output voltage, while output current is increased substantially to support follow on sensor load. It will be appreciated that any suitable amplifier may be chosen to amplify the drive/pore voltage as necessary. FIG.3Bis a schematic diagram illustrating an exemplary IVC310in accordance with some embodiments. In the example ofFIG.3B, the IVC310includes a transimpedance amplifier330and a voltage generator340. The transimpedance amplifier330includes an operational amplifier (op-amp)330, a feedback resistor (Rf)332connected between a first input terminal330aand an output terminal330cof the operational amplifier (op-amp), and a feedback capacitor (Cf)334connected in parallel to the feedback resistor (Rf)332. The voltage generator340is connected to a second input terminal330bof the operational amplifier (op-amp)330, is programmable in this embodiment. Voltage generator340is configured to receive an input signal (IN2) and to generate an input voltage (Vin) applied to the second input terminal330bof the operational amplifier (op-amp) that corresponds to the input signal (IN2). As with IN1, IN2may be a control signal provided according to one or more control circuits110A in semiconductor circuit layer110, or by an external control device. As with IN1, generating an output of Vin according to a control signal is well known in the art and is not discussed further. IN2allows for greater controller of the IVC310by allowing external control circuits (not shown) to control the amplifier as dictated by a designer according to design considerations that are beyond the scope of this disclosure other than that IN2may be employed to control Vin as desired. As with the relationship between IN1and Vref explained above, voltage generator340takes IN2which may be of a first voltage domain and outputs Vin may be of a second domain and based on control signal IN2as required by circuit design considerations. In one exemplary control scheme, when IN2is 1V Vin is 10 mV and when IN2is 2V Vin is 35 mV, but one will appreciate that IN2may control Vin in any suitable fashion. In other embodiments IN1may be an external reference, e.g. VDD or ground or 0V, in which case Vin may likewise be VDD, or ground, or 0V from an external reference. The transimpedance amplifier330is configured to provide at the first input terminal330athereof the bias voltage (Vbias1/Vbias2) substantially equal to the input voltage (Vin). In this way, the bias voltage may be controlled by modulation of IN2as desired according to design considerations. The transimpedance amplifier330is further configured to receive the drive/pore current (Idrv/Ipore1) and to provide at the output terminal330cthereof the second drive/pore voltage (Vdrv/Vpore1). As one of skill in the art will readily appreciate, the gain of the transimpedance amplifier310is established by the magnitude of the resistance of resister332, and the capacitance of capacitor334may be selected as needed to provide a low pass filter in the feedback path to increase circuit stability, thereby offsetting any capacitance, e.g. Cpore, experienced by the ionic current iB. FIG.3Cis schematic diagram illustrating an exemplary pore-forming module230in accordance with some embodiments. The pore-forming module230includes a pulse generator350and a voltage booster360. The pulse generator350is programmable in this embodiment and is configured to receive an input signal (IN3) and to generate the pore-forming pulses (Vpulse) that correspond to the input signal (IN3). IN3, like IN1and IN2, is a control signal and is utilized to control pore formation according to known principles. Pore-forming pulses may have any suitable magnitude as explained in reference toFIG.2B, e.g. for embodiments including a graphene nanoribbon Vpulse may varying between 7V during pore formation and 5V during pore enlargement, or according to other embodiments other suitable voltages may be employed according to design considerations. Signal IN3may further be provided by any suitable semiconductor structure within the circuit layer110. In embodiments, signal IN3is supplied via a route to a voltage source implemented in CMOS structures. As explained above, IN3for example may be a control signal supplied by external control circuitry. IN3may comprise a range of suitable voltages. In some embodiments IN3may be a toggle signal that toggles between two predetermined voltages (e.g. when IN3=50 mV, Vpulse=5V and when IN3=70 mV, Vpulse=7V). Or, IN3may have dynamic qualities that determine the characteristics of Vpulse such that as IN3takes many voltages on demand between various voltages, IN3causes VPulseto vary on demand as well in a like manner according to a linear function (e.g. Vpulse=A*IN3). For example, IN3may vary between two or more voltages in a first power domain (e.g. 0v-5v), such that the variation in IN3voltage correspond to voltage changes in Vref in a second power domain (0-100 mV). Control signal IN3may also be a current domain signal, such that Vref varies in response to variations in current supplied by IN3. This pulse generator350may be programmed to control Vref in response to IN3according any suitable function. The pore-forming pulses (Vpulse) may alternate between the reference voltage (Vref1) level and a voltage level higher than the reference voltage (Vref1) level. This variation may be controlled via control signal IN3as discussed above. The voltage booster360is connected to the pulse generator330and is configured to step up a level of the pore-forming pulses (Vpulse). Voltage booster may increase Vpulse by a predetermined factor, e.g. a factor of ten). In this way, Vpulse is within the range of voltages that will cause an enlarge nanopore formation. It will be appreciated that the voltages discussed above are by way of example only, and in particular embodiments will be dictated by design considerations and materials. As explained in detail in reference toFIG.2B, in one example, higher voltages are first employed in a series of pulses to cause dielectric break down, then lower voltage pulses are employed to increase pore size over a period of time, such that the longer low voltage pulses are employed the larger the diameter of the nanopore. In an embodiment, nanopores are formed in a graphene membrane using electrical pulse fabrication, as described in reference toFIG.2B. A transmembrane current is measured to monitor pore size. A series of 250 ns 7v electrical pulses are applied across the graphene membrane to nucleate the pore. In an embodiment, 7v pulses are applied for approximately 25 sec at which point an appreciable increase in transmembrane current (e.g. from 0 nA to 0.2 nA) may be observed. Then, a series of low voltage 5V electrical pulses are applied to increase the diameter of the pore. This low voltage soaking signal, in an embodiment, applied over the course of 40 seconds, with 1 second interval pauses for measurement of the transmembrane current, causes the diameter of the pore to increase from approximately 0.2 nm to 2.3 nm in nearly linear fashion (e.g. by observing an increase in transmembrane current from 0.2 nA to approximately 1.8 nA). It will be appreciated that the voltages and times discussed above are by way of example only, and in particular embodiments will be dictated by design considerations and materials. The structures and circuitry depicted inFIG.2A-2Bare not intended to be limiting and in embodiments further include additional circuit components configured to sense biomolecule characteristics as the biomolecule passes through the pore140A. FIG.4is a functional block diagram illustrating an exemplary second circuit400formed in semiconductor layer110. Second circuit400is formed for controlling a voltage applied across a nanopore layer (e.g. between terminals150B,150C). Second circuit may sense a transistor current iTpassing through a nanopore layer (i.e. a current comprising currents such as Isense or Ipore2that pass between ‘source/drain’ regions of a 2D transistors connected to the electrode layers150B,150C) in accordance with some embodiments. The second circuit400is configured to apply a sense voltage across the electrode layers150B,150C. And second circuit400is configured to detect a sense current through the electrode layers150B,150C that is associated with the sense voltage. As a biomolecule is driven through the pore140a,RC characteristics of the nanopore layer140between electrodes150B,150C change in a predicable manner associated with individual sensed portions of a biomolecule (e.g., nucleotides). As the RC characteristics of the nanopore layer change, such changes affect a sensed current, e.g. variations in Isense. Thus, alternatively to sensing drive currents passing through a nanopore, the sense currents detected by the second circuit400may be used to determine one or more characteristics of the biomolecule. It will be appreciated that all values of currents, voltages, and sizes are exemplary and according to various embodiments, but in other embodiments these values will take any suitable value depending on design considerations. The second circuit400can also be configured to detect the diameter of the pore140A in conjunction with (e.g., as a corroborating activity), or in the alternative to, the first circuit200detecting the diameter. First circuit and second circuits are each respectively configured to detect orthogonal currents iB, iTpassing through nanopore layer140. Each current may differ from the other, while also being deterministically affected by changes to the RC characteristics of the nanopore layer. Therefore sensing iTand iBvariations may complement each respective sensing. That is, instead of forming and detecting a size of a nanopore using ionic currents passing through the nanopore and sensed, e.g., by an electrode150A or620, a nanopore size may be monitored using currents created and monitored by second circuit400, e.g. Ipore2. Or, both an ionic current iBand a transistor current iTmay be used in forming and sizing a nanopore (e.g. an ionic current, e.g. Ipore1, may form the nanopore while the size of the nanopore is monitored using transistor currents like Ipore2). In any such case, second circuit400may be configured apply a sensing voltage across the electrode layers150b,150c,and also to detect a pore current, Ipore2, through the electrode layers150b,150c,where Ipore2is associated with the sensing voltage and a corresponding resistance of the nanopore layer140, that changes as a nanopore is enlarged. Thus, aside from the pore currents detected by the first circuit200, the pore currents detected by the second circuit400may alternatively be used to estimate a diameter/width of the pore140a.When the nanopore layer, e.g.140, is electrically conductive, instead of sensing and measuring a current passing through the chemical solution, and through the nanopore itself as with first circuit200, alternatively, electrodes150band150cmay be coupled to the nanopore layer, e.g.140,250, and a current/voltage relationship across the nanopore layer, e.g.140,250, may be measured to determine nanopore diameter. For any given configuration, and selection of nanopore layer, the IV relationship across a conductive nanopore layer as a nanopore is formed, or enlarged, may be obtained by prior direct measurement in order to calibrate the sensor system. The is a functional block diagram ofFIG.4illustrates an exemplary second circuit400connected to the electrode layers150b,150cin accordance with some embodiments. The second circuit400includes a voltage generator410and a sensing module420. The voltage generator410is connected to the electrode layer150b,is programmable in this embodiment, and is configured to receive an input signal (IN4) as a control signal to generate a reference voltage (Vref2) applied to the electrode layer150bthat corresponds to an input control signal (IN4) supplied, e.g. by an external control device or circuit. Signal IN4and reference voltage Vref2may be provided by any suitable semiconductor structure within the circuit layer110. In embodiments, signal IN4is supplied via a route to a voltage source implemented in CMOS structures. The particular mechanisms for controlling a voltage generator for generating a desired reference voltage using a control signal IN4are known in the art, as discussed above, and need not further be discussed. IN4for example may be a control signal supplied by external control circuitry. IN4may be a toggle signal that toggles between two predetermined Vref2(e.g. IN4may toggle between 50 mV and 70 mV), or IN4may have dynamic qualities that determine the characteristics of Vref2. For example IN4may vary between two voltages in a first power domain (e.g. 0v-5v), such that the variation in IN4voltage correspond to voltage changes in Vref2in a second power domain (−100 mV-100 mV). In other embodiments IN4, as with IN1, IN2, may be an external reference, e.g. VDD or ground or 0V, in which case Vref2may likewise be VDD, or ground, or 0V. Control signal IN4may also be a current domain signal, such that Vref2varies in response to variations in current supplied by IN4. This voltage generator410may be programmed to control Vref2in response to IN4according to a function, which may be a linear function, or any suitable control function, e.g. Vref2=A*IN4, where A is a scalar. Like pulse generator230, voltage generator410may be programmable to employ a voltage booster configured to step up a level of the of Vref2. Voltage booster may increase Vref2by a predetermined factor, e.g. a factor of ten. It will be appreciated that the voltages discussed above are by way of example only, and in particular embodiments will be dictated by design considerations and materials. In embodiments, the sensing module420is connected to the electrode layer150cand is configured to generate a bias voltage (Vbias3) applied to the electrode layer150c,as a biomolecule is driven through the pore140a.The reference voltage (Vref2) at the electrode layer150band the bias voltage (Vbias3) at the electrode layer150cresult in a sense voltage (Vsense) across the electrode layers150b,150cwhich varies with the RC characteristics of the nanopore device140. This causes proportional variation on a sensed current, e.g. Isensesensed by sensing module420. The sensing module420is further configured to detect a sense current (Isense) through the electrode layers150b,150cthat is associated with the sense voltage Vsenseand a corresponding resistance of the nanopore layer140. One or more characteristics of the biomolecule may be determined using the sense currents (Isense) detected by the sensing module420. The sensing module420is further configured to generate a bias voltage (Vbias4) applied to the electrode layer150c.Voltage Vbias4may be generated by a voltage source implemented in CMOS structures, or by any suitable means. In embodiments, sensing module420includes a voltage source that may generate Vbias4as a constant voltage or sensing module420may, like Vref2, be programmable such that Vbias4may be varied according any suitable function. The reference voltage (Vref2) at the electrode layer150band the bias voltage (Vbias4) at the electrode layer150cresult in a sense voltage across the electrode layers150b,150c. The sensing module420is further configured to detect a pore current (Ipore2) through the electrode layers150b,150cthat is associated with a sense voltage and a corresponding resistance of the nanopore layer140. A diameter/width of the pore140amay be estimated using the pore currents (Ipore2) detected by the sensing module420. FIG.5is schematic diagram illustrating an exemplary sensing module420in accordance with some embodiments. The sensing module420includes a current-to-voltage converter (IVC)510and an analog-to-digital converter (ADC)520. The IVC510and the ADC520may each be formed in circuit layer110using CMOS techniques according to any suitable semiconductor device forming process, the details of which are beyond the scope of this disclosure. The IVC510, e.g., a transimpedance amplifier circuit, is configured to generate the bias voltage (Vbias3), to detect the sense current (Isense), and to convert the sense current (Isense) to a sense voltage (Vsense). In embodiments, sensing module420includes a voltage source creating a constant Vbias3voltage having an associated current Isensethat varies with the resistance between electrodes150B and150C. As the resistance changes responsive to a passing biomolecule, current Isensevaries corresponding in the change in resistance caused by the biomolecules passing through nanopore140A. The ADC520is connected to the IVC510and is configured to convert the sense voltage (Vsense), which is in an analog format, into a digital format for transmission to an external biomolecule characterization device. By converting to a digital format in situ, within semiconductor layer110, the distance between biomolecule sensor, e.g. nanopore layer140, and sensing circuitry is minimized thereby minimizing noise and distortion interfering with the sensed analog signal. One or more characteristic of the biomolecule may be determined using the sense voltages (Vsense) provided by the ADC520. During pore-formation, the IVC510is further configured to generate the bias voltage (Vbias4). Vbias4may be a constant voltage having an associated current Ipore2that varies with the size of the pore. While Vbiasis applied, IVC510is configured to detect the pore current (Ipore2) and to convert the pore current (Ipore2) to a pore voltage (Vpore2) which differs in relation to the size of the pore140A. The ADC520is further configured to convert the pore voltage (Vpore2), which is in an analog format, into a digital format. A diameter/width of the pore140A may be estimated using the pore voltages (Vpore2) provided by the ADC520. In embodiments Vbias4is the same as Vbias3, but as will be appreciated Vbias3and Vbias4may be tailored to a particular application. In embodiments, Vbias3is generated during sensing of a biomolecule's characteristics by sensing Isense, and Vbias4is generated during pore formation to characterize pore size by sensing Ipore. In an alternative embodiment, the sensing module420further includes an amplifier, e.g., a cascade amplifier, connected between the IVC510and the ADC520and configured to amplify the sense/pore voltages (Vsense/Vpore2) provided by the IVC510prior to receipt by the ADC520. In various embodiments the signals created and sensed by first circuit200and second circuit400may be independently sensed and transmitted off chip to an external biomolecule characterization device for analysis of the sensed signals in order to characterize the sensed biomolecule or in feedback to one or more control signals or functions (e.g. CS1, IN1, IN2, IN3, IN4). In embodiments these sense signals may be analyzed singularly, or an analysis may rely on multiple sensed signals, (e.g., ipore1, ipore2, iB, iT, idrv, isense), or an analysis may rely on differences between two signals (e.g., A*ipore1-B*ipore2), or an analysis may rely on any suitable function of the sensed signals. FIG.6is an alternate cross-sectional view illustrating aspects of an exemplary semiconductor device100in accordance with some embodiments and highlighting aspects of circuit layer110and wafer630. As in exemplary embodiment described inFIG.1B, the semiconductor device100includes a first chamber130aand second chamber130b,each configured to receive a work fluid or solution. The first chamber130ais above the second chamber130band is in spatial communication with the second chamber130bthrough the pore140a.As illustrated inFIG.6, the second chamber130bincludes the electrode layer620, a wafer630, and the circuit layer110. The second chamber cavity130bextends through the electrode layer620, the wafer630, and the circuit layer110. The wafer630is above the electrode layer620and, in the example ofFIG.6, is a semiconductor-on-insulator (SOI) wafer and includes a bulk substrate630aand a buried oxide (BOX)630b,e.g., SiO2, above the bulk substrate630a.In this embodiment, the bulk substrate630ahas a thickness of, e.g., about 200 um. Examples of materials for the bulk substrate630ainclude, but are not limited to, Si, Ge, other suitable elementary substrate material, SiC, GaAs, GaP, InP, other suitable compound substrate material, and the like. In an alternative embodiment, the wafer630is a bulk wafer, a ceramic wafer, a quartz wafer, a glass wafer, or the like. A second chamber130bmay take a variety of forms, for example, as illustrated inFIG.6, the second chamber130bincludes a cavity that extends through the wafer630has a width (W1), e.g., of about 500 um, and a height (H1), e.g., of about 200 um. The second chamber cavity130bextends through the circuit layer110has a width (W2), e.g., about 20 um, and a height (H2), e.g., about 5 um. The circuit layer110includes the first circuit200and the second circuit400, described above, that are implemented using transistors, e.g., FETs. That is, in embodiments, voltage generator210, driving module220(including IVC310and ADC320), pore-forming module230, and switch240of first circuit200may be one or more semiconductor structures formed within circuit layer110and interconnected by one or more metal layers. Similarly, in embodiments, voltage generator410, and sensing module420(including IVC510or ADC520) may be one or more semiconductor structures formed within circuit layer110and interconnected by one or more metal interconnections610, e.g. which may be of the same composition as interconnections111depicted inFIG.1B. For simplification of illustration,FIG.6illustrates an exemplary FET transistor comprising source drain regions610a,610b,and gate610c,but it will be appreciated that according to fabrication techniques many FETs may be formed and interconnected to create the components of first circuit200and second circuit400in circuit layer110. For illustration, circuit layer110is formed with a source region610a, a drain region610b,and a gate structure610cabove a channel region between the source and drain regions610a,610b.The source and drain regions610a,610band the gate structure610cconstitute an exemplary FET that may be a component structure of first circuit200or second circuit400. The circuit layer110further includes a plurality of exemplary interconnects, e.g., interconnect610d,that in various embodiments connect the transistors thereof to each other, thereby realizing the first and second circuits200and400within circuit layer110as explained above. The interconnects610dfurther connect, as with interconnections111, the components structures forming first circuit200and the electrode layers150a,620to each other, or interconnects connect the component structures forming second circuit and the electrode layers150b,150cto each other. In this way, components comprising voltage generator210(e.g.,310,330, and340) may be formed in circuit layer110, and may supply Vref1to second chamber610via interconnections, such as through substrate via (TSV)640, to electrode620. Similarly, driving module220components (such as IVC310and ADC320) may be formed in circuit layer110and may supply and receive Vbias1/Vbias2and Idrv/Ipore1to first chamber130a(through switch240(also formed in circuit layer110)) through interconnection like610d,e.g.111, to electrode150a.Similarly, pore-forming module230may be formed of various semiconductor structures as described above, and may provide Vpulse to first chamber130avia switch240and interconnections, e.g.610dor111, to electrode150a. In embodiments, the second circuit400may also be formed of various semiconductor components within circuit layer110and interconnected through interconnections610d.Sensing module420(including component IVC510and ADC520) may be formed of one or more semiconductor structures formed in circuit layer110, and may provide Vbias3/Vbias4and Isense/Ipore2to pad150c(coupled to a nanopore layer, e.g.140,250) via interconnections610d.Similarly, voltage generator410may be formed of one or more semiconductor structures within circuit layer110and capable of providing Vref2to pad150b(also coupled to a nanopore layer, e.g.140) via interconnections610d.The circuit layer110further includes at a top surface thereof a passivation layer610ethat covers/insulates the interconnects610d. The second chamber130bfurther includes a through substrate via (TSV)640that extends from circuit layer110to a bottom surface of the wafer640and that connects the first circuit200and the electrode layer620to each other. The circuit layer110may further include other active components, e.g., diodes and other type of transistors such as a bipolar junction transistor (BJT), and passive components, e.g., resistors, capacitors, inductors, and the like. FIG.7is a flow chart illustrating an exemplary method700of manufacturing a semiconductor device, e.g., semiconductor device100, in accordance with some embodiments. In operation710, a chamber, e.g., second chamber610, is formed. In an embodiment, the chamber includes a circuit layer, e.g., circuit layer110, and defines a chamber cavity, e.g., second chamber cavity610a,that extends through the circuit layer. In operation720, a nanopore layer, e.g., nanopore layer140, is transferred from a source substrate to the circuit layer110, thereby the nanopore layer may form a boundary between chamber cavities130a,130bwhere they meet, e.g. orifice142A. FIGS.8A-8Rare sectional views of a semiconductor device, e.g., semiconductor device100, at various stages of manufacturing in accordance with some embodiments, e.g., as produced using operations described above with reference to method700. Method700will now be described with further reference toFIGS.1,2,3A,3C,4,5, and8A-8Rfor ease of understanding. It is understood that method700is applicable to structures other than those ofFIGS.1,2,3A,3C,4,5, and8A-8R. Further, it is understood that additional operations can be provided before, during, and after method700, and some of the operations described below can be replaced or eliminated, for other embodiments of method700. FIG.8Aillustrates an exemplary structure resulting after receiving/providing a wafer630. In embodiments wafer630comprises an oxide layer over a SOI substrate. The oxide layer may comprise BOX.FIG.8Billustrates an exemplary structure resulting after formation of a TSV640that extends from a top surface to a bottom surface of the wafer630. The formation of the TSV640includes: performing a lithographic patterning and etching to form a TSV opening through the wafer630; coating with a TSV liner, e.g., oxide, a TSV sidewall that defines the TSV opening; and filling the TSV opening with a TSV material, e.g., polysilicon. The TSV opening is filled with the TSV material using a deposition process, such as chemical vapor deposition (CVD), atomic layer deposition (ALD), physical vapor deposition (PVD), other suitable depositing/forming/filling/growing process, derivatives thereof, or a combination thereof. FIG.8Cillustrates an exemplary structure resulting after performance of operation710and depicts a circuit layer110above the wafer630. The formation of the circuit layer110includes: depositing a semiconductor material, e.g., silicon, germanium, other suitable semiconductor material, or a combination thereof, on the top surface of the wafer630to form an active layer; form transistors over the active layer; depositing inter-layer dielectric (ILD) material, e.g., SiO2 or any other low-K dielectric material, on the active layer; performing lithographic patterning and etching to form openings in the ILD; filling the openings with a conductive material to form interconnects, e.g., interconnect150d,that connect the transistors to each other, resulting in a first and second circuits, e.g., first and second circuits200,400, respectively; and depositing a passivation layer material, e.g., SiN, on the ILD. FIG.8Dillustrates an exemplary structure resulting after formation of an electrode layer620on the bottom surface of the wafer630.FIG.8Eillustrates an exemplary structure resulting after formation of a chamber cavity130bthat extends through the electrode layer620, the wafer630, and the circuit layer110. Next, formation of a nanopore layer140is described hereinafter.FIG.8Fillustrates an exemplary structure resulting after growth of a nanopore device layer140, on a metal catalyst810, e.g., a copper-based metal catalyst. The growth of the nanopore layer140includes: receiving/providing the copper-based metal catalyst; annealing the copper-based metal catalyst at a temperature of, e.g., greater than about 800° C., to clean the copper-based metal catalyst; depositing nanopore layer140material on the copper-based metal catalyst, using e.g., Ar, CH4, and H2, such as by CVD at a temperature of, e.g., about 1000 degrees Celsius or plasma enhanced CVD (PECDV) at a temperature of, e.g., greater than about 450 degrees Celsius; and annealing the resulting structure at a temperature of, e.g., greater than about 600 degrees Celsius. FIG.8Gillustrates an exemplary structure resulting after coating, e.g., spin coating, the nanopore layer140with a source substrate820, e.g., a thin layer of polymer, such as poly methyl methacrylate (PMMA). The source substrate820protects the nanopore layer140from cracks during the transfer of the nanopore layer140to the structure ofFIG.8E, as described below. FIG.8Hillustrates an exemplary structure resulting after removal, such as by dry or wet etching, of the copper-based metal catalyst810from the structure ofFIG.8G.FIG.81illustrates an exemplary structure resulting after sizing the nanopore layer140to a length of, e.g., about 200 um, and a width of, e.g., about 200 um, using e.g., lithographic, e.g., electron beam lithographic (EBL), patterning and etching. FIG.8Jillustrates an exemplary structure resulting after performance of removing the graphene layer form source substrate820and then performing a transferring operation, e.g., operation720.FIG.8Jdepicts the nanopore layer140transferred, unformed, from the source substrate820to the structure ofFIG.8E. In this embodiment, the transfer operation includes removing the graphene layer from the source substrate820and then bonding the nanopore layer140to the circuit layer110. FIG.8Killustrates an exemplary structure resulting after resizing the nanopore layer140to a shorter length, e.g., about 100 nm to about 160 nm, and a narrower width, e.g., about 80 nm, such as by lithographic, e.g., EBL, patterning and etching. FIG.8Lillustrates growing a sacrificial layer635on the structure ofFIG.8K.FIG.8Millustrates performing lithographic patterning and etching636to form openings637that extend through the sacrificial layer; andFIG.8Nillustrates the openings created in reference toFIG.8Mwith conductive material e.g.150a,150b; subsequently, the sacrificial layer is removed as shown inFIG.8O.FIG.8Oillustrates an exemplary structure resulting after formation of electrode layers150a,150b,150c.In embodiments, the formation of the electrode layers150a,150b,150cmay include standard techniques for forming electrode layers over a semiconductor circuit layer. In one embodiment this includes the steps shown inFIGS.8L-8N: FIG.8Pillustrates an exemplary structure resulting after formation of an insulating layer160that covers the electrode layers150b,150cand first and second end portions of the nanopore layer140. The formation of the insulating layer160includes: conformably depositing a material, e.g., A12O3, over the electrode layers150b,150cand the nanopore layer140; and removing the insulating layer160on an intermediate portion of the nanopore layer140. Next, formation of a chamber wall, e.g., chamber wall130, is described hereinafter.FIG.8Qillustrates an exemplary structure resulting after patterning a first layer830, e.g., polymer, over a second layer130, e.g., silicon, glass, other suitable material for a chamber wall, and the like.FIG.8Rillustrates an exemplary structure resulting after etching the second layer130using the first layer830as a mask to form a chamber cavity130ain the second layer130.FIG.8Sillustrates an exemplary structure resulting after bonding the second layer130to the structure ofFIG.8M, e.g. using the first layer830as an adhesive. Next, formation of a pore140ain the nanopore layer140is described hereinafter.FIG.8Tillustrates an exemplary structure resulting after filling the chamber cavities130b,130awith a chemical solution800.FIG.8Uillustrates an exemplary structure resulting after formation of a pore140ain the nanopore layer140, e.g. by the process described above in reference to first circuit200. The formation of a pore140amay include: the voltage generator210receiving an input signal (IN1) instructing generation of a reference voltage (Vref1), e.g., about 0V, applied to the electrode layer620that corresponds to the input signal (IN1); the switch240receiving a control signal (CS1) and selectively connecting the pore-forming module230to the electrode layer150ain response to the control signal (CS1); the pore-forming module230receiving an input signal (IN3) instructing generation of high pore-forming pulses, e.g., about 7.0 V, applied to the electrode layer150a,thereby forming the pore140a;subsequently the pore-forming module230receiving an input signal (IN3) instructing generation low pore-forming pulses, e.g., about 5.0 V, applied to the electrode layer150a,thereby adjusting a diameter/width of the pore140a.It will be appreciated that the voltages described above are by way of example only and in practice a suitable voltage is selected based on the solution and the nanopore layer device, e.g.140. In some embodiment, method700includes: the switch240receiving a control signal (CS1) and selectively connecting a driving module220to the electrode layer150ain response to the control signal (CS1); the driving module220generating a bias voltage (Vbias2) applied to the electrode layer150athat results in a pore voltage across the electrode layers620,150a;the driving module220detecting a pore current (Ipore1) through the electrode layers620,150athat is associated with the pore voltage and a corresponding resistance of the nanopore layer140; the driving module220converting the pore current (Ipore1) to a pore voltage (Vpore1); and the driving module220converting the pore voltage (Vpore1), which is in an analog format, into a digital format. A diameter/width of the pore140amay be estimated using the pore voltages (Vpore1). In other embodiments, method700includes: the voltage generator410receiving an input signal (IN4) and generating a reference voltage (Vref2), e.g., about 0V, applied to the electrode layer150bthat corresponds to the input signal (IN4); The sensing module420generating a bias voltage (Vbias4) applied to the electrode layer150cthat results in a pore voltage across the electrode layers150b,150c;the sensing module420detecting a pore current (Ipore2) through the electrode layers150b,150cthat is associated with the pore voltage and a corresponding resistance of the nanopore layer140; The sensing module420converting the pore current (Ipore2) to a pore voltage (Vpore2); and the sensing module420converting the pore voltage (Vpore2), which is in an analog format, into a digital format. A diameter/width of the pore140amay be estimated using the pore voltages (Vpore2). FIG.9is a flow chart illustrating an exemplary method900of determining a biomolecule characteristic in accordance with some embodiments. Method900will now be described with further reference toFIGS.1A-1D,2A,3A-3C,4, and5for ease of understanding. It is understood that method900is applicable to structures other than those ofFIGS.1A-1D,2A,3A-3C,4, and5. Further, it is understood that additional operations can be provided before, during, and after method900, and some of the operations described below can be replaced or eliminated, for other embodiments of method900. In operation910, the first and second chamber cavities130a,130bare filled with a chemical solution. Next, a biomolecule is placed in the chemical solution in the first chamber cavity130a,or the second chamber130b. In operation920, the first circuit200applies across the electrode layers620,150aa drive voltage for driving the biomolecule from the first chamber cavity130ato the second chamber cavity130bthrough the pore140a(or visa versa). In this embodiment, operation920includes: the voltage generator210receiving an input signal (IND and generating a reference voltage (Vref1) applied to the electrode layer620that corresponds to the input signal (IN1); the switch240receiving a control signal (CS1) and connecting the driving module220and the electrode layer150ato each other; the driving module220generating a bias voltage (Vbias1) applied to the electrode layer150athat results in the drive voltage. In operation930, driving module220detects a drive current (Idrv) through the electrode layers620,150athat is associated with the drive voltage and a corresponding resistance of the nanopore layer140. In operation940, one or more characteristics of the biomolecule are determined using the drive currents (Idrv). In this embodiment, operation940includes: the driving module220, which in embodiments may be implemented in CMOS structures formed in circuit layer110. As illustrated voltage Vbias1is shown as applied to150afor illustrative purposes, as it will be appreciated that the voltage is supplied via the interconnected semiconductor structures within circuit layer110. Similarly as illustrated voltage Vref1is shown as applied to layer620. Vref1may be generated by voltage generator210, which is similarly implemented in circuit layer110. By applying these voltages at their respective terminals, establishing a voltage Vbias-Vref1across the nanopore barrier which drives a biomolecule through the nanopore. As the biomolecule passes through the nanopore, the resistance varies along the length of the biomolecule as it passes. Vbias-Vref1is associated with a transmembrane current Idrvthat varies in proportion to the varying resistance caused by the passing of the biomolecule. In embodiments, Idrvis sensed by, converting the drive current (Idrv) to a drive voltage (Vdrv) with IVC310. IVC310converts Idrvto a voltage, the drive voltage (Vdrv). In embodiments, the drive voltage Vdrv, which in an analog format, is then converted into a digital format, which is then formatted to a biological characteristic determining device, which then determines the biomolecule characteristic based on the drive voltages (Vdrv). In an embodiment, method900further includes: the voltage generator410receiving an input signal (IN4) and generating a reference voltage (Vref2) applied to the electrode layer150b.And sensing module420generating a bias voltage (Vbias3) applied to the electrode layer150cthat results in a sense voltage across the electrode layers150b,150c.Sensing module420detects a sense current (Isense) through the electrode layers150b,150cthat is associated with the sense voltage and a corresponding resistance of the nanopore layer140. In embodiments, the sensing module420converts the sense current (Isense) to a sense voltage (Vsense). The sense voltage (Vsense) is then converted into a digital format, and may be forwarded to a biological characteristic sensing device, and one or more characteristics of the biomolecule may be determined using the sense voltages (Vsense). Although the semiconductor device100is exemplified using one first chamber130a,highlighted by square120, it should be understood that, after reading this disclosure, the number of first chambers130amay be increased. For example,FIG.10is a cross sectional view illustrating an exemplary semiconductor device1000in accordance with some embodiments. When compared to the semiconductor device100, the semiconductor device1000includes aspects of device100and further includes a chamber1030a,highlighted in square1020. The construction of the chamber1030ais similar to that of the chamber130a.In particular, the chamber1030aincludes a chamber wall1030, a nanopore layer1040, electrode layers1050a,1050b,1050c,and an insulating layer1060. The chamber wall1030is formed on the circuit layer110, such as by bonding the chamber wall1030to the circuit layer with the use of an adhesive. The chamber wall1030defines a chamber cavity1030atherein configured to receive a chemical solution (not shown).The chamber wall1030may be, for example one or more silicon caps. The nanopore layer1040is disposed in the chamber cavity1030a,is formed in situ by semiconductor circuits in circuit layer110, (e.g. a first circuit200) and has first and second end portions and an intermediate portion between the first and second end portions and formed with a pore1040atherethrough. The electrode layer1050ais disposed in the chamber cavity1030aand is formed over the circuit layer110. The electrode layers1050b,1050care disposed in the chamber cavity1030a,are formed over the circuit layer110, and are connected to first and second end portions of the nanopore layer1040, respectively. The insulating layer1060covers the electrode layers1050b,1050cand the first and second end portions of the nanopore layer1040. Further, the circuit layer110includes, instead of the first and second circuits200,400, third and fourth circuits1100,1200, shown inFIGS.11and12, respectively.FIGS.11and12are schematic diagrams illustrating exemplary third and fourth circuits1100,1200, respectively, in accordance with some embodiments. When compared with the first circuit200, the switch240of the third circuit1100comprises a driving module220, a pore-forming module230, and is coupled to electrode layers150a,1050a.A switch240of the third circuit1100is configured to receive a control signal (CS1) to selectably connect either a driving module220or a pore-forming module230to either the electrode layer150aor the electrode layer1050ain response to the control signal (CS1). When compared with the second circuit400, the fourth circuit1200further includes switches1210,1220. The switch1210is connected to the voltage generator410and the electrode layers150b,1050band is configured to receive a control signal (CS2) to selectably connect a voltage generator410to either of an electrode layer150bor an electrode layer1050bin response to the control signal (CS2). Switch1220is connected to the sensing module420and the electrode layers150c,1050cand is configured to receive a control signal (CS3) to selectably connect the sensing module220and either the electrode layer150cor the electrode layer1050cto each other in response to the control signal (CS3). FIG.13is a flow chart illustrating a method1300of determining a biomolecule characteristic in accordance with some embodiments. Method1300will now be described with further reference toFIGS.10-12for ease of understanding. It is understood that method1300is applicable to structures other than those ofFIGS.10-12. Further, it is understood that additional operations can be provided before, during, and after method1300, and some of the operations described below can be replaced or eliminated, for other embodiments of method1300. In operation1310, the chamber cavities130a,610a,1030aare filled with a chemical solution. Next, first and second biomolecules (not particularly illustrated, but seeFIG.14for exemplary biomolecule1408suspended in solution passing through a nanopore) are placed in the chemical solution in the chamber cavities130a,1030a, respectively. In operation1320, the third circuit1100applies a drive voltage across the electrode layers620,150afor driving the first biomolecule from the chamber cavity130ato the chamber cavity610athrough the pore140a.In an embodiment, operation1320includes: the voltage generator210receiving an input signal (IND and generating a reference voltage (Vref1) applied to the electrode layer620that corresponds to the input signal (IN1); the switch240receiving a control signal (CS1) and connecting the driving module220and the electrode layer150ato each other; and the driving module220thereby generating a bias voltage (Vbias1) applied to the electrode layer150athat results in the drive voltage. In operation1330, the driving module220detects a drive current (Idrv) through the electrode layers620,150athat is associated with the drive voltage and a corresponding resistance of the nanopore layer140. In operation1340, one or more characteristics of the first biomolecule, e.g. DNA (not particularly illustrated, but seeFIG.14for exemplary biomolecule1408suspended in solution passing through a nanopore), are determined from variations in drive currents (Idrv) that are responsive to a characteristics of a biomolecule passing through nanopore140a.In an embodiment, operation1340includes: the driving module220converting the drive current (Idrv) to a drive voltage (Vdrv); the driving module220converting the drive voltage (Vdrv), which is in an analog format, into a digital format; and determining the first biomolecule characteristic using the drive voltage (Vdrv). In operation1350, the first circuit1100applies a drive voltage across the electrode layers620,1050afor driving the second biomolecule, e.g. RNA, from the chamber cavity1030ato the chamber cavity610athrough the pore1040a.In this embodiment, operation1350includes: the voltage generator210receiving an input signal (IN1) and generating reference voltage (Vref1) applied to the electrode layer620that corresponds to the input signal (IN1); the switch240receiving a control signal (CS1) and connecting the driving module220and the electrode layer1050ato each other; and the driving module220generating a bias voltage (Vbias1) applied to the electrode layer1050athat results in the drive voltage. In operation1360, the drive module220detects a drive current (Idrv) through the electrode layers620,1050athat is associated with the drive voltage and a corresponding resistance of the nanopore layer1040. In operation1370, one or more characteristics of the second biomolecule are determined determined from variations in drive currents (Idrv) that are responsive to a characteristics of a biomolecule passing through nanopore1040a.In this embodiment, operation1370includes: the driving module220converting the drive current (Idrv) to a drive voltage (Vdrv); the driving module220converting the drive voltage (Vdrv), which is in an analog format, into a digital format; and determining the second biomolecule characteristic using the drive voltages (Vdrv). In this embodiment, method1300further includes: the switch1210receiving a control signal (CS2) and selectably connecting the voltage generator410and an electrode layer150bor1050bto each other; the voltage generator410receiving an input signal (IN4) and generating a reference voltage (Vref2), applied to the electrode layer150bor1050b,that corresponds to the input signal (IN4); the switch1220receiving a control signal (CS3) and selectably connecting the sensing module420and an electrode layer150cor1050cto each other; the sensing module420generating a bias voltage (Vbias3) applied to the electrode layer150cor1050c,respectively, that results in a sensed voltage between the electrode layer150bor1050band the electrode layer150cor1050c;the sensing module420detecting a sense current (Isense) through the electrode layer150bor1050band the electrode layer150cor1050c,respectively, that is associated with the sense voltage and a corresponding resistance of the nanopore layer140or1040respectively; the sensing module420converting the sense current (Isense) to a sense voltage (Vsense); the sensing module420converting the sense voltage (Vsense), which is in an analog format, into a digital format; and one or more characteristics of the first/second biomolecule are determined using the sense voltages (Vsense). FIG.14illustrates a PDMS (polydimethylsiloxane) measurement cell1400that may be utilized for various biosensors, for example a portable handheld biosensor, comprising (as illustrated) three semiconductor devices, which in embodiments may include semiconductor device100, in each of cell1404a,1404b,1404c.It will be appreciated that the measurement cell, e.g. a microfluidic chip such as microfluidic chip196, may be formed of any material suitable for microfluidic applications, for example glass, silicon or other suitable polymers. An array of nanopore cells1402includes a plurality of nanopore cells1404a,1404b,1404cseparated from each other by silicon caps, e.g. silicon cap1406. Each nanopore cell1404a,1404b,1404cincludes a semiconductor device100with integrated nanopore device140. While the exemplary array of nanopore cells1402is illustrated with three exemplary nanopore cells1404a,1404b,1404c,any number of nanopore cells may be used without exceeding the scope of this disclosure. Each nanopore cell1404a,1404b,1404cis electrically separated from each other by the silicon caps1406, and each nanopore (e.g.140A) in each nanopore cell1404a,1404b,1404cis formed in situ, electrically, and independently of the other, according to the present disclosure. The nanopore cell array1402is inserted into the PDMS measurement cell1400with microfluidic channels (e.g.194, not particularly depicted inFIG.14) that feed into and form a reservoir1410for containing a working fluid1412in contact with either side of the nanopore cell array1402(which may be an embodiment of array180). Each nanopore cell, e.g.1404b,may individually sense the properties of individual biomolecules, e.g. biomolecule1408, present in or introduced into the fluid1412, as it passes through the nanopore cell's1404bnanopore (e.g. nanopore140A). In embodiments sensing circuitry and the decoding circuitry, e.g. circuitry110A,110A1,200,400, formed in semiconductor layer110of each semiconductor device100of each nanopore cell1404a,1404b,1404c,are localized in each nanopore cell, e.g.1404b,each nanopore cell is able to reliably and accurately report its sensed biomolecule characteristics (e.g. based on varying resistance or capacitance of the nanopore as a biomolecule passes through the nanopore, e.g.140), to a biosequencing device. FIG.15illustrates a simplified device schematic for controlling the programming and sensing of an array of nanopore cells, e.g. array1402according to various embodiments. Each nanopore cell device may include an embodiment of semiconductor device100, and may receive control signals via control circuitry formed in semiconductor device layer110, or applied to IO electrodes170A,170B. Each nanopore cell response to, among other signals, four control signals Isel1502, Isen/Iprog1504, Gsel1506, and Gsen1508. Isel1502, for a given combination nanopore/semiconductor IC device1520, which may be device100, enables selection of a respective ion channel signal (e.g. a signal associated with iBin path155). Isen/Iprog1504provides either a programming voltage for forming a nanopore (as described herein in reference toFIGS.1A-1D2A-2B,3A-3C,4,5), or Isen/Iprog1504activates a sensing current which may be selected by mux1510in order to convert a sensed current to a digital signal for output to an external device (like a portable biosensor, or other type of biosensor). Alternatively, Gsel1506may be asserted, which selects the membrane channel, (e.g. channel associated with iT), and Gsen1508may provide a sensing signal that may be selected, by mux1512, for analog to digital conversion by ADC1514for transmission to an external device for processing, e.g. via output198to a biomolecule characterization device. FIGS.16A,16B,16Cillustrate various aspects of embodiments of an exemplary nanopore cell array1630according to various embodiments.FIG.16Aillustrates a cross section of a single nanopore cell1600and includes an equivalent circuit1610for sensing a biomolecule. Nanopore cell1600includes a semiconductor device, e.g. a semiconductor device100, as a sensor device (e.g. 2D transistor140sensor with integrated sensing circuitry) and sensing circuitry e.g. a first and second circuit200,400formed in a semiconductor device layer110. A cis program-and-sense ring1602(e.g. an electrode such as an embodiment of electrode150A) encircles the nanopore140A and nanoproe layer140on the cis side of a membrane1606(e.g., a graphene transistor, or a nanopore layer), and a trans program-and-sense-ring1604(e.g. an electrode such as an embodiment of electrode620) encircles the nanopore and nanopore layer140on the trans side of a membrane1606, thereby providing contacts between, e.g., first circuit200and the fluid present on each side of the membrane1606. This contact allows a control circuit, e.g. first circuit200, to establish a voltage difference across a membrane1606having a nanopore1620through which a biomolecule1608may pass. As biomolecule1608passes through nanopore1620the resistance and capacitance of the ionic circuit varies, which causes a varying voltage to appear across the membrane1606. This varying voltage causes a varying current between the membrane source1614and the membrane drain1612(as illustrated by equivalent circuit1610), which may be converted into a digital representation by local CMOS circuitry, e.g. in circuit layer110, for transmission to an external device for further processing. The membrane drain1612and membrane source1614may be electrically coupled to other cells in the array1630, and to control circuitry (not shown in detail), through line1622and line1624respectively. In embodiments signals Gsen1508and Gsel1506may be provided to the membrane transistor through lines1622,1624(in a manner analogous to a word line or a bit line in a memory cell array). Similarly, signals Isel1502and Isen/Iprog1504may be provided to the ionic channel by way of trans prog and sense ring1604and cis prog and sense ring1602. As in the embodiments illustrated inFIG.16Ca trans ring of each individual nanopore cell1600is couple to a transring of each other nanopore cell, and likewise each cisring is coupled together, in this way each a solutions surrounding each nanopore is maintained at a similar potential during application of drive or sensing voltages. Additionally, control lines GD1622and GS1624for controlling currents applied across each nanopore device, e.g. nanopore cell1600, may be routed to a single control circuit that is separate from each cell's sensing circuitry, e.g. sensing circuitry disposed in a semiconductor layer1650, local to each nanopore cell1600, and which may be independently coupled to a transchamber by TSV1640. In this way, control circuit of the array, e.g. control circuit110B, may be separate and distinct from sensing circuitry formed in a semiconductor layer, e.g.1650. FIG.17is a flow chart illustrating an exemplary method1700of determining a biomolecule characteristic in accordance with some embodiments. At step1702a semiconductor device, e.g.100, is formed having a circuit layer, e.g.110, coupled to first, second, and third electrodes, e.g.150A,150B,150C, formed on a first surface of the semiconductor, the circuit layer, e.g.110, further coupled to a fourth electrode, e.g.620, formed on a second surface of the semiconductor device. At1704, an unformed nanopore layer (having no nanopore, e.g. nanopore device140prior to pore formation) is coupled between the first and second electrodes, e.g.150B,150C, such that the nanopore layer, e.g.140, forms a membrane between first and second chambers, e.g.130A,130B, defined in part by the semiconductor device, e.g.100and each chamber, e.g.130A,130B, containing a solution, e.g.800. At step1706, a voltage is applied between the third electrode, e.g.150A, in the first chamber and the fourth electrode, e.g.620in the second chamber, thereby forming a nanopore, e.g.140A, in the unformed nanopore layer to obtain a formed nanopore layer, e.g.140. At1708a semiconductor device, e.g.100, is disposed in a biomolecule detection device, e.g. microfluidic chip196. And a biomolecule is disposed, at1710, in a solution suspected in a first chamber, e.g.130A, or a second chamber, e.g.130B. A driving voltage is applied, in step1712, between the third, e.g.150A, and fourth electrodes, e.g.620, to drive the biomolecule from the first chamber or the second chamber through the nanopore, e.g.140. A sensing voltage is applied between the first electrode, e.g.150A, and the second electrode, e.g.150B, at1714, and at1716a current flowing between the first and second electrodes is sensed, e.g. by an integrated circuit formed in circuit layer110. The sensed current is encoded in a digital signal at1718, and at1720the digital signal is transmitted, e.g. via output198, to a biomolecule characterization device capable of characterizing the biomolecule based on the digital signal. From the above, the semiconductor device of the present disclosure includes a circuit layer and a nanopore layer. The circuit layer includes a circuit configured to drive a biomolecule through a pore in the nanopore layer and to detect a drive/sense current associated with a resistance of the nanopore layer. The circuit is a driving/sensing circuit that is built-in to the semiconductor device such that parasitic capacitances associated with the drive/sense currents detected by the circuit are reduced. Thus, a relatively accurate biomolecule characteristic can be obtained from the drive/sense currents provided by the semiconductor device. Devices and methods in accordance with this disclosure provide many benefits including enabling an integrated semiconductor nanopore process structure. This semiconductor nanopore process structure may contain CMOS transistors, interconnects, thin film membranes for forming nanopores, a liquid chamber in which formation occurs, and programming electrodes. A nanopore may be formed by in-silicon (or generally in situ) by programming circuits. An in-silicon sense amplifier circuit and signal processing circuits, such as ADC, DAC, IVC, may be integrally formed with a sensing device, and may be used to digitize the signal and to communicate the signal to an external computing device. To digitize the signal means to convert an analog sensed signal into a digital form, e.g. a 16-bit, or 32-bit, or 64-bit word, prior to transmitting the sensed signal to an external computing device. One of skill in the art will appreciate any digital encoding of the analog signal may be employed. Thus, the signal to noise ratio is significantly improved at the receiving device, e.g. a biomolecule characterizing device. The membrane, e.g. nanopore layer140, may be formed using 2D FETs such as Graphene FETs or MOS2 FETs. The devices and methods disclosed herein are useful for DNA sequencing. For forming nanopore cell boundaries, silicon caps with pre-drilled holes may be bonded to a silicon substrate to provide liquid isolation and thereby isolate each cell. The semiconductor device of the present invention provides many benefits. Devices in accordance with this disclosure are useful in handheld biometric devices. For example, a handheld biological molecule detection and characterization device would employ an integrated semiconductor nanopore process structure. Because of its integrated nature, the nanopore sensor SNR is greatly reduced by eliminating noise introduced by long sensor wires, thus enabling a reliable sensing device. The semiconductor nanopore process structure contains CMOS transistors, interconnects, a thin film nanopore, a liquid chamber formation, and program electrodes as described above. The nanopore is formed by in-silicon programming circuits as described above. Additional, in-silicon sense amplifier circuits and signal processing circuits, such as analog-to-digital converters, or digital to analog converters, or current to voltage signal converters. These components are used to digitize the signal and communicate the digitized signal to the handheld biological molecule detection and characterization device. In embodiments, 2D FETS such as graphene FETS or MOS2 FETs are used for biological detection and sequencing purposes. In embodiments, an array of semiconductor nanopore process structures are employed; each semiconductor nanopore process is isolated from others in the array by a cavity structure having silicon caps with pre-drilled holes which may be bonded to silicon substrate to provide liquid isolation. In an embodiment, a semiconductor device comprises a circuit layer and a nanopore layer. The nanopore layer is formed on the circuit layer and is formed with a pore therethrough. The circuit layer includes a circuit unit configured to drive a biomolecule through the pore and to detect a current associated with a resistance of the nanopore layer, whereby a characteristic of the biomolecule can be determined using the currents detected by the circuit unit. In another embodiment, a method of manufacturing a semiconductor device comprises forming a circuit layer, transferring a nanopore layer from a source substrate to the circuit layer, and forming a chamber on the circuit layer such that the nanopore layer is disposed in a chamber cavity in the chamber. In another embodiment, a method comprises filling a chamber formed on a circuit layer with a chemical solution and enabling a circuit of the circuit layer to drive a biomolecule from the chamber through a pore in a nanopore layer. In embodiments, an array is formed by metal and souree/drain as interconnects. A membrane transistor is connected together to form integrated circuits. The programing, sensing and driving electrodes are formed by metal rings around each cell on both the cis side and trans side of the membrane. A cell to be programmed is selected by a CMOS circuit addressing signal in a similar manner as techniques employed in SRAM or other memory addressing methods. A duration of a program pulse is controlled electronically by integrated decoding circuits as described herein. A soaking signal, after programming, is also controlled electronically to provide soaking pulses having soaking pulse widths for a number of pulse. A pore is formed during programming, and a pore's size is monitored continuously by continuous monitoring of the ion current, which is correlated with the nanopore size as described herein (e.g. correlated by prior direct measurement). Once a specified ion current is reached, a desired pore size is achieved and soaking of the pore is terminated. A to-be-detected biomolecule, e.g. a particular DNA strain, is then deposited in a cells fluid and a bias voltage is applied (smaller than programming and soaking voltage) to drive the biomolecule through the nanopore. While the DNA strain passes through the nanopore, the ion current from cis and trans terminals will change according to the type of nucleotides. At the same time, the membrane transistor source/drain is affected significantly, and a measured current is larger and of better SNR quality than in known methods. The membrane transistor may be a GNR transistor. The membrane transistor current iTand the ion current through cis-trans terminal may be compared for more accuracy detection. In embodiments, disclosed process technology combines SOI CMOS technology, MEMS technology and Graphene Nano-Ribbon transistor technology. This combination may be employed to form nanopore cells and sensing components, e.g. first circuit200and second circuit400in semiconductor layer110as CMOS circuits, e.g.110A,110A1, according to the following summarized process, more fully described below. First, top size silicon and through substrate vias (TSV) are formed. Then SOI CMOS transistors are formed, e.g. in circuit layer110. Then thin SIN is deposited over the circuit layer110. A 2D transistor (e.g. graphene) layer is then transferred onto a surface of the SIN and graphene/metal contacts and silicon contacts are formed coupling the 2D transistor layer140to the circuit layer110components. Backside silicon metal is formed, and etched in order to form a cavity (e.g.130B). Backside SiN is then patterned or etched as desired, and finally silicon caps are formed and then bonded to sensor wafers. The foregoing outlines features of several embodiments so that those skilled in the art may better understand the aspects of the present disclosure. Those skilled in the art should appreciate that they may readily use the present disclosure as a basis for designing or modifying other processes and structures for carrying out the same purposes and/or achieving the same advantages of the embodiments introduced herein. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure. | 112,742 |
11860153 | DETAILED DESCRIPTION In the example illustrated in the figures, the storage device1according to the invention comprises a cartridge10, which is covered with a cover11, and a dispensing member12. This storage device forms part of an assembly furthermore comprising a plurality of strips13intended to be accommodated in the cartridge10of the device1. FIG.1illustrates the closed storage device1. InFIG.2, the assembly1is open and the cover11has been removed. This figure therefore shows the cartridge10covered with a capsule101making it possible to maintain the leaktightness of the cartridge even though the cover11has been removed. The capsule101covers the entirety of the upper face of the cartridge10. Opening the capsule101, as illustrated inFIG.3, uncovers the cartridge10comprising a cavity102, in which a plurality of analysis strips13are accommodated, and a dispensing member12. The cover11of the assembly1is illustrated in more detail inFIG.4. The cover has a circumferential edge111intended to cooperate with the cartridge10by shape complementarity and preferably by clipping. More particularly, the cover comprises a first tongue112aat one end, intended to cooperate with a first engagement orifice104aformed on the cartridge10, and a second tongue112bpositioned at the opposite end and intended to cooperate with a second engagement orifice104bformed on the cartridge10. Advantageously, the fact that the first engagement orifice104aand the second engagement orifice104bare not identical is referred to as a poka-yoke. Specifically, each has an opening with a different size, so that the cover11can be engaged on the cartridge10in only one direction respectively via the first tongue112aand the second tongue112b. For these purposes, the first tongue112aof the cover11can cooperate only with the first engagement orifice104aand the second tongue112bof the cover11can engage only with the second engagement orifice104b. Furthermore, the cover11comprises a face113intended to face toward the cartridge10, said internal face113comprising two ribs112extending longitudinally on said internal face113. The two ribs112are arranged parallel to one another and preferably substantially in the middle of the internal face113of the cover11. The two ribs112are positioned so as to be able to hold the strips13in the cavity102of the cartridge10when the cover11covers the cartridge10. In a variant which is not represented, the internal face113has a single rib112arranged transversely and substantially in the middle of said internal face113. Moreover, the cover11comprises a joint114having a shape substantially complementary with a longitudinal cavity102which is formed in the cartridge10and is configured in order to accommodate the strips13. Advantageously, the joint114is fitted on the internal face113of the cover11, as may be seen inFIG.4. Furthermore, as may be seen particularly inFIG.4, the cover11furthermore comprises at least one transverse holding rib115which is arranged on the internal face of the cover11, facing toward the longitudinal cavity12, and is configured in order to make it possible to hold the strips inside the cavity when the cover11is positioned on the cartridge10. The cartridge10according to the invention has a substantially rectangular shape. The cartridge10comprises a longitudinal cavity102which is preferably centered, as may be seen particularly inFIG.5. The cavity102comprises a bottom103. Furthermore, the cavity102comprises a first portion102ashaped in order to receive at least one strip13and preferably a plurality of strips13, and a second portion102bshaped in order to at least partly accommodate the dispensing member12, as illustrated inFIG.6. In the example illustrated inFIG.6, the first portion102aof the cavity and the second portion102bof the cavity102are connected by an inclined intermediate portion102c, the second portion102bof the cavity102being deeper than the first portion102awith respect to an axis D-D substantially perpendicular to the bottom103of the cavity102. In other words, the part of the bottom103of the cavity102at the level of the second portion102bis offset downward, that is to say away from the upper face of the cartridge10, along the axis D-D substantially perpendicular to the bottom103. Advantageously, the inclined intermediate portion has a slope of between 25° and 35°. Moreover, as may be seen particularly inFIG.5, the cartridge10comprises a first engagement orifice104aformed on a first end of the cartridge and intended to cooperate with the first tongue112aof the cover11. The cartridge10also comprises a second engagement orifice104bon a second end of the cartridge, opposite the first end, which is intended to cooperate with the second tongue112bof the cover11. According to the invention, the cartridge10may comprise lateral indentations106which are formed symmetrically on either side of the cartridge and are configured in order to allow it to be held in position in the tool for picking up the strips. Furthermore, the cartridge10comprises at least one retaining tab105positioned at one end of the cavity102of the cartridge10. The retaining tab105is formed on the first portion102aof the cavity102. The retaining tab105is configured in order to prevent the strips from leaving the cavity102. The retaining tab105also makes it possible to facilitate gripping at the middle of the strip by an instrument, as may be seen inFIG.13. The retaining tab105holds the second end132of the strips13. The dispensing member12is illustrated particularly inFIGS.7to9. The dispensing member is configured in order to cooperate with the cartridge10, said dispensing member12being at least partially accommodated in a second portion102bof the cavity102of the cartridge10, as illustrated particularly inFIGS.10and11. As may be seen inFIGS.7and8, the dispensing member12comprises a protruding part121having a contact surface122shaped in order to cover at least a first end131of each strip13, the contact surface122facing toward the bottom103of the cavity102of the cartridge10and overhanging the inclined intermediate portion102c. Furthermore, the dispensing member12comprises a second part123which is widened with respect to the protruding part121. More particularly, the protruding part121extends radially or laterally (depending on the shape of the second part) with respect to the second part123. Furthermore, the dispensing member has a front-to-rear cross section of substantially triangular shape along the longitudinal axis, as illustrated inFIG.9. This shape makes it possible to promote tilting of the dispensing member12forward, that is to say toward the protruding part121. Specifically, as may be seen inFIGS.8and9, the second part123of the dispensing member12has a bearing surface125followed by a shoulder, followed by a plane surface substantially parallel to the bearing surface125. An inclined portion124connects the plane surface to the protruding part121of the dispensing member12. The object of the invention is, in particular, to facilitate individual gripping of the strips stored together in the same container. For this purpose, the dispensing member12is configured in order to be mobile in rotation between:a resting position illustrated inFIG.10, in which the protruding part121extends at a distance from the inclined intermediate portion102c, andan operating position illustrated inFIGS.11,12,13, in which the protruding part121converges toward the inclined intermediate portion102cso that the first end131of each strip13is at least pressed against the inclined intermediate portion102c, and is preferably pinched between the protruding part121and the inclined intermediate portion102c. As may be seen inFIG.10, the strips13are covered on a first end131by the protruding part121of the dispensing member12. During the tilting/rotation of the dispensing member12, in the operating position, the protruding part121bears on the end131of the strips against the inclined intermediate portion102cof the cartridge, thereby causing tilting of the strips as illustrated inFIG.12. When the pressure exerted on the dispensing member is manually released, the retaining tab105is no longer capable of retaining the second end132of the strips13, which thus allows individual manual gripping of said strips13. Moreover, when the pressure exerted on the dispensing member is less strong than a standard manual pressure, the retaining tab105retains the second end132of the strips13, causing the strips13to curve as illustrated inFIG.13, which allows gripping by an instrument200of the automatic type, for example by suction of the center of the strip13. Furthermore, this curvature makes it possible to create a friction movement between the gripped strip and the strip located directly below, which could be adhesively bonded, and to separate them. In both cases, the full tilting or curvature of the strips allows detachment of the strips from one another and makes it possible to grip a single strip at a time, irrespective of the instrument used. This furthermore makes it possible to ensure that the strip located below remains in place in the cartridge and does not turn over. InFIG.12, a manual instrument or the hand of an operator may be used, while inFIG.13an automatic instrument (suction cup or any other gripping system) may be used. Of course, the invention is not limited to the embodiments described and represented in the appended figures. Modifications remain possible, particularly in terms of the construction of the various elements or by substitution of technical equivalents, without thereby departing from the protective scope of the invention. | 9,673 |
11860154 | DETAILED DESCRIPTION OF THE INVENTION Aside from the preferred embodiment or embodiments disclosed below, this invention is capable of other embodiments and of being practiced or being carried out in various ways. Thus, it is to be understood that the invention is not limited in its application to the details of construction and the arrangements of components set forth in the following description or illustrated in the drawings. If only one embodiment is described herein, the claims hereof are not to be limited to that embodiment. Moreover, the claims hereof are not to be read restrictively unless there is clear and convincing evidence manifesting a certain exclusion, restriction, or disclaimer. There is shown inFIG.1, one embodiment of the method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos. The method includes acquiring a first plurality of images of a region of interest including one or more capillaries of a predetermined area of a human subject from non-invasive capillary videos captured with an optical device, step10,FIG.1. First plurality of images12,FIG.2, are preferably derived from non-invasive capillary videos acquired or captured with optical device14, e.g., a high-resolution camera, an imager or imaging device as disclosed in the '277 patent and/or the '221 patent application cited supra and incorporated by reference, or similar type device. In this example, first plurality of images12includes images or frames16,18,20,22and24of region of interest (ROI)26,FIGS.3and4, which includes one or more capillaries, e.g., capillary28,FIGS.2and4of a predetermined area of a human subject. In this example, first plurality of images12includes five images or frames16,18,20,22, and24. In other examples, first plurality of images12may include more or less than five images or frames16-24as depicted in this example. In one example, the predetermined area of the human subject may be the nailfold of a finger, e.g., nailfold40,FIG.3, of finger42of human subject44,FIG.5. Nailfold40,FIG.3, is one preferred area of the human subject because one or more capillaries are more easily detected by optical device14because the capillaries are in a more longitudinal position, e.g., as shown by capillary28,FIG.6. In other examples, the predetermined area of human subject44,FIG.5, may include a toe, a tongue, a gum, a lip, a retina, an earlobe, or any similar body part determined area of human subject44. FIG.4shows in further detail one example of ROI26of a predetermined area of the human subject where images of one or more capillaries may be acquired or captured with optical device14. As disclosed in the '221 patent application and/or the '277 patent, in one design, a light source50emits light52which is reflected by mirror54such that light52penetrates nailfold56of40in ROI26and reflected light60is detected by optical device14coupled to processing subsystem70,FIGS.2and4, to create non-invasive capillary videos which include first plurality of images12,FIG.2, with images or frames16-24. The method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos also includes processing first plurality of images12to determine one or more OAGs located in the capillary, step60.FIG.1. As discussed in the Background Section above, an OAG is an area within a capillary that is depleted of red blood cells and does not absorb light at the wavelengths at which absorption occurs in hemoglobin (e.g., about 400 nm to about 600 nm) and indicates the presence of one or more white blood cells, e.g., as disclosed in the '221 patent application and/or the '277 patent. In one example, processing subsystem70coupled to optical device14, similar to the processor disclosed in the '221 patent application and/or the '277 patent, or similar type processing subsystem, processes first plurality of images12and detects one or more OAGs, e.g., OAGs64,FIG.2, in capillary28in images or frames16,18,20,22, and24. The method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos also includes annotating the first plurality of images12with an indication of any OAG detected in the plurality of images, step62,FIG.1. In one example, first plurality of images12,FIG.2, are input to processing subsystem70which outputs annotated information72associated with any OAG detected. In one example, annotated information72preferably includes OAG reference data, e.g., OAG reference table74,FIG.7, or similar type OAG reference data, that preferably includes frame identifier76and an indication of any optical gap detected in each of images or frames16,18,20,22, and24of the first plurality of images12, indicated at78. In this example, OAG reference table74includes frame identifiers t0, t1, t2, t3, t4. . . tn, for each image or frame and an OAG identifier for each image or frame, e.g., a 1 to indicate an OAG has been detected. Annotating first plurality of images12,FIGS.2and7, with an indication of any OAG detected in first plurality of images12to generate annotated information72may be performed by a trained human operator or by processing subsystem70,FIGS.2and4. The method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos also includes acquiring a second plurality of images of the same region of interest of the same capillary with an advanced optical device capable of resolving cellular structure of white blood cells and white blood cell subtypes, step80,FIG.1. FIG.2shows one example of second plurality of images82which includes images or frames84,86,88,90,92, and94of the same ROI26,FIGS.3and4, which includes one or more capillaries, e.g., capillaries28of a predetermined area of the human subject, e.g., nailfold40, acquired or captured with advanced optical device96,FIGS.2and6, capable of resolving cellular structure of white blood cells and white blood cell subtypes. In one example, advanced optical device96may include a spectrally-encoded confocal microscopy (SECM) device, a swept confocally aligned planar excitation (SCAP) microscopy device, a scattering confocally aligned oblique plane imaging (SCOPI) device, or an oblique black illumination microscopy (OBM) device, e.g., as disclosed in Golan et al.,Noninvasive Imaging of Flowing Blood Cells Using Label-Free Spectrally Encoded Flow Cytometry, Biomedical Optics Express, Vol. 3 No. 6 (2012), Bouchard et al.,Swept Confocal-Aligned Planar Excitation(SCAP)Microscopy for High-Speed Volumetric Imaging of Behaving Organisms, Nature Photonics, Vol. 9 (2015), McKay et al.,High-Speed Imaging of Scattering Particles Flowing Through Turbid Media With Confoncially Aligned, Oblique Plane Illumination, SPIE Bios, San Francisco, CA (2019), McKay et al.,Imaging Human Blood Cells In Vivo With Oblique Back-Illumination Capillaroscopy, Biomedical Optics Express, Vol. 11 (5) (2020), and Ford, T., N. and Mertz, J.,Video-Rate Imaging of Microcirculation With Single-Exposed Oblique Black Illumination Microscopy, Journal of Biomedical Optics, Vol. 18 (6) (2013), all incorporated by reference herein. In one example, the cellular structure of white blood cells and white blood cell subtypes resolved by advanced optical device96may include the subtype of any white blood cells detected, e.g., a granulocyte, a neutrophil, a lymphocyte, a monocyte, an eosinophil, or a basophil. Image100,FIG.2, shows one example of the cellular structure of a white blood cell and/or white blood cell subtype in images86and88resolved by advanced optical device96, e.g., in this example, resolved by a spectrally-encoded confocal microscopy (SECM) or similar type advanced optical device. The method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos also includes spatiotemporally annotating second plurality of images with an indication of any white blood cell detected and/or a subtype of any white blood cell detected in the second plurality of image, step102,FIG.1. Spatiotemporally annotating the second plurality of images82,FIG.2, may include indicating one or more of a size, a granularity, a brightness, a speed, an elongation, and/or a margination of any white blood cells detected and/or a change in density of red blood cells located upstream or downstream from the location of any white blood cells detected. In one example, second plurality of images82,FIG.2, is spatially annotated using spatiotemporally annotated data, e.g., spatiotemporally annotated look-up table108,FIG.7, that includes frame identifier110, e.g., t0, t1, t2, t3, t4. . . tnfor each of images or frames84,86,88,90,92, and an indication of the white blood cell subtype associated with each frame identifier, e.g., a granulocyte, a neutrophil, a lymphocyte, a monocyte, an eosinophil, or a basophil, exemplary indicated at112. Spatiotemporally annotating second plurality of images82with an indication of any white blood cell detected and/or a subtype of any white blood cell detected in the second plurality of image82may be performed by a trained human operator or by processing subsystem70,FIGS.2and4, and preferably generates annotated information120,FIG.2, associated with second plurality of images82, and spatiotemporally annotated second plurality of images122. The method to detect white blood cells and/or white blood cell subtypes also includes inputting first plurality of images12,FIG.2, annotated information72from the first plurality of images12and annotated information120from spatiotemporally annotated second plurality of images122into machine learning subsystem124configured to determine a presence of white blood cells and/or subtype of any white blood cells present in the one or more optical absorption gaps in the first plurality of images12, step128,FIG.1. In one example, machine learning subsystem124may be neural network a support vector machine, a machine learning subsystem utilizing a Random Forest learning method, an AdaBoost meta-algorithm, a Naïve Bayes classifier, or deep learning, as known by those skilled in the art. Preferably, machine learning subsystem122may be configured to determine the presence of white blood cells in OAGs and determine a full white blood cell differential measurements and/or partial white blood cell differential measurements. In one example, first plurality of images12,FIG.2, is preferably temporally aligned with spatiotemporally annotated second plurality of images122. In this example, temporally aligning includes creating the same region of interest, e.g., ROI26,FIGS.2and4, using the same objective lens170on both optical device14and advanced optical device96,FIG.4. In other examples, temporally aligning first plurality of images12with spatiotemporally annotated second plurality of images122includes creating the same ROI26for optical device14and advanced optical device96,FIG.4, e.g., by focusing optical device28and advanced optical device90at the same location in the capillary, e.g., focusing on ROI26and capillary28, as shown. In other examples, temporally aligning first plurality of images12with spatiotemporally annotated second plurality of images122may use image alignment processing methods, e.g., registration or similar image alignment processing methods as known by those skilled in the art. See e.g., Oliveira, F. P. and Travares, J. M. R., et al.,Medical Image Registration: A Review, Computer Methods in Biomechanics and Biomedical Engineering, 17 (2) (2014), incorporated by reference herein. In one embodiment, the method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos preferably includes aligning first plurality of images12,FIG.2, to spatiotemporally annotated second plurality of images122. In one example, temporally aligning first plurality of images12with spatiotemporally annotated second plurality of images122includes aligning each frame identifier76,FIG.7, e.g., t0, t1, t2, t3, t4. . . tn, in optical absorption gap reference table74to each frame identifier110, e.g., t0, t1, t2, t3, t4. . . tn, in spatiotemporally annotated look-up table108. Plots129,FIG.8, show one example of OAG signal132output by optical device14,FIGS.2and4, and input to processing subsystem70. In this example, OAG signal132includes peaks140,142, and144which each indicate the presence of an OAG indicative of a white blood cell in a capillary e.g., OAG64,FIGS.2and9, in capillary28. Plots129,FIG.8, also show an example of advanced optical output signals134,136, and138output by advanced optical device96, in this example a SECM device, which are input to processing subsystem70,FIGS.2and4. Each of advanced optical signals134,136, and138preferably include a peak that indicates the subtype of a white blood cell that corresponds to the presence or detection OAG in a capillary. For example, peak146of advanced optical signal134indicates a white blood cell subtype of a monocyte, peak148indicates a white blood cell subtype of a lymphocyte, and peak150indicates a white blood cell subtype of a granulocyte, a neutrophil. Peaks146,148, and150of advanced optical signals134,136, and138, respectively, are for exemplary purposes only, as advanced optical signals134,136, and138may have peaks which represent other types of white blood cell subtypes. Plots129may also include additional advanced optical signals with peaks indicating additional white blood cell subtypes, e.g., eosinophils, basophils, or other white blood cellular structures. In this example, processing subsystem70temporarily aligns peak146of advanced optical signal134with peak140of OAG signal132as shown which indicates a monocyte is present in OAG64,FIGS.2and9, in capillary28. Similarly, processing subsystem70temporarily aligns peak148of advanced optical signal136with peak142of OAG signal132as shown which, in this example, indicates a lymphocyte is present in OAG64in capillary28. Processing subsystem70also temporarily aligns peak150of advanced optical signal138with peak144of OAG signal132as shown which indicates a neutrophil is present in OAG64in capillary28. In a similar manner, processing subsystem70may temporarily align a peak of one or more additional advanced optical signals each having a peak indicating additional white blood cell subtypes, e.g., granulocyte, eosinophils, basophils, or other white blood cell structures with additional peaks on OAG signal132. FIG.9shows one example of first plurality of images12with images of frames16,18,20,22, and24at frame identifiers, t0, t0+68 ms, t0+136 ms, t0+204 ms, and t0+272 m, respectively, and second plurality of images82with images or frames84,86,88,90, and92at frame identifies t0, t0+68 ms, t0+136 ms, t0+204 ms, and t0+272 m, respectively, which are temporarily aligned as shown. In this example advanced optical device96,FIGS.2and4, acquires second plurality of images82,FIG.9, using an SECM device that utilizes a line scan of capillary28, indicated at180. Other advanced optical devices may be used as disclosed above. In one example, first plurality of images12,FIG.2, annotated information72from the first plurality of images, e.g., OAG reference data74,FIG.7, e.g., a table or similar type data and annotated information from the second plurality of images120.FIG.2, e.g., spatiotemporally annotated look-up data108,FIG.7, e.g., a table or similar type data are input to machine learning subsystem124,FIG.2, which outputs results data170, e.g., a table of similar type results data, which indicates any white blood cell detected and/or the subtype of any white blood cell detected. Machine learning subsystem124then preferably compares results data170to ground truth data172, e.g., a table or similar type data to determine and improve the accuracy of the white blood cells detected and/or the white blood cell subtypes determined. As known by those skilled in the art, “ground truth” is a term relative to the knowledge of the truth concerning an ideal expected result. In one embodiment, machine learning subsystem122may output results data174, e.g., a table of similar type data, that includes any white blood cells detected and/or a subtype of any white blood cells detected for each OAG in first plurality of images12and compares results data174to ground truth data172data to determine and improve the accuracy of the white blood cells detected and/or the white blood cell subtypes determined. Once machine learning subsystem124,FIG.2, efficiently and effectively learns and determines the presence of white blood cells and/or the subtype of white blood cells present in one or more optical absorption gaps using the annotated information from the second plurality of images acquired with the advanced optical device, the method to detect white blood cells and/or white blood cell subtypes from non-invasive capillaries of another embodiment using similar techniques as discussed above with reference to one or more ofFIGS.1-9, may include acquiring a first plurality of images of a region of interest including one or more capillaries of a predetermined area of a human subject from non-invasive capillary videos captured with an optical device, step190,FIG.10. The method may also include processing the first plurality of images to determine one or more optical gaps located in the capillary, step192. The method may also include annotating the first plurality of images with an indication of any optical gap detected in the first plurality of images, step92, and determining a presence of white blood cells and/or the subtype of any white blood cells present in the one or more optical absorption gaps using the first plurality of images and annotated information from the first plurality of images and information from a machine learning subsystem which has learned and determined the presence of white blood cells and/or the subtype of white blood cells present in one or more optical absorption gaps using annotated information from a second plurality of images acquired with an advanced optical device, step194. The result is the method to detect white blood cells and/or white blood cell subtypes from non-invasive capillary videos accurately, efficiently, and quantitatively determines white blood cell differential measurements and/or partial white blood cell differential measurements to assist medical personnel in treating various diseases and conditions associated with dangerously low levels of white blood cells, e.g., neutropenia, AIDs, autoimmune diseases, organ transplantation, patients treated with immunosuppressant drugs for various conditions, and the like. Once the machine learning subsystem efficiently and effectively learns and determines the presence of white blood cells and/or the subtype of white blood cells present in one or more optical absorption gaps using the annotated information from the second plurality of images acquired with the advanced optical device, the claimed method can then utilize a simple, portable and cost-effective imaging device, e.g., a capillaroscope to determine the presence of white bloods in OAGs and the subtype of the white blood cells and does not need to further utilize the advanced and expensive optical imaging system, e.g., SECM, SCAP, SCOPI, OPBM, and the like. Using similar techniques as discussed above with reference to one or more ofFIGS.1-9, the method to determine density of red blood cells from non-invasive capillary videos of one embodiment of this invention includes acquiring a first plurality of images of a region of interest including one or more capillaries of a predetermined area of a human subject from non-invasive capillary videos captured with an optical device, step200,FIG.10. The first plurality of images is processed to determine one or more areas of hemoglobin optical absorption located in the capillary, step202. The first plurality of images is annotated with an indication of any areas of hemoglobin optical absorption detected in the first plurality of images, step204. A second plurality of images of the same region of interest of the same capillary is acquired with an advanced optical device capable of resolving cellular structure of red blood cells, step206. The second plurality of images with an indication of a density of any red blood cell detected is spatiotemporally annotated in the second plurality of images, step208. The first plurality of images and annotated information from the first plurality of images and annotated information from the spatiotemporally annotated second plurality of images are input into a machine learning subsystem configured to determine the density of any red blood cells present in the one or more optical absorption gaps in the first plurality of images, step210. In one example, red blood cell count may be determined from the density of red blood cells. Although specific features of the invention are shown in some drawings and not in others, this is for convenience only as each feature may be combined with any or all of the other features in accordance with the invention. The words “including”, “comprising”, “having”, and “with” as used herein are to be interpreted broadly and comprehensively and are not limited to any physical interconnection. Moreover, any embodiments disclosed in the subject application are not to be taken as the only possible embodiments. Other embodiments will occur to those skilled in the art and are within the following claims. In addition, any amendment presented during the prosecution of the patent application for this patent is not a disclaimer of any claim element presented in the application as filed: those skilled in the art cannot reasonably be expected to draft a claim that would literally encompass all possible equivalents, many equivalents will be unforeseeable at the time of the amendment and are beyond a fair interpretation of what is to be surrendered (if anything), the rationale underlying the amendment may bear no more than a tangential relation to many equivalents, and/or there are many other reasons the applicant cannot be expected to describe certain insubstantial substitutes for any claim element amended. | 22,194 |
11860155 | DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods and materials are described herein. As used here, the term “flocculant” is intended to mean a molecule that has a cationic charge that is capable of facilitating the coalescence of RBCs in a fluid at room temperature, and form a settled RBC mass with less than 30 minutes at room temperature without centrifugation. Reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.” As used herein, “patient” or “subject” means an individual having symptoms of, or at risk for, cancer or other malignancy. A patient may be human or non-human and may include, for example, animal such a horse, dog, cow, pig or other animal. Likewise, a patient or subject may include a human patient including adults or juveniles (e.g., children). Moreover, a patient or subject may mean any living organism, preferably a mammal (e.g., human or non-human) from whom a blood volume is desired to be determined and/or monitored from the administration of compositions contemplated herein. As used herein, “about” means within a statistically meaningful range of a value or values such as a stated concentration, length, molecular weight, pH, sequence identity, timeframe, temperature or volume. Such a value or range can be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art. The following examples are presented to demonstrate preferred embodiments of the invention. Example 1—RBC Flocculants The present example presents materials that may be used as RBC flocculants in fluid containing or suspected to contain blood. The following chemicals in Table 1 may be provided as RBC flocculants. TABLE 1ChemicalsDescriptionsGelatinA solution of electrostatic charged poly-peptides having a widerange of molecular weight. Literature indicates gelatin is able toincrease RBC aggregation after adsorption on a RBC surface.Dextran 80 + CaC12Literature indicates that Dextran 80 plus a divalent cation likeCa2+Mg2+, and Ba2+will increase aggregation of RBCsAcid TreatmentThe isoelectric point of blood is at pH = 4.75-5. The pH changeof blood samples to less than 4.75 will convert RBCs fromnegative to positive surface charge.Polyethylenimine (PEI) withPEI is a polymer composed of large number of positivelydifferent molecular weightcharged amine groups, which is expected to attract RBCsto cause RBC settlement.Polyacrylamide (PAM) withPAM is widely used as a flocculants for water treatment. It candifferent molecular weightbe configured to either positive or negative charge. Positivelycharged PAM is toxic to aquatic wildlife.Aluminum sulfate (Alum)Alum is positively charged at neutral pH.PolydiallyldimethylammoniumPolyDADMAC is a positively charged water-soluble polymerchloride (PolyDADMAC) withdifferent molecular weight Lab Test and Down Selection: All the identified chemicals listed in Table 1 were tested and evaluated for suitability. Bovine whole blood purchased from Innovative Research (Novi, MI) was used to determine the suitability of each chemical above as a RBC flocculant. The whole blood contained with sodium citrate as the anticoagulant. The whole blood was diluted with saline to provide the following blood concentrations: 20%, 30%, 40%, 50%, 65%, and 80%, which were examined in the present studies. Gelatin induced RBC aggregation but did not cause sedimentation. At the end, gelatin converted the entire blood/saline mixture samples into a gel structure. Without separating RBCs from the plasma and saline, gelatin is not suitable for this application. Dextran 80+CaCl2also converted the blood/saline mixture samples into gels instead of separating the RBCs from the blood plasma and saline. Acid treatments of the blood/saline mixture samples were performed by adding 1.8% of high concentration acid6N HCL. The pH of the samples was changed to about 4.5. The acid treatment caused aggregation and sedimentation of the RBCs. However, using relatively large amount of strong acid is not cost-effective for this application and it is difficult to handle strong acid in the process. Thus, acid treatment may be used to increase ESR. PEI, a positively charged polymer, caused RBC sedimentation from the blood plasma and saline. The PEI must be dissolved under an acidic condition (i.e., with pH<7.0). In other words, a blood/saline mixture sample at neutral pH has to first have acid added to lower the pH before PEI can be used to facilitate RBC sedimentation in a biological fluid containing blood. The relative effectiveness of PEI and polyDADMAC were compared. The ESR (erythrocyte settling) induced by PEI was found to be lower than that of polyDADMAC. PEI is an acceptable flocculant for RBCs in a blood containing biological material (fluid). PAM is a polymer that can be either negatively or positively charged. The negatively charged polymer will not induce the negatively charged RBCs to aggregate and settle. The positively charged PAM may be expected to be used for this application. Alum is a positively charged molecule. It did facilitate a lower ESR, but at a much lower rate and amount. The relative molecular weight of a flocculant is considered for use as an appropriate flocculant because it enhances settlement of RBCs from gravity. In the studies with PEI and polyDADMAC, it was found that the higher molecular weight of those polymers caused faster RBC sedimentation. PolyDADMAC is another positively charged polymer. It is also water soluble at neutral pH. PolyDADMAC in the presence of blood/saline mixture samples has the ability to rapidly cause RBC sedimentation. Only a small amount by weight of polyDADMAC is needed to induce the sedimentation. Quantitative studies are described below to determine the fraction of polyDADMAC needed proportionally to the amount of blood in a biological mixture containing blood. These studies also suggested that too great an amount of polyDADMAC may hinder, or even stop, RBC sedimentation. This may be because the RBCs become coated and surrounded by the positively charged polyDADMAC, and as such, the coated RBCs repel each other, by positive charges instead of negative charges, thereby hindering and/or preventing aggregation and sedimentation. The low-cost, high molecular weight (at least 100 KDa, 200 KDa, 300 KDa or about 400 KDa), positively charged polyDADMAC was identified as a preferred, cost-effective flocculant to quickly induce RBC sedimentation. Other high molecular weight, positively charged polymer flocculants (e.g., PEI, PAM, etc.) would also be useful in the practice of the present methods and creation of container vessels. It is expected that an acid treatment would also be useful as part of the method to provide RBC sedimentation. Example 2—Blood Loss Estimation The present example demonstrates the material polyDADMAC as an effective flocculant for RBCs contained in a fluid, and the use of this flocculant for estimating total blood volume in a fluid that contains blood and other liquids, including saline. High-molecular weight (at least 100, 200, 300 or 400 KDa) research-grade polyDADMAC was obtained from Sigma-Aldrich (Catalog number 409022 or 409030) along with industrial-grade polyDADMAC. The industrial-grade product is commercially available from Kemira Chemical under the product name “Superfloc™ C-591”, which contains a 20% concentration of polyDADMAC. This material is used in water treatment. Studies were performed to compare the effectiveness between the research-grade and industrial-grade products. The studies revealed that the Superfloc™ C-591 produced similar results in RBC sedimentation as the research-grade product. The cost of the industrial-grade product is lower than that of the research-grade product. The purity of C-591 varied, resulting in some differences in RBC flocculation. In the use of research grade and industrial grade polyDADMAC, the 20 wt. % in water stock solutions were diluted with water to 6.67 wt. % working solution to reduce the viscosity of the solution and to enhance ease of handling. The dry weight of flocculant for a 1200 ml collection canister was about 600 mg. of polyDADMAC. Amount of Flocculant Needed to Facilitate RBC Sedimentation. Superfloc™ C-591 (obtained from the manufacturer (viscous liquid, 20 wt. % in H2O) was used as a RBC flocculant to test a bovine blood and saline mixture. The amount of flocculant needed to facilitate a more rapid RBC sedimentation rate was examined. It was discovered only about 0.4% (v/v %) of the Superfloc™ C-591 working solution added to the total blood/saline mixture volume was needed to provide an acceptably rapid (within 15 minutes) sedimentation of RBCs in the mixture. For example, in a 1-liter blood/saline mixture sample, only 4 ml of the Superfloc™ C-591 working solution was needed, regardless of the blood concentration in the mixture. This translates to about 320 mg of polyDADMAC flocculant for a 1.2 liter canister. FIG.1illustrates three 50 ml-volume blood/saline mixture samples with the blood concentrations (from left to right in the figure) of 20%, 40%, and 65%, respectively. The addition of the same amount of 200 μl (or 0.4% of 50 ml) of the Superfloc™ C-591 working solution induced red blood sedimentation at all of these blood concentrations. However, the sedimentation rate was found to be different depending on the concentration of blood in the particular mixture, as discussed below. Tests also revealed lower or higher concentrations of the Superfloc™ C-591 working solution may be used. For example, concentrations as low as 0.3% (v/v %) and as high as 0.6% (v/v %) were found effective, however, the exact upper and lower limits were not determined. It was found that a 6% (v/v %) concentration of the Superfloc™ C-591 working solution added to blood/saline mixture sample with a 20% blood concentration induced minimal settlement, but that may not be the upper limit for sedimentation. Aspect Ratio of Blood Container (Receptacle or Canister). During the experiments, it was found that the RBC sedimentation rate depends, in part, on the aspect ratio D:H of the container, where D is the diameter and His the height of the container as shown, for example, inFIG.2. The aspect ratio D:H depends on the shape of the container. The sedimentation rate is higher when D:H is higher because a larger D provides a larger area or space for the aggregated RBCs to settle by gravity. Therefore, the sedimentation rate will be different when different blood collection containers (or canisters) are used. Another finding during the experiments is that the Superfloc™ C-591 flocculant sedimented RBCs if it is placed in the container before adding the blood/saline mixture samples as well as when added to the container after it is already filled with the blood/saline mixture samples. Sedimentation Rate and Blood Concentrations. The present studies indicated that the blood concentration in the blood/saline mixture also affects the RBC sedimentation rates, with a faster sedimentation rate in samples having a lower concentration of blood versus samples containing a higher concentration.FIGS.3A,3B, and3C, for example, illustrate the sedimentation rates of the RBCs in the 30%, 40%, and 65% blood/saline mixture samples, respectively. A graduated glass cylinder (FIG.3D) was used in these studies to determine difference in the sedimentation rates. Three 60 ml blood/saline mixture samples, with blood concentrations of 30%, 40%, and 65%, were used in the experiments. A drop of 240 μl, (or 0.4% of 60 ml) of the Superfloc™ C-591 working solution was added to the glass cylinder first. Then a 60 ml blood/saline mixture sample was added to the glass cylinder. The separation of the RBCs was visually observed by a clear differentiation of fluid layers within the glass cylinder, with the settling RBCs being the more opaque fluid layer toward the bottom of the cylinder and the blood plasma/saline layer being the clearer fluid layer above the settling RBC layer. The settled RBC volume within the cylinder was recorded by reference to the cylinder graduated markings and recorded every minute. The settling RBC volume was set to 60 ml at time 0 since no visual separation had started at time 0. After time 0, the RBCs begin settling from the blood plasma/saline layer and initially settle toward the bottom of the container at a high sedimentation rate, but the sedimentation rate decreases over time. In this regard, as the amount of RBCs separated from the blood plasma/saline layer increases there are fewer RBCs to separate from the blood plasma and saline. Eventually, the RBC sedimentation rate is very low, so that the rate of RBC volume change is less than 0.5% per minute. At this point, the volume of settled RBCs is considered stable. In other words, a stable sedimentation is achieved when the volume change of the settled RBCs is less than 0.5% per minute. The “packed RBC volume,” which is denoted as Vm, is the volume of the RBC layer at the bottom of the container when the RBC sedimentation is considered stable. FIG.3Aillustrates that the blood/saline sample mixture with 30% blood concentration started RBC sedimentation almost instantly after mixing with the flocculant. Most of the sedimentation was complete within one minute. A stable RBC sedimentation was achieved around minutes after the sample is mixed with the flocculant. The stable packed RBC volume Vmwas about 9.5 ml. For the blood/saline mixture sample with 40% blood concentration, the sedimentation rate slowed slightly (FIG.3B). The most sedimentation of the majority of RBC's occurred at two minutes instead of the one minute observed for the 30% blood concentration sample. A stable sedimentation for the 40% concentration sample was achieved at about fifteen minutes and the Vmis about 14 ml. For the 65% blood concentration sample, the sedimentation was slower. (FIG.3C). It took over 60 minutes before a stable sedimentation was observed. The methods for determining blood volume in a blood/saline mixture were optimal and consistent in liquids containing 50% or less blood. Rapid sedimentation is characterized as achieving a stable sedimentation of RBCs in a saline/blood mixture within fifteen minutes of addition of the flocculant at room temperature (no agitation of the mixture during settlement). To ensure that the blood concentration in a fluid remains below about 50%, blood (for optimal RBC separation), additional saline or other appropriate blood diluent may be added. It was found that blood/saline mixtures with high concentrations of blood can still achieve a rapid sedimentation. One way in which sedimentation may be reinitiated should the sedimentation rate appear to be slowing down, one may add an additional volume of saline so as to further dilute the blood. Three 10 ml blood/saline mixture samples with 80% blood (bovine blood containing sodium citrate anti-coagulant) concentration were prepared in three bottles, as shown inFIG.4. All samples were mixed with 30 μl, (about 0.3% of 10 ml) of the Superfloc™ C-591 working solution (about 2 mg dry weight Superfloc™ C-591). No sedimentation was observed before the samples were diluted. Each bottle had saline added to dilute the blood concentration and, going from left to right inFIG.4, 5 ml, 10 ml, and 20 ml of saline was added, which diluted the samples to 53.3%, 40%, and 26.7%, concentrations of blood, respectively. The container was agitated slightly to mix the added saline with the sample. Upon dilution, the RBCs began to sediment. The 26.7% blood concentration mixture and the 40% blood concentration mixture achieved a stable sedimentation at around 10 minutes. The 53.3% blood concentration mixture achieved a stable sedimentation at about 16 minutes. As shown inFIG.4, the final packed RBC volume Vm(i.e., the darker bottom layer at the bottom of the container) in all three samples is the same, even in the presence of different amounts of saline. This result was achieved because the initial 10 ml of 80% blood concentration mixture contains the same amount of blood volume, 8 ml. This study indicated that 1) more saline can be added to dilute a blood/saline mixture to help increase the RBC sedimentation rate and 2) the total blood volume can be estimated through the sedimentation of the RBCs, since the volume of RBCs aggregated at the bottom of the container (i.e., the packed RBC volume) is not affected by the amount of saline in a blood/saline mixture. It should be noted that with other flocculants or other chemical treatments discussed above, further testing using the testing protocol previously outlined may be followed to determine optimal flocculant concentrations and blood/saline ratios. Repeatability. The RBC sedimentation study using the 40% blood (bovine blood with sodium citrate anticoagulant) concentration mixture (FIG.10A) was repeated three times. Standard deviation error bars are shown at various points on the line in theFIG.10Aplot. The repeatability of the experiments was very good, especially at the points on the line when the sedimentation approaches stability. Relatively high variations occurred initially as shown by the longer standard deviation error bars at those points on the line, but these variations are probably due to visual reading errors caused by the rapidly changing volume of the settling RBC layer at those points. The data variation is very small when the sedimentation approaches stability. In other words, the variation of the packed RBC volume Vmis small when the sedimentation is stable. Total Blood Volume Estimation. To demonstrate the feasibility of estimating the total blood volume, an algorithm was developed to estimate the total blood volume in a blood/saline mixture based on the sedimented RBC volume Vm. If the actual RBC volume is denoted as Vc(as determined through centrifugation), the total blood volume is denoted as Vb, and the patient's hematocrit is denoted as Hct, then the following relationships are created: η=Vm/Vc,Hct=Vc/Vb→Vb−Vm/(Hct×η) Since Hct is available from the patient data measured prior to surgery and Vmis measurable based on the method described above, the total blood volume Vbcan be estimated when the value of the packing ratio (η) between Vmand Vcis determined experimentally beforehand (methods and examples are presented herein, demonstrating the method for determining packing ratio η). The values of the packing ratio η may be different for different blood types (e.g., human versus non-human). The packing ratio value is also likely to be container-specific in that it may vary according to the size and shape (aspect ratio) of the container used to collect the blood. In this regard, recall tests showed the container shape affected the ESR (Erythrocyte sedimentation rate). Thus, the packing ratio η should remain the same where the same blood type and container shape for blood measurement are used. The packing ratio η is determined empirically by using a known hematocrit value and a known volume of blood within a blood/saline mixture. Preferably, the value of the packing ratio will be determined by finding an average packing ratio value from numerous blood/saline mixture samples with different known blood concentrations. In this regard, by running a study with blood from the same species of animal (bovine, equine, human), and a container having a defined size and shape, in the presence of the flocculant as described herein, where different known blood/saline concentration mixtures are examined, an appropriate packing ratio η may be calculated. The greater the number of different blood/saline mixtures examined, the more statistically reliable the average packing ratio value derived will be. As discussed above, the blood concentration of a collected fluid in some embodiments should be about 50% or less blood. Additionally, an average hematocrit may be determined from a number of hematocrit values obtained from a representative number of subjects (animal/human). Hematocrit may be calculated using traditional capillary centrifugation methods, as known by those of skill in the art. The average hematocrit value for a group of animals, such as a group of humans or a group of horses, etc., will provide a value Vcthat can be used in the formulas and methods described herein when the blood volume is known during the particular collection episode. As noted, the value of the packing ratio η is preferably an average packing ratio value calculated from a group of blood/saline mixture samples in a defined collection container. The actual RBC volume Vcand the settled RBC volume (by ordinary gravity, at room temperature), Vm, facilitated in the presence of an RBC flocculant, will be determined for a number of individual samples, and an average packing ratio value determined. To determine the actual RBC volume Vc, the hematocrit of the blood added to a mixture sample is multiplied by the volume of blood added to that sample. Next, the packed RBC volume Vmmay be determined for each mixture sample using conventional volumetric graduated markings on a container, as described above. That is, with the flocculant present, the Vmmay be determined by reading the volume of the RBC sedimentation. As part of the method, the same type and amount (concentration in the total fluid mixture volume) of RBC flocculant should be added to a collection container. As shown in table 2, even when the flocculant, polyDADMAC concentration was changed, the packing ratio η, remained relatively consistent. Thus, a range of flocculant concentrations (range of flocculant of about 0.3%, 0.4%, and 0.75% flocculant in the total liquid volume) can be used to induce RBC sedimentation without significantly affecting the packing ratio. In addition, the data in Table 2 demonstrates that the packing ratio is relatively insensitive to the amount of blood volume in the liquid mixture (blood/saline), when the flocculant concentration remains relatively the same. With the actual RBC volume Vcand the settled, packed RBC volume Vmfor each mixture sample, a packing ratio value, η value, for each sample (i.e., each different blood concentration mixture) can be determined. Then, an average packing ratio value may be calculated. For bovine blood, for example, the average packing ratio calculated was 1.61 (See Table 2). To demonstrate the empirical determination of the packing ratio value, the value of the packing ratio η was determined for bovine blood purchased from a commercial vendor, these blood materials containing sodium citrate (for anticoagulant). Plastic canisters that were marked for volume (ml) were used, and are shown inFIG.4. The results are illustrated in the following table 2. Before the experiments, the average hematocrit Hct of bovine blood was calculated from a number of bovine blood samples, and determined to have an average of 37.3%. This average Hct is used in the table below, and was used in the present approximation of blood volume in a sample. A traditional capillary centrifugation method was used for determining individual Hct values. TABLE 2ExperimentsHctVb(ml)Vc(ml)Vm(ml)η value30 ml 20% blood concentration + 90 μl37.3%6 ml2.243.51.56flocculant (i.e., 0.3% (v/v %)concentration of polyDADMAC workingsolution (6 mg.)30 ml 40% blood concentration + 180 μl37.3%12 ml4.487.51.67flocculant (i.e., 0.6% (v/v %)concentration of polyDADMAC workingsolution (12 mg)50 ml 20% blood concentration + 200 μl37.3%10 ml3.736.01.60flocculant (i.e., 0.4% (v/v %)concentration of poyDADMAC workingsolution (13.3 mg)50 ml 40% blood concentration + 200 μl37.3%20 ml7.4612.01.61flocculant (i.e., 0.4% (v/v %)concentration of polyDADMAC workingsolution (13.3 mg)Average1.61Coefficient of Variance2.8% The packing ratio values that resulted from these experiments showed relatively small variations. In these experiments, the blood concentration of all blood/saline mixture samples in the chart above is less than 50% since mixtures with a higher amount of blood can be diluted to provide a mixture with less than 50% blood by adding saline. After determining that the Hct is 37.3%, and the mean packing ratio η value equals 1.61, the total blood volume in a blood/saline mixture can be estimated using the following formula (derived from Equation 1): Vb=Vm/(37.3%×1.61)=1.67×Vm. The number 1.67 in the above formula is the calculated value of the 1/37.3% (average Hct)×1.61. (average η value from Table 2). The following Table presents a comparison between the actual blood volume known to be present in each sample compared to the approximate volume (in ml) of blood in the sample determined through the calculation of blood volume determined using the formula above and techniques described here. The results demonstrate that the formula and techniques provided here may be used to provide an approximation of blood volume in a liquid sample containing mammalian blood (for example, bovine blood), in the presence of a RBC flocculant (for example, a polymeric flocculant such as polyDADMAC). In addition, the data shows that the approximated blood volume present in a collected mixed blood/saline liquid sample closely correlates with the actual blood volume in the liquid. TABLE 3VmApproximateActual VbExperiments(ml)Vb(ml(ml)30 ml 20% blood3.55.85 ml6 ml30 ml 40% blood7.512.53 ml12 ml50 ml 20% blood6.010.02 ml10 ml50 ml 40% blood12.020.04 ml20 ml The 20% blood noted in Table 2 is composed of 200 ml bovine blood and 800 ml saline in every 1000 ml of fluid mixture. Example 3—Fluid Collection Canister The present example demonstrates the preparation of a particular fluid collection container with a RBC flocculant. Blood Containing Liquid Collection Container. The fluid collection container used in the following examples was a 1200-ml suction canister, shown inFIG.7B. To this canister (1), a flocculant was applied, polyDADMAC, which appears as a film of dispersed flocculant particles (2) on the bottom and walls of the canister. Flocculant—PolyDADMAC. Kemira's “Superfloc™ C-591” was used as the source of polyDADMAC. The quality or purity of this product was not consistent. Therefore, the Sigma-Aldrich version of high-molecular weight (200-350 KDa) 20% polyDADMAC (Sigma Catalog #409022) was used. The results indicate that the Sigma version of polyDADMAC significantly improve the testing results. However, it is anticipated that virtually any number of different sources flocculants may be used in the practice of the present invention, as well as in the fabrication of the herein described flocculant containing and treated fluid collection containers. Example 4—Optimization of PolyDADMAC as the Flocculant for a 1200 ml Collection Canister for Blood Volume Approximation in a Mixture Containing Blood In the operating room, a biological fluid waste collection canister may be used to collect a volume of fluid, which will include an unknown volume of blood. The volume of blood in a canister volume of 1200 ml can vary anywhere from 10 ml to 1200 ml. The volume of blood in a collected fluid collected may also vary depending on specific species of animal, the gender and weight of the patient, as well as the particular medical procedure, being performed. However, the volume of blood in a collected fluid in a collection canister during a typical adult human surgical procedure is generally contains about 20% to about 50% blood. Conventional techniques for blood volume determination used in a standard hospital operating settings provide only gross estimations of blood volume that are most times inaccurate by at least 50%-75%, and are not available until hours after a surgical procedure has been completed. In most cases, the amount of blood in an aspirated fluid during a surgical procedure is 50% or less. In those cases where the fluid contains more than 50% blood, the present methods and devices may be used to accurately determine blood volume by adding saline or other diluent to the fluid to lower the blood concentration, so as to facilitate the sedimentation of RBC's in the fluid in the presence of a RBC flocculant, polyDADMAC. The present example demonstrates that a relatively constant amount of RBC flocculant, such as polyDADMAC, may be used to achieve a relatively accurate estimation of blood volume is a fluid containing about 50% or less blood concentration. This is achieved by using a visual reading of the volume of settled RBCs in a calibrated canister containing a RBC flocculant. This volume of settled RBCs is then used in a calculation to determine the volume of blood in the mixture. The settled RBC volume value alone is insufficient to accurately approximate blood volume in a mixture. A test of 40% of blood at three different volumes of a blood/saline mixture was tested. The volumes of 40% blood/saline mixtures tested were: 200 ml, 800 ml, and 1200 ml. A Sigma-Aldrich polyDADMAC (20 wt. % in water) solution was diluted with saline to provide a working solution of 6.67 wt. % of polyDADMAC in water. This working solution was used to provide the appropriate amount of flocculant in this study. Table 4 provides the optimal amount of flocculant for each volume of the 40% blood/saline mixture examined. The optimal amount of flocculant was identified as the amount of flocculant required to provide the fastest rate of red blood sedimentation out of the mixture. The RBCs in each of the mixture volumes examined achieved a visually discernable level of sedimentation in the canister, with a relatively clear fluid being observed above the visually discernable meniscus of settled RBCs at about 15 minutes after the blood/saline mixture had been combined with the flocculant at room temperature. It was observed that the rate of sedimentation varied depending on the amount of flocculant provided in the mixture. From Table 4, an average of about 0.75 ml to about 1.5 ml of the flocculant working solution (about 50 mg to about 100 mg dry weight polyDADMAC) was optimal for promoting rapid RBC sedimentation in a 100 ml volume of the blood/saline mixture. For a larger 1,200 ml canister having a volume of about 1200 ml, about 9 ml of the polyDADMAC flocculant working solution (or about 600 mg dry weight polyDADMAC) would be provided in the bottom of the canister or on the canister walls to promote rapid RBC sedimentation is a volume of up to 1,200 ml of the blood/saline mixture. In the following studies, about 0.75 ml to about 1.5 ml of the flocculant working solution was used per 100 ml (or 0.75% v/v) of a blood/saline mixture. This concentration value is slightly larger than the 0.4% and 0.6% used in the studies described herein when using the industrial grade flocculant Superfloc™ C-591 version of polyDADMAC. Based on the identified 0.75% v/v polyDADMAC concentration, a 1,200 ml canister that can collect up to 1,200 ml of a blood-containing fluid will need about 9 ml of the polyDADMAC working solution (or about 600 mg dry weight polyDADMAC). TABLE 4Optimal Amount ofNeed of flocculant perTest blood/1/3 diluted 20%100 ml of blood salinesaline volumepolyDADMACmixture200 ml of 40% blood2-3 ml1-1.5 ml800 ml of 40% blood6-8 ml0.75~1 ml1200 ml of 40% blood9 ml0.75 ml Table 4 demonstrates the amount of flocculant that is optimal for achieving sedimentation of RBCs for different volumes of a 40% blood/saline mixture. As shown, 9 ml of the RBC flocculant polyDADMAC (about 600 mg dry weight) provided optimal RBC sedimentation in a volume of 1200 ml of the blood saline mixture. Example 5—Method of Preparing a Flocculant Treated Canister The present example describes various methods in which a flocculant may be provided and distributed within and/or on a fluid collection container, particularly a fluid containing blood. While the specific polymeric flocculant, polyDADMAC, is used in the present example, many other polymeric and non-polymeric flocculant may be used in the practice of the present invention for providing the methods and devices described herein. Vertical Band Coating on Canister Wall. A 1,200 canister was coated with a 9 ml volume of material that contained 600 mg flocculant. The canister was coated in the form of 1″ wide vertical band in the canister. This way, only flocculant immersed by the blood/saline mixture is dissolved or released into the mixture to cause RBC sedimentation. In other words, the vertical band of polyDADMAC coating can provide a control release of flocculant proportional to the volume of blood/saline mixture. Two methods were used to apply the vertical band coating. One is to use a brush to provide a 1″ band directly on the canister wall. Another is to apply a flocculant coating on a 1″ wide clear tape, and then to adhere the clear tape to the canister wall. The canister has a hydrophobic surface such that it can be difficult for the flocculant solution to stay on the wall. An ozone treatment was developed to change the hydrophobic canister wall to hydrophilic. The flocculant adherence using this technique was improved. After the coating of the vertical flocculant band, the canister was tested with bovine blood purchased from a commercial vendor. The canister with the flocculant band did facilitate RBC sedimentation. However, the sedimentation rate was somewhat slower. It took more than 20 minutes for the RBC to settle at bottom of the canister. Generally, a faster rate of RBC sedimentation is desired. While not intending to be limited to any theory or specific mechanism of action, the slower rate may be related at least in part to the time needed for the flocculant coating to dissolve and spread throughout the entire blood/saline volume. It takes more time to spread the flocculant from single-band coating to the entire blood/saline mixture. A thinner coating that is distributed throughout the entire canister is expected to accelerate the dissolution and distribution of the flocculant. Uniform Coating on Entire Canister using the Ultrasound Atomization Technology. An ultrasound based method was used to coat the interior of a blood collection canister. Ultrasound atomization coating is a pressureless, low-velocity (typically on the order of 3-5 inches per second) coating that differentiates itself from spray coating. Piezoelectric transducers convert electrical input into mechanical energy in the form of vibrations. The high frequency sound vibration atomizes liquid into a fine mist spray (FIG.6). The unpressurized, low-velocity spray significantly reduces the amount of overspray since the drops tend to settle on the substrate, rather than bouncing off it. The mist spray pattern can be controlled and shaped precisely. Spray patterns from as small as 0.070 inches wide to as much as 1-2 feet wide can be generated using these types of specialized spray-shaping equipment. The atomization device used in this process had a 60 kHz ultrasound nozzle. FIG.7Aillustrates the manual coating process of a canister using the ultrasound atomizer. The atomizer tip moves along the canister wall starting from the bottom of the canister gradually moving up to the top of the canister. Regions coated by misty spray turn clear wall to foggy surface, which guides the manual coating process to cover the entire canister wall and to make the coating as uniform as possible based on visual observation. An automatic coating process can be developed in future to make uniform coating. Multiple parameters were tested, using different concentrations of polyDADMAC, and incorporating high vaporized alcohol, like methanol and ethanol, into the spray solution to accelerate the drying process. The polyDADMAC working solution (prepared in DI (de-ionized) water) provided an optimal misty spray to deposit the flocculant using an ultrasound atomization device at the intensity setting of “10” (FIG.7A). The syringe pump setting was 60 ml/h. About 4.5 ml of the flocculant coating solution was needed to cover the entire 1,200 ml canister wall. Therefore, to apply 9 ml of the flocculant solution onto the canister, two batches of the flocculant coatings were needed. A 9 ml volume of the flocculant solution contains about 600 mg polyDADMAC. Between each coating, the canister was allowed to dry completely, either inside an oven for couple of hours or under room temperature for 24 hours.FIG.7Billustrates a canister applied two batches of coating in its entire wall. Use of a more powerful ultrasound atomization device will permit a greater concentration of the coating solution. In this manner, a single batch of coating may be used to deliver all the polyDADMAC contained in 4.5 ml of the solution, instead of in a 9 ml volume, to provide the flocculant concentration described above. The flowing protocol provides the manual steps for preparing the flocculant treated (coated) canisters: Preparation for the Coating1. Prepare polyDADMAC working solution by mixing one part of 20 wt. % polyDADMAC (Sigma Catalog #409022) with two parts of DI water.2. Fill a 60 ml syringe with the polyDADMAC working solution.3. Set the ultrasound atomization device at the intensity setting of “10”.4. Set the syringe pump rate at 60 ml/hr., and volume deliver of 4.5 ml. Manual Coating Process1. Turn on the ultrasound atomization device, start moving the ultrasound nozzle from the bottom of the canister applying misty spray on the wall and bottom of the canister.2. Gradually moving the ultrasound nozzle from bottom to the top of the canister following the spiral path and ensuring everywhere on the wall is coated with polyDADMAC. The regions coated with the polyDADMAC mist show misty looking as seen in the following picture.3. Typically, the 4.5 ml diluted polyDADMAC is sufficient to provide one coating to the entire canister.4. Let the coated canister to dry overnight at the room temperature.5. After the dry of the first layer of coating, a second layer is applied to the canister in the same way as described above. A total of 9 ml of diluted polyDADMAC will be applied on the canister after two layers of coating.6. Dry the canister again overnight, then the canister is ready to be used. Example 6—Estimation of Packing Ratio (II) of the PolyDADMAC Coated 1200 ml Canister The polyDADMAC coated 1200 ml canister was prepared using a canister having a volume of 1,200 ml, and having the dimensions of a biological waste canister employed in operating rooms. A typical organization of such a canister in an operating room setting is provided inFIG.5. First, a packing ratio η associated with this coated canister was determined. Bovine blood purchased from a commercial vendor was used in the present study. This blood had been refrigerated, and then warmed to 23° C. at the time of testing. In addition, the bovine blood contained sodium citrate in order to prevent coagulation. Bovine blood and saline were added to a flocculant treated canister so as to achieve a defined ratio of blood/saline. To simulate the process in which a liquid containing blood would be provided into a collection canister during a routine operation, for example, a 600 ml volume of a blood/saline mixture (40% blood) was delivered to a flocculant treated canister according to the following technique. A container A was prepared to include 240 ml of blood, and a container B was prepared to contain 360 ml of saline. A serological pipet was used as the aspiration probe. The aspiration probe was connected to an inlet port (the patient port) on the canister via tubing. The canister was further connected to a vacuum line using a second port on the canister, and used to impart a vacuum in the canister. Under vacuum, the pipet was placed in the container including blood or the container including saline to aspirate the respective fluid alternatively into the flocculant containing canister (polyDADMAC) (FIG.8). A total fluid volume of 600 ml of the 40% blood was therefore provided in the canister. After all of the blood and saline had been aspirated into the third canister, the mixture was monitored to assess separation/settlement of the RBCs apart from the plasma and saline, in the presence of the flocculant. The volume of the RBC settlement line was recorded every minute for 20 minutes. The study was conducted using different volumes of the 40% blood/saline mixture solutions (200 ml, 400 ml, 800 ml and 1200 ml). Each study was repeated three times.FIGS.10A-10Dillustrate the change of RBC settlement volume along the time of 20 minutes with the 40% blood mixture. The studies were also performed three times with a 20% blood/saline mixture solution at volumes of 200, 400, 800 and 1200 ml. (FIGS.9A-9D). In order to obtain an average packing ratio η that would work for different volumes and different blood concentration mixtures in a fluid collected in this canister, two blood concentrations of 20% and 40%, and four different volumes of these, 200 ml to 1200 ml, were examined. With a 20% blood mixture (20% blood/80% saline), the RBCs settled very quickly in the flocculant treated canister. A visually observable settlement of RBCs of 30 ml (Vm) was reached within 10 to 15 minutes at room temperature (FIG.11A). The average Vmfrom three repeated experiments and the calculated Vic(based on the measured bovine blood hematocrit (35.4%), and the unknown blood volumes used in the study), of all four different total mixture volumes of the 20% and 40% blood preparations are listed in Table 5. These experiments demonstrate the polyDADMAC coating facilitated the rapid sedimentation of RBCs out of the blood/saline mixture at room temperature within 15 minutes. The sedimentation of RBCs in these blood/saline mixtures at room temperature in the absence of a flocculant would have required 3-6 hours. Estimation of the Packing Ratio η. Using the data from the above experiments, the packing ratio η (see Equation 1) between the settled RBC volume (Vm) and the actual RBC volume (V s) can be determined empirically. Table 5 illustrates the empirically determined packing ratio for the 1200 ml Medi-Vac canister, which has the average value of 1.20. Since the packing ratio covers a large range of volumes from 200 to 1200 ml as well as a large range of blood concentrations, the variance of the packing ratio is relatively high, above 13%. These variations in packing ratio also affect the variance in blood volume loss estimation. The following packing ratio η values were calculated for each of the respective blood mixtures. An average value η was then calculated. TABLE 5Empirically Determined Packing Ratio:AverageobservedAverageηBlood MixturesVm(ml)Vc (ml)(Vm/Vc)1. 200 ml 20% blood1814.581.232. 400 ml 20% blood3029.161.033. 800 ml 20% blood6058.321.034. 1200 ml 20% blood10087.481.145. 200 ml 40% blood3028.011.076. 400 ml 40% blood7056.031.257. 800 ml 40% blood160112.051.438. 1200 ml 40% blood233.3168.081.39Average1.20Coefficient of13%Variance In this study, the average hematocrit value of bovine blood used was 35.4%, and the derived average packing ratio η was 1.2. Using the formula at Equation 1, the following formula was created to estimate the volume of blood in a fluid collected in the flocculant containing canister: Vb=Vm/35.4%×1.2=2.35×Vm The estimate of blood loss for each of the blood mixtures 1-8 are presented in the following Table 6, and compared to the known amount of blood in the sample. TABLE 6EstimatedActualVmbloodbloodBlood Mixtures(ml)volume(ml)volume(ml)1. 200 ml of 20% blood1842.30402. 400 ml of 20% blood3070.50803. 800 ml of 20% blood60141.01604. 1200 ml of 20% blood100235.02405. 200 ml of 40% blood3070.5806. 400 ml of 40% blood70164.51607. 800 ml of 40% blood160376.033208. 1200 ml of 40% blood233.3548.3480 As demonstrated in the table above, the amount of blood loss calculated using the present formula was correlated with the actual amount of blood in the fluid. The present methods and devices therefore are demonstrated to provide a contemporaneous visual indicator tool of blood volume loss for the physician/health care professional in a surgical setting, which is more accurate than conventional approaches (saline bag use assessment and/or post-surgery estimation from total patient fluid collection). Based on an observed volume (in ml) of settled RBCs in a graduated canister in the presence of a flocculant, without the requirement of any electrical, temperature, or other material manipulating procedure, a Blood Indicator Panel was devised using the above formula, that provides an immediate visual tool for total blood volume approximation in a collected fluid. Example 7—Creation of a Blood Volume Indicator Panel for Blood Volume Assessment in a Biological Fluid Receptacle For a user to easily estimate blood volume via a visual inspection of settled RBCs in a receptacle (such as a flocculant containing canister), a blood volume indicator panel with calibrated markings is provided for a fluid collection receptacle, and will indicate a total blood volume approximation in a collected fluid, from the level of settled RBCs in the presence of a flocculant in the collection receptacle. It is envisioned that these collection receptacles may or may not include conventional volumetric measures. To create the graduated marking for the blood volume indicator panel of the present receptacles (canisters, etc.), the following Equation 2 is used: Vm=Vb/(Hct×η) The Equation 2 will employ an average Hct calculated from a number of animals/human from the same species, and of the same approximate age and gender. For example, for an adult human male, the average Hct is about 45%. In this study, a blood indicator panel for a large mammal was created using the formula: Vm=0.43×VbEquation 3: The formula will use an average Hct in bovine blood of 35.4%, and an average packing ratio η=1.20, as calculated for bovine blood (Table 5). A 50 ml estimated blood volume mark is provided on the blood volume indicator panel, which corresponds to a visually discernable settled RBC volume of about 21.5 ml. using the Equation 3 (See Table 7). A 100 ml estimated blood volume calibrated mark can be generated on the blood volume indicator panel that corresponds to about the 43.02 ml of the settled RBC volume line of the receptacle, and so forth. The graduated markings of the blood volume indicator panel provide a series of visually identifiable markings that do not relate to a measure of the volume of material in the canister, but instead to an approximation of the blood volume in the fluid collected in the canister. An illustration of a typical blood indicator panel (BIP), is provided inFIG.15(See Left Panel6, calibrated markings at 50 ml, 100 ml, 200 ml, 400 ml, 600 ml). Inclusion of a Blood Indicator Panel on a conventional collection vessel having standard volumetric markings (SeeFIG.16, Right Panel1), will provide an immediately visual estimation of blood volume in a collected fluid, without the necessity of performing mathematical calculations or other manipulations of collected or sedimented materials. As shown, the blood volumes identified with the calibrated markings of the BPI do not coincide with the conventional volume of settled RBCs in the fluid. Instead, the volume of settled RBCs is used as part of a calculation together with hematocrit and the defined aspect ratio to provide an approximation of blood volume. In the absence of a flocculant, the volume of blood in a liquid would not be possible to approximate within less than about 3-6 hours because, among other things, RBCs do not begin to settle until well after 3-6 hours. In addition, the presence of flocculant, alone, while facilitating rapid RBC sedimentation, does not immediately approximate the amount of blood in a liquid. As demonstrated in the present results, the volume of settled RBCs in a liquid was less than about 50% of the actual blood volume known to be contained in a test fluid containing a known amount of blood. The volume of settled RBCs in the presence of flocculant has to be further corrected to accommodate blood average hematocrit and packing ratio, to provide an approximate blood volume in a liquid. The BIP Panel is created based on the derivation of an average hematocrit (Hct), for example, the average Hct for human, bovine, equine, etc. To correct for Hct differences in individual patients/animals, such as differences in individual Hct due to gender, species, age, etc., the approximate blood volume value indicated on the BIP may be adjusted by a factor that corrects for significantly higher or lower individual hematocrit values. For example, if the measured Hct value from a patient is lower, for example, 80% of the typical Hct of an adult human male, then the blood volume indicator value on the panel observed for that patient will be divided by 80%, so as to provide an even closer approximation of the estimated blood volume in the receptacle. More particularly, if the blood volume indicator value on the panel is 50 ml according to the graduated markings on the indicator panel, the actual blood volume in the biological material collected from this patient would be calculated to be 62.5 ml where the patient's Hct is 80% of the typical Hct value. Similarly, if the measured Hct value of a patient is higher, for example 110% of a typical adult male hematocrit value, then the blood volume indicator value on the blood volume indicator panel would be divided by 110%, which will yield a lower blood volume. For example, if the blood volume indicator value on the blood volume indicator panel is 100 ml, then the actual blood volume loss for the patient would be calculated to be 90.9 ml, so as to correct for the patient's higher than average Hct. (e.g., 10% higher).FIG.8illustrates a 1200 ml canister with a blood volume indicator panel. The particular Blood Indicator Panel as placed on a collection canister is shown inFIG.12. The BIP was created to provide a blood volume estimation in a volume of a 20% blood/saline mixture or in a 40% blood/saline mixture using bovine blood. The Blood Indicator Panel may also be useful for assessing the volume of human blood in a liquid sample. This is because both bovines and humans are mammals, and blood from bovines and humans share many characteristics, including similar average hematocrit. Table 7. Example of the Blood Indicator Panel markings to be used on a1200 ml canister. These calibrated blood volume markings correspond to an estimate of the volume of blood contained in a fluid sample, compared in the table to the corresponding sedimented RBC volume (indicated by the value Vmin the table). (Bovine blood with sodium citrate) TABLE 7Example of the Blood Indicator Panelmarkings to be used on a 1200 ml canister.CalibratedGraduated Volume MarkingApproximationof Sedimented RBCs (Vm)of Blood Volume(about 20% blood to 50%(Vb) BIPBlood containing Fluids)50 ml21.5 ml100 ml43.0 ml200 ml86.0 ml400 ml172.0 ml600 ml258.0 mlThese calibrated blood volume markings correspond to an estimate of the volume of blood contained in a fluid sample, compared in the table to the corressponding sedimented RBC volume (indicated by the Vmin the table). (Bovine blood with sodium citrate) This study successfully developed the prototype for a BIP using a RBC flocculant, polyDADMAC coated biological fluid collection canister (1200-ml). This size canister is used in operating rooms for human patients, adult and pediatric. The evaluation of the prototype using bovine blood has shown a quick sedimentation of RBCs in the presence of this exemplary RBC flocculant, and the achievement of stable RBC sedimentations within 20 minutes. Calibrated markings on the specially designed BIP were designed for this collection canister, and may be used to provide a visual estimation of total blood volume in a collected mammalian liquid. If the patient's measured hematocrit is different from the typical hematocrit used to create the calibrated BIP markings, the calibrated blood volume can be corrected to accommodate the percent blood hematocrit to the individual blood volume amount. The following provides the average hematocrit for an adult man and for an adult woman: Normal Hct Values: Men—42-52% (Average Hct, 47); Women—37-47% (Average Hct, 42). Example 8—Creation of 100 ml Canister with Flocculant A 100 ml canister was prepared, to contain about 50 mg flocculant. In this example, the RBC flocculant used was polyDADMAC. The flocculant was provided in a volume of the polyDADMAC working solution described herein. In a typical operating room setting, smaller volumes of fluid containing blood and other materials (tissue, urine, non-blood fluid, etc.) will be aspirated from a surgical field. The aspiration of these fluids results in an undetermined loss of blood from the patient. A smaller container may be prepared according to the present invention to accommodate the estimation of blood loss in these small, sometimes critical, volumes of collected fluid. Therefore, these 100 ml receptacles containing a RBC flocculant, such as polyDADMAC, are provided and are especially useful for determining blood volume in small amounts of collected fluid. These devices may be used, for example, in pediatric applications (infant) as well as in low volume critical fluid collection procedures. The aspect ratio of the 100 ml collection device was calculated to be 0.96. The small 100 ml container with the RBC flocculant was used in the study described in Example 9. Example 9—Fresh Blood Loss Estimation The present example is provided to demonstrate the utility of the methods and devices for use for estimating blood loss in a fluid containing mammalian fresh blood (no anti-coagulants). In this example, a non-blood material present in the fluid was saline. The present example examines a technique for estimating blood loss using fresh, never refrigerated, volume of mammalian blood. Further, the blood did not contain calcium citrate, or any other anti-coagulants. In the present study, the fresh blood specimen was obtained from an adult horse. Thus, the devices and methods are especially useful in the approximation of blood loss in mammals, including humans and veterinary animals (horses, dogs, cats, cows, bulls, sheep, pigs, etc.). In this example, blood was collected from a live, adult horse (approximately 12 years old, weight 1,200 pounds), having no known clinical pathologies, and not on any known medications. The animal was being treated for a lame foot, and was being given a nerve block to manage pain. Unlike blood collected from a commercial vendor, which contains an anticoagulant such as sodium citrate (used in the prior examples), no anticoagulants or other drugs were present in the blood collected from the horse used in this study. A total of twelve (12) canisters having a total volume capacity of 100 ml were used in the present study. The canisters were marked with demarcations along the side of the canisters at 50 ml and 100 ml increments. The aspect/ratio of the 100 ml container, D:H (Diameter vs. Height) was calculated to be about 0.96. Comparatively, the aspect ratio of the 1200 ml canister is about 0.61. Typically, the larger the aspect ratio of the collection device, the more quickly RBCs contained within any blood in the collected liquid will settle out in the collection device. Therefore, the rate of RBC sedimentation in the 100 ml collection device was expected to be more rapid compared to the sedimentation rate of RBCs in a 1200 ml canister, in the presence of the RBC flocculant, under similar conditions. The 100 ml dry canisters received 50 mg of the RBC flocculant, polyDADMAC, research grade (Sigma Catalog #409022). (See Example 7). Other RBC flocculants, as well as industrial grade versions of these flocculants, including PEI, PAM and others, may be expected to be useful in the present methods and devices. The amounts of fresh equine blood and saline indicated in Table 8 were then added to each of the canisters, and RBC sedimentation volume was recorded every minute for 20 minutes: TABLE 8Fresh Equine Blood Study: Rate of Sedimentation of RBCRBC Sedimentation/ml (Vm)Blood/Saline1 min2 min4 min5 min10 min15 min20 minWith Flocculant1. 10% Blood10 ml/90 ml50403010 ml10 ml10 ml10 ml2. 20% Blood20 ml/80 ml605050202020203. 30% Blood30 ml/70 ml909060303030304. 40% Blood40 ml/60 ml10010080454530405. 50% Blood50 ml/50 ml10010075454545456. 65% Blood65 ml/35 ml1001007560656560Controls: WithoutFlocculant7. 10% Blood10 ml/90 mlNDNDNDNDNDNDND8. 20% Blood20 ml/80 mlNDNDNDNDNDNDND9. 30% Blood30 ml/70 mlNDNDNDNDNDNDND10. 40% Blood40 ml/60 mlNDNDNDNDNDNDND11. 50% Blood50 ml/50 mlNDNDNDNDNDNDND12. 65% Blood65 ml/35 mlNDNDNDNDNDNDND*ND = Non-detectable by visual inspection. Blood from an adult horse was drawn, and a volume of the fresh blood at body temperature was added to each of the canisters in the amounts indicated above. The top on each of the canisters was then put in place, the contents mixed so as to assure proper mixture of saline, blood and flocculant. Each canister was allowed to sit undisturbed at room temperature, and observed. The time at which sedimentation of RBCS was observed was recorded at 1 minute intervals up to 30 minutes.FIG.18presents the RBC sedimentation rates of the various mixtures. Essentially no visually detectable RBC sedimentation was observed with the blood mixtures in the absence of flocculant. In contrast, RBC sedimentation was rapidly observed in all blood mixtures containing flocculant within 5 minutes at room temperature. Table 9 presents the hematocrit of various large animals. The values for mean corpuscular hemoglobil (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), and packed cell volume (PCV), and may be employed in providing an appropriate customized Blood Indicator Panel and blood volume approximation method as described in the present disclosure. TABLE 9Normal Values for Erythron Data in Ruminants and the HorseCattleSheepGoatsHorsesPCV (%)24-4627-4522-3832-53Erythrocytes (×106/L)5-109-158-186.7-12.9Hemoglobin (g/dl)8-159-158-1211-19MCV (fl)40-6028-4016-2537-58.5MCH (pg)11-178-125.2-812.3-19.7MCHC* (g/dl)30-3631-3430-3631-38.6Reticulocytes0<0.5%00Erythrocyte4-83.2-62.5-3.95-6diameter (m)Erythrocyte fragility0.52-0.660.58-0.760.740.54(percent NaCl)Minimum(beginning hemolysis)Maximum0.44-0.520.40-0.550.440.34(complete hemolysis)Erythrocyte01-2.5050-60sedimentation rate(mm/1 hour)Erythrocyte life span160140-150125140-150(days)(MCH, Mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume.) TABLE 10Normal Values for Leukogram Data (Adult Aminals)Biological ComponentCattleSheepGoatsHorsesWhite blood cells4-124-124-135.4-14.3(×103/μl)Neutrophils (×1.03/μl)0.6-40.7-61.2-7.22.3-8.6Bands (×103/μl)0-0.12RareRare0-1Lymphocytes (×103/μl)2.5-7.52-92-91.5-7.7Monocytes (×103/μl)0.025-0.840-0.750-0.550-1Eosinophils (×103/μl)0-2.40-10.05-0.650-1Basophils (×103/μl)0-0.20-0.30-0.120-0.29Neutrophil/lymphocyte0.3-0.60.3-0.70.6-3.60.8-2.8(N:L) ratio TABLE 11Normal Values for Hemostatic Data in Ruminants and the HorseBlood ComponentCattleSheepGoatsHorsesPlatelet count (×10−3/L)100-800250-750300-600100-600Fibrinogen (mg/dl)200-500100-500100-400200-400Prothrombin time(s)22-55—*9.5-12.57-9Activated partial44-64—28-5237-54thromboplastin time(s)Fibrin/fibrinogen<8<8—<32degradation products(μg/ml)(Modified from Duncan JR et al: Veterinary laboratory medicine, Ed 2, Ames, Iowa, 1986, Iowa State University Press; and Kaneko JJ: Clinical biochemistry of domestic animals, Ed 3, New York, 1980.) Example 10—Blood Volume Estimation in a Fluid Using a Canister with Fresh Equine Blood (No Anti-Coagulant) in the Presence of a Flocculant The present example was performed with fresh blood drawn from an adult horse (12 years old, about 1,200 pounds). The horse blood did not contain any anticoagulant. The hematocrit of horse blood is between 32% and 53%. The horse blood was used immediately after having been drawn, and was at body temperature (about 101° F., 38.3° C.) at the time it was combined with saline in the presence of flocculant, polyDADMAC (600 mg). A 1,200 ml canister that had been coated by spray application of about 9 mls of polyDADMAC working solution as the flocculant was used. Thus, the 1,200 ml canister was coated evenly with a total of about 600 mg dry weight polyDADMAC as the flocculant. Because the flocculant was evenly distributed along the walls of the canister, the amount of flocculant is released in proportion to the volume of fluid provided in the treated canister. The following volumes of settled RBCs were recorded over time. A volume of 400 ml fresh horse blood (not refrigerated, body temperature, no anti-coagulants), was placed in the treated canister. A known volume of 250 ml saline was added to the canister. The total volume was 650 ml of fluid in the canister. This provided a 61.5% blood solution. The volume of settled RBCs (gravity only, no centrifugation), Vm, was recorded immediately upon mixing, up to a period of 30 minutes. After being allowed to settle undisturbed for 30 minutes, the canister was manually agitated, and then allowed to sit at room temperature again. The agitated blood containing canister was again observed for evidence of settlement of RBCs at room temperature. TABLE 12Time (min)RBC (settled RBC)0 sec600 ml20 sec600 ml30 sec575 ml1 min500 ml1.5 min500 ml2.0 min450 ml2.5 min440 ml3.0 min410 ml4.0 min400 ml5 min395 ml6 min360 ml7 min360 ml8 min350 ml9 min350 ml10 min350 ml11 min340 ml12 min330 ml13 min320 ml14 min310 ml15 min310 ml16 min305 ml17 min305 ml18 min305 ml19 min300 ml20 min300 ml21 min300 ml22 min300 ml23 min295 ml24 min295 ml25 min295 ml30 min295 ml At the end of the 30 minute observation period, the canister was manually shaken, and allowed to sit. The re-sedimentation of RBCs occurred, and was observed every minute for 30 minutes, and resulted in the observation of settled RBC levels as indicated in Table 14. TABLE 14TimeRBC (settled RBC)00 ml1 min0 ml2.50 ml3.0 min350 ml4.0 min300 ml5.0 min300 ml8 min300 ml10300 ml20300 ml30300 ml40300 ml50 min250 ml From this study it is demonstrated that the settled volume of RBC's in a solution containing whole fresh blood remains relatively stable up to about 30-40 minutes at room temperature. With agitation, it appears that RBCs in the fresh blood sample again settled to provide a discernable RBC sedimentation volumetric line, Vm, very quickly (3 to 4 minutes verses 16-19 minutes, settled RBC volume about 250 ml to 300 ml.). The actual known volume of fresh blood present in the fluid was 400 ml. The total approximation of blood volume may be calculated by the formula using the average hematocrit of the type of animal (42.5%, horse average Hct), the Vm(observable settled RBC volume in ml), and a new packed ratio value (n) determined for horse blood via experimentation with multiple equine blood sample assessments of settled RBC volume (Vm) and Hct information, a visual Blood Indicator Panel may be created and provided along the vertical axis of a collection canister for use with large animals, such as horses. This would provide an immediately visually discernable approximation of blood volume in a biological fluid containing horse blood. The development of a Blood Indicator Panel for a treated canister or other receptacle may be used to provide a visual indicator of equine blood volume loss, and especially for assessing a more exact equine blood loss, than is presently available. An equine Blood Indicator Panel may be developed employing the information and results presented here, by one of ordinary skill in the veterinary arts, without more than ordinary and routine experimental optimization trial and error. Example 11—Human Pediatric Applications for Use in Estimating Blood Loss The present example is provided to provide canisters and methods for efficient measurement of blood loss in a pediatric patient. As used in the present example, a pediatric patient is defined as an individual up to 12 years of age having a body weight of up to 70 to 80 pounds. A person's total blood volume (TBV) is related to body weight. The TBV of a child is around 75-80 ml/kg and is higher in the neonatal period (from 85 ml/kg it rises to a peak of 105 ml/kg by the end of the first month and then drops progressively over ensuing months). Thus, the TBV of a 3.5-kg 2-week-old will be about 350 ml while that of a 10-kg 15-month-old will be about 800 ml. Because of the much reduced total volume of blood in a pediatric patient, it is especially important to provide a blood collection and blood loss estimation system and device that are designed for estimating blood loss accurately from a smaller volume of blood collected from a pediatric patient. The specifically designed pediatric blood loss estimation devices of the present invention are therefore crafted with a container having the herein described flocculant and canister demarcations with a total volume capacity of less than 1000 ml, such as about 500 ml or even about 250 ml, in the case of an infant or neonate. A large acute loss of blood volume in a pediatric patient may compromise the circulation, and therefore blood loss should be carefully monitored so as to be able to detect a volume of blood loss of about 12% of the TBV (around 10 ml/kg) of the specific pediatric patient, assuming the child is in a stable condition and has a normal blood hemoglobin (Hb) level at the beginning of a procedure. By way of example, a suitable pediatric blood loss collection device would, in some embodiments, have a capacity of 250 mls. The canister would preferably provide an appropriate aspect ratio of D:H for a typical pediatric blood loss volume. The D (diameter) of the device would typically be between 2 and 3 inches, and have an H (height) of about 2 inches to about 3 inches. With these smaller dimensions, a collected blood loss volume would provide a reasonably rapid yet monitorable sedimentation rate of RBCs so as to alert an attending physician if an amount of blood loss has reached a volume where transfusion to the pediatric patient is in order. It would be preferred that a sedimentation rate would be achieved that provides for RBC sedimentation within 15 minutes of blood collected in the canister. In some embodiments, the 250 ml container has a conical shape (FIG.14). The flocculant will be provided to the container either at the time of the surgical intervention event, or may be provided as a pretreatment to the canister (such as by a spray coating). The amount of flocculant to be added to a 250 ml collection device would be about 50 mg to about 150 mg, or about 125 mg, or an amount sufficient to achieve at least a 0.3%, 0.4% or 0.75% of total volume of the solution. For sake of description, the following average total blood volume in a pediatric group of patients may be used in calculating when a 12% or greater blood loss has occurred. An average hematocrit value may also be calculated for the class/group (premature neonate, full term neonate, infant) of pediatric patients, and a marking provided alongside one axis of the canister, of average hematocrit values for these patient groups, so as to provide a ready visual reference for the attending physician or anesthesiologist to refer to and compare as against the hematocrit obtained for the patient undergoing the procedure:Premature Neonates 95 ml/kgFull Term Neonates 85 ml/kgInfants 80 ml/kg The total approximation of blood volume may be calculated by the formula using the average hematocrit of for a human child of a particular weight range and/or age, or for a human adult male or adult female, the Vm(observable settled RBC volume in ml), and the packed ratio value (n) determined for human blood. With multiple human blood sample assessments of settled RBC volume (Vm) and Hct information, a visual blood volume indicator panel to be located along the vertical axis of the collection canister may be prepared for the human, and especially for a pediatric human model. This would provide an immediately visually discernable approximation of blood volume in volumes less than about 250 ml, contained in a biological fluid containing human blood. The development of a vertical canister or other receptacle having a Blood Indicator Panel for human blood volume assessment in a liquid, and especially for assessing small volumes of human blood loss, may be developed by one of ordinary skill in the art given the teachings provided herein, without more than a routine and ordinary amount of trial and error. Example 12—Collapsible Treated Containers for Blood Loss Collection The present example presents a collapsible plastic-like container (bag) that may be used to collect biological fluid loss, and used to estimate blood loss. Such a collection device is envisioned to be especially useful in combat situations, or any other situation where space for medical equipment is limited. It is envisioned that the plastic bag containers will include an amount of an RBC flocculant suitable for providing the RBC sedimentation and the blood loss estimate features described herein. In some aspects, the bag could be placed within a supporting container, such as a box, canister, or other structure. The bag may also include a number of markings along the vertical axis of the bag, corresponding to volumetric measures (such as milliliters). In some aspects, a clear plastic bag having a volume capacity of about 1,000 ml containing between about 300 milligrams and about 4,700 milligrams of a flocculant, such as polyDADMAC, will be placed in the bag. The bag will include, in some embodiments, calibrated demarcations at a 50 ml, 100 ml, 200 ml, 250 ml, 400 ml, 500 ml, 600 ml, 750 ml and 1 liter marker. The bag may also include a BIP, such as in the form of an adhesive strip, which may be placed on the bag and used to provide a visually discernable indicator of approximated blood volume in a liquid based on the settled RBC level in the collection bag/container. An exemplary rendition of this embodiment is provided atFIG.13. An insert bag having the RBC flocculant and calibrated BIP for human blood designed for a 1200 ml collection canister, such as the canister shown atFIG.11A, is also provided. In such embodiments, the canister itself need not be treated with RBC flocculent, and instead, the insert bag will contain the RBC flocculent. The insert bag may also optionally also include a calibrated BIP for human blood. Example 13—Blood Loss Collection Kit The flocculant containing canisters (1.2 ml, 500 ml., 250 ml., 10 ml), that includes a blood volume indicator panel, may be provided together as a kit with a length of aspiration tubing and a second length of tubing suitable for adding saline into a canister and/or collapsible envelope. An instructional insert may be provided as part of the kit for the end user. Example 14—Stability Testing High Temperature Ageing This example demonstrates the stability of the RBC flocculant polyDADMAC and retained activity for providing RBC coalescence (flocculation) in a fluid containing blood, after exposure of the polyDADMAC coated canister to high temperatures. In the present study, the flocculant used was polyDADMAC provided as a coating on a canister for collecting a material, such as a biological liquid material collected during a surgical procedure that will contain a component of blood. The coated canisters were incubated at 55° C. for 6 weeks (equivalent to one-year shelf life at room temperature). The coated canisters, after high temperature aging, were then compared in the function test with the coated canister without going through the high temperature aging test. Materials:1) Control Group—four Canisters (Cardinal Health), coated with 600 mg of polyDADMAC (FIG.1), allowed to dry overnight at room temperature (z 22° C.).2) Experimental Group—four Canisters (Cardinal Health), coated with 600 mg of polyDADMAC, allowed to dry overnight at room temperature (z 22° C.). Then the canisters were incubated in a convection oven set at 55° C. for six weeks.3) Bovine whole blood (Innovative Research Lot #24301), stored in refrigerator and warm up to room temperature before the experiment.4) Isotonic Saline (Thermo Scientific, Lot #994448) at room temperature. Methods:1) Four canisters were chosen randomly from the experimental and control lots, respectively.2) Bovine whole blood purchased from a commercial vender (sodium citrate containing), was mixed with isotonic saline at room temperature to the concentrations of 20% and 40% v/v of blood, respectively. The 20% blood mixture had a total volume of 1000 ml and the 40% blood mixture had a total volume of 500 ml.3) Canisters were tested two at a time: one experimental vs. one control. Mixed blood and saline solutions were introduced into the canister via vacuum aspiration.4) The settlement of the red blood volume was recorded every minute for 20 minutes, according to the existing graduations on the canister.5) After 20 minutes, images were taken comparing the two groups.6) Data was charted as a function of RBC settlement volume vs. time for comparison. Results:FIGS.16A and16Bcompares the settlement of RBCs after blood saline mixtures were introduced to the control and experimental (heat-treated) canisters. The tests were performed using 1000 ml of 20% blood with saline mixture (FIG.16A), and 500 ml of 40% blood with saline mixture (FIG.16B). Both contain 200 ml of bovine blood. Each test was repeated once. The data illustrates a closely overlay of the volume change curves of RBC settlement in the 20% blood test. All RBC volume settlements were stabilized around 125 ml around 15 minutes after the mixtures were introduced the into the control and experimental canisters. In the 40% blood test, although the volume settlement of one experiment was lagged, all the volume settlements were stabilized around 125 ml after 15 minutes.FIG.17illustrates the pictures of the settled RBCs in both control and experimental canisters.FIG.17A(Panels I and II) andFIG.17B(Panels I and II) compares the settlement of RBCs after blood and saline mixtures were introduced to the control and experimental (heat-treated) canisters. The tests were performed using 1000 ml of 20% bovine blood with saline mixture (FIG.17A), and 500 ml of 40% blood with saline mixture (FIG.17B). Both fluids were known to contain 200 ml of bovine blood. Each test was repeated once. The data illustrates a closely overlay of the volume change curves of RBC settlement in the 20% blood test. All RBC volume settlement levels were stabilized around 125 ml at about 15 minutes after the mixtures were introduced into the control (FIG.17A) and experimental (FIG.17B) canisters. In the 40% blood test, although the volume settlement of one experiment lagged, the volume of settled RBCs was stabilized at about the 125 ml volumetric mark after 15 minutes.FIGS.17A and17Billustrate the settled RBC volumes in the control (17A) and experimental (17B) canisters. The studies demonstrated no discernable difference in the function between the control and experimental canisters after heat treatment. The polyDADMAC coated canisters, after six-week of aging test under 55° C., show no functional degradation of the polyDADMAC or decrease in effectiveness for facilitating floculation of RBCs in a liquid. The floculent coated canisters (polyDADMAC coated canisters) are expected to have at least a one-year shelf life without loss of the flocculant activity to provide blood volume estimation in a fluid stored at room temperature. The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the methods for prediction of the selected modifications that may be made to a biomolecule of interest, and are not intended to limit the scope of what the inventors regard as the scope of the disclosure. Modifications of the above-described modes for carrying out the disclosure can be used by persons of skill in the art, and are intended to be within the scope of the following claims. It is to be understood that the disclosure is not limited to particular methods or systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims. | 77,692 |
11860156 | DESCRIPTION OF EMBODIMENTS This disclosure is drawn to single measurement methods to detect and quantify antibody and drug components of antibody drug conjugates (ADCs) that robustly measure total antibody and antibody-conjugated drug quantity from a single sample preparation thereby providing drug to antibody ratio (DAR) calculation and significant time and resource savings. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and are consistent with: Singleton et al, (1994) “Dictionary of Microbiology and Molecular Biology”, 2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, et al (2001) “Immunobiology”, 5th Ed., Garland Publishing, New York. When trade names are used herein, the trade name product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product are also included. Definitions The term “biological sample” is any component derived or separated from an animal and includes blood, plasma, serum, cells, urine, cerebrospinal fluid (CSF), milk, bronchial lavage, bone marrow, amniotic fluid, saliva, bile, vitreous, tears, or tissue. The term “digestive enzyme” is an enzyme capable of cleaving or hydrolyzing peptides or proteins into fragments in either a specific or generic, random manner. A digestive enzyme can form a digested antibody sample from an antibody where the antibody is a component of a biological sample. Digestive enzymes include proteases such as trypsin, papain, pepsin, endoproteinase LysC, endoproteinase ArgC,Staph aureusV8, chymotrypsin, Asp-N, Asn-C, PNGaseF, endoproteinase GluC, and LysN. The term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fc, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); WO 93/16185; U.S. Pat. Nos. 5,571,894; 5,587,458. For discussion of Fab and F(ab′)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life (U.S. Pat. No. 5,869,046). Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific (EP 404097; WO 1993/01161; Hudson et al. (2003) Nat. Med. 9:129-134; Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448). Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134. Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (U.S. Pat. No. 6,248,516). Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g.E. colior phage), as described herein. The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. “Framework” or “FR” refers to constant domain residues other than hypervariable region (HVR) residues. The FR of a constant domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein. A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Humanized antibodies and methods of making them have been extensively reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337; 7,527,791; 6,982,321; 7,087,409; Kashmiri et al. (2005) Methods 36:25-34 (describing SDR (a-CDR) grafting); Padlan, (1991) Mol. Immunol. 28:489-498 (describing “resurfacing”); Dall'Acqua et al. (2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn et al, (2005) Methods 36:61-68; Klimka et al. (2000) Br. J. Cancer 83:252-260 (describing the “guided selection” approach to FR shuffling). Human framework regions that may be used for humanization include but, are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623); human mature (somatically mutated) framework regions or human germline framework regions (Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and framework regions derived from screening FR libraries (see, e.g., Baca et al. (1997) J. Biol. Chem. 272:10678-10684; and Rosok et al. (1996) J. Biol. Chem. 271:22611-22618). Human antibodies are described generally in van Dijk and van de Winkel, (2001) Curr. Opin. Pharmacol. 5: 368-74; Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., (1991) J. Immunol., 147: 86). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al. (2006) Proc. Natl. Acad. Sci. USA, 103:3557-3502. Additional methods include those described in: U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines); Ni, (2006) Xiandai Mianyixue, 26(4):265-268 (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, (2005) Histology and Histopathology, 20(3):927-937 and Vollmers and Brandlein (2005) Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91. Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. A “human consensus framework” is a framework region of an antibody which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup, as in Kabat et al. supra. In an exemplary embodiment, for the VL, the subgroup is subgroup kappa I. In another exemplary embodiment, for the VH, the subgroup is subgroup III. A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. An exemplary “chimeric” antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region (U.S. Pat. No. 4,816,567; Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Another exemplary chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity. Antibodies of this disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in McCafferty et al. (1990) Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al. (1992) J. Mol. Biol. 222: 581-597; Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al. (2004) J. Mol. Biol. 338(2): 299-310; Lee et al. (2004) J. Mol. Biol. 340(5): 1073-1093; Fellouse, (2004) Proc. Natl. Acad. Sci. USA 101(34): 12467-12472; and Lee et al. (2004) J. Immunol. Methods 284(1-2): 119-132. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. (1994) Ann. Rev. Immunol., 12: 433-55. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., (1993) EMBO J, 12: 725-734. Finally, naive libraries can also be made synthetically by cloning un-rearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388. Human antibody phage libraries are described in U.S. Pat. Nos. 5,750,373; 7,985,840; 7,785,903; 8,679,490; 8,054,268; and US 2005/0079574; US 2007/0117126; US 2007/0237764; US 2007/0292936. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments for the purposes of this disclosure. An antibody may be a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. One of the binding specificities may be for one antigen while the other is for a second antigen. Alternatively, bispecific antibodies may bind to two different epitopes of the same antigen. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express an antigen. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (see, e.g., Ortiz-Sanchez et al., Expert Opin. Biol. Ther. (2008) 8(5):609-32). Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 1993/08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (e.g., Kostelny et al. (1992) J. Immunol. 148(5):1547-1553); using “diabody” technology for making bispecific antibody fragments (e.g., Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA, 90:6444-448); and using single-chain Fv (sFv) dimers (Gruber et al. (1994) J. Immunol., 152:5368); and preparing trispecific antibodies (Tutt et al. (1991) J. Immunol. 147: 60). Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (e.g. US 2006/0025576). The antibody or fragment herein also includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to an antigen as well as another, different antigen (e.g., US 2008/0069820). Antibody Variants Amino acid sequence variants of the antibodies provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. Antibodies include fusion proteins comprising an antibody and a protein, drug moiety, label, or some other group. Fusion proteins may be made by recombinant techniques, conjugation, or peptide synthesis, to optimize properties such as pharmacokinetics. The human or humanized antibodies may also be a fusion protein comprising an albumin-binding peptide (ABP) sequence (see, Dennis et al (2002) J Biol. Chem. 277:35035-35043 at Tables III and IV, page 35038; US Pat. Pub. No. 2004/0001827 at [0076]; and WO 01/45746 at pages 12-13, all of which are incorporated herein by reference). Substitution, Insertion, and Deletion Variants Antibody variants having one or more amino acid substitutions are provided for use and analysis in the methods of this disclosure. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Substantial changes are provided in the following table under the heading of “exemplary substitutions,” and are further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) or CDC. OriginalPreferredResidueExemplary SubstitutionsSubstitutionsAla (A)Val; Leu; IleValArg (R)Lys; Gln; AsnLysAsn (N)Gln; His; Asp, Lys; ArgGlnAsp (D)Glu; AsnGluCys (C)Ser; AlaSerGln (Q)Asn; GluAsnGlu (E)Asp; GlnAspGly (G)AlaAlaHis (H)Asn; Gln; Lys; ArgArgIle (I)Leu; Val; Met; Ala; Phe; NorleucineLeuLeu (L)Norleucine; Ile; Val; Met; Ala; PheIleLys (K)Arg; Gln; AsnArgMet (M)Leu; Phe; IleLeuPhe (F)Trp; Leu; Val; Ile; Ala; TyrTyrPro (P)AlaAlaSer (S)ThrThrThr (T)Val; SerSerTrp (W)Tyr; PheTyrTyr (Y)Trp; Phe; Thr; SerPheVal (V)Ile; Leu; Met; Phe; Ala; NorleucineLeu Amino acids may be grouped according to common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro;(6) aromatic: Tip, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity). Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, (2008) Methods Mol. Biol. 207:179-196), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted. In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may be outside of HVR “hotspots” or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions. A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-85. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties. Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Glycosylation Variants In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region (Wright et al. (1997) TIBTECH 15:26-32). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties. In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry (see, e.g., WO 2008/077546). Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function (U.S. Pat. Pub. Nos. 2003/0157108; US 2004/0093621). Patent publications describing “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. (2004) Biotech. Bioeng. 87:614. Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (Yamane-Ohnuki, et al. (2004) Biotech. Bioeng. 87:614; Kanda, et al. (2006) Biotechnol. Bioeng., 94(4):680-688; WO2003/085107). Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Publications describing such antibody variants having bisected oligosaccharides include WO 2003/011878; U.S. Pat. No. 6,602,684; and US Pat. Pub. 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided and may have improved CDC function. Publications describing such galactose residue antibody variants include WO 1997/30087; WO 1998/58964; WO 1999/22764. Site-Specific Antibody Drug Conjugates As noted above, one of the main challenges in ADC design is the homogeneity of currently available ADCs that may have zero to eight drug molecules linked to each antibody or antibody fragment. This heterogeneity in ADC species adversely influences analytical methods of evaluating and monitoring stability, consistency, pharmacokinetics, and in vivo performance of ADC compositions. For this reason, conjugation strategies have been identified that permit chemical installation of the drug onto an antibody at pre-determined site(s), to ensure stability of the conjugate following production and, while in circulation, in vivo. These site-specific ADCs, also referred to as immunoconjugates, rely on emerging site-specific conjugation strategies that includes the use of engineered cysteines (e.g., THIOMAB™, Genetech Inc.), unnatural amino acids, selenocysteine residues, enzymatic conjugation through glucotransfersase and transglutaminasesl, and other techniques. In particular, THIOMAB™-drug conjugates (TDCs) can be controlled to produce a homogeneous DAR2. 1) Cysteine Engineered Antibody Drug Conjugates Cysteine-engineered antibodies (e.g., a THIOMAB™, Genentech, Inc.), comprise one or more residues of an antibody substituted with cysteine residue(s). The substituted residues may occur at accessible sites of the antibody, such that reactive thiol groups are positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create a site-specific ADC. Examples of such THIOMAB™ (Genentech, Inc.) antibodies include cysteine engineered antibodies in which any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region, and S121, and K149 of the light chain. Methods of making cysteine engineered antibodies include, but are not limited to, the methods described in U.S. Pat. Nos. 7,521,541; 9,000,130. Thus, the methods of this disclosure may be applied to antibody-drug conjugates comprising cysteine engineered antibodies wherein one or more amino acids of a wild-type or parent antibody are replaced with a cysteine amino acid (THIOMAB™, Genentech, Inc.) antibody. Any form of antibody may be so engineered. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab, and a parent monoclonal antibody may be engineered to form a cysteine engineered monoclonal antibody. It should be noted that a single site mutation yields a single engineered cysteine residue in a Fab antibody fragment, while a single site mutation yields two engineered cysteine residues in a full length antibody, due to the dimeric nature of the IgG antibody. Mutants with replaced (“engineered”) cysteine (Cys) residues are evaluated for the reactivity of the newly introduced, engineered cysteine thiol groups. The thiol reactivity value is a relative, numerical term in the range of 0 to 1.0 and can be measured for any cysteine engineered antibody. Thiol reactivity values of cysteine engineered antibodies of the invention are in the ranges of 0.6 to 1.0; 0.7 to 1.0; or 0.8 to 1.0. Cysteine amino acids may be engineered at reactive sites in the heavy chain (HC) or light chain (LC) of an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al. (2008) Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485; WO2009/052249, Shen et al (2012) Nature Biotech., 30(2):184-191; Junutula et al (2008) J. Immuno. Methods 332:41-52). The engineered cysteine thiols may react with linker reagents or the linker-drug intermediates of the present invention which have thiol-reactive, electrophilic pyridyl disulfide groups to form ADC with cysteine engineered antibodies and the drug moiety. The specific location (i.e., site) of the drug moiety in these engineered ADCs can thus be designed, controlled, and known. The drug loading can therefore be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or linker-drug intermediates in high yield. Engineering an antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody. A drug loading (DAR) near 2 can be achieved, with nearly complete homogeneity in these site specific conjugated ADCs. 2) Unnatural Amino Acid Engineered Antibody Drug Conjugates Similar to cysteine-engineered antibodies, the incorporation of unnatural amino acids (UAAs) into proteins provides a flexible method of site-specifically engineering a bioorthogonal functionality (see, e.g., Agarwal and Bertozzi, Bioconjugate Chem. 2015, 26:176-92; Sochaj, et al., Biotech. Advances (2015) 33:775-84). To design and specifically introduce a non-natural amino acid into a protein such as an antibody or antibody fragment, a mutant protein encoded by a gene with the amber stop codon (TAG) at the site of the desired UAA may be expressed in cells, along with a corresponding orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair capable of installing the UAA at the amber stop codon site (see, e.g., Liu and Schultz (2010) Annu. Rev. Biochem. 79:413-44). One unnatural amino acid incorporated inE. coliUAA expression systems was p-acetylphenylalanine, chosen for the bioorthogonal reactivity of its ketone (Wang, et al., (2003) Proc. Natl. Acad. Sci. U.S.A. 100:56-61.). This unnatural amino acid was conjugated to an aminooxy-auristatin F, and the resulting trastuzumab antibody displayed superior pharmacokinetic properties in mice (Tian, et al., (2014) Proc. Natl. Acad. Sci. U.S.A. 111: 1766-1771). This site-specific engineering methodology can be expanded to include more than one biorthogonal functional group into the protein. This approach, based on the incorporation of one or more unnatural amino acids into the protein, may provide antibody-drug conjugates with a specific number of known unnatural amino acid substitutions that are easily and consistently conjugated to a therapeutic moiety, such as an anti-cancer drug, producing a very homogenous ADC composition with drug conjugation(s) limited to precisely designed and identified sites in the protein. 3) Selenocysteine Engineered Antibody Drug Conjugates Selenocysteine is a natural, but rare, amino acid that exists in all kingdoms of life as a component of selenoproteins, of which only 25 are currently known in mammals. Selenocysteine contains selenium in the place of sulfur, which makes it more reactive towards electrophiles in acidic conditions than cysteine. This chemical property was used to selectively couple maleimide- and iodoacetamide-containing agents to antibodies containing genetically engineered selenocysteine residues (Hofer, et al. (2009) Biochemistry 48:12047-57; Li, et al., (2014) Methods 65:133-38). Selenocysteine was used to conjugate fluorescent probes, biotin and biotin polyethylene glycol (biotin-PEG) to antibodies, resulting in the fully functional conjugates having specifically defined sites and stoichiometries of agent attachment, demonstrating the production of homogenous ADCs based on selenocysteine residue engineering. (see, e.g., Agarwal and Bertozzi, (2015) Bioconjugate Chem. 26:176-92; Sochaj, et al. (2015) Biotech. Advances 33:775-84). 4) Glycan Modified Antibody Drug Conjugates Human IgG molecules have a conserved glycosylation site at each N297 residue in the CH2 domain, making these pendant N-glycans a convenient target for site-specific conjugation. This glycosylation site is sufficiently far from the variable region that conjugation of drug moieties to attached glycans should not impact antigen binding. One method of linking therapeutic moieties to these glycans includes oxidative cleavage of the vicinal diol moieties contained in these glycans with periodate to generate aldehydes that can be reductively aminated and conjugated to hydrazide and aminooxy compounds (O'Shannessy, et al. (1984) Immunol. Lett. 8:273-77). Another method includes increasing the fucosylation of the N-acetylglucosamine residues in these glycans. Oxidation of these fucose residues produces carboxylic acid and aldehyde moieties that can be used to link drugs and fluorophores to these specific sites on the antibody (Zuberbuhler, et al. (2012) Chem. Commun. 48:7100-02). Another method includes modifying sialic acid in these glycans (as well as increasing the sialic acid content in these glycans) followed by oxidation of the sialic acid and conjugation with aminooxy-drugs to form oxime-linked conjugates (Zhou, et al. (2014) Bioconjugate Chem. 25:510-20). Alternatively, a sialyltransferase may be used to incorporate a modified sialic acid residue containing a bioorthogonal functional group into these glycans. The bioorthogonal functional group may then be modified to attach therapeutic moieties to the site of the glycan (Li, et al. (2014) Angew. Chem. Int. 53:7179-82). Another approach to modifying these glycan sites is the use of glycosyltransferases to link galactose, or galactose analogues containing ketones or azides, to the N-acetylglucosamine in these glycans, and linking drugs or radionucleotides to the galactose molecules (Khidekel, et al., (2003) J. Am. Chem. Soc. 125:16162-63; Clark, et al., (2008) J. Am. Chem. Soc. 130:11576-77; Boeggeman, et al. (2007) Bioconjugate Chem. 18:806-14). Another approach relies on the introduction of modified sugars into these glycans at the time of expression of the antibody by metabolic oligosaccharide engineering (Campbell, et al. (2007) Mol. BioSyst. 3:187-94; Agard, et al., (2009) Acc. Chem. Res. 42:788-97). This approach has been utilized with the introduction of fucose analogues followed by drug linking/modification at the fucosylation site (Okeley, et al. (2013) Bioconjugate Chem. 24:1650-1655; Okeley, et al., (2013) Proc. Natl. Acad. Sci. U.S.A. 110:5404-09.). 5) Probody Drug Conjugates Probodies (PROBODY™, Cytomx Therapeutics LLC, South San Francisco, Calif.) are recombinant, proteolytically-activated antibody prodrugs, comprised of a monoclonal antibody in which the amino terminus of the antibody light chain is extended with a protease-cleavable linker and a masking peptide designed to block antibody binding to an antigen (U.S. Pat. No. 8,563,269; Desnoyers, et al., Sci Transl Med. 2013 16; 5(207):207ra144; Polu and Lowman, Expert Opin Biol Ther. 2014, 14(8):1049-53; Wong, et al., Biochimie. 2016 122:62-7). Cleavage of the linker by specific tumor-associated proteases leads to dissociation of the mask and release of an antibody competent to bind to antigen in the tumor. Probodies are designed to exploit the fundamental dysregulation of extracellular protease activity that exists within the tumor microenvironment, relative to healthy tissue, thereby binding only minimally to antigen in healthy tissue where there are insufficient active proteases present to remove the mask. Within a tumor, in the presence of sufficient dysregulated protease activity, the mask is removed by cleavage of the linker, and antigen binding proceeds. Probody Drug Conjugates (PDCs) have been engineered to bind a probody to the microtubule inhibitor MMAE (Weidle, et al., Can Gen & Proteom 2014, 11:67-80; Sagert, et al., Abstract 2665, AACR Annual Meeting 2014). 6) Polymer or Peptide Conjugates Antibody drug conjugates are also formed using antibodies, or antibody fragments, linked to hydrophilic polymers or peptides that are comprised of natural amino acids, which can themselves be attached to therapeutic peptides, proteins or small therapeutic molecules. Thus, the polymer or peptide essentially serves as a linker between the antibody and the therapeutic moiety (drug), but this linker provides a means to attach multiple therapeutic moieties, thereby significantly increasing DAR for each ADC molecule. Using these constructs, DAR of 14-18, or even higher, are possible while maintaining the site-specific conjugation attributes of a site-specific ADC. Exemplary ADCs that include such peptide/polymer conjugates include ADCs linked to the XTEN™ peptides conjugate (Amunix, Mountain View, Calif.) at specific, engineered amino acid residues in the light chain of the antibody, such as the cysteine-engineered antibodies described above. These peptides are substantially homogeneous polypeptides that are useful as conjugation partners to link to one or more payloads via a cross-linker reactant resulting in an XTEN-payload ADC conjugate. These peptide linkers are polypeptides composed of non-naturally occurring, substantially non-repetitive sequences having a low degree, or no secondary or tertiary structure under physiologic conditions, and typically have from about 36 to about 3000 amino acids, of which the majority or the entirety are small hydrophilic amino acids with defined numbers of orthogonal pendant reactive groups conjugated to one or more molecules of a targeting moiety that serves as a ligand to a cell-surface receptor and one or more molecules of an effector drug (U.S. Pat. Pub. 2015/0037359). 7) Fc Fusion Proteins There are a broad variety of antibody-cytokine fusion proteins that have been developed as biopharmaceutical products and approved by the FDA for use as drugs in the United States. Most of these fusion proteins target tumor antigens with a protein construct in which different cytokines have been fused to full-length antibodies or their derivatives (see, e.g., Ortiz-Sanchez et al. (2008) Expert Opin. Biol. Ther. 8(5):609-32; Sochaj, et al. (2015) Biotechnology Advances 33:775-84). Each cytokine can be fused at the amino- or carboxy-terminus of the antibody depending on the structure of the cytokine and antibody, in order to conserve the biological activity of both components. Between the growing number of antibody derivatives, and the different cytokines that can be combined with them, the quantity of different antibody-cytokine fusion proteins is very large. Additionally, Fc-fusion constructs are being developed for non-cancer clinical indications such as autoimmune conditions. These proteins may directly compete with antibodies that target self proteins. Generally, these Fc-fusion protein constructs have been categorized into four groups based on ligand specificity (binding to one or multiple epitopes on a ligand molecule) and valency (stoichiometry of binding to ligand molecules): bivalent with single-ligand specificity; monovalent with multi-ligand specificity; multivalent with single-ligand specificity; and monovalent with single-ligand specificity. Other than the Fc-fusion protein constructs, these site-specific, engineered immunoconjugates may retain the antigen binding capability of their wild type, parent antibody counterparts. Thus, the site-specific antibody conjugates are capable of binding, preferably specifically, to antigens. Such antigens include, for example, tumor-associated antigens (TAA), cell surface receptor proteins, and other cell surface molecules, transmembrane proteins, signaling proteins, cell survival regulatory factors, cell proliferation regulatory factors, molecules known or suspected to contribute functionally to tissue development or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in vasculogenesis, and molecules known or suspected to contribute functionally to angiogenesis. The tumor-associated antigen may be a cluster differentiation factor (i.e., a CD protein). An antigen to which a cysteine engineered antibody is capable of binding may be a member of a subset of one of the above-mentioned categories, wherein the other subset(s) of said category comprise other molecules/antigens that have a distinct characteristic (with respect to the antigen of interest). The site specific antibody conjugates used in the methods of this disclosure include immunoconjugates useful in the treatment of cancer including, but not limited to, antibodies against cell surface receptors and tumor-associated antigens (TAA). Tumor-associated antigens are known in the art, and can be prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal, non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. Examples of tumor-associated antigens TAA include, but are not limited to, TAA (1)-(53) listed below. Information relating to these antigens, which are known in the art, is listed below and includes names, alternative names, Genbank accession numbers, and primary reference(s), following nucleic acid and protein sequence identification conventions of the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to TAA (1)-(53) are available in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references. For example, a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed. The sequences and disclosure in the reference specifically recited herein are expressly incorporated by reference. Tumor-Associated Antigens (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203) ten Dijke, P., et al Science 264 (5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1 (Page 38-39); WO2002102235 (claim 13; Page 296); WO2003055443 (Page 91-92); WO200299122 (Example 2; Page 528-530); WO2003029421 (claim 6); WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; Page 183); WO200254940 (Page 100-101); WO200259377 (Page 349-350); WO200230268 (claim 27; Page 376); WO200148204 (Example; FIG. 4) NP_001194 bone morphogenetic protein receptor, type D3/pid=NP_001194.1—Cross-references: MIM:603248; NP_001194.1; AY065994; (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486) Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998), Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV); WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (Example 2); WO200299074 (claim 19; Page 127-129); WO200286443 (claim 27; Pages 222, 393); WO2003003906 (claim 10; Page 293); WO200264798 (claim 33; Page 93-95); WO200014228 (claim 5; Page 133-136); US2003224454 (FIG. 3); WO2003025138 (claim 12; Page 150); NP_003477 solute carrier family 7 (cationic amino acid transporter, y+system), member 5/pid=NP_003477.3—Homo sapiensCross-references: MIM:600182; NP_003477.3; NM_015923; NM_003486_1; (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449) Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004065577 (claim 6); WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (Example 5); US2003185830 (Example 5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page 618-619); WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173, Example 2; FIG. 2A); NP_036581 six transmembrane epithelial antigen of the prostate. Cross-references: MIM:604415; NP_036581.1; NM_012449_1; (4) 0772P (CA125, MUC16, Genbank accession no. AF361486) J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004045553 (claim 14); WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; Page 116-121); US2003124140 (Example 16); U.S. Pat. No. 798,959. Cross-references: GI:34501467; AAK74120.3; AF361486_1; (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823) Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; Page 287-288); WO2002101075 (claim 4; Page 308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57); Cross-references: MIM:601051; NP_005814.2; NM_005823_1; (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284 (1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (claim 2); EP1394274 (Example 11); WO2002102235 (claim 13; Page 326); EP875569 (claim 1; Page 17-19); WO200157188 (claim 20; Page 329); WO2004032842 (Example IV); WO200175177 (claim 24; Page 139-140); Cross-references: MIM:604217; NP_006415.1; NM_006424_1; (7) Sema 5b (F1110372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; Page 50); WO200188133 (claim 1; Page 41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737; (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al (2002) Cancer Res. 62:2546-2553; US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (Example 5); WO2003105758 (claim 12); US2003206918 (Example 5); EP1347046 (claim 1); WO2003025148 (claim 20); Cross-references: GI:37182378; AAQ88991.1; AY358628_1; (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463); Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991; Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991; Arai H., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J. Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J. Cardiovasc. Pharmacol. 20, sl-S4, 1992; Tsutsumi M., et al Gene 228, 43-49, 1999; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J. B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996; Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998; Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et al (2002) Hum. Genet. 111, 198-206; WO2004045516 (claim 1); WO2004048938 (Example 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (claim 12; Page 144); WO200198351 (claim 1; Page 124-125); EP522868 (claim 8; FIG. 2); WO200177172 (claim 1; Page 297-299); US2003109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34); WO2004001004; (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accession no. NM_017763); WO2003104275 (claim 1); WO2004046342 (Example 2); WO2003042661 (claim 12); WO2003083074 (claim 14; Page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; FIG. 93); WO200166689 (Example 6); Cross-references: LocusID:54894; NP_060233.2; NM_017763_1; (11) STEAP2 (HGNC 8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138) Lab. Invest. 82 (11):1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; FIG. 1); WO200272596 (claim 13; Page 54-55); WO200172962 (claim 1; FIG. 4B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG. 10); WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; FIG. 10); Cross-references: GI:22655488; AAN04080.1; AF455138_1; (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636). Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820 (2003)); US2003143557 (claim 4); WO200040614 (claim 14; Page 100-103); WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); WO200230268 (claim 27; Page 391); US2003219806 (claim 4); WO200162794 (claim 14; FIG. 1A-D); Cross-references: MIM:606936; NP_060106.2; NM_017636_1; (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP_003203 or NM_003212). Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003224411 (claim 1); WO2003083041 (Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; Page 52-53); WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; Page 94-95, 105); WO200222808 (claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18); U.S. Pat. No. 5,792,616 (FIG. 2); Cross-references: MIM:187395; NP_003203.1; NM_003212_1; (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004). Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol. 35, 1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320; WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (claim 9); WO2004045520 (Example 4); WO9102536 (FIGS. 9.1-9.9); WO2004020595 (claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1; (15) CD79b (CD79B, CD7913, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626 or 11038674). Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (claim 2, FIG. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401 (claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15); U.S. Pat. No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO 99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B); WO200055351 (claim 11, pages 1145-1146); Cross-references: MIM:147245; NP_000617.1; NM_000626_1; (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764, AY358130). Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; FIG. 18D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25); Cross-references: MIM:606509; NP_110391.2; NM_030764_1; (17) HER2 (ErbB2, Genbank accession no. M11730) Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T., et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol. 165, 869-880, 2004; Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15, 426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIG. 1A-B); WO2003025228 (claim 37; FIG. 5C); WO200222636 (Example 13; Page 95-107); WO200212341 (claim 68; FIG. 7); WO200213847 (Page 71-74); WO200214503 (Page 114-117); WO200153463 (claim 2; Page 41-46); WO200141787 (Page 15); WO200044899 (claim 52; FIG. 7); WO200020579 (claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (claim 3; Col 31-38); WO9630514 (claim 2; Page 56-61); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1; (18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (Example 2); WO200286443 (claim 27; Page 427); WO200260317 (claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728; (19) MDP (DPEP1, Genbank accession no. BC017023) Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9); Cross-references: MIM:179780; AAH17023.1; BC017023_1; (20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971); Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19, 2001; Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al (2003) Biochemistry 42:12617-12624; Sheikh F., et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11); US2004005320 (Example 5); WO2003029262 (Page 74-75); WO2003002717 (claim 2; Page 63); WO200222153 (Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59); WO200146232 (Page 63-65); WO9837193 (claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1; (21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053). Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG. 52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1); (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (claim 12); WO200053216 (claim 1; Page 41); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (Page 128-132); WO200053216 (claim 1; Page 42); Cross-references: MIM:600997; NP_004433.2; NM_004442_1; (23) ASLG659 (B7h, Genbank accession no. AX092328). US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3); US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235 (claim 13; Page 299); US2003091580 (Example 2); WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7b); WO200202624 (claim 13; FIG. 1A-1B); US2002034749 (claim 54; Page 45-46); WO200206317 (Example 2; Page 320-321, claim 34; Page 321-322); WO200271928 (Page 468-469); WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318; (24) PSCA (Prostate stem cell antigen precursor, Genbank accession no. AJ297436) Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274 (Example 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; Page 164); WO2003003906 (claim 10; Page 288); WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b); WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; Page 97); WO9851824 (claim 10; Page 94); WO9840403 (claim 2; FIG. 1B); Accession: 043653; EMBL; AF043498; AAC39607.1; (25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1—Homo sapien Species:Homo sapiens(human) WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example 3, claim 20); US2003194704 (claim 45); Cross-references: GI:30102449; AAP14954.1; AY260763_1; (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1—Homo sapiens. Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33); WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; Page 615-616); WO200294852 (Col 136-137); WO200238766 (claim 3; Page 133); WO200224909 (Example 3; FIG. 3); Cross-references: MIM:606269; NP_443177.1; NM_052945_1; AF132600; (27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al (1991) J. Exp. Med. 173:137-146; WO2003072036 (claim 1; FIG. 1); Cross-references: MIM:107266; NP_001762.1; NM_001771_1; (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession No. NP_001774.10); WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No. 5,644,033; Ha et al (1992) J. Immunol. 148(5):1526-1531; Mueller et al (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud'homme et al (1992) Clin. Exp. Immunol. 90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637; Sakaguchi et al (1988) EMBO J. 7(11):3457-3464; (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession No. NP_001707.1); WO2004040000; WO2004015426; US2003105292 (Example 2); U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188 (claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1, pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58); WO9217497 (claim 7, FIG. 5); Dobner et al (1992) Eur. J. Immunol. 22:2795-2799; Barella et al (1995) Biochem. J. 309:773-779; (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) that binds peptides and presents them to CD4+T lymphocytes); 273 aa, pI: 6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_002111.1) Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989) Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol. 228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766; Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse et al (2002) Tissue Antigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No. 6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat. No. 6,011,146 (col 145-146); Kasahara et al (1989) Immunogenetics 30(1):66-68; Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119; (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No. NP_002552.2); Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al (2000) Genome Res. 10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (page 82); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2), pI: 8.66, MW: 40225 TM: 1 [P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1); WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655 (pages 105-106); Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903; (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pI: 6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No. NP_005573.1); US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al (1996) Genomics 38(3):299-304; Miura et al (1998) Blood 92:2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages 24-26); (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains, may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No. NP_443170.1); WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7); (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pI: 6.88 MW: 106468 TM: 1 [P] Gene Chromosome: 1q21, Genbank accession No. Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse:AK089756, AY158090, AY506558; NP_112571; WO2003024392 (claim 2, FIG. 97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG. 18B-1-18B-2); (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/heregulin family of growth factors and follistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5; Genbank accession No. AF179274; AY358907, CAF85723, CQ782436; WO2004074320; JP2004113151; WO2003042661; WO2003009814; EP1295944 (pages 69-70); WO200230268 (page 329); WO200190304; US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579; Horie et al (2000) Genomics 67:146-152; Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int J Cancer. October 15; 94(2):178-84; (37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20; gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R. P. et al (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736; Kummer, M. P. et al (2009) J. Biol. Chem. 284 (4), 2296-2306; (38) TMEFF1 (transmembrane protein with EGF-like and two follistatin-like domains 1; Tomoregulin-1); H7365; C9orf2; C9ORF2; U19878; X83961; NM_080655; NM_003692; Harms, P. W. (2003) Genes Dev. 17 (21), 2624-2629; Gery, S. et al (2003) Oncogene 22 (18):2723-2727; (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847; BC014962; NM_145793 NM_005264; Kim, M. H. et al (2009) Mol. Cell. Biol. 29 (8), 2264-2277; Treanor, J. J. et al (1996) Nature 382 (6586):80-83; (40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E,SCA-2,TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A. G. et al (2003) Int. J. Cancer 103 (6), 768-774; Zammit, D. J. et al (2002) Mol. Cell. Biol. 22 (3):946-952; (41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); NP_001007539.1; NM_001007538.1; Furushima, K. et al (2007) Dev. Biol. 306 (2), 480-492; Clark, H. F. et al (2003) Genome Res. 13 (10):2265-2270; (42) Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); NP_067079.2; NM_021246.2; Mallya, M. et al (2002) Genomics 80 (1):113-123; Ribas, G. et al (1999) J. Immunol. 163 (1):278-287; (43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5; GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al (2009) Am. J. Epidemiol. 170 (5):537-545; Yamamoto, Y. et al (2003) Hepatology 37 (3):528-533; (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12; Hs.168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4; Tsukamoto, H. et al (2009) Cancer Sci. 100 (10):1895-1901; Narita, N. et al (2009) Oncogene 28 (34):3058-3068; (45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al (2007) Cancer Res. 67 (24):11601-11611; de Nooij-van Dalen, A. G. et al (2003) Int. J. Cancer 103 (6):768-774; (46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1; NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105 (1-2):162-164; O'Dowd, B. F. et al (1996) FEBS Lett. 394 (3):325-329; (47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); NP_115940.2; NM_032551.4; Navenot, J. M. et al (2009) Mol. Pharmacol. 75 (6):1300-1306; Hata, K. et al (2009) Anticancer Res. 29 (2):617-623; (48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982); NP_859069.2; NM_181718.3; Gerhard, D. S. et al (2004) Genome Res. 14 (10B):2121-2127; (49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP_000363.1; NM_000372.4; Bishop, D. T. et al (2009) Nat. Genet. 41 (8):920-925; Nan, H. et al (2009) Int. J. Cancer 125 (4):909-917; (50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627); NP_001103373.1; NM_001109903.1; Clark, H. F. et al (2003) Genome Res. 13 (10):2265-2270; Scherer, S. E. et al (2006) Nature 440 (7082):346-351; (51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T. A. et al (2003) Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764; Takeda, S. et al (2002) FEBS Lett. 520 (1-3):97-101; (52) CD33, a member of the sialic acid binding, immunoglobulin-like lectin family, is a 67-kDa glycosylated transmembrane protein. CD33 is expressed on most myeloid and monocytic leukemia cells in addition to committed myelomonocytic and erythroid progenitor cells. It is not seen on the earliest pluripotent stem cells, mature granulocytes, lymphoid cells, or nonhematopoietic cells (Sabbath et al., (1985)J. Clin. Invest.75:756-56; Andrews et al., (1986)Blood68:1030-5). CD33 contains two tyrosine residues on its cytoplasmic tail, each of which is followed by hydrophobic residues similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors; (53) CLL-1 (CLEC12A, MICL, and DCAL2), encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined. This gene is closely linked to other CTL/CTLD superfamily members in the natural killer gene complex region on chromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9 (5):585-90; van Rhenen A, et al., (2007) Blood 110 (7):2659-66; Chen C H, et al. (2006) Blood 107 (4):1459-67; Marshall A S, et al. (2006) Eur. J. Immunol. 36 (8):2159-69; Bakker A B, et al (2005) Cancer Res. 64 (22):8443-50; Marshall A S, et al (2004) J. Biol. Chem. 279 (15):14792-802). CLL-1 has been shown to be a type II transmembrane receptor comprising a single C-type lectin-like domain (which is not predicted to bind either calcium or sugar), a stalk region, and a transmembrane domain and a short cytoplasmic tail containing an ITIM motif. Antibody Derivatives An antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc. Conjugates of an antibody and a non-proteinaceous moiety may be formed by selectively heating by exposure to radiation. The non-proteinaceous moiety of such conjugate may be a carbon nanotube (Kam et al. (2005) Proc. Natl. Acad. Sci. USA 102:11600-605). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non-proteinaceous moiety to a temperature at which cells proximal to the antibody-non-proteinaceous moiety are killed. The term “hypervariable region” or “HVR,” as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops (“hypervariable loops”). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the “complementarity determining regions” (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, (1987) J. Mol. Biol. 196:901-917). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat numbering). With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise “specificity determining residues,” or “SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. An “isolated” antibody is one which has been separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For review of methods for assessment of antibody purity, see, e.g., Flatman et al. (2007) J. Chromatogr. B 848:79-87. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody within a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. A “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel or fluorophore. The naked antibody may be present in a pharmaceutical formulation. “Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Multispecific Antibodies An antibody provided herein may be a multispecific antibody, e.g. a bispecific antibody. The term “multispecific antibody” as used herein refers to an antibody comprising an antigen-binding domain that has polyepitopic specificity (i.e., is capable of binding to two, or more, different epitopes on one molecule or is capable of binding to epitopes on two, or more, different molecules). In example embodiments, multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigen binding sites (such as a bispecific antibody). The first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind the two epitopes within one and the same molecule (intramolecular binding). For example, the first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind to two different epitopes on the same molecule. In certain embodiments, the two different epitopes that a multispecific antibody binds are epitopes that are not normally bound at the same time by one monospecific antibody, such as e.g. a conventional antibody or one immunoglobulin single variable domain. The first antigen-binding domain and the second antigen-binding domain of the multispecific antibody may bind epitopes located within two distinct molecules (intermolecular binding). For example, the first antigen-binding domain of the multispecific antibody may bind to one epitope on one molecule, whereas the second antigen-binding domain of the multispecific antibody may bind to another epitope on a different molecule, thereby cross-linking the two molecules. The antigen-binding domain of a multispecific antibody (such as a bispecific antibody) may comprise two VH/VL units, wherein a first VH/VL unit binds to a first epitope and a second VH/VL unit binds to a second epitope, wherein each VH/VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full length antibodies, antibodies having two or more VL and VH domains, and antibody fragments (such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently). A VH/VL unit that further comprises at least a portion of a heavy chain variable region and/or at least a portion of a light chain variable region may also be referred to as an “arm” or “hemimer” or “half antibody.” A hemimer may comprise a sufficient portion of a heavy chain variable region to allow intramolecular disulfide bonds to be formed with a second hemimer. In some embodiments, a hemimer comprises a knob mutation or a hole mutation, for example, to allow heterodimerization with a second hemimer or half antibody that comprises a complementary hole mutation or knob mutation. Knob mutations and hole mutations are discussed below. A multispecific antibody provided herein may be a bispecific antibody. The term “bispecific antibody” as used herein refers to a multispecific antibody comprising an antigen-binding domain that is capable of binding to two different epitopes on one molecule or is capable of binding to epitopes on two different molecules. A bispecific antibody may also be referred to herein as having “dual specificity” or as being “dual specific.” Exemplary bispecific antibodies may bind both and any other antigen. One of the binding specificities may be for HER2 and the other is for CD3. See, e.g., U.S. Pat. No. 5,821,337. Bispecific antibodies may bind to two different epitopes of the same molecule. Bispecific antibodies may bind to two different epitopes on two different molecules. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express cancer-associated antigens. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello,Nature305: 537 (1983), WO 93/08829, and Traunecker et al.,EMBO J.10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168, WO2009/089004, US2009/0182127, US2011/0287009, Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, and Kontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term “knob-into-hole” or “KnH” technology as used herein refers to the technology directing the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact. For example, KnHs have been introduced in the Fc:Fc binding interfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, Zhu et al., 1997, Protein Science6:781-788, and WO2012/106587). In some embodiments, KnHs drive the pairing of two different heavy chains together during the manufacture of multispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprises different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions). The term “knob mutation” as used herein refers to a mutation that introduces a protuberance (knob) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation. The term “hole mutation” as used herein refers to a mutation that introduces a cavity (hole) into a polypeptide at an interface in which the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation. A “protuberance” refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of a second polypeptide) so as to stabilize the heteromultimer, and thereby favor heteromultimer formation over homomultimer formation, for example. The protuberance may exist in the original interface or may be introduced synthetically (e.g., by altering nucleic acid encoding the interface). In some embodiments, a nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one “import” amino acid residue which has a larger side chain volume than the original amino acid residue. There can be more than one original and corresponding import residue. The side chain volumes of the various amino residues are shown, for example, in Table 1 of US2011/0287009. A mutation to introduce a “protuberance” may be referred to as a “knob mutation.” Import residues for the formation of a protuberance may be naturally occurring amino acid residues selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Exemplary import residues are tryptophan or tyrosine. The original residue for the formation of the protuberance may have a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. A “cavity” refers to at least one amino acid side chain which is recessed from the interface of a second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). In some embodiments, nucleic acid encoding the interface of the second polypeptide is altered to encode the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. Import residues for the formation of a cavity may be naturally occurring amino acid residues selected from alanine (A), serine (S), threonine (T) and valine (V). An import residue may be serine, alanine or threonine. The original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. A mutation to introduce a “cavity” may be referred to as a “hole mutation.” The protuberance is “positionable” in the cavity which means that the spatial location of the protuberance and cavity on the interface of a first polypeptide and second polypeptide respectively and the sizes of the protuberance and cavity are such that the protuberance can be located in the cavity without significantly perturbing the normal association of the first and second polypeptides at the interface. Since protuberances such as Tyr, Phe and Trp do not typically extend perpendicularly from the axis of the interface and have preferred conformations, the alignment of a protuberance with a corresponding cavity may, in some instances, rely on modeling the protuberance/cavity pair based upon a three-dimensional structure such as that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using widely accepted techniques in the art. An exemplary knob mutation in an IgG1 constant region is T366W (EU numbering). Exemplary hole mutations in an IgG1 constant region may comprise one or more mutations selected from T366S, L368A and Y407V (EU numbering). An exemplary hole mutation in an IgG1 constant region may comprise T366S, L368A and Y407V (EU numbering). An exemplary knob mutation in an IgG4 constant region is T366W (EU numbering). An exemplary hole mutation in an IgG4 constant region may comprise one or more mutations selected from T366S, L368A, and Y407V (EU numbering). An exemplary hole mutation in an IgG4 constant region comprises T366S, L368A, and Y407V (EU numbering). “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes this disclosure, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (Portolano et al. (1993) J. Immunol. 150:880-887; Clarkson et al. (1991) Nature 352:624-628). “Tumor-associated antigens” (TAA) are known in the art as provided in the list of exemplary TAAs provided above, and can prepared for use in generating human or humanized antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies. Examples of TAA include, but are not limited to, those described in U.S. Pat. Nos. 8,679,767 and 8,541,178, which are expressly incorporated herein. The antibody components of the ADCs useful in the methods of this disclosure may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. Isolated nucleic acids encoding such antibodies described herein are provided. Such nucleic acids may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). One or more vectors (e.g., expression vectors) comprising such nucleic acid are also provided. A host cell comprising such nucleic acid is also provided. A host cell may comprise (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. The host cell may be eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). Thus, methods of making an antibody are provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of an antibody, nucleic acid encoding an antibody, may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; 5,840,523; Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., (2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern (Gerngross, (2004) Nat. Biotech. 22:1409-1414; Li et al. (2006) Nat. Biotech. 24:210-215). Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperdacells. Plant cell cultures can also be utilized as hosts (U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; 6,417,429, describing PLANTIBODIES™ technology for producing antibodies in transgenic plants). Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al. (1977, J. Gen Virol. 36:59); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, (1980) Biol. Reprod. 23:243-251); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells, as described, e.g., in Mather et al. (1982) Annals N.Y. Acad. Sci. 383:44-68; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al. (1980) Proc. Natl. Acad. Sci. USA 77:4216); and myeloma cell lines such as Y0, NS0 and Sp2/0. A review of certain mammalian host cell lines suitable for antibody production is provided in, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). The antibody components of an ADC may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art. An antibody may be tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc. Competition assays may also be used to identify an antibody that competes with another known antibody for binding to antigen. A competing antibody may bind to the same epitope (e.g., a linear or a conformational epitope) that is bound by the known antibody. Detailed exemplary methods for mapping an epitope to which an antibody binds are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, N.J.). Exemplary antibodies forming the site-specific ADC may include, but are not limited to, trastuzumab, ocrelizumab, pertuzumab, anti-PD1, anti-PD-L1, anti-neuropilin-1, anti-MUC16, rituximab, anti-mesothelin, anti-LY6E, anti-STEAP1, anti-FcRH5, anti-CD22, anti-B7H4, anti-LGR5, anti-CD79b, and anti-Napi2b. Drug moieties which form the drug component of the ADC may be covalently attached to antibodies through a linker unit to form antibody-drug conjugates for targeted therapeutic effects. An exemplary embodiment of an ADC compound comprises an antibody (Ab) which targets, e.g., a tumor cell, cytotoxic or cytostatic drug moiety (D), and a linker moiety (L) that attaches Ab to D. The antibody is attached through the one or more amino acid residues, such as lysine and cysteine, by the linker moiety (L) to D; the composition having the Formula: Ab-(L-D)p, where p is 1 to about 20, or from about 2 to about 5. The number of drug moieties which may be conjugated via a reactive linker moiety to an antibody molecule may be limited by the number of cysteine residues, including free cysteine residues present in the antibody or which may be introduced by methods described herein, or native cysteines that form the interchain disulfide bonds of the antibody. The drug moiety (D) of an ADC may include any therapeutic compound, moiety or group, especially a group that has a cytotoxic or cytostatic effect. Exemplary drug moieties may impart such cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and topoisomerase. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. Exemplary drug moieties include, but are not limited to, a peptide (including therapeutic peptides comprising one or more non-natural amino acids, such as cyclic peptides, beta peptides, stables peptides, and cysteine knot peptides), a polyamide, a maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine (PBD), PNU-159682, anthracycline, duocarmycin, vinca alkaloid, taxane, trichothecene, CC1065, duocarmycin, camptothecin, elinafide, an antibiotic including a rifamycin or rifamycin-analog, a fluorophore, a radioisotope, and stereoisomers, isosteres, analogs or derivatives thereof, including derivatives of these drugs that have cytotoxic activity. Fc Region Variants One or more amino acid modifications may be introduced into the Fc region of an antibody forming a site specific ADC provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions. The invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII and Fc(RIII FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet,Annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al.Proc. Nat? Acad. Sci. USA83:7059-7063 (1986)) and Hellstrom, I et al.,Proc. Nat'l Acad. Sci. USA82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al.,J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al.,J. Immunol. Methods202:163 (1996); Cragg, M. S. et al.,Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al.,Int'l. Immunol.18(12):1759-1769 (2006)). One or more amino acid modifications may be introduced into the Fc portion of the antibody in order to increase IgG binding to the neonatal Fc receptor. The antibody may comprise the following three mutations according to EU numbering: M252Y, S254T, and T256E (the “YTE mutation”) (U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006). The YTE mutation does not affect the ability of the antibody to bind to its cognate antigen. The YTE mutation may increase the antibody's serum half-life compared to the native (i.e., non-YTE mutant) antibody. The YTE mutation may increase the serum half-life of the antibody by 2- to 10-fold compared to the native (i.e., non-YTE mutant) antibody. See, e.g., U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006). The YTE mutant may provide a means to modulate ADCC activity of the antibody. The YTEO mutant may provide a means to modulate ADCC activity of a humanized IgG antibody directed against a human antigen. See, e.g., U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry 281(33):23514-23524 (2006). The YTE mutant may allow the simultaneous modulation of serum half-life, tissue distribution, and antibody activity (e.g., ADCC of an IgG antibody). See, e.g., U.S. Pat. No. 8,697,650; see also Dall'Acqua et al., Journal of Biological Chemistry (2006) 281(33):23514-24. Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 according to EU numbering (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327 according to EU numbering, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine according to EU numbering (i.e., D265A and N297A according to EU numbering) (U.S. Pat. No. 7,332,581). In certain embodiments the Fc mutant comprises the following two amino acid substitutions: D265A and N297A. In certain embodiments the Fc mutant consists of the following two amino acid substitutions: D265A and N297A. The proline at position329 (EU numbering) (P329) of a wild-type human Fc region may be substituted with glycine or arginine or an amino acid residue large enough to destroy the proline sandwich within the Fc/Fcγ receptor interface, that is formed between the P329 of the Fc and tryptophane residues W87 and W110 of FcgRIII (Sondermann et al.: Nature 406, 267-273 (20 Jul. 2000)). In a further embodiment, at least one further amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S and still in another embodiment said at least one further amino acid substitution is L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, all according to EU numbering (U.S. Pat. No. 8,969,526 which is incorporated by reference in its entirety). A polypeptide may include the Fc variant of a wild-type human IgG Fc region wherein the polypeptide has P329 of the human IgG Fc region substituted with glycine and wherein the Fc variant comprises at least two further amino acid substitutions at L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, and wherein the residues are numbered according to the EU numbering (U.S. Pat. No. 8,969,526 which is incorporated by reference). The polypeptide comprising the P329G, L234A and L235A (EU numbering) substitutions may exhibit a reduced affinity to the human FcγRIIIA and FcγRIIA, for down-modulation of ADCC to at least 20% of the ADCC induced by the polypeptide comprising the wildtype human IgG Fc region, and/or for down-modulation of ADCP (U.S. Pat. No. 8,969,526 which is incorporated by reference). The polypeptide comprising an Fc variant of a wildtype human Fc polypeptide may include a triple mutation: an amino acid substitution at position Pro329, a L234A and a L235A mutation according to EU numbering (P329/LALA) (U.S. Pat. No. 8,969,526 which is incorporated by reference). In example embodiments, the polypeptide comprises the following amino acid substitutions: P329G, L234A, and L235A according to EU numbering. Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al.,J. Biol. Chem.9(2): 6591-6604 (2001).) An antibody variant may include an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering). Alterations may be made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. (2000)J. Immunol.164: 4178-4184. Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al.,J. Immunol.117:587 (1976) and Kim et al.,J. Immunol.24:249 (1994)), are described in US2005/0014934. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826) according to EU numbering. See also Duncan & Winter,Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fc region variants. Cysteine Engineered Antibody Variants It may be desirable to create cysteine engineered antibodies, e.g., “THIOMAB™ (Genentech, Inc.) antibody,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug intermediates, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A140 (EU numbering) of the heavy chain; L174 (EU numbering) of the heavy chain; Y373 (EU numbering) of the heavy chain; K149 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. In specific embodiments, the antibodies described herein comprise the HC-A140C (EU numbering) cysteine substitution. In specific embodiments, the antibodies described herein comprise the LC-K149C (Kabat numbering) cysteine substitution. In specific embodiments, the antibodies described herein comprise the HC-A118C (EU numbering) cysteine substitution. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. Nos. 7,521,541; 9,000,130. The antibody may comprise one of the following heavy chain cysteine substitutions: ChainEU MutationKabat Mutation(HC/LC)ResidueSite #Site #HCA118114HCT114110HCA140136HCL174170HCL179175HCT187183HCT209205HCV262258HCG371367HCY373369HCE382378HCS424420HCN434430HCQ438434 The antibody may comprise one of the following light chain cysteine substitutions: ChainEU MutationKabat Mutation(HC/LC)ResidueSite #Site #LCI106106LCR108108LCR142142LCK149149LCC205205 Linkers A “Linker” (L) is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties (D) to an antibody (Ab) to form an ADC of Formula I. In some embodiments, ADC can be prepared using a Linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, in some embodiments, the cysteine thiol of a cysteine-engineered antibody (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC. A linker may have functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent disulfide bond (See, e.g., the conjugation method at page 766 of Klussman, et al (2004),Bioconjugate Chemistry15(4):765-773, and the Examples herein). Exemplary spacer components include valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), and p-aminobenzyloxycarbonyl (a “PAB”). Various linker components are known in the art. A linker may have a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method at page 766 of Klussman, et al (2004),Bioconjugate Chemistry15(4):765-773, and the Examples herein. A linker may have a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. A heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting examples of such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. A linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“MCC”). Various linker components are known in the art, some of which are described below. A linker may be a “cleavable linker,” facilitating release of a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020). A linker may comprise one or more spacer units between the disulfide group and the drug moiety. An example includes a linker having the following formula -Aa-Ww—Yy— wherein A is a “stretcher unit”, and a is an integer from 0 to 1; W is an “amino acid unit”, and w is an integer from 0 to 12; Y is a “spacer unit”, and y is 0, 1, or 2; and Ab, D, and p are defined as above for Formula I. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298, which is expressly incorporated herein by reference. A linker component may include a “stretcher unit” that links an antibody to another linker component or to a drug moiety. Exemplary stretcher units are shown below (wherein the wavy line indicates sites of covalent attachment to an antibody, drug, or additional linker components): The linker may be a peptidomimetic linker such as those described in WO2015/095227, WO2015/095124 or WO2015/095223, which documents are hereby incorporated by reference. Drug Moieties The site specific ADC compounds of the invention comprise an antibody conjugated to one or more drug moieties, including the following: Maytansine and Maytansinoids In some embodiments, an ADC comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, and are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrubMaytenus serrata(U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. Maytansinoid drug moieties are attractive drug moieties in antibody-drug conjugates because they are: (i) relatively accessible to prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation through non-disulfide linkers to antibodies, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines. Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or produced using genetic engineering techniques (see, e.g., Yu et al (2002) Proc. Natl. Acad. Sci. U.S.A. 99:7968-7973). Maytansinoids may also be prepared synthetically according to known methods. Exemplary maytansinoid drug moieties include, but are not limited to, those having a modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746) (prepared, for example, by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, for example, by demethylation usingStreptomycesorActinomycesor dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared, for example, by acylation using acyl chlorides), and those having modifications at other positions of the aromatic ring. Exemplary maytansinoid drug moieties also include those having modifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, for example, by the reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl(demethoxy/CH2OR)(U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No. 4,450,254) (prepared, for example, fromNocardia); C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866) (prepared, for example, by the conversion of maytansinol byStreptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (for example, isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, for example, by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by the titanium trichloride/LAH reduction of maytansinol). Many positions on maytansinoid compounds are useful as the linkage position. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group. The linkage is formed at the C-3 position of maytansinol or a maytansinol analogue. Maytansinoid drug moieties include those having the structure: where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to a linker of an ADC. Each R may independently be H or a C1-C6alkyl. The alkylene chain attaching the amide group to the sulfur atom may be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020; Chari et al (1992)Cancer Res.52:127-131; Liu et al (1996)Proc. Natl. Acad. Sci USA93:8618-8623). All stereoisomers of the maytansinoid drug moiety are contemplated for the ADC of the invention, i.e. any combination of R and S configurations at the chiral carbons (U.S. Pat. Nos. 7,276,497; 6,913,748; 6,441,163; 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al (2006) J. Med. Chem. 49:4392-4408, which are incorporated by reference in their entirety). In some embodiments, the maytansinoid drug moiety has the following stereochemistry: Exemplary embodiments of maytansinoid drug moieties include, but are not limited to, DM1; DM3; and DM4, having the structures: wherein the wavy line indicates the covalent attachment of the sulfur atom of the drug to a linker (L) of an antibody-drug conjugate. Immunoconjugates containing maytansinoids, methods of making the same, and their therapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020 and 5,416,064; US 2005/0276812 A1; and European Patent EP 0 425 235 B1, the disclosures of which are hereby expressly incorporated by reference. See also Liu et al.Proc. Natl. Acad. Sci. USA93:8618-8623 (1996); and Chari et al.Cancer Research52:127-131 (1992). Antibody-maytansinoid conjugates may be prepared by chemically linking an antibody to a maytansinoid molecule without significantly diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which is hereby expressly incorporated by reference). ADCs with an average of 3-4 maytansinoid molecules conjugated per antibody molecule have shown efficacy in enhancing cytotoxicity of target cells without negatively affecting the function or solubility of the antibody. In some instances, even one molecule of toxin/antibody is expected to enhance cytotoxicity over the use of naked antibody. Exemplary linking groups for making antibody-maytansinoid conjugates include, for example, those described herein and those disclosed in U.S. Pat. No. 5,208,020; EP Patent 0425235; Chari et al.Cancer Research52:127-131 (1992); US 2005/0276812; and US 2005/016993, the disclosures of which are hereby expressly incorporated by reference. Auristatins and Dolastatins Drug moieties may include dolastatins, auristatins, and analogs and derivatives thereof (U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin-10. While not intending to be bound by theory, dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother.45(12):3580-3584) and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother.42:2961-2965). The dolastatin/auristatin drug moiety may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172; Doronina et al (2003)Nature Biotechnology21(7):778-784; Francisco et al (2003)Blood102(4):1458-1465). Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties disclosed in U.S. Pat. Nos. 7,498,298 and 7,659,241, the disclosures of which are expressly incorporated by reference in their entirety: An exemplary auristatin embodiment of formula DEis MMAE, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate: An exemplary auristatin embodiment of formula DFis MMAF, wherein the wavy line indicates the covalent attachment to a linker (L) of an antibody-drug conjugate: Other exemplary embodiments include monomethylvaline compounds having phenylalanine carboxy modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008848) and monomethylvaline compounds having phenylalanine sidechain modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007/008603). Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (see, e.g., E. Schröder and K. Lübke, “The Peptides”, volume 1, pp 76-136, 1965, Academic Press). Auristatin/dolastatin drug moieties may be prepared according to the methods of: U.S. Pat. Nos. 7,498,298; 5,635,483; 5,780,588; Pettit et al (1989)J. Am. Chem. Soc.111:5463-5465; Pettit et al (1998)Anti-Cancer Drug Design13:243-277; Pettit, G. R., et al.Synthesis,1996, 719-725; Pettit et al (1996)J. Chem. Soc. Perkin Trans.1 5:859-863; and Doronina (2003)Nat. Biotechnol.21(7):778-784. Auristatin/dolastatin drug moieties of formulas DEsuch as MMAE, and DF, such as MMAF, and drug-linker intermediates and derivatives thereof, such as MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, may be prepared using methods described in U.S. Pat. No. 7,498,298; Doronina et al. (2006)Bioconjugate Chem.17:114-124; and Doronina et al. (2003)Nat. Biotech.21:778-784 and then conjugated to an antibody of interest. Calicheamicin The calicheamicin family of antibiotics, and analogues thereof, are capable of producing double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., (1993)Cancer Research53:3336-3342; Lode et al., (1998)Cancer Research58:2925-2928). Calicheamicin has intracellular sites of action but, in certain instances, does not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody-mediated internalization may, in some embodiments, greatly enhances their cytotoxic effects. Nonlimiting exemplary methods of preparing antibody-drug conjugates with a calicheamicin drug moiety are described, for example, in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; and WO 2017/068511. The drug moiety conjugated to the antibody is a calicheamicin compound having the formula: wherein X is Br or I; L is a linker; R is hydrogen, C1-6alkyl, or —C(═O)C1-6alkyl; and Rais hydrogen or C1-6 alkyl. Pyrrolobenzodiazepine An ADC may comprise a pyrrolobenzodiazepine (PBD) drug moiety. PDB dimers may recognize and bind to specific DNA sequences. The natural product anthramycin, a PBD, was first reported in 1965 (Leimgruber, et al., (1965)J. Am. Chem. Soc.,87:5793-5795; Leimgruber, et al., (1965)J. Am. Chem. Soc.,87:5791-5793). Since then, a number of PBDs, both naturally-occurring and analogues, have been reported (Thurston, et al., (1994) Chem. Rev. 1994, 433-465 including dimers of the tricyclic PBD scaffold (U.S. Pat. Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; 7,557,099). Without intending to be bound by theory, it is believed that the dimer structure imparts the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986)Acc. Chem. Res.,19:230-237). Dimeric PBD compounds bearing C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res.70(17):6849-6858; Antonow (2010)J. Med. Chem.53(7):2927-2941; Howard et al (2009)Bioorganic and Med. Chem. Letters19(22):6463-6466). PBD compounds can be employed as prodrugs by protecting them at the N10 position with a nitrogen protecting group which is removable in vivo (WO 00/12507; WO 2005/023814). PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (US 2010/0203007). Nonlimiting exemplary linkage sites on the PBD dimer include the five-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598). A linker may be attached at one of various sites of the PBD dimer drug moiety, including the N10 imine of the B ring, the C-2 endo/exo position of the C ring, or the tether unit linking the A rings (see structures C(I) and C(II) below). In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A-1: wherein n is 0 or 1. In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A-2: wherein n is 0 or 1. In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A-3: wherein REand RE″are each independently selected from H or RD, wherein RDis defined as above; andwherein n is 0 or 1. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, REand/or RE″is H. In some embodiments, REand RE″are H. In some embodiments, REand/or RE″is RD, wherein RDis optionally substituted C1-12alkyl. In some embodiments, REand/or RE″is RD, wherein RDis methyl. In some embodiments, an exemplary PBD dimer component of an ADC has the structure of Formula A-4: wherein Ar1and Ar2are each independently optionally substituted C5-20aryl; wherein Ar1and Ar2may be the same or different; andwherein n is 0 or 1. An exemplary PBD dimer component of an ADC has the structure of Formula A-5: wherein Ar1and Ar2are each independently optionally substituted C5-20aryl; wherein Ar1and Ar2may be the same or different; andwherein n is 0 or 1.Ar1and Ar2may each, independently, be selected from optionally substituted phenyl, furanyl, thiophenyl and pyridyl. In some embodiments, Ar1and Ar2are each independently optionally substituted phenyl. In some embodiments, Ar1and Ar2are each independently optionally substituted thien-2-yl or thien-3-yl. In some embodiments, Ar1and Ar2are each independently optionally substituted quinolinyl or isoquinolinyl. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. In some embodiments, the quinolinyl is selected from quinolin-3-yl and quinolin-6-yl. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. In some embodiments, the isoquinolinyl is selected from isoquinolin-3-yl and isoquinolin-6-yl. An exemplary PBD dimer component of an ADC has the structure of Formula A-6: Further nonlimiting exemplary PBD dimer components of ADC have Formula B: and salts and solvates thereof, wherein:the wavy line indicates the covalent attachment site to the linker;the wavy line connected to the OH indicates the S or R configuration;RV1and RV2are independently selected from H, methyl, ethyl and phenyl (which phenyl may be optionally substituted with fluoro, particularly in the 4 position) and C5-6heterocyclyl; wherein RV1and RV2may be the same or different; andn is 0 or 1.RV1and RV2may, independently, be selected from H, phenyl, and 4-fluorophenyl. Nonlimiting exemplary PBD dimer components of ADC include tether-linked Formulas C(I) and C(II): Formulas C(I) and C(II) are shown in their N10-C11 imine form. Exemplary PBD drug moieties also include the carbinolamine and protected carbinolamine forms as well, as shown in the table below: wherein:X is CH2(n=1 to 5), N, or O;Z and Z′ are independently selected from OR and NR2, where R is a primary, secondary or tertiary alkyl chain containing 1 to 5 carbon atoms;R1, R′1, R2and R′2are each independently selected from H, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C5-20aryl (including substituted aryls), C5-20heteroaryl groups, —NH2, —NHMe, —OH, and —SH, where, in some embodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;R3and R′3are independently selected from H, OR, NHR, and NR2, where R is a primary, secondary or tertiary alkyl chain containing 1 to 5 carbon atoms;R4and R′4are independently selected from H, Me, and OMe;R5is selected from C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C5-20aryl (including aryls substituted by halo, nitro, cyano, alkoxy, alkyl, heterocyclyl) and C5-20heteroaryl groups, where, in some embodiments, alkyl, alkenyl and alkynyl chains comprise up to 5 carbon atoms;R11is H, C1-C8alkyl, or a protecting group (such as acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), 9-fluorenylmethylenoxycarbonyl (Fmoc), or a moiety comprising a self-immolating unit such as valine-citrulline-PAB);R12is H, C1-C8 alkyl, or a protecting group;wherein a hydrogen of one of R1, R′1, R2, R′2, R5, or R12or a hydrogen of the —OCH2CH2(X)nCH2CH2O— spacer between the A rings is replaced with a bond connected to the linker of the ADC. An ADC comprising a PBD dimer described herein may be made by conjugating a linker-drug intermediate including a pyridine leaving group via a sulfur atom with a cysteine thiol of an antibody to form a disulfide linkage. Further, in some embodiments, an ADC comprising a PBD dimer described herein may be made by conjugating a linker-drug intermediate including a thiopyridyl leaving group, wherein the pyridine ring is substituted with one or more nitro groups. In some embodiments, the pyridyl ring is monosubstituted with —NO2. In some embodiments, the —NO2monosubstitution is para relative to the disulfide. In some embodiments, the PBD dimer is connected through the N10 position. For example, non-limiting exemplary ADC comprising a PBD dimer may be made by conjugating a monomethylethyl pyridyl disulfide, N10-linked PBD linker intermediate (shown below) to an antibody: PBD dimers and ADCs comprising PBD dimers may be prepared according to methods known in the art. See, e.g., WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; WO 2011/130598; WO 2013/055987. Anthracyclines A site specific ADC of this disclosure may comprise an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. While not intending to be bound by theory, anthracyclines may operate to kill cells by a number of different mechanisms, including: 1) intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; 2) production by the drug of free radicals which then react with cellular macromolecules to cause damage to the cells, and/or 3) interactions of the drug molecules with the cell membrane (see, e.g., C. Peterson et al., “Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia” inAnthracycline Antibiotics In Cancer Therapy; N. R. Bachur, “Free Radical Damage” id. at pp. 97-102). Because of their cytotoxic potential anthracyclines have been used in the treatment of numerous cancers such as leukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma and sarcomas (see e.g., P. H-Wiernik, inAnthracycline: Current Status and New Developmentsp 11). Exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al (2006)Current Med. Chem.13:477-523; Jeffrey et al (2006)Bioorganic&Med. Chem. Letters16:358-362; Torgov et al (2005)Bioconj. Chem.16:717-721; Nagy et al (2000)Proc. Natl. Acad. Sci. USA97:829-834; Dubowchik et al (2002)Bioorg. &Med. Chem. Letters12:1529-1532; King et al (2002)J Med. Chem.45:4336-4343; EP 0328147; U.S. Pat. No. 6,630,579). The antibody-drug conjugate BR96-doxorubicin reacts specifically with the tumor-associated antigen Lewis-Y and has been evaluated in phase I and II studies (Saleh et al (2000)J. Clin. Oncology18:2282-2292; Ajani et al (2000)Cancer Jour.6:78-81; Tolcher et al (1999)J. Clin. Oncology17:478-484). PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri, et al. (2005) Clinical Cancer Research 11(4):1608-1617). Nemorubicin is a semi synthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin and has been under clinical evaluation (Grandi et al (1990) Cancer Treat. Rev. 17:133; Ripamonti et al (1992) Brit. J. Cancer 65:703;), including phase II/III trials for hepatocellular carcinoma (Sun et al (2003) Proceedings of the American Society for Clinical Oncology 22, Abs1448; Quintieri (2003)Proceedings of the American Association of Cancer Research,44:1st Ed, Abs 4649; Pacciarini et al (2006) Jour. Clin. Oncology 24:14116). In some embodiments, the nemorubicin component of a nemorubicin-containing ADC is PNU-159682. wherein the wavy line indicates the attachment to the linker (L). Anthracyclines, including PNU-159682, may be conjugated to antibodies through several linkage sites and a variety of linkers (US 2011/0076287; WO2009/099741; US 2010/0034837; WO 2010/009124), including the linkers described herein. Exemplary ADCs may be made by conjugating a pyridyl disulfide PNU amide (shown below) to an antibody: to produce a disulfide-linked PNU-159682 antibody-drug conjugate: The linker of PNU-159682, maleimide acetal-Ab is acid-labile, while the linkers of PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer(R1R2)-Ab are protease-cleavable. 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) Dimer Drug Moieties In some embodiments, an ADC comprises 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI). The 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (amino CBI) class of DNA minor groove alkylators are potent cytotoxins (Atwell, et al (1999) J. Med. Chem., 42:3400), and have been utilized as effector units in a number of classes of prodrugs designed for cancer therapy. These have included antibody conjugates, (Jeffrey, et al. (2005) J. Med. Chem., 48:1344), prodrugs for gene therapy based on nitrobenzyl carbamates (Hay, et al (2003) J. Med. Chem. 46:2456) and the corresponding nitro-CBI derivatives as hypoxia-activated prodrugs (Tercel, et al (2011) Angew. Chem., Int. Ed., 50:2606-2609). The CBI and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) pharmacophores have been linked together by an alkyl chain (Tercel et al (2003)J Med. Chem46:2132-2151). A site-specific ADC may comprise a 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer (WO 2015/023355). The dimer may be a heterodimer wherein one half of the dimer is a CBI moiety and the other half of the dimer is a PBD moiety. An exemplary CBI dimer comprises the formula: whereR1is selected from H, P(O)3H2, C(O)NRaRb, or a bond to a linker (L);R2is selected from H, P(O)3H2, C(O)NRaRb, or a bond to a linker (L);Raand Rbare independently selected from H and C1-C6alkyl optionally substituted with one or more F, or Raand Rbform a five or six membered heterocyclyl group;T is a tether group selected from C3-C12alkylene, Y, (C1-C6alkylene)-Y—(C1-C6alkylene), (C1-C6alkylene)-Y—(C1-C6alkylene)-Y—(C1-C6alkylene), (C2-C6alkenylene)-Y—(C2-C6alkenylene), and (C2-C6alkynylene)-Y—(C2-C6alkynylene);where Y is independently selected from O, S, NR′, aryl, and heteroaryl;where alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with F, OH, O(C1-C6alkyl), NH2, NHCH3, N(CH3)2, OP(O)3H2, and C1-C6alkyl, where alkyl is optionally substituted with one or more F;or alkylene, alkenylene, aryl, and heteroaryl are independently and optionally substituted with a bond to L;D′ is a drug moiety selected from: where the wavy line indicates the site of attachment to T;X1and X2are independently selected from O and NR3, where R3is selected from H and C1-C6alkyl optionally substituted with one or more F;R4is H, CO2R, or a bond to a linker (L), where R is C1-C6alkyl or benzyl; andR5is H or C1-C6alkyl. Amatoxin and Amanitin The site specific ADCs may comprise one or more amatoxin molecules. Amatoxins are cyclic peptides composed of 8 amino acids. They can be isolated fromAmanita phalloidesmushrooms or prepared synthetically. Amatoxins specifically inhibit the DNA-dependent RNA polymerase II of mammalian cells, and thereby also the transcription and protein biosynthesis of the affected cells. Inhibition of transcription in a cell causes stop of growth and proliferation. See e.g., Moldenhauer et al. JNCI 104:1-13 (2012), WO2010115629, WO2012041504, WO2012119787, WO2014043403, WO2014135282, and WO2012119787, which are hereby incorporated by reference in its entirety. The one or more amatoxin molecules may be a-amanitin molecules. Other Drug Moieties Drug moieties may also include geldanamycin (Mandler et al (2000)J. Nat. Cancer Inst.92(19):1573-1581; Mandler et al (2000)Bioorganic&Med. Chem. Letters10:1025-1028; Mandler et al (2002)Bioconjugate Chem.13:786-791); and enzymatically active toxins and fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,Aleurites fordiiproteins, dianthin proteins,Phytolaca americanaproteins (PAPI, PAPII, and PAP-S),Momordica charantiainhibitor, curcin, crotin,Sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, e.g., WO 93/21232. Drug moieties may also include compounds with nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease). It is to be understood that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADC compounds with a distribution of one or more drug moieties attached to an antibody. Linker-drug intermediates may be prepared by coupling a drug moiety with a linker reagent, and according to the procedures of WO 2013/055987; WO 2015/023355; WO 2010/009124; WO 2015/095227, and conjugated with a protein, including cysteine engineered antibodies, described herein. Methods of Analyzing and Quantifying Antibody and Drug Moieties in Site Specific ADCs ADCs are targeted anti-cancer therapeutics designed to reduce nonspecific toxicities and increase efficacy relative to conventional small molecule and antibody cancer chemotherapy. They employ the powerful targeting ability of monoclonal antibodies to specifically deliver highly potent, conjugated small molecule therapeutics to a cancer cell. To evaluate properties such as efficacy, stability, homology, pharmacokinetics and toxicity of these ADCs, it is useful to accurately characterize and quantify the antibody component and drug moiety from solution, plasma, urine, and other biological samples, via sample analytical analyses. This disclosure provides reproducible, accurate, and efficient analytical methods for quantification and analysis of characteristics of antibody and drug components of site specific ADC therapeutic constructs.FIG.2shows a cartoon of the work flow in an ADC sample assay of this disclosure, including the optional affinity capture of an ADC from a sample, site specific enzymatic digestion, which may include drug cleavage and release from the ADC, and subsequent analysis of the drug and peptide fragments by chromatography and/or spectrometry methods. In these methods, the site specific ADC construct is digested with one or more specific enzymes to form a digested ADC composition containing at least one peptide fragment that is not linked to the drug moiety, and at least one peptide fragment that is linked to the drug moiety. Either one or both of the drug and digested antibody component(s)/fragment are then analyzed by chromatography/spectrometry to determine characteristics of the ADC, which may include, but are not limited to, the protein concentration of the ADC composition, the total antibody concentration of the ADC, the drug concentration and/or the average DAR of the ADC, ADC metabolite or catabolite structures, and the extinction coefficient of the ADC. The protein component of the ADC sample may be digested with a proteolytic enzyme such as an IdeS protease, an IdeZ protease, an IgdE protease, a SpeB protease, a gingipain protease, an endoglycosidase, and combinations of these enzymes. IdeS is an extracellular cysteine proteinase produced byS. pyogenesand available commercially from Promega (Madison, Wis.) and Genovis AB (Cambridge, Mass.). This enzyme, designated IdeS for Immunoglobulin G-degrading enzyme ofS. pyogenes, cleaves human IgG below the hinge region with a high degree of specificity yielding a homogenous pool of F(ab′)2 and Fc fragments. Thus, other human proteins, including immunoglobulins M, A, D and E, are not digested by IdeS. The enzyme efficiently cleaves IgG antibodies bound to streptococcal surface structures, thereby inhibiting the killing ofS. pyogenesby phagocytic cells, leading to identification of this enzyme as a determinant of bacterial virulence, and a potential therapeutic target (von Pawel-Rammingen, et al. EMBO J. (2002) 21(7):1607-15). The proteolytic cleavage site of the IdeS enzyme is shown in the following table: IgG Species and SubclassesIdeS cleavage siteSEQ ID No.Human IgG1. . . CPAPELLG/GPSVF . . .1Human IgG2. . . CPAPPVA/GPSVF . . .2Human IgG3. . . CPAPPVA/GPSVF . . .2Human IgG4. . . CPAPPVA/GPSVF . . .2Mouse IgG1Does not cutMouse IgG2a. . . CPAPPVA/GPSVF . . .2Mouse IgG2bDoes not cutMouse IgG3. . . CPAPPVA/GPSVF . . .2Rat IgG2b. . . CPAPPVA/GPSVF . . .2Rhesus Monkey. . . CPAPPVA/GPSVF . . .2Rabbit. . . CPAPPVA/GPSVF . . .2 SEQ ID NO.: 1HTCPPCPAPELLGGPSVFSEQ ID NO.: 2HTCPPCPAPPVAGPSVF IdeZ Protease (IgG-specific) is an antibody-specific protease cloned fromStreptococcus equisubspecieszooepidemicusthat recognizes all human, sheep, monkey, and rabbit IgG subclasses, specifically cleaving at a single recognition site below the hinge region, yielding a homogenous pool of F(ab′)2 and Fc fragments, and is commercially available from New England Biolabs (Ipswich, Mass.), Promega (Madison, Wis.), and Genovis AB (Cambridge, Mass.). IdeZ Protease has significantly improved activity against mouse IgG2a and IgG3 subclasses compared to the IdeS Protease. IgdE is a protease ofStreptococcus suisthat exclusively targets porcine IgG. This enzyme, designated IgdE for immunoglobulin G-degrading enzyme ofS. suis, is a cysteine protease distinct from streptococcal immunoglobulin degrading proteases of the IdeS family and cleaves the hinge region of porcine IgG with a high degree of specificity (Spoerry, et al., J Biol Chem. (2016) 291(15):7915-25). SpeB is a cysteine protease isolated fromStreptococcus pyogenes, which degrades IgA, IgM, IgE, and IgD, and cleaves IgG antibodies in the hinge region after reduction, i.e., cleaves IgG molecules in a reduced state, e.g., in the presence of dithiothreitol (DTT), (3-mercaptoethanol, or L-cysteine (Persson, et al., Infect. Immun. (2013) 81(6):2236-41). Gingipain Kgp (also referred to as Lys-gingipain) is a cysteine protease secreted byPorphyromonas gingivalis, which cleaves human IgG1 in the upper hinge region (between K223 and T224) fragments under mild reducing conditions, producing a homogenous pool of Fab and Fc. A recombinant form of Gingipain Kgp is commercially available from Genovis AB (Cambridge, Mass.). Endoglycosidases represent a family of enzymes expressed byStreptococcus pyogenescapable of releasing the terminal sialic acid residues from glycoproteins such as immunoglobulins, and Asp279 of IgG in particular. EndoS is a specific endoglycosidase used to deglycosylate antibodies. Additional endoglycosidases that may be useful in the methods of this disclosure include one or more of Endo S2, EndoH, EndoA, EndoM, EndoF, EndoF1, EndoF2, and EndoF3. Endoglycosidases may be used in the assays of this disclosure in combination with one or more of the proteases described above in preparation of the digested site specific ADC for chromatographic/spectrometric analysis. The proteolytic enzyme(s) used to digest the site specific ADC construct may be chosen to produce a unique peptide fragment for detection and quantitation. One or more of the peptide fragments unique to the antibody of the ADC is detected and quantified, thereby eliminating background or non-specific proteins or other contaminants that may be present in the analysis sample applied to the chromatography or spectrometry, that do not form part of the ADC. In example embodiments, the ADC sample is not digested with trypsin, papain, pepsin, endoproteinase LysC, endoproteinase ArgC,Staph aureusV8, chymotrypsin, Asp-N, Asn-C, PNGaseF, endoproteinase GluC, LysN, or any combinations of these enzymes. Thus, in example embodiments, the ADC sample, in the digestion or analysis procedures, contains no detectable amounts of trypsin, papain, pepsin, endoproteinase LysC, endoproteinase ArgC,Staph aureusV8, chymotrypsin, Asp-N, Asn-C, PNGaseF, endoproteinase GluC, or LysN. Depending upon the identity of the linker component of the ADC and the chemical treatment applied to reduce, denature, and/or digest the protein component of the sample, the drug moiety of the ADC may be cleaved from the antibody/peptide component of the ADC and may therefore be detected and quantified as an unconjugated drug component in the LC-MS/MS analysis. Alternatively, or additionally, the drug moiety component of the ADC may remain linked to the antibody/peptide component of the ADC following reduction and denaturation of the ADC, and may therefore be detected and quantified as a peptide-bound drug moiety in the analysis. The sample containing the ADC for analysis/quantification may be subjected to digestion (and optionally reduction and/or denaturation) without any preliminary sample clean up or enrichment (i.e., “direct digestion” of the sample). Alternatively, or additionally, the sample containing the ADC may be enriched or concentrated for further analysis, prior to digestion. Such concentration of low-abundance peptides or drugs may include enrichment techniques such as size exclusion chromatography, dialysis, selective precipitation, differential centrifugation, filtration, gel electrophoresis, liquid chromatography, reversed-phase phase chromatography, immunoprecipitation, SPINTRAP™ (Cytiva, Inc.) spin columns including protein A and protein G, NHS and streptavidin iron or phosphorus or immobilized antibodies or lectin, paramagnetic beads, immuno-depletion, fractionation, solid phase extraction, phosphopeptide enrichment, polyacrylamide gel electrophoresis, desalting, and the like. The ADC may be reduced by contact with a composition that includes at least one reductant, for example dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine (TCEP). The ADC may also be denatured by contact with a composition that includes at least one denaturant, for example formamide, dimethylformamide, acetonitrile, SDS, urea, guanidine, sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate (ProteaseMax™), and/or an acid labile surfactant(s) such as those containing a dioxolane or dioxane functional group, such as RapiGest™-SF-surfactant (as described in U.S. Pat. Nos. 7,229,539 and 8,580,533; which are incorporated herein by reference). The ADC may be simultaneously reduced and denatured by contact with a composition that includes at least one reductant and at least one denaturant. Such compositions may include additional solvents, buffers and/or pH modifying agents, such as acetonitrile, methanol, ethanol, HCl, ammonium bicarbonate, ammonium acetate, and/or formic acid, dephosphorylating agents including phosphatases such as calf intestinal alkaline phosphatase, bovine intestinal alkaline phosphatase, or lambda protein phosphatase. The ADC presented for analysis may also be present in a solution or suspension, such as a pharmaceutical composition formulated for administration to an animal or human, or in cell culture or supernatant that may be present in a production step of the ADC, or in a biological sample obtained from an animal or a human. Thus, the ADC may be present in a matrix selected from a buffer, whole blood, serum, plasma, cerebrospinal fluid, saliva, urine, lymph, bile, feces, sweat, vitreous, tears, and tissue. Biological samples that are frequently presented for analysis of various safety, efficacy and pharmacokinetic/biodistribution parameters of ADCs include human, cynomolgus monkey, rat, and mouse plasma and tissue samples, as well as biological samples from other non-human species. When presented as part of such biological samples, the ADC may be contacted with an affinity capture media. Affinity capture is a widely used method to enrich/isolate intact proteins, to identify binding partners and protein complexes, or to investigate post-translational modifications. The protein or protein complexes may be separated by non-specific means (e.g., gel electrophoresis, Protein A or G media, type 1 antineuronal nuclear autoantibody (ANNA-1, also known as “anti-Hu”), or specific means (e.g., extracellular domain (ECD) antibodies, or anti-idotypic antibodies). The ADC may then be eluted from the affinity capture media as a means of sample cleanup prior to digestion (optionally including reduction and/or denaturation), and subsequent chromatography/spectrometry analysis of the digest. Alternatively, or additionally, the ADC sample is analyzed with an affinity capture by bead- or resin-supported Protein A/G, followed by on-bead digestion (which may include proteolysis, deglycosylation, dephosphorylation, reduction, and/or denaturation) prior to elution of an enriched, digested antibody sample from the affinity capture media, and subsequent chromatography/spectrometry analysis. Methods to detect and screen antibody-drug conjugates by Immunoaffinity membrane (IAM) capture and mass spectrometry have been disclosed (U.S. Pat. No. 7,662,936), including bead-based affinity capture methods (U.S. Pat. No. 8,541,178). The analysis sample(s) (or at least a portion thereof) comprising one or both of the drug (or peptide-linker-drug) and the digested antibody components of the site specific ADC is then applied to a detection methodology that may include high performance liquid chromatography (HPLC), reverse-phase liquid chromatography (RP-LC), mass spectrometry (MS) or tandem mass spectrometry (MS/MS), RP-LC/MS and LC-MS/MS, to detect and quantify both the drug and antibody component of the ADC. Mass spectrometry may be used to establish the mass to charge ratio of at least one peptide fragment of the digested antibody, and/or the mass to charge ratio of the drug (or peptide-linker-drug) moiety of the ADC. The molar extinction coefficient (or mass attenuation coefficient) is equal to the molar attenuation coefficient times the molar mass. The molar extinction coefficient of a protein at 280 nm depends almost exclusively on the number of aromatic residues, particularly tryptophan, and can be predicted from the sequence of amino acids. Thus, if the molar extinction coefficient is known, it can be used to determine the concentration of the protein in solution. Each publication or patent cited herein is incorporated herein by reference in its entirety. The disclosure now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present disclosure. The examples are not intended to limit the disclosure, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed disclosure. EXAMPLES Materials. Human lithium heparin plasma was purchased from BioreclamationIVT (New York, U.S.A.). Streptavidin-coated Dynabeads M-280 were purchased from Invitrogen (CA, U.S.A.). IdeS, i.e. FabRICATOR, was purchased from Genovis, Inc. (Cambridge, Mass.). Other reagents included HBS-EP buffer containing 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Polysorbate 20 (GE Healthcare; Little Chalfont, U.K.) and the peptide N-glycosidase F (PNGase F; ProZyme; CA, U.S.A.). All TDCs and specific ADC capture reagents, for example, ECD, were produced at Genentech (South San Francisco, Calif., U.S.A.). ECD was biotinylated with a 10 mol equiv of Sulfo-NHS-LC-biotin (Pierce/Thermo Fisher Scientific, Rockford, Ill., U.S.A.) to ECD for 60 min at room temperature in 10 mM sodium phosphate/150 mM NaCl, pH 7.8. Excess unbound biotin was removed using Zeba spin desalting column (Pierce/Thermo Fisher Scientific), as per the manufacturer's protocol. Biotinylated ECD concentration was determined spectrophotometrically by measuring the absorbance at 280 nm using GeneQuant 1300 (GE Healthcare). Animal Plasma Samples. All animal studies were carried out in compliance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Genentech, Inc. For PK studies, female C.B-17 SCID mice (Charles River Laboratories) were administered a single dose of ADC intravenous bolus injection, and whole blood was drawn from animals via terminal cardiac puncture. Blood samples were collected into tubes containing lithium heparin and were allowed to sit on wet ice until centrifugation (within 15 min of collection). The collected plasma samples were stored at −70° C. until analysis. Plasma samples from Sprague-Dawley rats were obtained in a similar way. Instrumentation. Affinity capture was carried out on a KingFisher 96 magnetic particle processor (Thermo Electron) using 2 mL square-top 96-deep well plates (Analytical Sales and Service, Pompton Plains, N.J., U.S.A.). The eluate was transferred to a VWR Dynablock 96-well 0.5 mL plate (VWR Scientific Products). Capillary RPLC-MS was carried out on a Waters nanoACQUITY UPLC system (Cambridge, Mass., U.S.A.) coupled to a Sciex TripleTOF® 5600 mass spectrometer (Redwood City, Calif., USA). Example 1: IdeS (2nd-Generation) Affinity Capture LC-MS Assay Design and Validation The affinity capture LC-MS assay was conducted for site-specific ADCs, including TDCs with conjugation sites in the Fab region (Su′ D. et al (2016) Anal. Chem., 88(23):11340-11346; Xu, K.; et al. (2013) Bioanalysis, 5, 1057-1071; U.S. Pat. No. 8,541,178; Xu, K.; et al Anal. Biochem. (2011) 412:56-66). In order to test and validate the multiplexing assays of this disclosure, IdeS protease (FIG.1) removed the glycan-containing Fc region at specific sites, thereby reducing the size of analytes and heterogeneity of ADC catabolites. On-bead digestion using IdeS was expected to quickly generate F(ab′)2 (about 100 kDa) for the final LC-MS analysis, instead of the intact ADC (about 150 kDa). The reduced size of analytes and quick digestion offered by IdeS resulted in improved sensitivity and resolution, and minimal artificial drug modification or decomposition and equal recovery of individual DAR species during the enrichment process, compared with a 1st-generation affinity capture LC-MS used to test in vivo stability and PK assessment of TDCs. This 2nd-generation affinity capture LC-MS showed surprising improvements for analyzing DAR and catabolite characterization of site-specific ADCs when tested on a variety of TDCs with different antibodies, linker-drugs, and conjugation sites. FIG.2provides a cartoon illustration of the 2nd-generation LC-MS assay tested in this Example, andFIG.3shows a cartoon illustration comparing embodiments of the 1stgeneration and 2ndgeneration assays that were tested and compared in this Example. In vivo plasma samples were collected from mouse, rat and cynomolgus monkey models that had been administered TDCs intravenously. All animal studies were performed in compliance with NIH guidelines for the care and use of laboratory animals. Plasma was purchased from BioreclamationIVT (New York, USA). All TDCs and specific ADC capture reagents, e.g., extracellular domain (ECD) and anti-human (Fab region) antibody, were produced at Genentech (South San Francisco, Calif., USA). ECD and anti-human (Fab region) antibody were biotinylated with 10 molar equivalent of Sulfo-NHS-LC-biotin (Pierce/Thermo Fisher Scientific, Rockford, Ill., USA) to ECD or anti-human (Fab region) antibody for 60 min at room temperature in 10 mM sodium phosphate/150 mM NaCl, pH 7.8. Excess unbound biotin was removed using ZEBA™ spin desalting column (Pierce/Thermo Fisher Scientific), per manufacturer's protocol. Biotinylated ECD or anti-human (Fab region) antibody concentration was determined spectrophotometrically by measuring absorbance at 280 nm using GENEQUANT™ 1300 (GE Healthcare). The assay experimental details of the 1st-generation affinity capture LC-MS have been described previously (U.S. Pat. No. 8,541,178 to Kaur et al; Xu, et al., Anal. Biochem. 2011, 412(1):56-66). For comparison testing of the 2nd-generation assay, 100 μL of streptavidin paramagnetic beads (Streptavidin-coated Dynabeads M-280; Invitrogen (CA, USA) were added to a 96-deep-well plate containing an excess amount of biotinylated specific capture reagents, e.g., ECD, in HBS-EP buffer (300 μL; 0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 (GE Healthcare; Little Chalfont, UK)) and incubated with agitation at room temperature (RT) for 1 h. TDC-containing plasma samples were then added (a maximum amount of 2 μg or 250 μL, whichever is less) to the ECD-immobilized beads to a total volume of 300-500 μL, and incubated with agitation at RT for 1.5 h. Affinity capture with a generic capture reagent, biotinylated anti-human IgG F(ab′)2 antibody is useful for capture for all different humanized therapeutic antibody ADC and is appropriate in a study with different therapeutic arms, or where no specific ECD is available. Results from the 2nd-generation LC-MS assay show that generic anti-human IgG F(ab′)2 is similar to specific ECD capture of ADC analytes. Affinity capture was carried out on a KingFisher 96 magnetic particle processor (Thermo Electron) using 2-mL square-top 96-deep-well plates (Analytical Sales and Service, Pompton Plains, N.J., USA). Elute was transferred to a VWR Dynablock 96 well 0.5 ml plate (VWR Scientific Products). Capillary RPLC-MS was carried out on a Waters nanoACQUITY UPLC (Cambridge, Mass., USA) coupled with AB Sciex 5600 triple time-of-flight (TOF) mass spectrometer (Redwood City, USA). Affinity captured TDCs were digested with IdeS protease (FABRICATOR™, Genovis AB) (40 units) in HBS-EP buffer (300 μL) at 37° C. for 1 h, in contrast to PNGase F (PROZYME™; CA) digestion for overnight as described in the 1st-generation method. All agitation was carried out carefully, ensuring that beads were well suspended in solution throughout the digestion procedure. Newly-generated F(ab′)2 fragments were washed sequentially with HBS-EP buffer, water and 10% ACN on beads, and then eluted by incubation in 50 μL of 30% acetonitrile with 1% formic acid at RT for 10 min. The subsequent F(ab′)2 elution was spun in a Bruker centrifuge at 4000 rpm for 10 min at RT and then transferred to a 96-well plate to remove residual beads with a magnet. Final elution was spun at 4000 rpm for 10 min at RT prior to LC-MS to avoid injection of any residual beads. An aliquot of 5 μL of F(ab′)2 elution was submitted for LC-MS analysis. Capillary LC-MS was performed on a TripleTOF 5600 mass spectrometer coupled with a Waters NanoAcquity. On-line desalting and pre-concentration were conducted on a PS-DVB monolithic column (500-μm i.d.×5 cm, Thermo Fisher Scientific, Waltham, Mass.) at 65° C. using a gradient condition at a flow rate of 15 μL/min with mobile phases A, 0.1% formic acid (FA) and B, acetonitrile (ACN) with 0.1% FA. The LC gradient was 0% B (0-4 min), 0-40% B (4-8 min), 40% B (8-11 min), 40-100% B (11-12.5 min), 100% B (12.5-13.5 min), 100-0% B (13.5-14.2 min), 0% B (14.2-15 min). LC flow was diverted to waste for the first 6 min. TripleTOF 5600 was operated with a DuoSpray Ion Source with the following key settings: ion source temperature, 425.0° C.; Ion source gas (GS)1, 40; GS2, 35; Curtain gas, 30; IonSpray Voltage Floating, 5000V; declustering potential, 250; collision energy, 20. Mass spectra were acquired in the intact protein mode, using ANALYST™ TF 1.6. Deconvolution was Performed with BIOANALYST™ 1.5.1. Relative ratios of individual TDC DAR species were obtained based on peak areas in the deconvoluted mass spectra. Calculated results within ±15% were considered not significantly different: Average DAR=Σ (% peak area×number of conjugated drugs)/100. For method development, direct LC-MS was tested and compared with both 1st-generation, and 2nd-generation, affinity capture LC-MS. Naked antibody (DAR0) and TDC standard (DAR2) were mixed as DAR0:DAR2≈1:1 at a total concentration of 100 μg/mL. An aliquot of 20 μL TDC mixture was spiked into 100 μL of human plasma to a final concentration of 20 μg/mL for the 1st-generation and 2nd-generation affinity capture LC-MS, respectively. Another aliquot of 10 μL TDC mixture was spiked into 50 μL of 30% ACN with 1% FA and directly submitted for LC-MS analysis. DAR profiling by direct LC-MS of TDC mixtures at known ratios allowed determination of whether all individual DARs have similar LC-MS response. For example, if the relative DAR ratios by direct LC-MS are consistent with the theoretical values, there is no significant bias against any individual DAR species in LC recovery and ionization efficiency. Consistency in DAR profiling by affinity capture LC-MS and direct LC-MS would suggest unbiased recovery of individual DAR species during the affinity enrichment process. FIGS.4-6shows a comparison among direct LC-MS (FIG.4), 1st-generation (FIG.5) and 2nd-generation (FIG.6) affinity capture LC-MS by injecting the same amount of starting TDC standard mixture. The example TDC contains a pyrrolobenzodiazepine dimer (PBD) as the cytotoxic drug payload. Similar retention time and charge envelopes suggested no significant differences in ionization efficiency of different TDC DAR species allowing for semi-quantification using relative ratios of individual DAR species based on their peak areas in the deconvoluted mass spectra. ADC catabolites contained glycations and/or other modifications. There was no significant difference in the relative ratio of DAR0 and DAR2 between direct LC-MS and the 2nd-generation affinity capture LC-MS, indicating unbiased capture of individual DAR species (DAR 0 and DAR2). The 2nd-generation affinity capture method was further tested with a large variety of TDC standards (DAR0 and DAR2) with different antibodies, conjugation sites in the Fab region, linkers (maleimide and disulfide), and toxins (DNA damaging agents including anthracyclines, CBI dimers and PBD dimers, and tubulin binders). Similar ionization efficiency and relative ratios of DAR0 and DAR2 were observed by the direct LC-MS approach, confirming that the 2nd-generation affinity capture LC-MS is applicable to a large variety of Fab site-specific antibody-drug conjugates. The PNGaseF digestion, 1st-generation, affinity capture LC-MS assay method uses a TDC (THIOMAB™, Genentech, Inc.) antibody drug conjugate) standard mixture that included a cysteine-engineered, anti-MUC16 antibody conjugated to the cytotoxic drug monomethyl auristatin E (MMAE) via a maleimido-caproyl-valine-citrulline-para-amino-benzyloxycarbonyl (MC-vc-PAB) linker. This method was later found to show different response to individual DARs depending on the linker-drugs and antibodies. Measured DAR0:DAR2 was compared with the theoretical value of 1 and was not significantly different with the calculated result within ±15%. The difference of measured DAR0:DAR2 from the theoretical value was pronounced by the 1st-generation and/or IdeS protease-overnight digestion affinity capture LC-MS, indicating that on-bead digestion for long hours (e.g., overnight) caused potential biased recovery of individual DARs during the affinity enrichment step. Reduced and optimized incubation time for ECD immobilization, ADC and ECD binding, and on-bead digestion steps in the 2nd-generation affinity capture LC-MS minimized potential biased capture of different DARs and therefore provided more accurate information on DAR profiling. Thus, the advantages of the 2nd-generation affinity capture LC-MS include: MS intensity by the 2nd-generation LC-MS (230 cps at maximum) was higher than by theist-generation analysis (48 cps at maximum). The 2nd-generation affinity capture LC-MS allows detection of TDCs as low as approximately 20 ng (0.2 μg/mL×100 μL). Adjacent MS peaks are better resolved by the 2nd-generation assay. More complete removal of glycans is observed with 2nd-generation capture LC-MS. Compared to deglycosylation by the PNGase F overnight digestion, Fc removal by the IdeS protease is completed within about 1 h, greatly improving the assay efficiency (1 day for the 2nd-generation LC-MS vs. 2 days for theist-generation LC-MS). Affinity capture LC-MS was specifically designed to identify ADC catabolites, characterize DAR profiles, and thereby understand the fate and PK behaviors of circulating ADCs. It is therefore important to retain ADC integrity throughout the sample preparation process, in order to accurately reflect in vivo biotransformations. However, using the PNGaseF digestion, 1st-generation affinity capture LC-MS assay, ADCs containing labile cytoxic drugs were observed to undergo unintended changes, such as ex vivo payload metabolism after incubation for long hours (FIG.9). Analysis of a labile TDC, TDC-L2 in rat plasma in vivo by affinity capture LC-MS intact antibody assay (left) and F(ab′)2 assay (FIG.10). Artificial partial drug loss (−PD) resulting from ex vivo payload metabolism was minimized by IdeS digestion at 37° C. for one hour and affinity capture LC-MS F(ab′)2 assay. TDC-L2 has a CBI dimer drug moiety and maleimide linker. The production of such artificial ADC catabolites was minimized in the 2nd-generation affinity capture LC-MS (FIG.10), in which IdeS digestion was complete within 1 h. The reduced digestion time enables minimal unintended changes in ADC integrity, and thus provides more accurate information on ADC biotransformation and PK behaviors in vivo. FIG.18Ashows DAR profiling a TDC (PBD dimer drug, disulfide linker) standard mixture (DAR0:DAR2=1:1) by direct LC-MS assay, affinity capture LC-MS F(ab′)2 assay, and affinity capture LC-MS intact antibody assay with a standard deviation of 0.13, 0.09, and 0.14 for 3 replicates, respectively. Human plasma (100 μL) containing spiked TDC standard mixture was used for the affinity capture and 5 μL eluent was injected for LC-MS analysis.FIG.18Bshows DAR (drug-antibody ratio) profiling of TDC standard mixtures (DAR0:DAR2=1:1) of TDC with PBD dimer (TDC1 and TDC4), anthracycline (TDC2) and CBI dimer (TDC5) drug moieties covalently attached to cysteine-engineered antibody with a disulfide linker by direct LC-MS, affinity capture LC-MS F(ab′)2 assay with IdeS digestion 1 hour, and affinity capture LC-MS F(ab′)2 assay with IdeS digestion overnight. Human plasma (100 μL) containing spiked TDC standard mixture was used for the affinity capture and 5 μL elute was injected for LC-MS analysis. The error of measured DAR0:DAR2 for TDC2 was pronounced by the IdeS overnight digestion affinity capture LC-MS, indicating that prolonged on-bead incubation (e.g., overnight digestion) led to potential biased recoveries of different drug-loaded TDC2 species during sample preparation. For instance, in the affinity capture LC-MS F(ab′)2 assay, the significantly reduced on-bead digestion time minimized potential biased recoveries of different ADC species and thereby provided more accurate information regarding DAR estimation. In the analysis of complex ADC catabolites, the adjacent deconvoluted MS peaks need near baseline separation to allow accurate assignment of ADC catabolite structures.FIGS.7and8show linker-drug deconjugation (−LD) by cleavage of the thiol-maleimide bond. Multiple TDC catabolites were generated in mouse plasma in vivo, due to loss of 42 Da from the drug molecule. Their MS peaks were not resolved by PNGaseF digestion, 1st-generation affinity capture LC-MS (FIG.7), but were near baseline-resolved by the IdeS digestion, 2nd-generation affinity capture LC-MS (FIG.8), which enabled confident catabolite identification and more accurate DAR calculation. This accurate information is helpful for understanding the ADC efficacy and toxicity profiles as well as the ADC drug metabolism, which in turn helps to optimize new cytotoxic ADC design, which is focused on new types of antibody platforms, conjugation chemistry, linkers, and drugs. The multiple drug and metabolism parameters that must be verified during the drug development of ADCs and complex catabolites in vivo poses challenges on bioanalytical analysis. For instance, the 1st-generation affinity capture LC-MS, a useful exploratory assay for DAR and catabolite characterization, was found not generally applicable to the next-generation ADCs due to its limited sensitivity, resolution, efficiency and potential biased response to certain DAR species. This PNGaseF digestion, 2nd-generation assay accommodates the low-dose and labile site-specific ADCs becoming predominant in drug development.FIGS.7and8show characterization of complicated TDC catabolites in mouse plasma in vivo by affinity capture LC-MS intact antibody assay (FIG.7) vs. affinity capture LC-MS F(ab′)2 assay (FIG.8). Partial drug loss by linker-drug deconjugation (−PD) significantly affected the potency of TDC-L1, leading to the reduction of DAR accordingly The 2nd-generation affinity capture method employed in this comparative example utilized the IdeS protease for deglycosylation by removing the Fc fragments where the majority of glycans are located. The resulting F(ab′)2 fragments (about 100 kDa) which retain the linker-drugs, are analyzed by LC-MS, instead of the traditional intact ADCs (about 150 kDa). Compared to deglycosylation by overnight digestion with PNGase F, rapid removal of Fc by on-bead IdeS digestion and the reduced size of the F(ab′)2 analytes results in an improved assay with higher sensitivity, resolution, and efficiency, as well as minimal unintended changes to ADC profiles and integrity during sample processing as summarized in the following table: TABLEChanges and Improvements to the 1st-generation AssayChanges to the AssayImprovements of the AssayReduced digestion time from overnight to 1 hrIncrease assay efficiency (2 days to 1 day)Reduced digestion time from overnight to 1 hrMinimized sample loss and therefore increasedthe assay sensitivityReduced and optimized incubation time for ECDMinimized unintended changes, e.g. DARimmobilization, ADC and ECD binding, and on-profile, drug modification or decomposition, andbead digestion stepstherefore kept the ADC integrityDecreased the analyte size from 150 kDa forIncreased LC-MS sensitivityintact ADC to 100 kDa for F(ab′)2 and m/z from2000-3200 to 1600-2800Decreased the analyte size from 150 kDa (intactIncreased LC-MS resolution for identification ofADCs) to 100 kDa [F(ab′)2 fragments]complex ADC catabolites The affinity capture LC-MS F(ab′)2 assay was extended to analysis of conventional ADCs, where drug is bound via inter-chain disulfides. Without IdeS protease digestion, after overnight deglycosylation by PNGase F, LC separation was needed to elute the Light Chain (25 kD) and Heavy Chain (50 kD) fragments since Light Chain fragments (smaller-size) suppress the ionization/MS signal of Heavy Chain fragments (larger-size). With IdeS protease digestion, the Light Chain and Heavy Chain are of similar size (about 23-29 kDa) and can be eluted and analyzed at the same time with minimal MS bias against the Heavy Chain. When utilizing a generic capturing reagent such as, biotinylated anti-human F(ab′)2 antibody, the affinity capture LC-MS F(ab′)2 assay allowed for parallel comparison in ADC biotransformations across ADCs with same drugs conjugated to different antibodies. This comparative example demonstrates the potential of the IdeS protease digestion, 2nd-generation assay to accommodate low-dose, labile, and complex site-specific ADCs, e.g., TDCs, for more accurate and detailed biotransformation and PK information. Such information helps to optimize cytoxic drug design, facilitate development of appropriate PK bioanalytical strategies, and leads to discovery of new ADC catabolites. The method is applicable to a variety of site-specific ADCs with conjugation sites in the Fab region, and to analysis of conventional ADCs via inter-chain disulfide conjugation. Example 2: Protein Concentration Determination An accurate protein concentration determination is essential for evaluating in vitro and in vivo efficacy, as well as toxicity, of protein-drug conjugates. The inventors have developed a method to determine protein concentration for ADCs comprising, for example, small molecule payloads that contribute to the protein's absorbance at 280 nm due to the presence of aromatic rings, and particularly when the extinction coefficient of the small molecule at 280 nm and/or its absorbance maximum are unknown. In this example, the protein concentration was determined independently of the conjugated payload by proteolytic digestion with Immunoglobulin-degrading enzyme ofStreptococcuspyrogenes (IdeS), and subsequent LC-MS analysis. IdeS cleaves human IgG1's with high specificity at a site below the hinge region generating F(ab′)2 and Fc fragments (FIG.1). These species can be chromatographically separated on reversed-phase (FIG.11). Non-covalent interactions between the two arms of the Fc are disrupted by the acidity and organic solvent concentration of the mobile phases, resulting in an Fc/2 peak of approximately 25 kD in size. In ADCs where the drug payload is conjugated to inter-chain disulfides or site-specifically to the F(ab′)2 or Fc region, the resultant antibody fragment that is free of drug, either the Fc/2 or F(ab′)2 fragment peak, can be used to quantitate the protein concentration of the sample. This method is useful to characterize both traditional ADCs conjugated via inter-chain disulfides as well as THIOMAB™ (Genentech, Inc.) antibody drug conjugates (TDC) that have two engineered cysteine residues per antibody located in either the Fab or the Fc region for site-specific conjugation. Antibody-drug conjugates are digested with IdeS and then injected on reversed-phase LC-MS with detection at an absorbance of 280 nm. In the case of TDCs conjugated on the Fab and traditional ADCs, the antibody fragments that contain drug are chromatographically separated from the Fc/2 fragment allowing for the Fc/2 fragment peak area to be used for protein concentration quantitation. This value is interpolated using the linear regression of a standard curve of antibody standards digested with IdeS where starting concentration is plotted against Fc/2 peak area (FIG.11). In the case of TDCs conjugated via engineered cysteines on the Fc, the drug containing Fc/2 fragment is chromatographically separated from the F(ab′)2 fragment allowing for the F(ab′)2 fragment peak area to be used for protein concentration quantitation. This value is interpolated using the linear regression of a standard curve of antibody standards digested with IdeS where starting concentration is plotted against F(ab′)2 peak area (FIG.11). Standard curves (Fc/2 peak areas vs. concentration;FIG.13A) (F(ab′)2 peak areas vs. concentration;FIG.13B) over a range of 0.5-20 mg/ml were generated using trastuzumab digested with IdeS protease. Protein concentration of TDCs site-specifically conjugated on the F(ab) can be determined using peak area of the Fc/2 of the TDC (FIG.13A) and the linear regression. Traditional ADCs conjugated on inter-chain disulfides can also be characterized using this method as the Fc/2 fragment is also without drug in these conjugates. Protein concentration of TDCs site-specifically conjugated on the Fc can be determined using peak area of the F(ab′)2 of the TDC (FIG.13B) and the linear regression. Method THIOMABs™ (Genentech, Inc.) with 2 engineered cysteine residues per antibody were incubated with a 3-fold molar excess of a thiol reactive linker-drug at pH 7.5 for 2 hours. Excess linker-drug was purified away by cation exchange and conjugates were formulated into a pH 5.5 buffer. The drug to antibody ratio (DAR) of the TDC was determined by LC-MS analysis using the abundance of the deconvoluted masses of drugged and un-drugged species (FIG.12). All conjugates examined had a DAR of >1.7. Linker-drug payloads ranged from 700-1500 Da in size. Thirty units (30 unit/μl) of IdeS (FABRICATOR™, Genovis AB) were added to 10 μl of antibody or antibody-drug conjugate that ranged in concentration from 0.52-20 mg/ml. The reaction mixtures were brought to a final volume of 50 μl with PBS with a final reaction pH of about pH 6.5. Samples where incubated at 37° C. for 1 hr before LC-MS analysis. Samples were analyzed on reversed phase high performance liquid chromatography (HPLC) using an HPLC system (Agilent 1260 infinity) coupled to an electrospray ionization time-of-flight mass spectrometer (Agilent 6224 TOF-LC). A volume of 10 μl of sample was injected on a PLRP-S 1000A°, 8 μm 50×2.1 mm column (Agilent) heated to 80° C. The gradient was generated using 0.05% trifluoroacetic acid (mobile phase A) and 0.05% trifluoroacetic acid in acetonitrile (mobile phase B) at a flow rate of 0.5 ml/min. The column was held at 5% B for 0.7 min, followed by a 4.3 min gradient from 30% B to 40% B. At 5 min, the concentration increased to 90% B where it was held for 1 min. The column was then re-equilibrated in 5% B for 2 min. Data was acquired and analyzed using Agilent MassHunter software. Deconvoluted mass spectral data was used to confirm that all antibodies and antibody drug conjugates had been digested to completion and contained no intact antibody. Standard curves were developed using Trastuzumab (HERCEPTIN™; anti-Her2 human IgG1 antibody) digested with IdeS at known concentrations. Site-specific Trastuzumab constructs were prepared having:1) linker-drug site-specifically conjugated on the F(ab) at engineered cysteine K149C (Conjugate A;FIG.14);2) linker-drug site-specifically conjugated on the Fc at engineered cysteine S400C (Conjugate B;FIG.15); or,3) linker-drug conjugated on inter-chain disulfides (Conjugate C;FIG.16). Trastuzumab was serially diluted by a factor of 1.5 from 20 mg/ml to 0.52 mg/ml in 20 mM histidine acetate pH 5.5, 240 mM sucrose. This buffer was chosen for dilutions as it is the buffer used for final formulation of many ADCs and mimics the conditions of an experimental sample in the assay. Samples were diluted in triplicate to a total of ten concentrations. A volume of 10 μl of each dilution was added to 39 μl of phosphate buffered saline pH 7.2, and 1 μl of IdeS (30 units/μl) to a total volume of 50 μl. The final pH of these samples was in the optimal activity range for IdeS activity. Samples where incubated at 37° C. for 1 hour. Samples were then run on LC-MS in order or increasing concentration. A volume of 10 μl of the antibody digests where injected onto a reversed phase column and gradient eluted to separate Fc/2 and F(ab′)2 peaks. Samples that resulted in an injection of >5 μg of antibody where followed by blank runs of the same LC-MS method with no injection to ensure there was no sample carry-over to the next run. Protein concentrations of antibody-drug conjugates were determined by proteolytic digestion with IdeS, and subsequent LC-MS analysis. A standard curve was developed using trastuzumab, digested with IdeS at known concentrations ranging from 0.52 mg/ml to 20 mg/ml (FIGS.14-16). Standards were digested and run in triplicate with minimal error. Starting concentrations were plotted against peak areas resulting in a linear regression for the set of standards (R2=0.9999) (FIG.17). This linear regression equation was used to determine the protein concentration of unknown samples, according to the following tables:1) for linker-drug site-specifically conjugated on the F(ab) (FIG.14): Fc/2Concen-AveragePeaktrationConcentrationCV,SampleReplicateArea(mg/ml)(mg/ml)%Conjugate A11555.7813.8013.751.0721530.5613.5831561.9713.862) for linker-drug site-specifically conjugated on the Fc (FIG.15) F(ab′)2Concen-AveragePeaktrationConcentrationCV,SampleReplicateArea(mg/ml)(mg/ml)%Conjugate B1719.983.233.220.392716.723.223714.163.213) for linker-drug conjugated on inter-chain disulfides (FIG.16) Fc/2Concen-AveragePeaktrationConcentrationCV,SampleReplicateArea(mg/ml)(mg/ml)%Conjugate C1428.213.813.830.792430.143.823434.913.87 The protein concentration of 81 TDCs was determined using this method as well as by the BCA, which is a widely accepted colorimetric assay for protein concentration determination. The concentration values determined by each method were plotted against each other showing a strong correlation, validating the accuracy and reproducibility of the IdeS digestion method (FIG.17). The concentration values determined by this method can then be used to calculate the extinction coefficient of the TDC or ADC at an absorbance of A280 using the Beer-Lambert law. FIG.18Ashows DAR profiling a TDC (PBD dimer drug, disulfide linker) standard mixture (DAR0:DAR2=1:1) by direct LC-MS assay, IdeS digestion, affinity capture LC-MS F(ab′)2 assay, and PNGaseF, affinity capture LC-MS intact antibody assay with a standard deviation of 0.13, 0.09, and 0.14 for 3 replicates, respectively. Human plasma (100 μL) containing spiked TDC standard mixture was used for the affinity capture and 5 μL eluent was injected for LC-MS analysis. FIG.18Bshows DAR (drug-antibody ratio) profiling of TDC standard mixtures (DAR0:DAR2=1:1) of TDC with PBD dimer (TDC1 and TDC4), anthracycline (TDC2) and CBI dimer (TDC5) drug moieties covalently attached to cysteine-engineered antibody with a disulfide linker by direct LC-MS, IdeS digestion affinity capture LC-MS F(ab′)2 assay with IdeS digestion 1 hour, and affinity capture LC-MS F(ab′)2 assay with PNGaseF digestion overnight (green). Human plasma (100 μL) containing spiked TDC standard mixture was used for the affinity capture and 5 μL elute was injected for LC-MS analysis. The error of measured DAR0:DAR2 for TDC2 was pronounced by the IdeS overnight digestion affinity capture LC-MS, indicating that prolonged on-bead incubation (e.g., overnight digestion) led to potential biased recoveries of different drug-loaded TDC2 species during sample preparation. These methods provide a rapid, robust, and reproducible assay for protein concentration determination of ADCs regardless of the spectral properties of their conjugated payload.—Antibodies conjugated with other experimental payloads such as fluorophores can also be analyzed using this method to determine their protein concentration independent of the fluorophore or absorbance. Protein concentration of conventional ADCs may also be determined by this method, particularly for ADCs in which the payload is conjugated to inter-chain disulfides, as the Fc/2 region is devoid of these conjugation sites. The assays of the invention include a relatively fast digestion step and continues to completion, without over-digestion. Thus, this methodology has significant advantages over the use of conventional proteases such as pepsin, papain, and endopeptidase Lysine C. For example, pepsin digestion of IgG1s is slow and occurs best below pH 5. Papain digestions are performed at neutral pH and the site of protein cleavage is not specific, and often leads to multiple protein cleavage events. Limited LysC digestion may over-digest the Fc region. IdeS has no risk of over-digestion as it cleaves IgG1 antibodies at one specific site on the heavy chain below the hinge region. In solution, IdeS is stable at 4° C. for up to one month. These characteristics contribute to the robust protein concentration assay demonstrated here. For routine concentration measurements, the method can be conducted on HPLC without a mass spectrometer in-line. For example, a trastuzumab standard is digested and analyzed alongside test samples for quality control. Complete digestion can be confirmed in this assay as intact antibody has a known retention time and can be detected without mass spectral analysis. The peak area of the absorbance at 280 nm is then correlated directly with protein concentration, without the need for MS analysis. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. | 191,772 |
11860157 | DETAILED DESCRIPTION Referring now to the drawings, wherein like reference numerals designate identical or corresponding parts throughout the several views, exemplary aspects of the present invention are shown in schematic detail. The matters defined in the description such as a detailed construction and elements are nothing but the ones provided to assist in a comprehensive understanding of the invention. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the exemplary aspects described herein can be made without departing from the scope and spirit of the invention. Also, well-known functions or constructions are omitted for clarity and conciseness. Some exemplary aspects of the present invention are described below in the context of commercial applications. Such exemplary implementations are not intended to limit the scope of the present invention, which is defined in the appended claims Aspects of the present invention are generally directed to a microfilter comprising a polymer layer formed from a photo-definable dry film, such as an epoxy-based photo-definable dry film. The microfilter includes a plurality of apertures each extending through the polymer layer. Further, the polymer layer is modified to be hydrophilic. In certain exemplary aspects, the microfilter may be formed by exposing the dry film to energy through a mask and developing the exposed dry film. In some exemplary aspects, the dry film may be exposed to energy in the form of ultraviolet (UV) light. In other exemplary aspects, the dry film may be exposed to energy in the form of X-rays. In certain exemplary aspects, the polymer layer has sufficient strength and flexibility to filter liquid. In some exemplary aspects, the apertures are sized to allow passage of a first type of bodily fluid cell and to prevent passage of a second type of bodily fluid cell. According to exemplary implementations of the present disclosure, a microfilter can comprise a polymer layer formed from an epoxy-based negative photo-definable dry film. According to further exemplary implementations of the present disclosure, the apertures of the microfilter can be essentially of any shape or configuration such as round, oval, racetrack, or rectangle, or any combination thereof. In yet other exemplary aspects, the polymer layer of the microfilter can have a uniform thickness of 5 to 100 microns. In still further exemplary aspects, the polymer layer of the microfilter can have a uniform thickness of 10 μm. In yet further exemplary aspects, the apertures can round with a diameter of 5-20 μm. Any combinations of such polymer layer, aperture features and configurations are within the scope for a microfilter structure of the present disclosure. Specifically, in certain exemplary aspects, the microfilter may be used to perform assays on bodily fluids. In some exemplary aspects, the microfilter may be used to isolate and detect large rare cells from a bodily fluid. In certain exemplary aspects, the microfilter may be used to collect circulating tumor cells (CTCs) from peripheral blood from cancer patients passed through the microfilter. In certain exemplary aspects, the microfilter may be used to collect circulating endothelial cells, fetal cells and other large cells from the blood and body fluids. In certain exemplary aspects, the microfilter may be used to collect large cells from processed tissue samples, such as bone marrows. In some exemplary aspects, cells collected using the microfilter may be used in downstream processes such as cell identification, enumeration, characterization, culturing, etc. More specifically, in certain exemplary aspects, multiple layers of photo-definable dry film, such as an epoxy-based photo-definable dry film, may be exposed to energy simultaneously for scaled production of microfilters. In some exemplary aspects, a stack of photo-definable dry film layers is provided, and all of the dry film layers in the stack are exposed to energy simultaneously. In some exemplary aspects, a dry film structure including photo-definable dry film disposed on a substrate is provided in the form of a roll. In such exemplary aspects, a portion of the structure may be unrolled for exposure of the dry film to energy. In certain exemplary aspects, portions of a plurality of rolls may be exposed to energy simultaneously. FIG.1is scanning electron micrograph (SEM) of microfilter fabricated based on the known techniques. The surface is smooth, shiny and hydrophobic. The contact angle is approximately 90 degrees. The hydrophobic property of the material allows performing assays with reagents staying above the filter without the reagents leaking through. However, the hydrophobic nature of the filter is also problematic when it is desired to have a filter through which fluids easily pass, e.g., a microfilter with hydrophilic surface characteristics. For some applications, it is desirable to modify a surface of the microfilter to have hydrophilic characteristics via, for example, increasing the surface energy of a surface of the microfilter and/or altering the surface topography of a surface of the microfilter through various methods of surface treatment. Surface Modification Methods and Resultant Microfilters The surface of a microfilter may be modified to impart a hydrophilic characteristic through methods of surface treatment. The most common methods of surface treatment are based on a principle of high voltage discharge in air without changing the topography of the surface. When the microfilter is placed in the discharge path, the electrons generated in the discharge impact the surface creating reactive free radicals. These free radicals in the presence of oxygen can react rapidly to form various chemical function groups on the microfilter surface. This raises the surface energy of the microfilter. It changes the microfilter from hydrophobic to hydrophilic. Surface treatment can improve wettability of the microfilter by raising the material's surface energy and positively affect adhesive characteristics by creating bonding sites. An example of high voltage discharge is corona discharge. Some of the applications of microfilters treated by corona discharge are: (i) flow of fluid through small pores with less resistance, (ii) cell morphologies may be better preserved when the use of small pores are required, (iii) better conjugation of analyte capture elements to the microfilter, (iv) attachment of various surface modification materials, and others. Four additional methods of surface treatment are provided herein that produce surface modifications on polymer microfilters and that serve to increase the hydrophilicity of the surface: (a) reactive ion etching, (b) energetic neutral oxygen atoms etching, (c) reactive ion etching through anodic aluminum oxide (AAO) template, and (d) surface imprinting. These methods make a surface of a polymer layer rougher in texture. The 3D surface features produced by each method are different but they share the characteristic that the surface of the polymer layer that has undergone treatment is rougher in texture than the surface prior to treatment. As with microfilters using polymer layers with surfaces treated using corona discharge, microfilters using polymer layers with surfaces treated to alter the 3D surface features also exhibit (i) increased flow of fluid through small pores with less resistance, (ii) better preservation of cell morphologies, (iii) better conjugation of analyte capture elements to the microfilter, and (iv) improved attachment of various surface modification materials. Reactive Ion Etching (RIE) Method. RIE utilizes chemically reactive plasma (high-energy ions) to remove material from the surface of a polymer layer. This results in the creation of a rough nanosurface on the polymer layer. Variations in the resulting etching of the surface are achieved depending on the material to be etched and on the settings of RIE parameters.FIGS.2A-Dare scanning electron micrographs (SEMS) of examples of surface modifications produced by RIE on photo-definable dry-film without pores.FIG.2Dshows nanostructures with two different length scales. RIE can be applied to microfilters, such as track etch microfilters, parylene microfilters, microfilters produced from photo-definable dry films, any filters made by polymer material as well as made from silicon wafers.FIG.3shows a SEM of a microfilter fabricated based on the method and material described in the cross reference patents, followed by treatment by RIE showing nanosurface topography and a pore. The surface treated by RIE becomes hydrophilic. The contact angle is almost zero. Energetic Neutral Oxygen Atom Etching. Another method to produce a rough nanosurface on a polymer layer is to apply energetic neutral oxygen atom etching on the polymer surface with or without pores. To create a rough nanosurface on microfilters, energetic neutral oxygen atom etching is performed after the microfilters are already formed but still attached to substrate.FIG.4shows SEM of a microfilter treated by energetic neutral oxygen atoms showing nanosurface topography and pores. RIE through a nanoporous AAO mask. Another method to produce a rough nanosurface on a polymer layer using a porous metal material as a mask. (i) One example of a mask is to utilize AAO. AAO template is fabricated on the resist surface by deposition and anodizing of ˜1 μm-thick Al film according to recipe.FIG.5Ais a SEM of the AAO template above the surface of the polymer material. Surface relief is obtained by RIE via AAO template followed by AAO removal in phosphoric acid solution. SEM of the resultant nanosurface structure is shown inFIG.5B. (ii) Another group of porous materials for RIE are micro magnetic beads and glass beads. Nanoimprinting. Another method to produce a rough nanosurface on a polymer layer is by imprinting the dry film on nanostructured surface. Using photo-definable dry films for microfilters, the substrate with rough nanosurface can be used.FIG.6is an SEM of microfilter produced by imprinting the dry film on the rough metal substrate. The nanosurface features is directly dependent on the mold. For some applications, it is desirable to have wells formed above the microfilters. For example of culture of cells in their individual well. A method to form the wells consists of laminated photo-definable dry films on surface of filter material with pores already formed. Microfilter-culture wells are fabricated using UV lithography, followed by development. After a hard bake, the microfilter device with wells can be released from substrate.FIG.7shows an SEM of a microfilter with square wells. 3D Culture Cell culture properties are highly dependent on the type of cell. It has been shown that some cells growing in culture in clumps (3D) express different markers than the same cell line grown in a flat layer (2D) on the culture plate. There has been a lot of research on finding conditions for 3D culture. A bladder cancer cell line, T24, was selected to illustrate the effect of 2D and 3D culture. When the microfilters or polymer materials of the present invention were coated with fetal bovine serum (FBS) and bovine serum albumin (BSA), the bladder cancer cell line T24 grew similar to culturing on the standard culture chamber slide. However, if FBS or BSA can be eliminated, the culture process can be simplified. When the polymer materials or microfilters of the present invention are uncoated, the T24 culture results become very different.FIG.8Ashows the microscope imaging of the nuclei stained by DAPI of T24 cells grown on chamber slide. The cells grew flat in 2D format. The cells are imaged after permeabilized and stained by cytokeratin (CK) 8 and 18 conjugated to FITC dye.FIG.8Bshows the microscope imaging combining DAPI (blue) and CK 8, 18 (green). The cells show very low or no CK 8 and 18. When T24 cells were cultured on photo-definable dry film polymer not treated by RIE, T24 cells grew in 2D format similar to the results of chamber slide. In contrast, T24 cells grew in 3D clumps on photo-definable dry film polymer treated by low dose RIE.FIG.9Ashows the microscope imaging of the clump of nuclei stained by DAPI of T24 cells grown on RIE treated films. The cells are permeabilized and stained by cytokeratins (CK) 8 and 18 conjugated to FITC.FIG.9Bshows the microscope imaging combining DAPI (blue) and CK 8, 18 (green). The cells show very strong CK 8, 18. It was also found that when cells were spiked into PBS followed by filtration using RIE treated microfilter,FIG.3, the cells grew in a 3D format. In summary, it has been shown that the photo-definable dry film polymer treated with RIE enabled 3D culture, and that the 3D cultured cells behaved differently than 2D cultured cells. Culture Plates and Devices Devices to implement 3D culture on chamber slides, and 6, 12, 24, 96 and 384 well culture plates were prepared. Some variations of implementation are possible.Place RIE etched polymers on the bottom of these wells. This includes RIE etched photo-definable dry film polymer.Place RIE etched polymers on the bottom of these wells coated with FBS or BSA. This includes RIE etched photo-definable dry film polymer.Place RIE etched microfilters on the bottom of these well. This includes RIE etched photo-definable dry film microfilter.Place RIE etched microfilters on the bottom of these well coated with FBS or BSA. This includes RIE etched photo-definable dry film microfilter. This includes RIE etched photo-definable dry film microfilter.Place fibroblast cells, fibroblast cell fragments, other cells, other cell fragments, or other culture reagents on the bottom of the culture wells. Place RIE etched microfilters above that.Cells can be captured on the RIE etched microfilter before placing into culture plates.Cells can be captured on FBS coated microfilters before placing into culture plates. Coating of Smooth Microfilters and Nanosurface Microfilters with Analyte Capture Elements As used herein, the term “analyte” is intended to mean a biological particle. Biological particles include, for example, cells, tissues, or organisms as well as fragments or components thereof. Specific examples of biological particles include bacteria, spores, oocysts, cells, viruses, bacteriophage, membranes, nuclei, golgi, ribosomes, polypeptides, nucleic acid and other macromolecules. “Analyte complex” is intended to mean a biological particle or a group of biological particles connected to analyte capture coating and/or other components, such as proteins, DNA, polymers, optical emission detection reagent, etc. “Analyte capture” coating or elements are useful for selectively attaching or capturing a target analyte to microfilter. Attachment or capture includes both solid or solution phase binding of an analyte to an analyte capture element. An analyte is attached or captured through a solid phase configuration when the analyte capture coating or element is immobilized to a microfilter when contacted with an analyte. An analyte is attached or captured through a solution phase configuration when the analyte capture coating or element is in solution when contacted with an analyte. Subsequent immobilization of a bound analyte-analyte capture coating or element complex to a microfilter completes attachment or capture to the microfilter. In either configuration, either direct or indirect immobilization of the analyte capture coating or element to a microfilter can occur. Direct immobilization refers to attachment of the analyte capture coating or element to a microfilter allowing for capture of an analyte from solution to a solid phase. Immobilization of the analyte capture coating or element can be directly to a microfilter surface or through secondary binding partners such as linkers or affinity reagents such as an antibody. Indirect binding refers to immobilization of the analyte capture coating or element to a microfilter. Analyte capture elements can form an analyte capture complex and become attached to the analyte capture surface on the microfilter. Moieties useful as an analyte capture coating or element in the invention include biochemical, organic chemical or inorganic chemical molecular species and can be derived by natural, synthetic or recombinant methods. Such moieties include, for example, macromolecules such as polypeptides, nucleic acids, carbohydrate and lipid. Specific examples of polypeptides that can be used as an analyte capture coating or element include, for example, an antibody, an antigen target for an antibody analyte, receptor, including a cell receptor, binding protein, a ligand or other affinity reagent to the target analyte. Specific examples of nucleic acids that can be used as an analyte capture coating or element include, for example, DNA, cDNA, or RNA of any length that allow sufficient binding specificity. Accordingly, both polynucleotides and oligonucleotides can be employed as an analyte capture coating or element of the invention. Other specific examples of an analyte capture coating or element include, for example, gangilioside, aptamer, ribozyme, enzyme, or antibiotic or other chemical compound. Analyte capture coatings or elements can also include, for example, biological particles such as a cell, cell fragment, virus, bacteriophage or tissue. Analyte capture coatings or elements can additionally include, for example, chemical linkers or other chemical moieties that can be attached to a microfilter and which exhibit selective binding activity toward a target analyte. Attachment to a microfilter can be performed by, for example, covalent or non-covalent interactions and can be reversible or essentially irreversible. Those moieties useful as an analyte capture coating or element can similarly be employed as an secondary binding partner so long as the secondary binding partner recognizes the analyte capture coating or element rather than the target analyte. Specific examples of an affinity binding reagent useful as a secondary binding partner is avidin, or streptavidin, or protein A where the analyte capture coating or element is conjugated with biotin or is an antibody, respectively. Similarly, selective binding of an analyte capture coatings or element to a target analyte also can be performed by, for example, covalent or non-covalent interactions. Specific examples of a biochemical analyte capture coating or element is an antibody. A specific example of a chemical analyte capture coating or element is a photoactivatable linker. Other analyte capture coatings or elements that can be attached to a microfilter and which exhibit selective binding to a target analyte are known in the art and can be employed in the device, apparatus or methods of the invention given the teachings and guidance provided herein. One exemplary form of microfilters manufactured in accordance with exemplary aspects of the present invention (i) standard microfilters and (ii) nanosurface topography microfilters are coated with analyte capture elements. One specific exemplary form of the microfilters are microfilters coated with antibodies against EpCAM, MUC-1, and other surface markers are to capture tumor cells from body fluids, such as blood, urine, bone marrow, bladder wash, rectal brushings, fecal matter, saliva, spinal and cerebral fluids, and other body fluids. Another specific exemplary form of the microfilters coated with antibodies against CD24, CD44, CD133, CD166, and/or other surface markers are to capture epithelial-mesenchymal transition (EMT) cells from body fluids, such as blood, urine, bone marrow, bladder wash, rectal brushings, fecal matter, saliva, cord blood, spinal and cerebral fluids, and other body fluids. Another specific exemplary form of the microfilters coated with antibodies against CD34, and/or other surface markers are to capture stem cells from body fluids, such as peripheral blood and cord blood. Filtration Applications of Smooth Microfilters and Nanosurface Microfilters Exemplary applications of the various forms of microfilters manufactured in accordance with exemplary aspects of the present invention (e.g. (i) standard microfilters, (ii) nanosurface topography microfilters, (iii) standard microfilters coated with analyte capture elements, and (iv) nanosurface microfilters coated with analyte capture elements) are for processing body fluids, such as blood, urine, bone marrow, bladder wash, rectal brushings, fecal matter, saliva, spinal and cerebral fluids, and other body fluids. The analyte of interests in the body fluids are circulating tumor cells, tumor cells, epithelial-mesenchymal transition (EMT) cells, CAMLs, white blood cells, B-cells, T-cells, circulating fetal cells in mother's blood, circulating endothelial cells, stromal cells, mesenchymal cells, endothelial cells, epithelial cells, stem cells, hematopoietic and non-hematopoietic cells, analytes bound to latex beads or an antigen-induced particle agglutination. Another exemplary application of the microfilters manufactured in accordance with exemplary aspects of the present invention (e.g. (i) standard microfilters, (ii) nanosurface topography microfilters, (iii) standard microfilters coated with analyte capture elements, and (iv) nanosurface microfilters coated with analyte capture elements) is capturing circulating cancer associated macrophage-like cells (CAMLs) from peripheral blood. CAMLs have the following characteristics:CAMLs have a large atypical nucleus; multiple individual nuclei can be found in CAMLs, though enlarged fused nucleoli approximately 14 μm to approximately 65 μm are common.CAMLs may express at least CK 8, 18 or 19, and the CK is diffused, or associated with vacuoles and/or ingested material. CAMLs express markers associated with the type of cancer. Those markers and CK are nearly uniform throughout the whole cell.CAMLs are most of the time CD45 positive.CAMLs are large, approximately 20 micron to approximately 300 micron in size.CAMLs come in five distinct morphological shapes (spindle, tadpole, round, oblong, or amorphous).If CAML express EpCAM, EpCAM is diffused, or associated with vacuoles and/or ingested material, and nearly uniform throughout the whole cell, but not all CAML express EpCAM, because some tumors express very low or no EpCAM.CAML express markers associated with the markers of the tumor origin; e.g., if the tumor is of prostate cancer origin and expresses PSMA, then CAML from this patient also expresses PSMA. Another example, if the primary tumor is of pancreatic origin and expresses PDX-1, then CAML from this patient also expresses PDX-1.CAMLs express monocytic markers (e.g. CD11c, CD14) and endothelial markers (e.g. CD146, CD202b, CD31). CAMLs also have the ability to bind Fc fragments. Another exemplary application of a microfilter manufactured in accordance with exemplary aspects of the present invention (e.g. (i) standard microfilters, (ii) nanosurface topography microfilters, (iii) standard microfilters coated with analyte capture elements, and (iv) nanosurface microfilters coated with analyte capture elements) is capturing circulating fetal cells in a mother's blood during weeks 11-12 weeks of pregnancy. Such fetal cells may include primitive fetal nucleated red blood cells. Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are 14-60 μm in diameter, larger than peripheral blood leukocytes. Enrichment of circulating fetal cells followed by genetic diagnostic can be used for noninvasive prenatal diagnosis of genetic disorders using PCR analysis of a DNA target or fluorescence in situ hybridization (FISH) analysis of genes. Another exemplary application of a microfilter manufactured in accordance with exemplary aspects of the present invention (e.g. (i) standard microfilters, (ii) nanosurface topography microfilters, (iii) standard microfilters coated with analyte capture elements, and (iv) nanosurface microfilters coated with analyte capture elements) is collecting or enriching stromal cells, mesenchymal cells, endothelial cells, epithelial cells, stem cells, hematopoietic and non-hematopoietic cells, etc. from a blood sample, collecting tumor or pathogenic cells in urine, and collecting tumor cells in spinal and cerebral fluids. Another exemplary application is using the microfilter to collect tumor cells in spinal fluids. Another exemplary application is using the microfilter to capture analytes bound to latex beads or antigen caused particle agglutination whereby the analyte/latex bead or agglutinated clusters are captured on the membrane surface. Another exemplary application of a microfilter formed in accordance with exemplary aspects of the present invention (e.g. (i) standard microfilters, (ii) nanosurface topography microfilters, (iii) standard microfilters coated with analyte capture elements, and (iv) nanosurface microfilters coated with analyte capture elements) is for erythrocyte deformability testing. Red blood cells are highly flexible cells that will readily change their shape to pass through pores. In some diseases, such as sickle cell anemia, diabetes, sepsis, and some cardiovascular conditions, the cells become rigid and can no longer pass through small pores. Healthy red cells are typically 7.5 μm and will easily pass through a 3 μm pore membrane, whereas a cell with one of these disease states will not. In the deformability test, a microfilter having 5 μm apertures is used as a screening barrier. A blood sample is applied and the membrane is placed under a constant vacuum. The filtration rate of the cells is then measured, and a decreased rate of filtration suggests decreased deformability. Another exemplary application of a microfilter formed in accordance with exemplary aspects of the present invention (e.g. (i) standard microfilters, (ii) nanosurface topography microfilters, (iii) standard microfilters coated with analyte capture elements, and (iv) nanosurface microfilters coated with analyte capture elements) is leukocyte/Red blood cell separation. Blood cell populations enriched for leukocytes (white blood cells) are often desired for use in research or therapy. Typical sources of leukocytes include whole peripheral blood, leukopheresis or apheresis product, or other less common sources, such as umbilical cord blood. Red blood cells in blood can be lysed. Then the blood is caused to flow through the microfilter with small pores to keep the leukocytes. Another exemplary application is using the microfilter for chemotaxis applications. Membranes are used in the study of white blood cell reactions to toxins, to determine the natural immunity in whole blood. Since immunity is transferable, this assay is used in the development of vaccines and drugs on white blood cells. Another exemplary application is using the microfilter for blood filtration and/or blood transfusion. In such applications, microfilters can be used to remove large emboli, platelet aggregates, and other debris. | 27,250 |
11860158 | GENERAL It is to be understood that the foregoing general description as well as the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Further, the use of the term “including” as well as other grammatical forms such as “includes” and “included”, is not limiting. In the same sense, the use of the term “comprising” as well as other grammatical forms such as “comprises” and “comprised” is not limiting. Section headings throughout the description are for organizational purposes only. They are in particular not intended as limiting for the various embodiments described therein, and it is to be understood that elements and embodiments described under one subheading may be freely combined with elements and embodiments described under another subheading. In the foregoing, subsequent description the claims, the features of any one embodiment are intended as being combinable with those of any other embodiment. Such combinations of one or more features in any one embodiment with one or more features in any other embodiment belong to the disclosure of the present application as filed. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, monographs, books, treaties and regulations, are hereby expressly incorporated by reference in their entirety for any purpose. DETAILED DESCRIPTION OF THE INVENTION As mentioned above, one aspect of the invention relates to a method of unmasking an endotoxin in a composition, preferably a pharmaceutical composition, comprising an endotoxin masker and suspected of comprising said endotoxin, said method comprising the step of adding to said composition a modulator capable of unmasking said endotoxin, e.g. by releasing said endotoxin, if present, from a complex between said endotoxin and said endotoxin masker. The pharmaceutical composition will in most cases be an aqueous composition. A further aspect of the invention relates to a method of detecting an endotoxin in a composition, preferably a pharmaceutical composition, comprising an endotoxin masker and suspected of comprising said endotoxin, said method comprising the steps of: adding to said composition a modulator capable of unmasking said endotoxin, e.g. by releasing said endotoxin, if present, from a complex between said endotoxin and said endotoxin masker; and detecting said endotoxin by means of a detection method. The pharmaceutical composition will in most cases be an aqueous composition. Endotoxin The term “endotoxin” refers to a molecule produced on the surface of bacteria in particular gram-negative bacteria, that is bacteria which, because of their thin peptidoglycan layer sandwiched between an inner cell membrane and a bacterial outer membrane, do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation and therefore evade positive detection by this method. Specifically, endotoxins are biologically active substances present in the outer membrane of gram-negative bacteria. One common class of endotoxins is lipopolysaccharides (LPS). For the purposes of the present application, the terms “endotoxin” and “LPS” are used interchangeably. As is discussed elsewhere herein, however, it is understood that there exist different types of LPS, e.g. derived from different sources, and that the terms “endotoxin” and “LPS” are intended to encompass these different types of LPS. Endotoxins are located on the surface of bacteria and, together with proteins and phospholipids, form the outer bacterial membrane. Generally, LPS is made up of two parts with different chemical and physical properties; a hydrophilic sugar domain (the polysaccharide) and a hydrophobic lipid domain (lipid A). Two distinct regions can be recognized in the polysaccharide: the core oligosaccharide and the O-specific polysaccharide (M. A. Freudenberg, C. Galanos, Bacterial Lipopolysaccharides: Structure, Metabolism and Mechanisms of Action, Intern. Rev. Immunol. 6, 1990). The lipid A is highly hydrophobic and is the endotoxically active part of the molecule. Lipid A is typically composed of a beta-D-GlcN-(1-6)-alpha-D-GlcN disaccharide carrying two phosphoryl groups. Up to four acyl chains are attached to this structure. These chains can then in turn be substituted by further fatty acids, which can vary quite considerably between species in their nature, number, length, order and saturation. Covalently attached to the lipid A is the core section of the molecule which can itself be formally divided into inner and outer core. The inner core is proximal to the lipid A and contains unusual sugars like 3-deoxy-D-manno-octulosonic acid (KDO). The outer core extends further from the bacterial surface and is more likely to consist of more common sugars such as hexoses and hexosamines. Onto this is attached, in most cases, a polymer of repeating saccharide subunits called the O-polysaccharide, also typically composed of common sugars. This O-polysaccharide is not ubiquitous, however, as it is seen to be truncated or lacking in a number of Gram-negative strains. In addition, certain strains carry mutations in the otherwise well-conserved locus and are termed “rough mutants” to differentiate them from the wild-type “smooth” strains which express O-polysaccharide bearing LPS (C. Erridge, E. Bennett-Guerrero, I. Poxton, Structure and function of lipopolysaccharides, Microbes and Infection, 2002). Copious information relating to endotoxins, e.g. LPS, as well as their impact on health may be found in the book “Endotoxin in Health and Disease”, edited by Helmut Brade, Steven M. Opal, Stefanie N. Vogel and David C. Morrison, 1999, published by Marcel Dekker, Inc., ISBN 0-8247-1944-1. As mentioned above, endotoxin may derive from different bacterial sources. The chemical nature of endotoxin may vary slightly from source to source. For instance, endotoxins derived from different bacterial sources may differ slightly in the length of the aliphatic chains in the aliphatic amides and aliphatic acid esters of the lipid A domain. However, despite slight variations in endotoxin structure from source to source, the same basic structure as described herein above applies for most if not all endotoxins, implying a similar mode of action, and a correspondingly similar mode of influencing endotoxin behavior regardless of the bacterial species of origin. Examples of known endotoxins include those derived from e.g.E. coli, e.g.E. coli O55:B5 (such as available from Sigma as product number L2637-5MG) orE. coliK 12; S. abortusequi (such as available from Acila as product number 1220302);Klebsiella pneumonia; Morganella morganii; Yersinia enterocolitica; Serratia marcescens; Neisseria, e.g.Neisseria meningitis; Acinetobacter baumanni; Enterobacter cloacae, e.g. naturally occurring endotoxin (NOE);Pseudomonas, e.g.Pseudomonas aeruginosa; Salmonella, e.g.Salmonellaenteric;Shigella; Haemophilusinfluenza;Bordatella pertussis; andVibrio cholerae. It is to be understood that this list is merely exemplary and in no way restricts the term “endotoxin” as used herein. Endotoxin Masker The term “endotoxin masker” refers to a substance which, in solution with the endotoxin, renders the endotoxin undetectable by available detection methods, e.g. by limulus amebocyte lysate (LAL) tests. Typically, endotoxin is detectable when it exists in solution in aggregated form, i.e. in a form in which multiple, or least two endotoxin moieties are held together in spatial proximity by non-covalent interactions such as electrostatic interactions, hydrophobic interactions, Van der Waals interactions or any combination thereof. However, endotoxin becomes significantly less active (undetectable) as measured by common detection systems, i.e. is masked, when its active aggregation state is changed such that the endotoxin becomes solubilized as individual molecules of endotoxin. It can be assumed that discrete molecular entities of endotoxin are stabilized, for example, by detergents present in the solution. Such detergents are assumed to stabilize individual endotoxin moieties by forming detergent micelles in which the individual endotoxin moieties become embedded and are no longer capable of reacting with Factor C in commercially available endotoxin assays. Certain proteins may also effect or contribute to stabilization of endotoxin in undetectable soluble form. For instance, such proteins may present the endotoxin with binding clefts offering individual endotoxin molecules a suitable environment for stable binding, thereby breaking up otherwise detectable endotoxin aggregates and/or preventing the endotoxin molecules from interacting with Factor C in available endotoxin assays. It is assumed that at least two molecules of endotoxin, that is at least two molecules of LPS, must form an aggregate in order to be detectable by commercially available endotoxin tests such as the EndoLISA® test kit available from Hyglos GmbH and LAL-based tests. In fact, several publications show that endotoxin aggregates are significantly more biologically active than disaggregated endotoxins (M. Mueller, B. Lindner, S. Kusumoto, K. Fukase, A, B. Schromm, U. Seydel, Aggregates are the biologically acitve units of endotoxin, The Journal of biological Chemistry, 2004; A. Shnyra, K. Hultenby, A. Lindberg, Role of the physical state ofSalmonellaLipopolysaccharide in expression of biological and endotoxic properties, Infection and Immunity, 1993). Furthermore, the activation of Factor C, described by Tan et al. (N. S. Tan, M. L. P. NG, Y. H Yau, P. K. W. Chong, B Ho, J. L. Ding, Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides, The FASEB Journal, 2000), is indicated as a cooperative binding mechanism. Here, as mentioned above, at least two LPS molecules are required for activation of Factor C, which is the key factor in limulus based detection methods such as the EndoLISA kit available from Hyglos GmbH. Examples of endotoxin maskers which are detergents include anionic detergents, cationic detergents, nonionic detergents and amphoteric detergents, and any combination thereof. Examples of anionic detergents which may function as detergent endotoxin maskers in the sense of the invention include alkyl sulfates such as for example ammonium lauryl sulfate or sodium lauryl sulfate (SDS); alkyl-ether sulfates such as for example sodium laureth sulfate or sodium myreth sulfate; cholesterol sulfate; sulfonates such as for example dodecylbenzensulfonate, sodiumlauryl sulfoacetate or xylene sulfonate; alkyl sulfo succinates such as for example disodium lauryl sulfosuccinate; sulfoxides such as for example dodecyl methyl sulfoxide; phosphates such as for example trilaureth-4 phosphate; and carboxylates such as for example sodium stearate or sodium lauroyl sarcosinate. Examples of cationic detergents which may function as endotoxin maskers in the sense of the invention include primary amines; secondary amines; tertiary amines; and quaternary ammonium cations such as for example alkyltrimethylammonium salts (e.g. cetyl trimethylammonium bromide (CTAB) or cetyl trimethylammonium chloride (CTAC)); cetylpyridinium chloride (CPC); quaternary ammonium detergents such as for example tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate (Quaternium 52); and hydroxyethylcellulose ethoxylate, quaternized (Polyquaternium-10). Nonionic detergents which may function as detergent endotoxin maskers in the sense of the invention include polyoxyethylene glycol sorbitan alkyl esters (polysorbates) such as for example polysorbate 20 (Tween-20), polysorbate 40, polysorbate 60 or polysorbate 80 (Tween-80); polyoxyethylene glycol alkyl ethers; polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers; polyoxyethylene glycol octylphenol ethers; polyoxyethylene glycol alkylphenol ethers; glycerol alkyl esters; sorbitan alkyl esters; block copolymers of polyethylene glycol and polypropylene glycol; cocamide MEA; sterols such as for example cholesterol; cyclodextrans; poloxamers such as for example Pluronic block polymers (for example HO—(CH2CH2O)n/2—(CH2CH(CH3)O)m—(CH2CH2O)n/2—H, with n=200 and m=65 for F127 and n=4.5 and m=31 for F61) and cocamide DEA. Amphoteric detergents which may function as detergent endotoxin maskers in the sense of the invention include CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate); sultaines, such as for example cocamidopropyl hydroxysultaine; betaines, such as for example cocamidopropyl betaine; amine oxides such as for example palmitamine oxide, laurylamine oxide and amine oxide of general formula R3N+O−, wherein R3is C8-C18alkyl, C8-C18alkenyl or C8-C18alkynyl; and lecithin. Specifically, R3in the above general formula R3N+O−may be any of C8alkyl, C9alkyl, C10alkyl, C11alkyl, C12alkyl, C13alkyl, C14alkyl, C15alkyl, Cm alkyl, C17alkyl or C18alkyl; or C8alkenyl, C9alkenyl, C10alkenyl, C11alkenyl, C12alkenyl, C13alkenyl, C14alkenyl, C15alkenyl, C16alkenyl, C17alkenyl or C18alkenyl; or C8alkynyl, C9alkynyl, C10alkynyl, C11alkynyl, C12alkynyl, C13alkynyl, C14alkynyl, C15alkynyl, Cm alkynyl, C17alkynyl or C18alkynyl. Alternatively or in addition to any of the endotoxin maskers indicated above (alone or in combination), the endotoxin masker may also be an active pharmaceutical ingredient (API). This API may exist in solution together with or without any of the detergent endotoxin maskers indicated above. If the API exists together with a detergent endotoxin masker in solution, the masking effect may be more pronounced, and more stringent measures may be necessary to liberate masked endotoxin from the endotoxin masker, as is discussed in greater detail below. APIs which may especially engender or augment the masking of endotoxin present in the solution are protein APIs, for example an antibody; an antibody fragment; a hormone; an enzyme; a fusion protein; a protein conjugate; and any combination thereof. When the protein API is an antibody fragment, the antibody fragment may be preferably chosen from the group consisting of: Fab; a Fab′; a F(ab′)2; an Fv; a single chain antibody; and any combination thereof. When the protein API is an antibody, the antibody may be preferably chosen from the group consisting of: a fully human antibody; an anti-idiotype antibody; a humanized antibody; a bispecific antibody; a chimeric antibody; a CDR-grafted antibody; a monoclonal antibody; a polyclonal antibody; and any combination thereof. Alternatively or in addition to the above, the API may also be a small organic molecule. The skilled person understands what is meant by the term “small organic molecule” or “small molecule”. This is a molecule with a molecular weight of no more than 300 g/mol, 400 g/mol, 500 g/mol, 600 g/mol, 700 g/mol, 800 g/mol, 900 g/mol or, preferably, 1000 g/mol. Generally, an endotoxin masker, whether a detergent or a protein, will have the characteristic of shifting the equilibrium between solubilized and aggregated endotoxin in the direction of solubilized endotoxin which is not detectable by available endotoxin assays. It is this shifting of endotoxin into an undetectable form which is referred to as “masking” herein. As mentioned above, the form in which the endotoxin is solubilized may for example include endotoxin a) being embedded in the lipid layer of a micelle formed by a detergent; b) being bound on or in a protein, e.g. in a suitable binding cleft of appropriate steric and electrostatic environment formed on the surface of an active pharmaceutical agent, e.g. a protein; or c) participating in a combination of these two possibilities. Regardless of the form in which endotoxin is solubilized so as to energetically disfavor the aggregate form, however, the net effect is that individual molecules of endotoxin which would otherwise be aggregated and therefore detectable, are individually stabilized and, in this individualized (solubilized) form, become and remain undetectable, i.e. masked. Although undetectable, however, such stabilized endotoxin molecules in solution can nevertheless engender and/or contribute to the sorts of pyrogenic and/or toxic reactions outlined above when administered to subjects. This danger is especially acute in pharmaceutical formulations, since pharmaceutical formulations often contain a detergent to solubilize an API, e.g. a protein API, which, without the detergent, would be insoluble at the concentrations provided in the pharmaceutical formulation. In rendering the API, e.g. protein API, soluble by including detergent, then, one often unwittingly destroys the very aggregation of endotoxin which is needed for detection of this endotoxin. Thus, when the endotoxin masker is a detergent, the very measure employed to formulate the API, e.g. protein API, in acceptable form and concentration also has the potential to mask endotoxin in solution. As mentioned above the endotoxin masker may also be a protein, for instance the API itself. This scenario may arise in conjunction with the presence of a detergent endotoxin masker or, in the event that no detergent is present, may also arise in the absence of a detergent endotoxin masker. In this latter case, the API, in particular a protein API, may offer the endotoxin a sufficient environment for stable binding on or in such protein such that the endotoxin is masked by the API alone, i.e. without any detergent being necessary to mask endotoxin, rendering it undetectable. In the event that the endotoxin masker is a protein, this protein may be the API itself, or may alternatively or additionally be a protein in solution which is different from the API. Generally, any protein having an appropriate steric and electrostatic environment to stabilize individual molecules of endotoxin, for instance individual molecules of LPS could potentially effect or contribute to the masking of endotoxin. It is a hallmark of the invention that when the endotoxin masker is a protein, either alone or together with an additional endotoxin masker such as e.g. a detergent endotoxin masker, unmasking the endotoxin leaves the protein endotoxin masker chemically unaltered following unmasking. In particular, unmasking the endotoxin does not cleave or otherwise degrade the protein endotoxin masker (e.g. protein API). In scenarios of the type described above, individual molecules of endotoxin which would otherwise remain in aggregates and therefore detectable, are stabilized at one or more such surface locations on or in said protein. As is the case for detergent micelles, such stabilization shifts the equilibrium existing between solubilized (undetectable) and aggregated (detectable) in the direction of solubilized (undetectable) endotoxin. As mentioned above, one may imagine such a shift of equilibrium toward the solubilized (undetectable) form as being especially pronounced in the event that a solution comprises both detergent and one or more proteins with the above characteristics, since in such cases the stabilization of individual molecules of endotoxin out of its aggregate form by the endotoxin masker may ensue both in the form of stabilization in micelles as well as on the surface of proteins. In such scenarios, more stringent measures may be required to shift said equilibrium toward the aggregate endotoxin form which is then detectable. These are discussed in more detail in the context of illustrative scenarios further below (FIGS.1-6). Modulator The term “modulator” as used herein refers to one or more compounds which, alone or in concert, render(s) a masked endotoxin susceptible to detection by an endotoxin assay (such as the EndoLISA® detection assay available from Hyglos GmbH). The term “modulator” as used herein may encompass both single as well as multiple components which achieve this end. In some instances herein below, reference is made to a “modulator system”, although the term “modulator” is sometimes used to designate multiple modulator substances which are intended to work in concert. This refers to a multi-component modulator comprising multiple substances which act in concert to render a masked endotoxin detectable by an endotoxin assay. The different components of a modulator system may be incorporated for different reasons, i.e. to take advantage of different functions of modulator substances which affect the stability of a complex between endotoxin and endotoxin masker in different ways. For ease of reference, one may for example refer to different kinds of modulator which may be employed alone or together to unmask endotoxin:“Disrupting modulator”: A “disrupting modulator” is a modulator which completely or partially breaks up a complex between an endotoxin masker and an endotoxin. When the endotoxin masker is a detergent, and the endotoxin is masked in solubilized form inserted in the lipid layer of a masking detergent micelle, then a modulator which disrupts such a detergent micelle so as to liberate the endotoxin would be referred to as a disrupting modulator. As discussed in greater detail below, 1-dodecanol is one such disrupting modulator. A disrupting modulator, for example 1-dodecanol, 1-decanoic acid or sodium octyl sulfate (SOS) may advantageously be used in a concentration range of 0.01-100 mM, preferably in a concentration range of 0.1-10 mM, preferably at a concentration of 10 mM in the unmasking process. In some cases, the disrupting modulator may also simultaneously function as a reconfiguring modulator, described below.“Adsorbing modulator”: An “adsorbing modulator” is a modulator which has the ability to bind substances which would otherwise stabilize the endotoxin in solubilized and therefore non-detectable form. For instance, when the endotoxin masker is a detergent as e.g. contained in some pharmaceutical compositions, then a modulator which binds molecules of the detergent and in this way contributes to the breakdown of endotoxin-stabilizing micelles would be referred to as an adsorbing modulator. As discussed in greater detail below, BSA is one such adsorbing modulator. An adsorbing modulator, for example BSA may advantageously be used in a concentration range of 0.1-20 mg/mL, preferably in a concentration range of 1-10 mg/mL, preferably at a concentration of 10 mg/ml in the unmasking process.“Displacing modulator”: A “displacing modulator” is a modulator which has the ability to completely or partially displace a molecule of endotoxin from its stable binding position in or on an endotoxin masker. For instance, when the endotoxin masker is a protein, and the endotoxin is bound in or on a protein which stabilizes the endotoxin in undetectable form, then a modulator which has the ability to replace the endotoxin in or on the protein, e.g. by means of hydrophobic interactions, would be referred to as a displacing modulator. As discussed in greater detail below, SDS is one such displacing modulator. A displacing modulator, for example SDS, may advantageously be used in a concentration range of 0.01-1 wt %, preferably in a concentration range of 0.05-0.5 wt %, preferably in a concentration range of 0.02-0.2 wt %, preferably at a concentration of 0.1 wt % in the unmasking process.“Reconfiguring modulator”: A “reconfiguring modulator” is a modulator which has the ability to transiently stabilize endotoxin following its liberation from the endotoxin masker (e.g. by a disrupting modulator or displacing modulator, as discussed above), thus helping the liberated, solubilized (undetectable) endotoxin to adopt an aggregated (detectable) form. With the help of the reconfiguring modulator, solubilized endotoxin becomes reconfigured as aggregated endotoxin. As discussed in greater detail below, 1-dodecanol is one such reconfiguring modulator. A reconfiguring modulator, for example 1-dodecanol, 1-decanoic acid or sodium octyl sulfate (SOS) may advantageously be used in a concentration range of 0.01-100 mM, preferably in a concentration range of 0.1-10 mM in the unmasking process. In some cases, the reconfiguring modulator may also simultaneously function as a disrupting modulator, described above. As will become clear herein below, the above types of modulator are not mutually exclusive; that is, it is possible for a given substance to have functionality as different kinds of modulators above. One example is 1-dodecanol, which may be classified as both a disrupting modulator (breaking up a detergent micelle) as well as a reconfiguring modulator (transiently stabilizing the micelle-liberated endotoxin so it can aggregate and become detectable). Similarly SDS may be classified as a disrupting modulator (breaking up existing micelles of another, non-SDS detergent) and a displacing modulator (liberating endotoxin from binding sites in or on any masking protein which may be present). The classification as to the type of modulator depends on the function that a substance in question plays in a particular composition. However, since it is assumed that reconfiguring of the endotoxin from solubilized into aggregated form will generally be required in order to render the endotoxin detectable, the modulator will normally comprise at least one component qualifying as a “reconfiguring modulator”. As a further example, a substance which functions as a “displacing modulator” when the endotoxin masker is a protein may in some cases function as a “disrupting modulator” when the endotoxin masker is a detergent. SDS is one example of such a substance, the classification of which as to the type of modulator component depends on the prevailing conditions. For instance, when the endotoxin masker is a protein, SDS will generally function as a displacing modulator, since it helps to displace the endotoxin bound in or on the masking protein. However, when the endotoxin masker is a detergent, then SDS, alone or together with another modulator component, may function more as a disrupting modulator, since in this case it promotes the liberation of endotoxin from the lipid layer of detergent micelles by disrupting the micelles. A modulator may contain one or more substances within the above classifications. For instance, a single component modulator may comprise only a disrupting modulator such as 1-dodecanol. A dual-component modulator may comprise a mixture of a disrupting modulator such as 1-dodecanol (also possibly functioning as a reconfiguring modulator) and, depending on the nature of the masking complex between endotoxin and endotoxin masker, one of an adsorbing modulator such as BSA or a displacing modulator such as SDS. A multi-component modulator may comprise a mixture of a disrupting modulator such as 1-dodecanol (also possibly functioning as a reconfiguring modulator) and, depending on the nature of the masking complex between endotoxin and endotoxin masker, one each of an adsorbing modulator such as BSA and a displacing modulator such as SDS. As will be discussed in detail below, the complexity of the modulator system chosen will depend on the nature of the complex between endotoxin and endotoxin masker, and the surrounding solution conditions which contribute to the stability of that complex. From the above, it is clear that each new composition to be analyzed for the presence of endotoxin may require its own customized modulator composition in order to render the masked endotoxin susceptible to detection. The identification of a suitable modulator for a given composition or formulation to be tested can however be accomplished by routine experimentation, as will be shown further below. As mentioned above, in its most general sense, the modulator is assumed to destabilize a complex between endotoxin and an endotoxin masker and to promote the liberation of the endotoxin from the endotoxin masker. In this way, the modulator or modulator system effectively shifts the equilibrium from a solubilized (undetectable) state toward an aggregated (detectable) state. The present inventors have surprisingly found that endotoxin which is present but undetectable in solution remains undetectable because, as assumed, the endotoxin remains stably solubilized in detergent micelles and/or bound to surface structures of proteins present in the solution. Individually stabilized in this form, the endotoxin molecules evade detection. However, the present inventors have found that solution conditions can be manipulated such that solubilized endotoxin is rendered into a form which can be detected. In some instances, multiple manipulations of solution conditions may be required to reach this end and the stringency of the measure or measures taken to effect the desired shift in equilibrium toward an aggregated state will vary depending on the degree to which the endotoxin masker stabilizes the endotoxin in solubilized form, as mentioned above. But generally, the manipulations performed in accordance with the invention as described herein should be understood in the context of the overall aim of shifting the equilibrium of endotoxin from a solubilized state to an aggregated state such that it can be detected. In order to accomplish the above, the “modulator” will generally include an amphiphilic molecule which competes for binding between the lipid component of endotoxin and the endotoxin masker, thus weakening the interaction between the former and the latter. Such competitive binding will generally be accomplished by providing at least one component of the modulator system in a form which is structurally similar to the (amphiphilic) lipid component of the endotoxin such that the former may displace the latter in its stabilized interaction with the endotoxin masker. For instance, in the event the endotoxin masker is a detergent, a suitable disrupting modulator will generally include an amphiphilic compound capable of stably inserting i.e. between the amphiphilic detergent molecules and the similarly amphiphilic lipid portion of the endotoxin. When the endotoxin masker is a detergent, an amphiphilic disrupting modulator will therefore elicit several effects in parallel which are conducive to an overall shift in equilibrium from a solubilized toward an aggregated form of endotoxin. First, providing a modulator system comprising at least one amphiphilic disrupting modulator disrupts the lipophilic interactions underlying the detergent micelles such that these micelles are broken up. Since endotoxin was previously solubilized (and therefore masked) by insertion of its lipid component into the lipid layer of the detergent micelles, the breakup of the micelles removes this stabilizing force and results in the liberation of previously embedded endotoxin. The role of the disrupting modulator in the event that the endotoxin masker is or includes a detergent is thus to break up detergent micelles. Further, the amphiphilic character of the disrupting modulator may also enable it to associate with the lipid component of the endotoxin, once the endotoxin is liberated from its detergent micelles as described above. This interaction between the amphiphilic disrupting modulator and the lipid component of the endotoxin has the effect of chaperoning the endotoxin in aqueous solution following its liberation from the stabilizing detergent micelles. In this event, the disrupting modulator would have a double function as a reconfiguring modulator. When the disrupting modulator is amphiphilic in character, it is not excluded that it may be capable for forming micelles of its own. However, the unmasking effect will generally be greatest when the amphiphilic disrupting modulator does not form micelles of its own which might simply swap one solubilized and therefore masked endotoxin state for another. A key role of the reconfiguring modulator is thus to transiently stabilize liberated endotoxin (albeit less than in its previous complex with the endotoxin masker), effectively chaperoning the endotoxin into an aggregated and therefore detectable state. Thus temporarily chaperoned in solution, the liberated endotoxin is then free to aggregate into a form which is detectable and therefore “unmasked”. Whether or not further manipulation of solution conditions beyond addition of the modulator or modulator system is necessary to shift equilibrium towards this aggregated, detectable form will generally depend on the conditions prevailing in solution and the initial stability of the endotoxin as complexed with the endotoxin masker. In another scenario already contemplated above, the endotoxin masker is not, or not only a detergent, but may also be or comprise a protein with binding clefts on its surface suitable to stably bind individual moieties of endotoxin such that it cannot be detected. In this event, similar considerations pertaining to the modulator apply as set out above. For instance, use of a disrupting (amphiphilic) modulator and/or a displacing modulator in the event that the endotoxin is or comprises a protein has the effect that the modulator disrupts the lipophilic interactions existing between lipophilic amino acid side chains of the protein (endotoxin masker) and the lipid component of the endotoxin. Because the disrupting modulator and/or displacing modulator is/are likely to be amphiphilic in character, the modulator(s) would also be able to disrupt electrostatic interactions existing between polar and/or ionized side chains within the protein (endotoxin masker) and polar groups within the core and/or O-antigen polysaccharide regions of the endotoxin. With these stabilizing interactions disrupted, the endotoxin which was previously masked by a protein endotoxin masker is thus displaced from its previous complex with the protein, and is chaperoned in solution into an aggregated state by association with a reconfiguration modulator as described above. As described above for the case in which the endotoxin masker is a detergent in the absence of a protein endotoxin masker, the liberated and reconfiguration modulator-chaperoned endotoxin is then free to aggregate into a form which is detectable and therefore “unmasked”. Whether or not further manipulation of solution conditions beyond addition of the components of the modulator system is necessary to shift equilibrium towards this aggregated, detectable endotoxin form will generally depend on the conditions prevailing in solution and the initial stability of the endotoxin as complexed with the endotoxin masker. The modulator, e.g. the disrupting modulator, the displacing modulator and/or the reconfiguring modulator may in certain embodiments comprise a first heteroatom-substituted aliphatic, wherein the main chain of the first heteroatom-substituted aliphatic comprises 8 to 16 carbon atoms. As used herein, the term “main chain” refers to the longest chain of the first heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms, as numbered by standard IUPAC nomenclature. Specifically, the main chain of the first heteroatom-substituted aliphatic may comprise 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms. As used herein, the term “heteroatom” refers to any atom other than carbon, to which a carbon atom in the first heteroatom-substituted aliphatic is covalently bound. Representative heteroatoms include oxygen, nitrogen and sulfur atoms. In a further preferred embodiment, the oxygen-substituted aliphatic is an aliphatic alcohol, in particular, 1-dodecanol, that is the molecule given by the formula HO—(CH2)11—CH3. As mentioned above, 1-dodecanol is especially well suited in many instances as a disrupting modulator as well as, in most if not all instances, as a reconfiguring modulator. The reconfiguring modulator is assumed to play an especially important, if not indispensible role in promoting an aggregated, detectable form of endotoxin. The reconfiguring modulator may be a heteroatom-substituted aliphatic comprising 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms in its main chain. The term “main chain” refers to the longest chain of the reconfiguring modulator, as numbered by standard IUPAC nomenclature. As used herein, the term “heteroatom” refers to any atom other than carbon, to which a carbon atom in the first heteroatom-substituted aliphatic is covalently bound. Representative heteroatoms include oxygen, nitrogen and sulfur atoms. It is especially suitable when the heteroatom is oxygen. Furthermore, the reconfiguring modulator may be branched or unbranched, with the branched variants comprising substitutions along the “main chain” as defined above. Said substitutions may be e.g. methyl, ethyl, propyl and/or butyl. An unbranched chain is preferred. The reconfiguring modulator may be saturated to various extents, and may for example comprise a C8alkyl, C9alkyl, C10alkyl, C11alkyl, C12alkyl, C13alkyl, C14alkyl, C15alkyl or C16alkyl moiety; or a C8alkenyl, C9alkenyl, C10alkenyl, C11alkenyl, C12alkenyl, C13alkenyl, C14alkenyl, C15alkenyl or C16alkenyl moiety; or a C8alkynyl, C9alkynyl, C10alkynyl, C11alkynyl, C12alkynyl, C13alkynyl, C14alkynyl, C15alkynyl or C16alkynyl moiety. Furthermore, the reconfiguring modulator may contain any mixture of single, double and triple carbon-carbon bonds. Especially suitable reconfiguring modulators are saturated, i.e. comprise C8alkyl, C9alkyl, C10alkyl, C11alkyl, C12alkyl, C13alkyl, C14alkyl, C15alkyl or C16alkyl. Especially suitable reconfiguring modulators comprise C12alkyl. Furthermore, the heteroatom may be of various oxidation states. For instance, when the heteroatom is oxygen, the oxygen may be in the form of an alcohol, an aldehyde or a carboxylic acid. Especially suitable as reconfiguring modulators are molecules in unbranched alkanols, in particular unbranched 1-alkanols. Among these, especially suitable are C12alkanols, especially 1-dodecanol having the formula HO—(CH2)11—CH3. In further embodiments, the modulator system may include other components in addition to said first heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms. For example, the modulator system may additionally comprise a second heteroatom-substituted aliphatic, e.g. as a disrupting modulator, a displacing modulator and/or a reconfiguring modulator, wherein the main chain of said second heteroatom-substituted aliphatic comprises 8 to 16 carbon atoms. The “main chain” of the second heteroatom-substituted aliphatic is defined as described above for the first heteroatom-substituted aliphatic. Specifically, the main chain of the second heteroatom-substituted aliphatic may comprise 8, 9, 10, 11, 12, 13, 14, 15 or 16 carbon atoms. The first heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms is different than the second heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms. In a preferred embodiment, the second heteroatom-substituted aliphatic which may be part of the modulator is an oxygen-substituted aliphatic. In certain preferred embodiments, this oxygen-substituted aliphatic is an aliphatic sulfate, wherein it is especially preferred that this aliphatic sulfate is sodium dodecyl sulfate (SDS). Thus, in a particularly preferred embodiment of the invention, the modulator system includes a first heteroatom-substituted aliphatic which is 1-dodecanol (e.g. as a disrupting modulator and/or a reconfiguring modulator), and a second heteroatom-substituted aliphatic which is SDS (as a further disrupting modulator and/or a displacing modulator). In a further embodiment, the modulator system as described above may further comprise a protein capable of binding a detergent so as to break up micelles formed by said detergent. Generally, the detergent bound will be the endotoxin masker (when said endotoxin masker is or comprises a detergent), and the principle by which the protein capable of binding a detergent binds the detergent is analogous to the principle described above, according to which a protein which functions as an endotoxin masker sequesters portions of the endotoxin molecule in or on its surface. In the present embodiment, the protein capable of binding a detergent, when used as part of the modulator, also bears on its surface areas of steric and electrostatic compatibility with a portion or portions of detergent molecules present in solution, which are sufficient to bind or sequester detergent molecules, thus rendering them unavailable for participation in micelles and thus breaking up any detergent micelles which may be harbor endotoxin, or which may serve to shift equilibrium away from an aggregated form of endotoxin. The inventors have found that albumin molecules are exceptionally good at binding detergent. Thus, it is contemplated that in addition to the first heteroatom-substituted aliphatic alone, or in addition to the first heteroatom-substituted aliphatic in combination with the second heteroatom-substituted aliphatic, the modulator may additionally comprise a protein (adsorbing modulator) capable of binding a detergent so as to break up micelles formed by said detergent. In certain embodiments, the protein component of the modulator may be an albumin, preferably human serum albumin (HSA), bovine serum albumin (BSA) or ovalbumin (OVA). It is additionally contemplated that the modulator may contain one or more of each of the first heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms, the said second heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms and said protein capable of binding a detergent so as to break up micelles formed by said detergent. In a preferred embodiment of the invention, the modulator comprises 1-dodecanol alone. In a further preferred embodiment of the invention, the modulator comprises 1-dodecanol and SDS. In a further preferred embodiment of the invention, the modulator comprises 1-dodecanol, SDS and HSA. In a further preferred embodiment of the invention, the modulator comprises 1-dodecanol, SDS and BSA. Composition As used herein, the term “composition” refers to a mixture comprising (at least) an endotoxin masker. The endotoxin, even if present and masked, remains undetectable in the composition. The composition is preferably a pharmaceutical composition, e.g. a composition comprising an active pharmaceutical ingredient, or API. The term “composition” may be e.g. an extract; vaccine; any composition suitable for parenteral administration, i.e. parentalia; any composition suitable for intraperitoneal, transdermal, subcutaneous or topical administration; a blood product; a cell therapy solution, e.g. intact, living cells, for example, T cells capable of fighting cancer cells; a gene therapy solution, e.g. a solution capable of nucleic acid polymer delivery into a patient's cells as a drug to treat disease; an implant or medical device; or a composition resulting from rinsing or wiping the surface of an object, said object for instance being a medical device, an implant or a filling machine. Detection Method As used herein, the term “detection method” refers to a method which is suitable for detecting endotoxin in solution. For example, suitable methods in this regard are limulus based detection methods, or is an enzyme linked immunosorbent assays (ELISA). The limulus methods can be performed classically by using natural derived lysate (J. Jorgensen, R. Smith, Perparation, Sensitivity, and Specificity of Limulus Lysate for Endotoxin Assay, Applied Microbiology, 1973) or recombinantly prepared Factor C (J. L. Ding, B. Ho, A new era in pyrogen testing, Trends in Biotechnology, 2001). The most promising of such methods are enzyme-linked affinitysorbent assays, using a solid phase for endotoxin capturing and subsequent detection by recombinant version of a protein in the LAL assay, Factor C. The EndoLISA® kit is one such affinitysorbent assay (H. Grallert, S. Leopoldseder, M. Schuett, P. Kurze, B. Buchberger, EndoLISA®: a novel and reliable method for endotoxin detection, Nature Methods, 2011). The EndoLISA® detection system is for example described in the book “Pharmazeutische Mikrobiologie-Qualitätssicherung, Monitoring, Betriebshygiene” by Michael Rieth, October 2012, Wiley-VCH, Weinheim, ISBN 978-3-527-33087-4. Agent which Influences Hydrogen Bonding Stability in Solution According to a further embodiment of the invention, the above methods of unmasking an endotoxin and/or the method of detecting an endotoxin may further comprise the step of adding to said composition an agent which influences hydrogen bonding stability in solution. Generally, as used herein, an agent which influences hydrogen bonding stability in solution modifies solution conditions so as to destabilize the complex in which an individual molecule or multiple molecules of endotoxin is/are solubilized and therefore masked. Not all complexes between endotoxin and endotoxin masker are the same. In particular, the energy minima governing endotoxin stabilization in certain masking complexes are different than those governing endotoxin stabilization in other masking complexes. All other things being equal, the lower an energy minimum governing the stabilization of endotoxin in a given complex with an endotoxin masker is, the more difficult it will be, i.e. the more stringent the modulator must be, to liberate endotoxin from its solubilized state. Yet as mentioned above, such liberation is an important step in the eventual aggregation of endotoxin into a detectable, i.e. unmasked, form. Thus, the more stable the complex between endotoxin and endotoxin masker, the more rigorous must be the measures taken to ultimately unmask the endotoxin. In instances where the complex between endotoxin and endotoxin masker is especially stable, addition of a single- or even multiple-component modulator may sometimes not be enough to destabilize the masking complex and liberate the endotoxin. It may in such instances be helpful to promote endotoxin liberation from its complex with endotoxin masker by adjusting solution conditions so as to destabilize the endotoxin-endotoxin masker complex. As mentioned above, an agent which influences hydrogen bonding stability in solution may assist in this aim. Some, if not most of the stabilization of endotoxin in complex with an endotoxin masker normally arises from non-covalent interactions between the endotoxin moiety and the endotoxin masker. These interactions may for instance take the form of hydrophobic, ionic, hydrogen bonding and/or Van der Waals interactions between regions of the endotoxin molecule and regions on the molecule or molecules of the endotoxin masker. As the strength of these endotoxin-endotoxin masker interactions is influenced by the surrounding hydrogen bonding network in solution, it conversely follows that influencing the hydrogen bonding stability in solution will modulate the strength of these interactions. Addition of an agent which influences hydrogen bonding stability in solution can therefore help to weaken the noncovalent bonding interactions between endotoxin and endotoxin masker, essentially raising the free energy of the complex and thus rendering it more susceptible to disruption by the modulator so that the endotoxin is liberated and rendered detectable. Besides the destabilizing effect discussed above, an agent which influences hydrogen bonding stability in solution may also have a further effect promoting endotoxin unmasking. By altering hydrogen bonding stability in solution, the agent may also foster aggregation of the endotoxin moieties once liberated from their complex with endotoxin masker. There will generally exist an equilibrium between endotoxin in solubilized and aggregated forms. The agent which influences hydrogen bonding stability in solution can be helpful in shifting this equilibrium towards the aggregated (and thus detectable). Suitable substances are those which would tend to decrease the hydrogen bonding stability in solution surrounding the chaperoned endotoxin moities, and/or compounds which tend to increase the ionic strength of the solution, thus driving the reconfiguring modulator-chaperoned endotoxin moieties together into a lipophilic aggregate. It should be noted that it may not always be necessary to add an agent which influences hydrogen bonding stability in solution. Whether or not addition of such an agent will be indicated will depend, for instance, on the stability of endotoxin in complex with the endotoxin masker and/or on the position of equilibrium between solubilized, chaperoned and aggregated forms of endotoxin moieties once liberated from the endotoxin masker. For instance, in solutions containing higher concentrations of salt, it is conceivable that the complex of the endotoxin and endotoxin masker may already be instable enough to be broken up by the disrupting modulator alone, and that the endotoxin moieties present in solution following liberation from the endotoxin masker will be instable enough so as to form aggregates without any further assistance. In such situations, an agent which influences hydrogen bonding stability in solution may not be required to achieve unmasking. On the other hand, there may exist situations, for instance in solutions containing lower concentrations of salt, where the endotoxin-endotoxin masker complex may be of such great stability that a disrupting modulator alone cannot break it up to liberate endotoxin, or where—even if liberated by disrupting modulator alone—the equilibrium between solubilized and aggregated endotoxin lies towards the solubilized form so that the aggregation needed for detection does not occur. In such situations incorporation of an agent which influences hydrogen bonding stability in solution may help to influence the energetics of complexation and/or aggregation so as to favor endotoxin in detectable form. In general, it can be said that the degree of destabilization of the complex between the endotoxin and endotoxin masker will depend on the amount of salt in solution, with this complex being destabilized to an extent directly proportional to the amount of salt present in solution. As a general rule though, reference may be made to the Hofmeister series, according to which the more chaotropic a salt is, the lower the amount of such a salt will be needed to destabilize a complex between endotoxin and endotoxin masker to a given extent. Merely as an illustrative example, in order to achieve approximately the same degree of destabilization of a complex between endotoxin and endotoxin masker achievable with, say, 100 mM CaCl2, one may need to use, say, 500 mM NaCl. In this example, CaCl2is more chaotropic than NaCl, so less CaCl2would be required to achieve the same degree of destabilization. In certain embodiments of the invention, the agent which influences hydrogen bonding stability in solution may be a chaotropic agent, a cation or a combination thereof. In certain embodiments, the chaotropic agent may be chosen from the group consisting of urea, guanidinium chloride, butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, a propanol (e.g. 1-propanol or 2-propanol, i.e. isopropanol) and thiourea. In certain embodiments, the cation is a divalent cation, for example Ca2+, Mg2+, Sr2+and/or Zn2+. An especially preferred divalent cation is Ca2+. The agent which influences hydrogen bonding stability in solution, e.g. a divalent cationic salt, e.g. CaCl2, may advantageously be used in a concentration range of 1-400 mM, preferably in a concentration range of 10-200 mM, preferably at a concentration range of 50-100 mM in the unmasking process. The agent which influences hydrogen bonding stability in solution, e.g. a chotropic agent, e.g. guanidinium chloride, may advantageously be used in a concentration range of 1 mM-1 M, preferably in a concentration range of 25-200 mM, preferably at a concentration range of 10-100 mM in the unmasking process. Without being bound by theory, and merely to illustrate the principles and possible mechanisms which the present inventors believe underlie the observed advantageous effect of unmasking endotoxin in solution, thereby rendering previously masked an undetectable endotoxin detectable, the following describes several mechanisms of interaction between endotoxin and further components of a given composition containing at least one endotoxin masker. To illustrate these mechanisms, reference is made toFIGS.1-6. Unmasking Endotoxin Masked by a Detergent Masker with a Single-Component Modulator, in which the Single Component Functions as Both a Disrupting Modulator and a Reconfiguring Modulator FIG.1depicts the scenario in which endotoxin resides in solution together with a detergent which is masking it in individualized form in a detergent micelle. Panel (a) ofFIG.1shows a single endotoxin moiety which is inserted in the lipid layer of such a detergent micelle via its lipid tail. The detergent molecules constituting the lipid layer of the detergent micelle are symbolized as open circles in panel (a). Because this single moiety of endotoxin is stably inserted in individual form in the lipid layer of the micelle rather than in multimeric, aggregated form, it evades detection using available detection methods (e.g. the EndoLISA® assay of Hyglos GmbH). If the solution shown in panel (a) ofFIG.1were a pharmaceutical formulation additionally containing an API, it would appear to be endotoxin-free and therefore safe for administration, even though endotoxin is present in the solution. Administering such an ostensibly endotoxin-free formulation to a patient would thus risk unwittingly eliciting the types of dangerous immunological and toxic responses to endotoxin mentioned above. Above the equilibrium arrows between panels (a) and (b) ofFIG.1, one sees the addition of a disrupting and reconfiguring modulator capable of releasing the endotoxin from a complex between the endotoxin and the endotoxin masker. In the scenario shown inFIG.1, this “complex” is the endotoxin embedded, via its lipid component, in the lipid layer of a detergent micelle. The disrupting and reconfiguring modulator shown here (an amphiphilic molecule used as a single-component modulator having capacity as both a disrupting and reconfiguring modulator) exhibits the dual properties of breaking up the detergent micelle so as to liberate inserted molecules of endotoxin, as well as of stabilizing the endotoxin once it is liberated from its complex with the endotoxin masker. This latter characteristic is schematically depicted in the upper portion of panel (b) ofFIG.1, showing a molecule of endotoxin stabilized by the disrupting and reconfiguring modulator such that the molecule of endotoxin can exist in chaperoned form outside of the micelles once these are broken up by the modulator. The lower portion of panel (b) makes clear that the disrupting modulator exists in equilibrium, associated with both the liberated endotoxin moiety and detergent previously making up the lipid layer of the detergent micelle prior to the micelles disruption by the disrupting (and reconfiguring) modulator. As mentioned above, in one embodiment of the present invention, the disrupting and/or reconfiguring modulator may be 1-dodecanol, bearing a polar alcohol moiety, followed by a saturated hydrocarbon tail of 12 carbon atoms. Both the steric and electrostatic configuration of 1-dodecanol is thus similar to that of the lipid moieties of the endotoxin, so that 1-dodecanol can efficiently interact with, and therefore stabilize, the endotoxin after it has been liberated from the detergent micelle. Another reason why 1-dodecanol is especially suitable for use as a disrupting and/or reconfiguring modulator is that 1-dodecanol, although amphiphilic, does not form micelles. Thus, once the detergent micelle depicted in panel (a) ofFIG.1is broken up by 1-dodecanol, new micelles of modulator do not reform, which might otherwise remask endotoxin by shifting equilibrium away from its aggregated form. The characteristic of the modulator that it does not form micelles itself thus contributes to the stabilization of endotoxin in solution, aided by the modulator, as depicted in panel (b) ofFIG.1. In the scenario depicted inFIG.1, the hypothetical prevailing solution conditions are such that equilibrium between the chaperoned moieties of endotoxin shown in panel (b) and the aggregated endotoxin shown in panel (c) already lies in the direction of the aggregate of panel (c). With the aggregate form of endotoxin favored, the endotoxin is already in, or predominantly in an aggregated form which is amenable to detection by known means, e.g. the EndoLISA® test kit of Hyglos GmbH. Overall, then,FIG.1shows the transition from individual endotoxin moieties (solubilized) which are stably inserted in and therefore masked by detergent micelles to a scenario in which the individual moieties of endotoxin have aggregated so as to become detectable. Previously masked endotoxin in panel (a) has been unmasked in panel (c), thereby allowing one to determine that a solution previously thought to be free of endotoxin actually contains this contaminant. Unmasking Endotoxin Masked by a Detergent Masker with a Dual-Component Modulator Comprising a Disrupting and Reconfiguring Modulator and an Adsorbing Modulator (Protein) The initial scenario depicted inFIG.2is much like that depicted inFIG.1: a single molecule of endotoxin is inserted in a detergent micelle (symbolized by a ring of open circles representing the individual detergent molecules) and, thus stably individualized, is masked such that it evades detection. Between panels (a) and (b), one sees the addition of a dual-component modulator system comprising both a non-protein component functioning simultaneously as a disrupting and reconfiguring modulator and a protein component functioning as an adsorbing modulator. The disrupting and reconfiguring modulator may be as described as above forFIG.1, e.g. 1-dodecanol, which helps to disrupt the detergent micelle and stabilize/reconfigure the liberated endotoxin, without forming micelles of its own. The adsorbing modulator may for example be added as part of the modulator in order to promote the disruption of detergent micelles which are more stable than those depicted inFIG.1, and for which a disruption modulator alone may not suffice to achieve the desired disruption. As explained above, the adsorbing modulator may for instance be bovine serum albumin (BSA) or human serum albumin (HSA), among other things. Such proteins have the ability to act as “molecular sponges” which adsorb on their surface molecules of the previously micelle-forming detergent. Of course, in the event that such an adsorbing modulator is employed, there will exist a certain equilibrium between other detergent-like molecules in solution, such as the disrupting and reconfiguring modulator. This would be expected to engender an equilibrium as shown in panel (b), in which the disrupting and reconfiguring modulator exists in forms bound to liberated endotoxin (right portion of panel (b)), bound to detergent previously constituting the detergent micelle, as well as bound to the surface of the adsorbing modulator, along with additional detergent from the (now disrupted) detergent micelle. Under the solution conditions prevailing in the scenario shown inFIG.2, endotoxin which has been liberated from the masking detergent micelle combine into detectable aggregates, shown in panel (c). In fact, the use of an adsorbing modulator as shown inFIG.2can promote such aggregate formation. This is assumed to be because the adsorbing modulator binds molecules of the disrupting and reconfiguring modulator on its surface, thereby removing these otherwise endotoxin-stabilizing species from solution such that equilibrium is driven to the right toward the aggregate of panel (c). Overall, then,FIG.2shows the transition from individual endotoxin moieties which are embedded in detergent micelles and, due to their individualization in these micelles, remain masked, to a scenario in which the individual moieties of endotoxin have been forced to aggregate so as to become detectable. That is, previously masked endotoxin in panel (a) has been unmasked in panel (c), thereby allowing one to determine that a solution previously thought (in panel (a)) to be free of endotoxin actually contains this contaminant (panel (c)). Unmasking Endotoxin Masked by a Detergent Masker with a Multi-Component Modulator System in Combination with an Agent which Influences Hydrogen Bonding Stability in Solution In the scenarios depicted inFIGS.1and2, the solution conditions were such that use of a modulator system alone suffices to disrupt masking detergent micelles. Looked at another way, neither of the masking micelles of detergent shown inFIGS.1and2have been so stable as to resist disruption using a disrupting modulator alone. In addition, the conditions inFIGS.1and2have also been such that the equilibria between the solubilized and aggregated forms of endotoxin lay toward the aggregated form, so that detection of this aggregated form was possible under the solution conditions shown without any further measures needing to be taken. The conditions underlying the scenario shown inFIG.3are now different. Here, individual molecules of endotoxin are inserted in the lipid layer of detergent micelles (again symbolized by a ring of open circles representing the individual detergent molecules), but whether due to solution conditions, the nature of the interaction of the masking detergent with the endotoxin, or a combination of these things, the endotoxin inserted in the detergent micelle in panel (a) is more stable, and therefore less resistant to disruption with disrupting modulator, than either of the initial situations inFIGS.1and2. Additional measures are required to destabilize the detergent-endotoxin complex so that, once destabilized, the modulator system can disrupt the micelle and liberate the inserted endotoxin. To this end, the scenario shown inFIG.3entails using an agent which influences hydrogen bonding stability in solution, symbolized by small squares added above the equilibrium arrows between panels (a) and (b), and shown in their interaction with the micelle-endotoxin complex in panel (b). As mentioned above, one substance useful as an agent which influences hydrogen bonding stability in solution is divalent calcium. With the complex between the detergent masker and the masked endotoxin thus destabilized, a modulator system comprising both an adsorbing modulator and a displacing modulator is added (see above equilibrium arrows between panels (b) and (c)) to displace the endotoxin from the already destabilized micelle of masking detergent. As mentioned above, the displacing modulator may be sodium-dodecyl sulfate (SDS), itself a detergent. The possibility that the modulator system contains a component which is itself a detergent and which may form new micelles of its own, is represented in panel (c) ofFIG.3by a dotted circle, in which the endotoxin is inserted. Under the conditions prevailing inFIG.3, however, any micelle formed by the displacing modulator is not as stable as the micelle formed by the masking detergent shown in panel (a). This is at least partly because the adsorbing modulator, e.g. BSA shown inFIG.3also binds the displacing modulator on its surface, establishing an equilibrium between protein-bound and micelle-forming populations of the displacing modulator which effectively destabilizes any micelle formed by the displacing modulator. The presence of a disrupting and reconfiguring modulator, for instance a non-micelle-forming amphiphilic species such as 1-dodecanol, is shown over the equilibrium arrows between panels (c) and (d) ofFIG.3. The remainder of the schematic shown inFIG.3is analogous to what has already been discussed in detail above in the context ofFIGS.1and2. Briefly, the disrupting and reconfiguring modulator shown between panels (c) and (d) ofFIG.3liberates and solubilizes endotoxin transiently inserted in micelles formed by the displacing modulator, at the same time establishing an equilibrium between solubilized (non-detectable) and aggregated (detectable) endotoxin species. This equilibrium may be shifted to the right (toward aggregated form) by the agent which influences hydrogen bonding stability in solution (e.g. a salt with a cation, preferably a divalent cation and/or a chaotropic agent). Overall,FIG.3shows the liberation of a masked molecule of endotoxin from a stable complex with a micelle of a detergent masker. It uses an agent which influences hydrogen bonding stability in solution to destabilize this complex, and a multicomponent modulator which in total disrupts this complex and chaperones the liberated endotoxin through a series of energetic minima in the ultimate direction of an aggregated and therefore detectable complex of endotoxin. Unmasking Endotoxin Masked by a Protein Masker with a Dual-Component Modulator Comprising a Displacing Modulator and a Disrupting and Reconfiguring Modulator FIG.4is a schematic depiction of a scenario in which an endotoxin is masked by a protein in solution. This is shown in panel (a) ofFIG.4. In the scenario depicted inFIG.4, the protein, which may for example be an API in a pharmaceutical formulation, exhibits a binding cleft which is both sterically and electrostatically suitable to stably bind endotoxin. In this way, the protein masker binds molecules of endotoxin, rendering them undetectable. Addition of a modulator component, symbolized by the displacing modulator added above the equilibrium arrows between panels (a) and (b) ofFIG.4, displaces the endotoxin from its binding site on the protein masker. This displacing modulator might for instance be a “second heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms” as discussed above. In the event that the displacing modulator would be e.g. sodium dodecyl sulfate, this displacing modulator might bind to the surface of the masking protein, displacing the molecule of endotoxin from its stable binding position within the protein's binding cleft. This is shown in the left portion of panel (b) ofFIG.4. In addition, as symbolized by the dotted circle in the right portion of panel (b), the displacing modulator component may also form transient micelles of its own, essentially chaperoning endotoxin liberated from the protein masker in a form stably inserted into the micelle's lipid layer. The exact position of the equilibrium shown in panel (b) ofFIG.4depends on the effectiveness with which the displacing modulator binds to the surface of the masking protein (left portion of panel (b)), as well as the stability of the micelle formed (right portion of panel (b)). Regardless of the exact position of this equilibrium, the important thing is that the displacing modulator depicted above the equilibrium arrows between panels (a) and (b) ofFIG.4tends to liberate the endotoxin from its energetically stable binding position in or on the masking protein. Once this endotoxin is freed from its masked state in or on the masking protein, a further modulator component (disrupting and reconfiguring modulator), depicted above the equilibrium arrows between panels (b) and (c) ofFIG.4shifts the energetic relationships prevailing in solution such that the most stable state for endotoxin is in freely solubilized form, chaperoned in solution by the disrupting and reconfiguring modulator. This disrupting and reconfiguring modulator may for example be a “first heteroatom-substituted aliphatic comprising 8 to 16 carbon atoms” as discussed above, which may for example be 1-dodecanol. As discussed above, this disrupting and reconfiguring modulator will typically have the property of disrupting existing micelles (for example formed by the displacing modulator, and show in the right portion of panel (b)), while not forming micelles of its own. With any previous micelles of the displacing modulator thus disrupted, and with the disrupting and reconfiguring modulator unable to form corresponding micelles of its own, the most energetically stable form of the endotoxin becomes the solubilized form shown in panel (c) ofFIG.4, chaperoned by the disrupting and reconfiguring modulator. The remainder ofFIG.4is as previously discussed for the final equilibrium step inFIGS.1and3. Briefly, there exists an equilibrium between individual, solubilized endotoxin (panel (c)) and aggregated endotoxin (panel (d)). To the extent that any appreciable population of aggregated endotoxin exists as part of this equilibrium, the endotoxin becomes detectable where, stably bound in or on the masking protein, it previously was not. Overall, endotoxin which was previously masked in individualized form by a protein has been unmasked and rendered detectable by adjusting the solution conditions such that the most energetically favorable state in which endotoxin can reside becomes its detectable aggregated form. As in previous figures discussed above, then, the “unmasking” is the result of manipulating solution conditions so as to shift equilibrium from a state in which endotoxin is stabilized in individualized form (“masked”) toward a state in which endotoxin is aggregated and detectable (“unmasked”). Unmasking Endotoxin Masked by a Protein Using a Multi-Component Modulator Comprising an Adsorbing Modulator (Protein), a Displacing Modulator and a Disrupting/Reconfiguring Modulator, in Combination with an Agent which Influences Hydrogen Bonding Stability The initial scenario shown inFIG.5corresponds to that shown inFIG.4: endotoxin is stably bound in or on a protein present in the composition. This protein in the composition, which may for example be an API, thus functions as an “endotoxin masker”. As already discussed in the context of the scenario depicted inFIG.3, the endotoxin is so stably complexed with the endotoxin masker in panel (a) ofFIG.5that simple addition of modulator cannot alone liberate it. InFIG.3, discussed above, the endotoxin masker was a detergent, which formed a micelle in which a single molecule of endotoxin was very stably inserted. Now inFIG.5, the endotoxin masker is a protein with a binding site amenable for stable endotoxin binding. But the principle remains the same: Whether inserted in the lipid layer of a detergent micelle (FIG.3) or residing stably in or on a protein, the endotoxin is stabilized to an extent that simple addition of a modulator is unable to overcome and the thus solubilized endotoxin remains undetectable. As explained above forFIG.3, this stable complex between endotoxin and endotoxin masker can be destabilized by addition of an agent which influences hydrogen bonding stability in solution, for example a salt or a chaotropic agent, for example divalent calcium. This agent which influences hydrogen bonding stability is symbolized inFIG.5by small squares starting over the equilibrium arrows between panels (a) and (b). This agent disrupts the hydrogen bonding network which is assumed to exist between endotoxin and the protein masker, thus raising the free energy of the complex to a level where the modulator components, which are shown above the equilibrium arrows between panels (b) and (c), can break up the complex to such an extent that the endotoxin is dislodged from the masking protein. Using a modulator system comprising both an adsorbing modulator (protein) and a displacing modulator as shown inFIG.5then is assumed to lead to the equilibrium situation shown in panel (c). In the left portion of panel (c) is the masking protein, now divested of the endotoxin previously bound. Molecules of the agent which influences hydrogen bonding stability in solution as well as of the displacing modulator, for example SDS, are shown bound to the surface of the masking protein, including in the binding site where endotoxin was previously bound. This depiction is intended to represent the fact that the displacing modulator effectively displaced endotoxin from its stable position in or on the masking protein. The middle portion of panel (c) ofFIG.5shows a micelle which might be formed by the displacing modulator (e.g. SDS), with a molecule of endotoxin transiently inserted into the lipid layer of the micelle. Molecules of the agent which influences hydrogen bonding stability in solution are also shown bound to endotoxin and micelle, and serve to further destabilize this micelle, ensuring that the micelle in fact remains transient and does not present the endotoxin with an energy binding minimum from which it cannot be dislodged by a further disrupting modulator. Finally, the right portion of panel (c) shows the adsorbing modulator (protein) acting, as described briefly above, as a “molecular sponge” which adsorbs both the agent which influences hydrogen bonding stability in solution as well as the displacing modulator on its surface. This effectively depletes these species in solution, destabilizing the transient micelle shown in the middle portion of panel (c) to the extent that the displacing modulator is depleted, while stabilizing it to the extent that the agent which influences hydrogen bonding stability in solution is depleted. Generally, however, the amount of the agent which influences hydrogen bonding stability in solution will be high enough to destabilize the initial complex between masking protein and endotoxin that enough of this agent will persist in solution despite depletion by the adsorbing modulator, so that the transient micelle shown in panel (c) will be destabilized as desired. Use of a disrupting and reconfiguring modulator, for example as shown over the equilibrium arrows between panels (c) and (d) ofFIG.5(e.g. 1-dodecanol), will then break up the transient micelle shown in panel (c) so as to liberate the molecule of inserted endotoxin. As already discussed above the thus solubilized endototoxin (panel (d)) will then enter into an equilibrium relationship with a reconfigured, aggregated form of endotoxin (panel (e)) which can be detected as discussed above. Unmasking Endotoxin Masked by Both Protein and Detergent Maskers with a Multi-Component Modulator Comprising an Adsorbing Modulator (Protein), a Displacing Modulator and a Disrupting/Reconfiguring Modulator, in Combination with an Agent which Influences Hydrogen Bonding Stability in Solution Many protein APIs, for example, antibodies, antibody fragments, hormones, enzymes, fusion proteins or protein conjugates are formulated and marketed at such high concentrations that detergents must be included in solution to avoid unwanted protein aggregation. The initial scenario shown inFIG.6is thus representative of one of the most relevant situations in the field of pharmaceutical formulation because both detergent and protein (e.g. API protein) maskers are present. The molecule of endotoxin is shown as inserted in the lipid layer of a detergent micelle (again symbolized by a ring of open circles representing the individual detergent molecules) as well as bound in or on the masking protein. In reality, these two species are likely to exist in equilibrium, with the relative position of this equilibrium, toward either a micelle- or a protein-bound species of endotoxin, being dictated by the relative stability of the respective complexes. All other things being equal, the complex of lower free energy, and therefore greater stability will generally prevail. The discussion ofFIG.6is analogous to that ofFIG.5above, with the only difference being that panel (b) ofFIG.6shows both the protein- and micelle-bound species of endotoxin in mutual equilibrium, each destabilized by the agent which influences hydrogen bonding stability in solution. Using an adsorbing modulator and a displacing modulator leads to the equilibrium situation depicted in panel (c) ofFIG.6. The discussion above for panel (c) ofFIG.5applies here correspondingly. The use of a further disrupting and reconfiguring modulator (shown over the equilibrium arrows between panels (c) and (d)) which is capable of disrupting the transient micelle of panel (c) without forming micelles of its own, frees the endotoxin from its transiently bound state in a micelle of displacing modulator (middle portion of panel (c)), and engenders the equilibrium relationship between soluble (non-detectable) and aggregated (detectable) forms of endotoxin as discussed above. As explained above for previous figures, the disrupting and reconfiguring modulator shown in panel (d) is shown in equilibrium between states bound to the liberated endotoxin (upper portion of panel (d)) and detergent previously constituting the detergent micelle shown in panel (a) (lower portion of panel (d)). It should be noted that the above scenarios are intended to illustrate the principles which the present inventors believe underlie the advantageous unmasking effect of the present invention in different situations. From the illustrativeFIGS.1-6, it will be clear that the processes discussed are all equilibrium processes, and that there is accordingly no prerequisite for the order of addition of different components of the modulator system or, if used, of the agent influencing hydrogen bonding stability and solution. The equilibria shown will thus be automatically established as soon as the components are present together in solution. The “order” of addition of these components as implied in the discussion above and shown inFIGS.2-6thus serves merely to illustrate the mechanisms which the present inventors believe underlie the advantageous technical effect of the present invention. Accordingly, unmasking a previously masked endotoxin might be accomplished by adding components at separate points in time as suggested byFIGS.2-6, however the desired unmasking effect is also achievable when the components depicted inFIGS.2-6are added all at once. In the most general sense, the scenarios depicted above inFIG.1-6and the corresponding discussion should illustrate the following general principles, which are intended as general guidelines to the skilled person in implementing the present invention. Many solutions which test negative for endotoxin by conventional methods actually contain endotoxin in masked form. Conventional methods detect endotoxin in its aggregated form, so the fact that many existing solutions, such as pharmaceutical formulations, test negative for endotoxin does not necessarily mean that these solutions contain no endotoxin, but rather that they contain no endotoxin in detectable form. In their most general form, the methods of the invention allow unmasking of endotoxin, e.g. by destabilizing complexes between endotoxin and endotoxin maskers so as to liberate, and ultimately aggregate individual molecules of endotoxin, thus rendering previously undetectable endotoxin detectable. Liberation of endotoxin from its masked complexes with endotoxin maskers may ensue directly using a disrupting and reconfiguring modulator to break up such complexes or, for especially stable complexes, these may be destabilized and then broken up with such a modulator or with a multi-component modulator system. However the bound endotoxin is liberated, the net effect is that endotoxin transitions from a stably bound form into a transient soluble form which may then aggregate. In its broadest sense, then, the methods of the present invention entail adjusting solution conditions as described above so as to usher previously masked endotoxin through a series of equilibria, wherein the final transition results in aggregation of endotoxin in a form which is detectable. Since the unmasking and/or detection of endotoxin according to the methods described herein depend on a final reconfiguration of liberated endotoxin in solubilized (undetectable) form into aggregated form (detectable) a reconfiguring modulator will generally be needed. This reconfiguring modulator (e.g. 1-dodecanol) will generally have the characteristic of not forming micelles on its own, while stabilizing individual molecules of endotoxin such that these can enter into an equilibrium with aggregated forms of endotoxin. As is clear from the above, a reconfiguring modulator will sometimes, but need not necessarily, also function as a disrupting modulator which is able to break up an initial complex between endotoxin and a micelle of masking detergent and/or a complex of between endotoxin and a transient micelle of displacing modulator. The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not construed as limiting the present invention. EXAMPLES Introduction Endotoxin masking is a common phenomenon in pharmaceutical composition, especially biopharmaceutical drug products. Masking of endotoxin is driven by several factors, leading in the end to the non-detectability or at least a decreased detectability of the endotoxin in the drug product. In one scenario, masking is not caused by the active pharmaceutical ingredient (API), e.g. protein, itself but by the formulation ingredients. Such ingredients are detergents, which are added to prevent aggregation of the protein, and buffer substances like citrate, phosphate, Tris, acetate, histidine, glycine which are added for pH-adjustment of the product. Unsurprisingly, the kinetics of masking is influenced by temperature, with masking proceeding faster at higher temperatures than at lower temperatures. Unless otherwise specified, all experiments described below were performed at room temperature. This is the temperature at which production process steps of the active pharmaceutical ingredient (API) are often performed, and is therefore the most relevant temperature for assessing the applicability of the inventive methods described herein to industrial processes. Example 1: Unmasking of Endotoxin from a Masking System of Polysorbate 20/Citrate Using a Disrupting and Reconfiguring Modulator (1-Dodecanol) Alone, and Together with a Further Adsorbing Modulator (BSA) A masking system of polysorbate 20/citrate was chosen for the first experiment because citrate and polysorbate 20 are often included in biopharmaceutical formulations. These experiments are intended to determine whether masked endotoxin can be released from a complex with detergent masker by addition of a disrupting and reconfiguring modulator as described herein. Materials and Methods Endotoxin masking was performed as follows. 1 ml aqueous aliquots of 10 mM Citrate pH 7.5 containing 0.05% (w/v) of polysorbate 20 were prepared in endotoxin-free glass test tubes. Subsequently, 10 μl of a 10,000 EU/ml LPS stock solution (LPS 055 B5, Sigma L2637-5MG) were added, the resulting solution was vortexed for 1 min and was stored at room temperature for at least 24 hours. As a positive LPS control containing non-masked LPS, 10 μl of a 10,000 EU/ml LPS stock solution was added to 1 ml of endotoxin-free water, mixed and identically incubated as the masking preparations, but without polysorbate 20. The LPS-water positive control is described in more detail below. Endotoxin unmasking was performed as follows. 100 μl of stock solutions of each of 1-dodecanol (disrupting and reconfiguring modulator) dissolved in 100% ethanol and 100 mg/ml BSA (adsorbing modulator) dissolved in endotoxin-free water were added. 1-dodecanol and BSA are used here as the two components of a dual-component modulator system. A separate unmasking experiment was performed identically as above, except that a single-component modulator was used. The single modulator in this experiment was 1-dodecanol alone, i.e. without BSA. Concentrations of the 1-dodecanol stock solutions were 400, 200, 100, 50, 25, 12.5 and 6.25 mM. For unmasking, the unmasking stock solutions of BSA and 1-dodecanol were sequentially added with 2 minutes mixing by vortexing after each addition. After mixing, the samples were incubated for 30 minutes at room temperature without mixing. Endotoxin content was analyzed using EndoLISA® (Hyglos GmbH) according to the kit instructions. Sample dilutions were 1:10 and 1:100 in endotoxin-free water. Endotoxin recovery was calculated as a percentage of recovery of a separate LPS-water control containing only water and LPS without any masking component. In the absence of any endotoxin masker, no LPS in this LPS.water control should be masked, that is all LPS present in this LPS-water control should be detectable. In this way, the LPS-water control serves as a standard to determine both qualitatively as well as quantitatively whether the EndoLISA® detection kit employed is functioning properly to detect LPS (qualitative control), and whether all LPS known to be present in the control is in fact detected (quantitative control). Results The recovery data inFIG.7and Table 1 (below) show that by the addition of BSA and/or 1-dodecanol in concentrations from 20 to 2.5 mM, masked endotoxin can be recovered to an extent greater than 100%. In the absence of BSA, 100% recovery cannot be achieved but, rather, greater than 50% in the range of 10 to 2.5 mM of 1-dodecanol with maximum recovery at 5 mM 1-dodecanol of approximately 90%. In this and following examples, recoveries of greater than 100% of LPS should be interpreted in light of the following: The activity of LPS has been found to depend on both LPS form (e.g. extent and orientation of aggregation) as well as LPS structure (this structure varying slightly in LPS deriving from different bacterial species). The inventive unmasking methods described herein have the potential to alter both the form and the orientation of LPS aggregation (indeed, it is due to such alteration as promoted by the modulator, especially the reconfiguring modulator, that unmasking of LPS is possible at all). The change in form and orientation of LPS aggregation between the LPS-water control (not unmasked) and the unmasked samples may in some cases cause the activity detected following unmasking to exceed that measured in the positive LPS-water control. This does not mean that performing the inventive unmasking methods as described herein generates new LPS not previously present, but rather than in some cases, performing the inventive unmasking methods as described herein alter the form of existing LPS such that the apparent measured activity for a given amount of LPS increases. TABLE 11-Dodecanol (mM)BSA (mg/ml)% LPS recovery40—2820—4610—605—892.5—651.25—310.625—7401070201015710101865101702.5101341.2510710.625100 The results clearly demonstrate that masked endotoxin can be unmasked by the addition of the modulator 1-dodecanol (disrupting and reconfiguring modulator) alone. The results further show that this unmasking effect can be improved by the addition of a further adsorbing modulator (BSA). In this latter case in which 1-dodecanol and BSA are added as a dual-component modulator, the BSA helps to adsorb detergent, thus destabilizing the detergent micelle masking the endotoxin, the modulator 1-dodecanol, is capable of disrupting detergent micelles (in its capacity as disrupting modulator) and reconfiguring liberated endotoxin into an aggregate structure (in its capacity as reconfiguring modulator). In the case of polysorbate 20 in the absence of BSA an almost quantitative recovery is possible (89% at 5 mM 1-dodecanol). This may be due to the similarity in the length of the alkyl chains of 1-dodecanol and the LPS-masking detergent polysorbate 20. The unmasking is improved by the addition of BSA, which is assumed to shift the equilibrium of LPS from solubilized to aggregated form (see e.g.FIG.2). Example 2: Unmasking of Endotoxin from a Masking System of Polysorbate 20/Citrate Using Alcohols of Different Alkyl Chain Length as Disrupting and Reconfiguring Modulators This experiment investigates the use of various alkyl alcohols as disrupting and reconfiguring modulators. One aim of the experiments described in this example was to investigate the relationship between alkyl chain length in the alcohol and unmasking efficiency. To this end, unmasking was performed by the addition of alcohols with carbon atom chain lengths from C8-C18 in different concentrations. Materials and Methods Endotoxin masking was performed as described in Example 1. Unmasking was performed by the addition of stock solutions of unbranched 1-alcohols of different alkyl chain lengths (C8, C10, C12, C14, C16, C18) as modulators (disrupting and reconfiguring modulators) as described in Example 1 for 1-dodecanol (having a 12-carbon alkyl chain). Each of the stock solutions was dissolved in 100% ethanol. In contrast to certain of the experiments described above in Example 1, no other modulator components, e.g. BSA, were included in the present unmasking experiments. Analysis of endotoxin concentrations was performed using the EndoLISA® kit (Hyglos GmbH), and the subsequent calculation of endotoxin recovery was expressed as a percent of the LPS in the LPS-water control sample. The LPS-water positive control is explained in detail in Example 1, above. Results Table 2 (below) show the percentage of unmasked endotoxin as dependent on alcohol concentration and the length of the alkyl chain in the alcohol. TABLE 2% LPS Recovery1-1-Conc.1-1-1-1-Hexa-Octa-(mM)OctanolDecanolDodecanolTetradecanoldecanoldecanol40002810ndnd200046361110006044315018928302.5056516031.25013125010.6251471021nd = no data Endotoxin recoveries of, i.e. unmasking endotoxin by, greater than 40% were achieved using 1-dodecanol and 1-tetradecanol. Recoveries using alcohols with alkyl chains lengths below or above C12 and C14 are below 10%. The above results imply that the alkyl chain length of the alcohol used as a disrupting and reconfiguring modulator should ideally match the alkyl chain length of the acyl chains in the endotoxin as closely as possible. In the present case, the lengths of the acyl chains in the Lipid A component of LPS are C12 and C14, and it was the 1-alcohols having alkyl chain lengths in that range which, when used as disrupting and reconfiguring modulators, most effectively unmasked the endotoxin. Example 3: Unmasking of Endotoxin from Masking Systems of Various Non-Ionic Surfactants Using 1-Dodecanol as a Disrupting and Reconfiguring Modulator Alone, and Together with the Adsorbing Modulator BSA To investigate the hypothesis that unmasking endotoxin from polysorbate 20 by 1-dodecanol alone is promoted by equivalent or similar alkyl chain length of the masking surfactant and 1-dodecanol, various experiments were designed using masking detergents of different chain lengths and different structure, and these were then unmasked using a disrupting and reconfiguring modulator of fixed alkyl chain length (1-dodecanol, with a 012 alkyl chain). To this end, masked samples were prepared in polysorbate 80 and Triton X-100 and these were subsequently unmasked with 1-dodecanol or BSA/1-dodecanol using different concentrations of 1-dodecanol. Materials and Methods Endotoxin masking was performed as follows: 1 ml aliquots of 10 mM citrate pH 7.5 containing 0.05% of polysorbate 20, polysorbate 80 or Triton X-100 were prepared in endotoxin-free glass test tubes. Subsequently, 10 μl of a 10,000 EU/ml LPS stock solution (LPS 055 B5, Sigma L2637-5MG) were added, vortexed for 1 min and stored at room temperature for at least 24 hours. As a positive LPS control, 10 μl of a 10,000 EU/ml LPS stock solution was added to 1 ml of endotoxin-free water, mixed and identically incubated as the masking preparations. The positive LPS-water control is discussed in detail above in Example 1. Unmasking was performed by the addition of stock solutions of 1-dodecanol (as a disrupting and reconfiguring modulator) in different concentrations as described in Example 1. Stock solutions of the respective alcohols were dissolved in 100% of ethanol. Unmasking was performed in both the absence and presence of 10 mg/ml BSA as described in Example 1. Analysis of endotoxin concentrations was performed with the EndoLISA® kit (Hyglos GmbH), with subsequent calculation of recovery of endotoxin expressed as a percent of the endotoxin in the LPS/water control sample. Results Table 3 (below) shows the recoveries of LPS after unmasking from the respective polysorbate 20/citrate, polysorbate 80/citrate and Triton X-100/citrate masking systems as dependent on the 1-dodecanol (disrupting and reconfiguring modulator) concentration in the absence or presence of BSA (adsorbing modulator). TABLE 3DodecanolBSA% LPS recovery(mM)(mg/ml)Polysorbate 20Polysorbate 80Triton X-10040—28.04.9nd20—46.27.53.410—60.511.5nd5—89.125.20.02.5—64.928.5nd1.25—31.212.10.00.625—7.20.0nd0.313—ndnd0.0401069.719.4nd2010156.836.42.01010186.169.9nd510170.586.923.02.510133.594.2nd1.251071.32.90.00.625100.012.9nd0.31310ndnd0.0nd = no data Unmasking with 1-dodecanol from the polysorbate 80/citrate masking system results in recovery of approximately 30% at an optimal concentration of 1-dodecanol of 2.5 mM. In the presence of BSA up to 90% can be recovered. Both unmasking approaches from the Triton X-100 masking system (i.e. with and without BSA) result in LPS recoveries below 20%, regardless of the concentration of 1-dodecanol. Thus, unmasking using 1-dodecanol alone (as a disrupting and reconfiguring modulator) is sufficient to unmask LPS from masking systems such as in the polysorbate 20 masking system. The addition of BSA (as an adsorbing modulator) to adsorb the masking detergent improves unmasking recoveries in the polysorbate 20 and polysorbate 80 masking systems. Unmasking from the Triton X-100 system is not highly efficient even when BSA is added together with 1-dodecanol. Adding a further modulator component such as e.g. SDS (as a displacing modulator) can help improve recovery of LPS from Triton-X-100 masking formulations. Example 4: Increasing Unmasking Efficiency by Addition of a Modulator and a Chaotropic Agent which Influences Hydrogen-Bonding Stability The weak recovery of LPS from the Triton X-100 masking system using the dual-modulator system of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) may be due to the high stability of the complex formed by Triton X-100 and LPS. This high stability may prevent the desired destruction of the endotoxin-masking micelles of Triton X-100 by the disrupting action of 1-dodecanol and adsorption of the detergent by BSA. For this reason, the present experiments invertigate the possibility of destabilizing the masking complex by addition of a chaotropic salt together with a multi-component modulator. The hope was that by destabilizing an otherwise stable detergent micelle, destruction of this micelle using a multi-component modulator system of 1-dodecanol (as disrupting and reconfiguring modulator), BSA (as adsorbing modulator) and SDS (as displacing modulator) would then become possible. Materials and Methods Endotoxin masking was performed as follows: 1 ml aliquots of 10 mM citrate pH 7.5 containing 0.05% of Triton X-100 were prepared in endotoxin-free glass test tubes. Subsequently, 10 μl of a 10,000 EU/ml LPS stock solution (LPS 055 B5, Sigma L2637-5MG) were added, vortexed for 1 min and stored at room temperature for at least 24 hours. As a positive LPS control, 10 μl of a 10,000 EU/ml LPS stock solution was added to 1 ml of endotoxin free water, mixed and incubated in an identical manner as the masking preparations. The positive LPS-water control is discussed in detail above in Example 1. Unmasking endotoxin was performed as follows: 100 μl of the following stock solutions were added as single component or as combinations to the 1 ml masked samples: 1 M CaCl2(dissolved in water), 100 mg/ml BSA (dissolved in water), 1% SDS (dissolved in water) and 50 mM 1-dodecanol (dissolved in 100% ethanol). In the case of addition of combinations, the agents were added sequentially, with a 2-minute vortexing step between each addition. The samples were then incubated at room temperature for 30 minutes without shaking. Endotoxin content was analyzed using the EndoLISA® kit (Hyglos GmbH) according to the kit instructions. Sample dilutions were 1:10 and 1:100 in endotoxin-free water. Endotoxin recovery was calculated and expressed as a percentage of recovery of the LPS-water control. The positive LPS-water control is discussed in detail above in Example 1. Results FIG.8shows the percentage of LPS recovery as dependent on the addition of combinations of CaCl2(C), BSA (B; adsorbing modulator), SDS (S; displacing modulator) and 1-dodecanol (D; disrupting and reconfiguring modulator). 1-Dodecanol as the sole (disrupting and reconfiguring) modulator does not efficiently unmask LPS from a Triton X-100 masking complex. Addition of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) as a dual-component modulator system results in approximately 20% recovery. Further addition of either a chaotropic salt such as CaCl2) or a further modulator such as SDS (displacing modulator) does not result in LPS recoveries greater than 20%. However, the addition of CaCl2, BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) results in LPS recoveries of greater 100%. Thus, additionally to BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator), a chaotropic salt and a further displacing modulator such as the detergent SDS help to break up the Triton X-100 masking complex. In this way, the combination of these 4 additives seems to break apart the masking complex and allows the formation of detectable LPS. Example 5: Comparison of Different Unmasking Approaches from Various Masking Systems As efficient unmasking from the Triton X-100 masking system was observed using a combination of CaCl2, BSA, SDS and 1-dodecanol, the question of unmasking efficiency of this approach starting from polysorbate masking systems remains. To answer this question, endotoxin was masked in polysorbate 20, 80 and Triton X-100/citrate masking systems and subsequently unmasked using 1-dodecanol alone; BSA and 1-dodecanol in combination; or CaCl2, BSA, SDS and 1-dodecanol in combination. In these experiments, 1-dodecanol is used as a disrupting and reconfiguring modulator, BSA is used as an adsorbing modulator, SDS is used as a displacing modulator and CaCl2) is used as an agent which influences hydrogen-bonding stability in solution. Materials and Methods Endotoxin masking was performed as follows: 1 ml aliquots of 10 mM citrate pH 7.5 containing either 0.05% polysorbate 20, or 0.05% polysorbate 80 or 0.05% Triton X-100 were prepared in endotoxin-free glass test tubes. Subsequently, 10 μl of a 10,000 EU/ml LPS stock solution (LPS 055 B5, Sigma Aldrich L2637-5MG) were added, vortexed for 1 min and stored at room temperature for at least 24 hours. As a positive LPS control, 10 μl of a 10,000 EU/ml LPS stock solution were added to 1 ml of endotoxin-free water, mixed and identically incubated as the masking preparations. The function of the positive LPS-water control is as described above in Example 1. Unmasking of endotoxin was performed as follows: Either 100 μl of a 50 mM 1-dodecanol stock solution; or 100 μl of 100 mg/ml BSA and 100 μl of a 50 mM 1-dodecanol stock solution; or 100 μl of a 1 M CaCl2) solution, 100 ml of a 100 mg/ml BSA solution, 100 μl of a 1% SDS solution and 100 μl of a 50 mM 1-dodecanol solution were added to the solution containing masked LPS. In the case of addition of combinations, the agents were added sequentially with a 2-minute vortexing step between each addition. The samples were then incubated at room temperature for 30 minutes without shaking. Endotoxin content was analyzed using the EndoLISA® kit (Hyglos GmbH) according to the kit instructions. Sample dilutions were 1:10 and 1:100 in endotoxin-free water. Endotoxin recovery was calculated as a percentage of recovery of the LPS-water control. Results Table 4 (below) andFIG.9show the percentages of LPS recovery using either 1-dodecanol alone; BSA and 1-dodecanol in combination; or CaCl2, BSA, SDS and 1-dodecanol in combination (CBSD) for unmasking from various detergent masking systems. TABLE 4% LPS recovery1-BSA/1-Masking detergentdodecanoldodecanolCBSDPolysorbate 2078170141Polysorbate 802894161Triton X-100023168 Efficient (˜80%) unmasking from the polysorbate 20 masking system is achieved by 1-dodecanol, BSA/1-dodecanol and CaCl2/BSA/SDS/1-dodecanol. In the case of a polysorbate 80 masking system, good unmasking efficiency is achieved in the presence of BSA/1-dodecanol and CaCl2/BSA/SDS/1-dodecanol. In the case of a Triton X-100 masking system, the addition of CaCl2/BSA/SDS/1-dodecanol results in good LPS recovery. Thus, dependent on the stability of the masking complex, efficient endotoxin recoveries can be achieved using different unmasking approaches. However, the unmasking approach involving the combination of CaCl2, BSA, SDS and 1-dodecanol may be the most universal method, due to its ability to achieve efficient unmasking, regardless of the masking system used. As is clear from the experiments described herein above, an optimal composition for unmasking LPS in any given formulation can be easily achieved by routine experimentation. Example 6: Unmasking of Endotoxin from Different Endotoxin Sources Endotoxin unmasking experiments in Examples 1-5 were performed with a commercially available, highly purified LPS preparation ofE. coli O55:B5. As only the conserved Lipid A part of LPS is responsible for toxicity and for detectability in Factor C-based detection methods, it can be assumed that the unmasking approaches described above will work equally well using LPS preparations from bacteria other thanE. coli O55:B5. However, the literature also describes differences in acyl chain length for the lipid A part of LPS, as well as modifications of side chains. Even more, the length of the O-sugar side chains of LPS could potentially impact the unmasking approach. Furthermore, it cannot be excluded that purified LPS and naturally occurring endotoxin (NOE) may differ in their unmasking behavior. To address these issues, and exclude the possibility, that the unmasking approaches are specific for the used LPS ofE. coli O55:B5, LPS from different bacteria, different length in core- and O-sugar chains and different purity were masked in various detergent masking systems and subsequently unmasked using either 1-dodecanol alone, BSA/1-dodecanol or CaCl2/BSA/SDS/1-dodecanol. Materials and Methods Masking of endotoxin was performed as follows: LPS samples of different types and from different sources were (approximately 50 EU/mL) added to 1 ml masking samples containing either 0.05% polysorbate 20, 0.05% polysorbate 80 or 0.05% Triton X-100 and 10 mM citrate pH 7.5. LPS source, type and the supplier are shown in Table 5 (below). NOEs were produced from bacterial culture supernatant after growth to stationary phase in LB media by sterile filtration. As a preservative, 0.05% sodium azide was added. Lyophilized LPS was dissolved in endotoxin-free water. LPS solutions for which the supplier in Tables 5-7 is indicated as “LMU” were kind gifts of Dr. A. Weser of the Ludwig-Maximilian University of Munich. Endotoxin content of the LPS stock solutions was determined using the EndoZyme® kit (Hyglos GmbH) and stock solutions of approx. 5000 EU/ml LPS in endotoxin-free water were produced. From these solutions 10 μl were added to 1 ml masking samples. Afterwards, the samples were allowed to mask the respective LPS for 7 days at room temperature. Unmasking of endotoxin was performed by addition of 100 μl of either a 100 mM 1-dodecanol stock solution, or addition of 100 μl of a 100 mg/ml BSA and 100 μl of 100 mM 1-dodecanol stock solution or by addition of 100 μl of each of 1 M CaCl2), 100 mg/ml BSA, 1% SDS and 100 mM 1-Dodecanol solutions. Unmasking and determination of endotoxin content were performed as described in Examples 1-5. Results Tables 5-7 (below) show the percent of LPS recovery after masking and after unmasking of LPS from different sources and types out of different detergent masking systems. Specifically, Table 5 shows the results obtained for a masking system of Tween20/Citrate; Table 6 shows the results obtained for a masking system of Tween80/Citrate; and Table 7 shows the results obtained for a masking system of Triton X-100/Citrate. TABLE 5Tween 20/Citrate masking systemMaskingCaCl2/BSA/SDS/controlDodecanolBSA/DodecanolDodecanolsupplier(% recovery)(% recovery)(% recovery)(% recovery)KlebsiellaLMU0.066128212pneumoniaMorganellaLMU0.081110120morganiiYersiniaLMU0.063174243enterocoliticaSerratiaLMU0.0128168182marcescensNeisseriaLMU0.092338meningitisAcinetobacterLMU0.00124655baumanni*EnterobacterHyglos0.055156187cloacae(NOE) *SalmonellaSigma0.0426376entericaE. coliK 12Invivogen3.07880137PseudomonasSigma0.0145179aeruginosa** Strains which are common water contaminants, and therefore more likely to be present in processes for the production of pharmaceutical compositions TABLE 6Tween 80/Citrate masking systemMaskingCaCl2/BSA/SDS/controlDodecanolBSA/DodecanolDodecanolsupplier(% recovery)(% recovery)(% recovery)(% recovery)KlebsiellaLMU0.012173353pneumoniaMorganellaLMU15.0153999morganiiYersiniaLMU7.022168309enterocoliticaSerratiaLMU0.0105199326marcescensNeisseriaLMU0.001142meningitisAcinetobacterLMU0.07337511baumanni*EnterobacterHyglos24.22774183cloacae(NOE) *PseudomonasSigma1.01190aeruginosa*SalmonellaSigma0.0181069entericaE. coliK 12Invivogen1.985106176* Strains which are common water contaminants, and therefore more likely to be present in processes for the production of pharmaceutical compositions TABLE 7Triton X-100/Citrate masking systemMaskingCaCl2/BSA/SDS/controlDodecanolBSA/DodecanolDodecanolsupplier(% recovery)(% recovery)(% recovery)(% recovery)KlebsiellaLMU9.82212162pneumoniaMorganellaLMU5.5352348morganiiYersiniaLMU0.01319236enterocoliticaSerratiaLMU3.5282080marcescensNeisseriaLMU0.05514161meningitisAcinetobacterLMU7.8057918baumanni*EnterobacterHyglos0.022685cloacae(NOE) *PseudomonasSigma0.011125aeruginosa*Salmonella entericaSigma0.02112234* Strains which are common water contaminants, and therefore more likely to be present in processes for the production of pharmaceutical compositions The above data clearly show that the ability to successfully unmask endotoxin from various masking systems is independent of the source and type of LPS used. These results are important because they show that the unmasking methods of the present invention represent a general teaching applicable to various types of endotoxin from various sources, under a variety of masking conditions. Example 7: Unmasking of Endotoxin from Protein Masking Systems The previous experiments have investigated the unmasking of LPS from detergent masking systems. However, as described herein above, detergents are not the only substances which can mask endotoxin from detection. Proteins (e.g. protein APIs) are also capable of masking endotoxin from detection when they contain binding sites on or within their structure in which endotoxin can bind, thus evading detection. The present experiments therefore relate to the masking of endotoxin (LPS) by a protein rather than a detergent. Lysozyme was used as the masking protein in these experiments because its ability to bind endotoxin is known (see e.g. Ohno & Morrison (1999). J. Biol. Chemistry 264(8), 4434-4441). Materials and Methods Endotoxin masking was performed as follows: 50 EU/ml of LPS (E. coli O55:B5) was incubated for seven days in 10 mM citrate buffer, pH 7.5 containing 1 mg/ml hen egg white lysozyme (Sigma Aldrich) at room temperature. Endotoxin unmasking was performed as follows: Unmasking was performed by addition of unmasking reagents (modulators as described in previous examples and agents influencing hydrogen bonding stability) in various combinations. Specifically, 100 μl of the following unmasking agents were added to 1 ml aliquots of the masked samples: 1-dodecanol, CaCl2, BSA, SDS. All stock solutions were dissolved in water except 1-dodecanol, which was dissolved in 100% ethanol. The added concentrations of the stock solutions were 100 mM 1 M CaCl2, 100 mg/ml BSA and 1% SDS, respectively. Unmasking was performed by sequential addition of the various components with a two-minute vortexing step after each addition. The samples were then incubated for 30 minutes at room temperature and subsequently diluted 1:10 and 1:100 in endotoxin-free water for analysis using the EndoLISA® kit (Hyglos GmbH). Results Table 8 (below) shows the efficiency unmasking from a protein masker (lysozyme) as dependent on the added components. TABLE 8% recoveryCaCl2BSASDS1-dodecanolLPS−−−−0+−−−0++−−4+++−33++++115++−+15+−++0+−+−4+−−+2−+−−9−++−0−+++1−+−+6−−+−0−−++0−−−+1 In the case of masking by lysozyme, use of 1-dodecanol (reconfiguring modulator) alone or together with a supporting detergent (displacing modulator) as a further component of the modulator system does not efficiently unmask. Here, the lysozyme-LPS masking complex seems to be more stable due to electrostatic interactions between the negatively charged LPS and the positively charged lysozyme. Improvement of unmasking may be achieved by the addition of salt, which disrupts the electrostatic interaction, thus rendering the lysozyme-LPS complex more labile and increasing its susceptibility to disruption with modulator. To this end, good results may be achieved by using a multi-component modulator system of BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (reconfiguring modulator), together with CaCl2) to lower the stability of the initial lysozyme-LPS complex. The combination of these components is able to break up the masking complex and lead to detectable LPS structures. This model may be taken as a general model of the measures which may be used to unmask endotoxin when it is masked, in whole or in part, by a protein, e.g. a protein API in a pharmaceutical composition. Example 8: Substances Other than 1-Alkyl Alcohols as Modulators for Unmasking As described herein above, 1-alkyl alcohols (used as reconfiguring modulators) have been found to promote the formation of detectable LPS structures. It was therefore desired to investigate whether other types of substances than 1-alkyl alcohols might also have the ability to promote similarly detectable forms of LPS. This example shows the results of a screening for substances other than 1-alkyl-alcohols which might be able to support formation of detectable LPS structures. Materials and Methods LPS (E. coli O55:B5, Sigma) 100 EU/ml was masked in polysorbate 20/citrate for 24 hours at room temperature. Unmasking was initiated by sequential addition of 1 part stock solutions of CaCl2(at 1 M), BSA (at 100 mg/mL), SDS (at 1%) and substance X into 10 parts of a solution of masked LPS, wherein “substance X” represented the substance other than a 1-alkyl alcohol, the ability of which as a reconfiguring modulator was to be tested. Substance X was titrated in different concentrations. After unmasking, samples were diluted 1:10 and 1:100 in endotoxin-free water and analyzed for detectable endotoxin using the EndoLISA® kit (Hyglos GmbH). Results Table 9 (below) shows the maximum LPS recoveries after unmasking as dependent on the substance used as modulator. Furthermore, suitable concentrations of stock solutions of the respective substances for unmasking are shown. TABLE 9Optimum stock% LPSconcentration ofSubstancesrecoverysubstance Xsodium octyl sulfate (SOS)2030 mM1-decanoic acid57100 mM As can be seen from the above, 1-alkyl alcohols are not the only class of compounds which may function as a reconfiguring modulator to promote the formation of a detectable form of LPS. Other substances containing higher oxidation states of oxygen (e.g. as in 1-decanoic acid) as well as other heteroatoms than oxygen (e.g. as in sodium octyl sulfate (SOS)) may also enable moderate to good unmasking. The results indicate that substances which are similar in structure to 1-alkylalcohols are also able to support unmasking to a certain extent. It appears that OH-derivatives of alkanes, preferably C8-C16alkanes, preferably C8-C12alkanes, preferably 012 alkanes serve best to render LPS susceptible to detection by Factor C-based assays. Example 9: Unmasking Using Albumins from Different Sources and 1-Dodecanol As part of the verification of the improvement in unmasking by the addition of bovine serum albumin (BSA) in masked samples containing polysorbate 80, albumins from different sources were tested. Materials and Methods Masked samples (1 ml) containing 50 EU/ml of LPS (O55:B5) in polysorbate 80/citrate buffer were unmasked by the addition of 100 μl of stock solutions with different concentrations of albumins (bovine serum albumin (BSA), very low endotoxin, Serva GmbH; human serum albumin (HSA, recombinantly produced inPichia pastoris(Sigma Aldrich); and Ovalbumin (Ova), EndoGrade Ovalbumin, Hyglos GmbH) and subsequent addition of 100 μl of a 100 mM 1-dodecanol stock solution). Concentrations of albumin stock solutions were 100, 33, 10, 3.3 and 1 mg/ml. Due to the lower solubility of ovalbumin in water, a 100 mg/ml solution of ovalbumin was not prepared. LPS recoveries were calculated following determination of detectable LPS content using the EndoLISA® kit (Hyglos GmbH). For EndoLISA® measurements the unmasked samples were 1:10 and 1:100 diluted in endotoxin-free water and subsequently measured according to the kit instructions. Results Table 10 (below) shows the unmasking efficiency from a polysorbate 80/citrate masking system, as dependent on albumins from different sources. TABLE 10[stock solution]protein(mg/ml)% LPS recoveryBSA10066.03346.21038.13.328.2130.9HSA10042.33394.510151.63.340.4134.3ovalbumin—nd3379.41059.03.333.0119.6nd = no data The data show that all albumins tested are able to support unmasking from a polysorbate 80 masking system. Suitable final concentrations in the unmasked samples are 10 mg/ml for BSA, 1 mg/ml for HSA and 3.3 mg/ml for ovalbumin. The differences in optimum concentrations may result from different affinities of the albumins to the detergent in the masked sample. Example 10: The Effect of Various Chaotropic Salts on Unmasking Efficiency Unmasking using the combination of substances CaCl2(agent influencing hydrogen bonding), BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (reconfiguring modulator) (this entire combination is referred to as “CBSD”) has been shown above to efficiently unmask LPS when masked by e.g. Triton X-100. The present experiments investigate the effect of the nature of the chaotropic salt (agent influencing hydrogen bonding stability) on unmasking efficiency. To this end, the following experiments employ salts of increasing chaotropic properties: Na+, Mg2+and Ca2+, in each case presented as the corresponding chloride salt. Materials and Methods Endotoxin masking was performed as follows: 50 EU/ml ofE. coliLPS O55:B5 was masked by allowing it to incubate for 3 days at room temperature in a 10 mM citrate buffer solution (pH 7.5) containing 0.05% Triton X-100. Here, Triton X-100 functioned as the detergent masker. Unmasking of endotoxin was performed as follows: 300, 100, 30, 10, 3 and 1 μl of either a 5 M sodium chloride (NaCl), 1 M magnesium chloride (MgCl2) or 1 M calcium chloride (CaCl2) stock solution were added to 1 ml aliquots of the masked samples and mixed. Subsequently, 100 μl of the other modulator components (BSA (adsorbing modulator), SDS (disrupting and displacing modulator) and 1-dodecanol (reconfiguring modulator)) were added as described in Examples 1-5. Results Table 11 (below) shows the percentage of endotoxin recovery as dependent on each chaotropic salt and the most suitable final concentration of each salt in the unmasked sample. TABLE 11LPS recoveryConcentrationsalt%(mM)NaCl96.7357MgCl2139.8188CaCl2142.572 The data show that all the salts tested were able to support efficient unmasking of LPS from the masking detergent Triton X-100 in combination with a multicomponent modulator system including BSA (as adsorbing modulator), SDS (here, as disrupting modulator) and 1-dodecanol (as disrupting and reconfiguring modulator). Furthermore, as described herein above, the amount of the salt required to achieve a comparable degree of unmasking efficiency decreased with increasing chaotropic properties. These results allow several general conclusions to be drawn. First, when using a salt to destabilize a masked complex between endotoxin and endotoxin masker, the chaotropic character of this salt is an important factor in achieving efficient unmasking. Second, the amount of salt required to achieve efficient unmasking will generally vary inversely with the chaotropic strength of the salt employed. Example 11: Unmasking of Endotoxin from Samples Containing Detergent and Phosphate Buffer Most formulations of drugs which contain a protein (e.g. antibody) as an active pharmaceutical ingredient (API) contain either non-ionic detergents like polysorbate 20 or 80 together buffered in either citrate or phosphate. In such formulations, the detergent concentration is usually above the respective detergent's critical micellar concentration (CMC). Furthermore pH-values of such formulattions are often adjusted in order to ensure optimum stability of the API. With the above in mind, the investigations set out in this Example sought to investigate the influence of pH value on unmasking efficiency. In order to approximate the conditions prevailing in pharmaceutical formulations containing a protein API as closely as possible, the detergents polysorbate 20 and polysorbate 80 were used as endotoxin maskers, and the solutions were phosphate-buffered. In view of the results described herein above, unmasking was performed using a combination of CaCl2) (chaotropic salt as an agent which influences hydrogen bonding stability), BSA (adsorbing modulator), SDS (here, as disrupting modulator) and 1-dodecanol (disrupting and reconfiguring modulator). As Ca2+and PO43−form non-soluble calcium-phosphate complexes, the calcium chloride solution was stabilized by addition of a two-fold molar excess of citrate, pH 7.5. Materials and Methods Masking of endotoxin was performed as follows: To 1 ml samples, each containing 10 mM of phosphate buffer of various pH-values and either 0.05% polysorbate 20 or polysorbate 80, were added 100 EU/ml ofE. coliLPS O55:B5. Masking was allowed to proceed by incubating these solutions for 7 days at room temperature. LPS-containing control samples of phosphate buffers lacking detergent were prepared, incubated and measured in parallel to the masking samples. Unmasking of endotoxin was performed as follows: A combination of CaCl2, BSA, SDS and 1-dodecanol was added to each of the samples as described in previous examples. To avoid calcium phosphate precipitation and to adjust the pH of the samples, a two-fold molar excess of citrate buffer pH 7.5 was added to each sample before addition of the unmasking components. Endotoxin content of the masked samples was determined using the EndoZyme® kit of Hyglos GmbH at time zero, and after 7 days. Endotoxin content of the unmasked samples was analyzed using the EndoLISA® kit of Hyglos GmbH. The percentage of LPS recovery after 7 days of masking and after unmasking was calculated in reference to control samples at time zero. Results Table 12 (below) andFIGS.10and11show the percentage of LPS recovery after 7 days of masking as dependent on the pH-value and the percentage of LPS recovery after unmasking of the masked samples. TABLE 12Polysorbate 20 maskerPolysorbate 80 maskerphosphaterecoveryrecoveryrecoveryrecoverybufferafterafterafterafter(pH-value)masking [%]unmasking [%]masking [%]unmasking [%]1.6811431041882.81461501791894.01563051302065.84158272377.0116002218.90156018712.131921128 The data show that masking in phosphate buffer solutions containing detergent is strongly pH dependent. At pH values below 4, no masking occurs after one week of sample incubation. At pH values above 4 a strong masking effect is seen, resulting in detectable LPS recoveries less than 1%. The data also show conclusively that the unmasking approach implemented renders the previously masked, undetectable LPS detectable. Independent of the pH-value and the extent of masking, 100% or more of LPS can be recovered, i.e. detected. Example 12: Unmasking Using Other Displacing Modulators than SDS As shown in the examples above, a combination of CaCl2/BSA/SDS/1-doedecanol efficiently unmasked endotoxin which is masked by Triton X-100 detergent. Several of the experiments described above suggests the importance of including SDS in this scheme to achieve efficient unmasking. The aim of the experiments described in the present example is to investigate whether the modulator component SDS (here, as disrupting modulator) can be exchanged for another detergent without negatively impacting the unmasking effect observed using SDS. Materials and Methods Masking of endotoxin was performed as follows: 1 ml aliquots of 10 mM citrate pH 7.5 containing 0.05% Triton X-100 were prepared in endotoxin-free glass test tubes. Subsequently, 10 μl of a 10,000 EU/ml stock solution of LPS (LPS 055 B5, Sigma L2637-5MG) were added, vortexed for 1 min and stored at room temperature for at least 24 hours. A positive LPS control in water was prepared as follows: 10 μl of a 10,000 EU/ml LPS stock solution was added to 1 ml of endotoxin-free water, mixed and identically incubated as the masking preparations. Further details regarding the positive LPS-water control are indicated in Example 1. Unmasking of endotoxin was performed as follows: To masked solutions of LPS, prepared as indicated above, CaCl2, BSA, detergent X and 1-dodecanol were added as described in the previous examples, where “detergent X” (disrupting modulator) was varied in identity and concentration. The following detergents were tested: dioctyl sulfosuccinate sodium salt (AOT), sodium dodecyl benzene sulfonate (SDBS), polyethylene glycol 4-nonylphenyl-3-sulfopropyl ether potassium salt (PENS) and p-xylene-2-sulfonic acid hydrate (XSA). Unmasking was performed as described in above examples, endotoxin content was determined using the EndoLISA® kit of Hyglos GmbH, and the percentage of LPS recovery was calculated with reference to the LPS-water positive control. Further details regarding the LPS-water positive control are described in Example 1 above. Results Table 13 shows the percentage of LPS recovery after unmasking using detergents other than SDS in the CaCl2/BSA/[detergent X]/1-dodecanol unmasking approach. TABLE 13ConcentrationLPS recoveryDetergentoptimum[%]AOT0.01%24SDBS0.01%34PENS0.10%23XSA0.05%26 The data show that other detergents besides SDS are able to support unmasking as a disrupting modulator in a CaCl2/BSA/[detergent X]/1-dodecanol unmasking approach. Furthermore, in the absence of 1-dodecanol no detergent was able to unmask LPS from Triton X-100. As mentioned above, this suggests that 1-dodecanol may play an important role (at least) as a reconfiguring modulator which may be crucial for mediating the transition of endotoxin from a solubilized (undetectable) to an aggregated (detectable) state. Example 13: Unmasking from Buffered Antibody Compositions as Dependent on the Masking Detergent The most commonly used formulations of protein-based drug products contain phosphate buffer and non-ionic detergents such as polysorbate 20 or polysorbate 80. Further, antibodies constitute one of the most commonly formulated pharmaceutical protein products. With this in mind, we sought to confirm whether the above unmasking approaches for detergents- or protein-masking systems are suitable for unmasking endotoxin in systems containing both detergent and protein, where the protein is an antibody buffered in phosphate. Polysorbate 20 and 80 were chosen as masking detergents in these experiments because these two detergents are the most commonly used detergents in protein drug formulations. Materials and Methods Endotoxin masking was performed as follows: 50 EU/ml of endotoxin (E. coli O55:B5; Sigma L2637-5MG) was added to 1 ml aliquots of an antibody solution containing 10 mg/ml of a bovine polyclonal IgG antibody preparation, dissolved in 10 mM sodium phosphate pH 7.5 and 50 mM NaCl. Subsequently, either polysorbate 20 or polysorbate 80 were added to a final concentration of 0.05%, and the solutions were incubated for 3 days at room temperature to allow masking to occur. Further, controls containing the buffer solution without detergent or antibody, as well as the buffer solution containing either the antibody or the respective polysorbate were prepared and treated like the masking samples. Each of the controls contained the same amount of LPS. Unmasking was performed as follows: Unmasking was performed by addition of either 1-dodecanol or BSA/1-dodecanol or CaCl2/BSA/SDS/1-dodecanol. 100 μl of the following stock solutions were added to 1 ml of sample solution: CaCl2) (1 M), BSA (100 mg/ml), SDS (1%) and 1-dodecanol (100, 10 or 1 mM). Furthermore, before addition of calcium chloride to a sample, the sample was stabilized against calcium phosphate precipitation by the addition of a final concentration of 200 mM sodium citrate pH 7.5. All stock solutions were added sequentially with two-minute mixing steps following each addition. After addition and mixing of the last component the samples were incubated for at least 30 minutes at room temperature. Afterwards, the samples were diluted 1:10 and 1:100 in endotoxin-free water and analyzed for endotoxin content using the EndoLISA kit (Hyglos GmbH). The percentage of LPS recovery was calculated with reference to the determined endotoxin content in the buffer control (discussed in more detail in Example 1). Results Table 14a (below) shows the percentage of LPS recovery of the water control, the buffer without detergent, the buffer containing antibody or detergent and the buffer containing antibody and detergent after 3 days of incubation at room temperature. TABLE 14apolysorbatepolysorbate2080LPS re-LPS re-sample typeingredientscovery (%)covery (%)water controlwater100100bufferbuffer without detergent10299masking controlbuffer + antibody3144masking controlbuffer + polysorbate02masking controlbuffer + polysorbate +09antibody Table 14b (below) shows the percentage of LPS recovery from an antibody solution after unmasking containing either polysorbate 20 or 80. Furthermore, it shows the concentrations of the added stock solutions. TABLE 14bpolysorbatepolysorbate2080[CaCl2][BSA][SDS][1-Dodecanol]LPS re-LPS re-(M)(mg/ml)(%)(mM)covery (%)covery (%)———10016.69.1———1019.96.8———10.05.0—100—10040.811.2—100—102.66.3—100—11.611.5110011004.83.0110011015.923.111001167.390.8 The data show that the buffer solutions without polysorbate 20 or 80 do not mask the added LPS. The buffer solutions containing antibody but no polysorbate mask ˜55% to 70% of the LPS, suggesting that the antibody protein contributes a masking effect of its own. The LPS recoveries from buffer solutions containing polysorbate or polysorbate and antibody are below 10% when no unmasking measures are taken. Thus, not only the detergent but also the antibody is responsible for masking of LPS. LPS recoveries after unmasking from the masking complexes containing LPS, detergent and antibody are low using 1-dodecanol alone (9 and 17% for polysorbate 80 and 20, respectively). Using a combination of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) allowed moderate LPS recoveries of 11 and 41% for polysorbate 80 and 20, respectively. Unmasking using a combination of CaCl2, BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (disrupting and reconfiguring modulator), results in recoveries of 67% and 91% of the masked LPS for polysorbate 20 and 80, respectively. Interestingly, unmasking was achieved using a 1-dodecanol stock solution with a concentration as low as 1 mM. Furthermore, in contrast to the unmasking from detergent systems lacking protein, using 1-dodecanol (disrupting and reconfiguring modulator) or BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) do not unmask with greater efficiency than 50%. As shown for lysozyme above, efficient unmasking was only be achieved in the presence of CaCl2, BSA, SDS and 1-dodecanol. Example 14: Unmasking from Compositions Containing Antibody and Polysorbate 20 as Dependent on the Buffer Substance It was determined in above Example 14 that the inventive unmasking approaches described herein are suitable for unmasking compositions which contain both detergent and buffered protein (antibody). In view of this, it was then desired to investigate the influence of buffer on unmasking efficiency. To this end, we chose 10 mM citrate or 10 mM phosphate buffer of pH 7.5, because these are the most commonly used buffers in protein drug formulations. Materials and Methods Endotoxin masking was performed as follows: 50 EU/ml of endotoxin (E. coli O55:B5; Sigma L2637-5MG) were added to 1 ml aliquots of an antibody solution containing 10 mg/ml of a bovine polyclonal IgG antibody preparation, dissolved in either 10 mM sodium phosphate containing 50 mM sodium chloride or 10 mM sodium citrate pH 7.5 containing 150 mM sodium chloride. Subsequently, polysorbate 20 was added to a final concentration of 0.05% and samples were masked for 3 days at room temperature. Further, positive controls containing the buffer solution without detergent or antibody, as well as the buffer solution containing either the antibody or the respective polysorbate were prepared and treated like the masking samples. Each of the positive controls contained the same amount of LPS. Endotoxin unmasking was performed as follows: Unmasking was performed by addition of either 1-dodecanol or a combination of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) or CaCl2, BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (disrupting and reconfiguring modulator). 100 μl of each of the following stock solutions were sequentially added to 1 ml of sample solution: CaCl2) (1M), BSA (10 mg/ml), SDS (1%) and 1-dodecanol (100, 10 or 1 mM). Furthermore, before addition of calcium chloride to a phosphate buffer-containing sample, this sample was stabilized against calcium phosphate precipitation by the addition of a final concentration of 200 mM sodium citrate pH 7.5. All stock solutions were added sequentially with two-minute mixing steps after each addition. After addition and mixing of the last component the samples were incubated for at least 30 minutes at room temperature. Afterwards, the samples were diluted 1:10 and 1:100 in endotoxin-free water and analyzed for endotoxin content using the EndoLISA kit (Hyglos GmbH). The percentage of LPS recovery was calculated with reference to the determined endotoxin content in the positive control (discussed in more detail in Example 1). Results Table 15 (below) shows the percentage of LPS recovery from an antibody solution after masking and unmasking containing either citrate or phosphate as buffer substance. TABLE 15citratephosphatebufferbufferLPSLPSsample typeingredientrecovery (%)recovery (%)water controlwater100100masking controlbuffer + antibody4031masking controlbuffer + polysorbate 2000masking controlbuffer + polysorbate 20 +10antibodyLPS[1-LPS[1-unmaskingre-dodec-re-dodec-sampleapproach/coveryanol]coveryanol]typeingredients(%)(mM)(%)(mM)unmasked1-dodecanol2610017100sample *unmaskedBSA/4910041100sample *1-dodecanolunmaskedCaCl2/87100671sample *BSA/SDS/1-dodecanol* “Unmasked” samples contained antibody. The data show that the buffer solutions containing antibody but no polysorbate, mask 60% to 70% of the LPS (based on the recovery of 40% and about 30% LPS for citrate and phosphate buffers, respectively). The LPS recoveries from buffer solutions containing polysorbate or polysorbate and antibody are below 1%. In these cases, masking is independent of the buffer present. LPS recoveries after unmasking from the compositions containing LPS, detergent and antibody are low using 1-dodecanol alone (17% and 26% for phosphate and citrate, respectively) and moderate using a combination of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) (41% and 49% for phosphate and citrate, respectively). Unmasking using a combination of CaCl2, BSA (adsorbing modulator), SDS (displacing modulator and 1-dodecanol (disrupting and reconfiguring modulator) results in recoveries of 67% and 87% of the masked LPS for phosphate and citrate, respectively. Interestingly, the necessary concentration of 1-dodecanol stock solution for efficient unmasking differs strongly between the buffer systems used (100 mM for antibody/detergent/citrate and 1 mM for antibody/detergent/phosphate). The data clearly show that efficient unmasking of endotoxin in compositions comprising both protein (antibody) and detergent can be achieved by adjustment of 1-dodecanol concentration. Example 15: Masking and Unmasking of an Antibody Solution Containing LPS from Unknown Source To show that unmasking is not only possible from solutions containing LPS from a known source, we tested a commercially available mouse monoclonal antibody for diagnostic use which contains an LPS contamination, where the source of the LPS is unknown. Furthermore, this antibody was dissolved in a buffer composition which corresponds to the formulation of the known antibody drug product Rituximab (MabThera®, Rituxan®). Materials and Methods Determination of endotoxin contamination: A mouse monoclonal antibody (MAB 33, Roche Diagnostics) was dissolved in a solution containing citrate and sodium chloride of pH 6.5 and stored at 4° C. The final concentrations of citrate, sodium chloride and antibody were 25 mM, 150 mM and 10 mg/ml, respectively. Directly after solubilization of the antibody, the endotoxin content was analyzed using EndoZyme® and EndoLISA® detection kits (Hyglos GmbH). The determined endotoxin content was 11 EU/mg of antibody. LPS masking was initiated by addition of polysorbate 80 to a final concentration of 0.07% and increasing the temperature to ambient conditions (22° C.). Afterwards, 1 ml aliquots of the samples were incubated at room temperature for 3 days to allow the endottoxin present to become masked. Unmasking was performed as follows: Unmasking was performed by addition of either 1-dodecanol (disrupting and reconfiguring modulator); or a combination of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator); or a combination of CaCl2, BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (disrupting and reconfiguring modulator). 100 μl of each of the following stock solutions were sequentially added to 1 ml of sample solution: CaCl2) (1 M), BSA (10 mg/ml), SDS (1%) and 1-dodecanol (100, 10 or 1 mM). All stock solutions were added sequentially with two-minute mixing steps after each addition. After addition and mixing of the last component the samples were incubated for at least 30 minutes at room temperature. Afterwards, the samples were diluted 1:10 and 1:100 in endotoxin free water and analyzed for endotoxin content using EndoLISA® (Hyglos GmbH). The percentage of LPS recovery was calculated in reference to the determined endotoxin content at time zero. Results Table 16 (below) shows the percentage of endotoxin recovery as dependent on the masking time, the presence or absence of polysorbate 80 and unmasking from antibody/polysorbate 80 solution. TABLE 16LPS recoverysample typeingredients(%)control t(0)buffer + antibody100masking control (3 days)buffer + antibody57masking control (3 days)buffer + polysorbate 800masking control (3 days)buffer + polysorbate 80 +3antibodyLPS[1-unmasking approach/recoverydodecanol]sample typeingredients(%)(mM)unmasked sample *1-dodecanol45100unmasked sample *BSA/1-dodecanol68100* “Unmasked” samples contained antibody. The data show that the buffer solution containing antibody but no polysorbate masks 40% of the LPS within 3 days of incubation at room temperature. However, incubation in buffer containing either polysorbate 80 or antibody and polysorbate 80, results in endotoxin recoveries smaller than 4%. Unmasking from the antibody/detergent samples results in recoveries of 45% using 1-dodecanol (disrupting and reconfiguring modulator); 68% using a combination of BSA (adsorbing modulator) and 1-dodecanol (disrupting and reconfiguring modulator); and 179% using a combination of CaCl2, BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (disrupting and reconfiguring modulator). In the latter case, the best recovery is achieved using a 1 mM 1-dodecanol stock solution. The experiments described in this example show that, when present, naturally occurring endotoxin (NOE) can be detected by a suitable endotoxin detection system. Furthermore, these experiments show that such NOE can be masked in the manner described herein above, i.e. the danger of masking applies not only for purified endotoxin, but also for NOE. The ability of the inventive methods as described herein to unmask such NOE further demonstrate their applicability to situations in which NOE has been masked, proving their effectiveness of the inventive methods to unmask masked NOE. These findings are relevant to the conditions prevailing in industry, where production processes often start with an expressed protein in the presence of NOE, and the latter is masked by incorporation of detergent to prevent unwanted protein aggregation. Overall, then, the results of the experiments described in this example demonstrate that the inventive methods are able to unmask endotoxin under conditions of relevance for the pharmaceutical industry. These data also clearly show that unmasking is independent of the source and purity of the LPS. In all three cases of masking in antibody solutions (Examples 13, 14 and 15), it can be seen that masking is not only due to the detergent component in the composition but also to some extent to the antibody itself. The most efficient unmasking approach is to use a combination of CaCl2, BSA (adsorbing modulator), SDS (displacing modulator) and 1-dodecanol (disrupting and reconfiguring modulator) to unmask the endotoxin. Here, analogies can be seen to the lysozyme case (discussed in Example 7 above), in which the protein itself plays a role as an endotoxin masker. Interestingly, in all cases, the concentration of 1-dodecanol should be optimized for efficient unmasking. Example 16: General Evaluation of Unmasking Approach as Applied to a New Composition in Question As shown in the above examples, the choice of the approach taken to unmask endotoxin suspected of being present, but masked in a composition will depend on a number of factors. For instance, as the foregoing examples have shown, it is sometimes possible to achieve efficient unmasking using a single-component modulator which doubles as a disrupting modulator and a reconfiguring modulator, as defined herein above. On the other hand, in some instances, the modulator should be a modulator system with two or more components, for instance a displacing modulator and/or an adsorbing modulator, depending on what measures are needed to destabilize and disrupt the endotoxin/endotoxin masker complex sufficiently such that the endotoxin is liberated and can be mediated into an aggregated form which can be detected. The above examples start from known, controlled solution conditions in order to illustrate concepts underlying the present invention. In a real-world scenario, however, in which the methods of the invention are to be applied to a new composition in question, it is necessary to first evaluate the approach of the methods of the invention before meaningful results can be obtained. The present example addresses such a validation, setting out a generic scheme by which the methods of the invention may be calibrated to a new composition in question. To this end, an iterative unmasking approach is necessary, starting with an initial screening for the best suited unmasking approach followed by subsequent improvement steps for adjustment of optimum unmasking component concentrations. General Description of an Evaluation Process for a Given Composition Generally,FIG.12shows a scheme which schematically sets out the steps which one would normally take in evaluating the inventive methods for a new, unknown composition. As will be clear from the above, ultimate detection of initially masked endotoxin depends on the ability to convert this endotoxin from stably bound (masked) form to an aggregated from which is unmasked and therefore detectable. The component of the modulator responsible for this final conversion is the reconfiguring modulator. The first step ofFIG.12reflects this, in that it specifies a first step of determining an optimal concentration of reconfiguring modulator (e.g. 1-dodecanol). Step 2 then optimizes the concentration of adsorbing modulator, if this modulator is included. Step 3 then optimizes the concentration of displacing modulator, if this modulator is included. It should be emphasized that not all three steps will always be needed. If one already sees that a composition, for example a pharmaceutical composition, in question contains significant amounts of endotoxin following step one, then this answer may already be enough to conclude that the composition thought to be endotoxin-free was really not. Specific Description of Evaluation Process for a Given Composition FIG.13shows the combinations and concentrations of stock solutions for selecting and optimizing the unmasking process. The unmasking approaches are divided into different possible scenarios A, B and C, depending on which substance or combination of substances is/are used in unmasking. Unmasking approach A describes an unmasking approach in which only 1-dodecanol is used as a modulator. Unmasking approach B describes an unmasking approach in which the modulator system is composed of 1-dodecanol and BSA. Unmasking approach C describes an unmasking approach in which the modulator system is composed of 1-dodecanol, BSA and SDS, and is performed in the presence of CaCl2. Procedure Add 100 μl of the unmasking component stock solutions to 1 ml of masked sample. After addition of one component, mix sample thoroughly by vortexing for 2 minutes. Then, add the next component and mix. After addition of all components and subsequent mixing, incubate samples for >30 minutes at room temperature. Afterwards, analyze samples for endotoxin content using an appropriate endotoxin testing method, e.g. the EndoLISA® kit of Hyglos GmbH. Example 17: Detection of Unmasked Endotoxin Using a Recombinant Factor C Assay This experiment investigates the effect of unmasking endotoxin using a multi-component modulator comprising CaCl2, BSA, SDS and dodecanol. Endotoxin content of the masked and unmasked samples was determined using the EndoZyme® kit of Hyglos GmbH. The experiment was performed in order to show that detection of unmasked endotoxin can be achieved using different detection assays. Materials and Methods Endotoxin (E. coli O55:B5, Sigma L2637-5MG) was masked in solutions containing 1×PBS-buffered 0.05 wt % Polysorbate 80 or 1×PBS buffered 0.05 wt % Polysorbate 20 for 3 days at room temperature. Unmasking was performed as follows: Unmasking was performed by a combination of sodium citrate, CaCl2, BSA, SDS and 1-dodecanol. 150 μL of sodium citrate and 100 μl of each of the following stock solutions were added to 1 ml of sample solution: sodium citrate (1.375 M pH 7.5), CaCl2) (1 M), BSA (10 mg/ml), SDS (1%) and 1-dodecanol (1 mM). 1-dodecanol was solubilized in 70% EtOH. In a separate masking control, no unmasking was performed. All stock solutions were added sequentially with two-minute mixing steps after each addition. After addition and mixing of the last component the samples were incubated for at least 30 minutes at room temperature. Subsequently, masked (masking control) and unmasked samples were diluted stepwise 1:10 and 1:5 in depyrogenated water (final dilution 1:50). A recombinant Factor C assay (EndoZyme®) was used for detection of endotoxin. Results Table 17 (below) shows the percent recovery, measured using a recombinant Factor C assay (EndoZyme®), of endotoxin recovered from the two masking systems specified above in this example. TABLE 17Detection of unmasked endotoxin using recombinant Factor CRecombinant Factor CPBS + P80PBS + P20Sample[EU/mL][EU/mL]Positive control9.36.8Recovery [%]Recovery [%]Masking control00After unmasking6566 The masking control showed no endotoxin recovery in either sample. Unmasking of endotoxin in polysorbate 80 or polysorbate 20 resulted in endotoxin recovery of 65% and 66%, respectively, with reference to the positive control (endotoxin content in depyrogenated water). The results indicate the efficient demasking of endotoxin using a multi-component modulator comprising Sodium citrate, CaCl2, BSA, SDS and dodecanol as detected by a recombinant Factor C detection system (EndoZyme®). This experiment proves that the detection of unmasked endotoxin is independent of the endotoxin detection system used. Accordingly, unmasked endotoxin may be detected using the endotoxin detection system employed in previous examples, but may also be detected using an endotoxin detection system differing from that used in previous examples. Example 18: Detection of Unmasked Endotoxin Using a Limulus Ameboecyte Lysate (LAL) Assay This experiment investigates the detection of unmasked endotoxin using a detection assay different from the recombinant Factor C assay (EndoZyme®), i.e. the Limulus Ameboecyte Lysate (LAL) assay. The experiment was performed in order to further corroborate that detection of endoxin unmasking does not depend on the detection assay. Materials and Methods Endotoxin (E. coli O55:B5, Sigma L2637-5MG). was masked in solutions containing 1×PBS-buffered 0.05 wt % Polysorbate 80 or 1×PBS buffered 0.05 wt % Polysorbate 20 for 3 days at room temperature. Unmasking was performed as follows: Unmasking was performed by a combination of sodium citrate, CaCl2, BSA, SDS and 1-dodecanol. 150 μL of sodium citrate and 100 μl of each of the following stock solutions were added to 1 ml of sample solution: sodium citrate (1.375 M pH 7.5), CaCl2(1 M), BSA (10 mg/ml), SDS (1%) and 1-dodecanol (1 mM). 1-dodecanol was solubilized in 70% EtOH. All stock solutions were added sequentially with two-minute mixing steps after each addition. After addition and mixing of the last component the samples were incubated for at least 30 minutes at room temperature. Subsequently, masked (masking control) and unmasked samples were diluted stepwise 1:10 and 1:5 in depyrogenated water (final dilution 1:50). A kinetic LAL-based chromogenic assay (kinetic-QCL®, Lonza) was used for detection of endotoxin. Masking control reflects the detectable endotoxin content without unmasking. In a separate masking control, no unmasking was performed. Results Table 18 (below) shows the percent recovery, measured using an LAL assay (kinetic QCL®, Lonza), of endotoxin recovered from the two masking systems specified above in this example. TABLE 18Unmasking using an LAL assayLALPBS + P80PBS + P20Sample[EU/mL][EU/mL]Positive control11.67.2Recovery [%]Recovery [%]Masking control30After unmasking9647 The masking control showed no endotoxin recovery in both samples. Unmasking of endotoxin in polysorbate 80 or polysorbate 20 resulted in endotoxin recovery of 96% and 47%, respectively, with reference to the positive control (endotoxin content in depyrogenated water). The data clearly demonstrate that unmasking of endotoxin can be detected with the LAL detection assay and that detection of endotoxin unmasking does not depend on the detection assay. Example 19: Variation of Alkanols (Aliphatic Alcohols) as Modulators for Unmasking Using a Multi-Component Modulator This experiment investigates unmasking of different endotoxins using different alkanols. The experiment was performed in order to investigate the unmasking efficiency of different alkanol compounds in the multi-component modulator. Materials and Methods Endotoxin fromE. coli O55:B5 (Sigma L2637-5MG),S. abortusequi (Acila 1220302) andK. pneumoniae(LMU) were masked in solutions containing 10 mM sodium citrate and 0.05 wt % Polysorbate 20 for three days at room temperature. Unmasking was performed as follows: Unmasking was performed by a combination of NaCitrate, CaCl2, BSA, SDS and 1-dodecanol. 150 μL of sodium citrate and 100 μl of each of the following stock solutions were added to 1 ml of sample solution: sodium citrate (1.375 M pH 7.5), CaCl2) (1 M), BSA (10 mg/ml), SDS (1%) and a certain concentration of 1-dodecanol. The alkanols and alkanol mixtures used in the multi-component modulator systems were solubilized in EtOH; concentrations are listed in Table 19a (below). In a separate masking control, no unmasking was performed. All stock solutions were added sequentially with two-minute mixing steps after each addition. After addition and mixing of the last component the samples were incubated for at least 30 minutes at room temperature. TABLE 19aUnmaskingConcentrationapproach:Alkanols (size)[mM]1Octanol (C8)1.02Decanol (C10)1.03Dodecanol (C12)1.04Tetradecanol (C14)1.05Hexadecanol (C16)1.06Octanol (C8)0.3Decanol (C10)0.3Dodecanol (C12)0.37Decanol (C10)0.3Dodecanol (C12)0.3Tetradecanol (C14)0.38Dodecanol (C12)0.3Tetradecanol (C14)0.3Hexadecanol (C16)0.3 Afterwards, the samples were diluted 1:10 and 1:100 in endotoxin free water and analyzed for endotoxin content using EndoLISA® (Hyglos GmbH). The percentage of LPS recovery was calculated in reference to the determined endotoxin content at time zero (summarized in Table 19b, below). Results Table 19b (below) shows the percent recovery after masking (masking control) and after unmasking using the EndoLISA® assay (Hyglos) from the above masking system by various unmasking approaches employing different alkanols (aliphatic alcohols) or alkanol mixtures (aliphatic alcohol mixtures) as specified above in Table 19a. TABLE 19bUnmasking of different endotoxins using Ca, BSA, SDS and varyingalkanols, as detected by the EndoLISA ® assayEndotoxinK. pneumoniae*S. abortusE. coliO55:B5[EU/mL]equi [EU/mL][EU/mL]Positive Control1915168Recovery [%]Recovery [%]Recovery [%]Masking Control000Unmaskingapproach(alkanol size)1 (C8)75022 (C10)52003 (C12)14762764 (C14)94108715 (C16)9983226 (C8, C10, C12)601467 (C10, C12, C14)126108438 (C12, C14, C16)12617343* For unmasking ofK. pneumoniae150 μL of CaCl2were added. The above results indicate that unmasking ofK. pneumoniaewas achieved with octanol (75% recovery), dodecanol (147%), tetradecanol (94%) and hexadecanol (99%), as well as with different combinations of alkanols (see e.g. unmasking approaches 7 and 8). Unmasking with decanol, however, was less efficient (52%). Unmasking of theS. abortusequi LPS was most efficient using tetradecanol (108%), hexadecanol (82%), dodecanol (62%), or different combinations of alkanols. Effective unmasking ofE. coli O55:B5 was observed for dodecanol (76%) and tetradodecanol (71%). No endotoxin recovery was observed for the masking controls. These results indicate that the most efficient unmasking (independent of the nature of the endotoxin) was achieved using dodecanol or tetradecanol, or using combinations of dodecanol and tetradecanol with a further alkanol (e.g. decanol in demasking 7). These results also indicate that all multi-component modulator systems with C12, C14and/or C16aliphatic alcohols exhibited efficient unmasking of endotoxin. The range of alkyl chain length of the fatty alcohols for efficient unmasking seems to depend on the endotoxin source. The differences in the unmasking efficiencies may depend to a certain extent on the heterogeneity in length of the acyl chains of the β-hydroxy-fatty acids which are present in the Lipid A portion of endotoxin. Between and within bacterial species, these acyl chains can vary in length from C10 to C28 (Endotoxin in health and disease, edited by H. Brade (1999), p98 et seq: “Chemical structure of Lipid A: Recent advances in structural analysis of biologically active molecules”; Marcel Dekker Inc, New York). However, most commonly β-hydroxy-fatty acids with chains length of C14 and C16 are appended to the diglucosamine of Lipid A. Thus, unmasking is in all cases most efficient in the presence of fatty alcohols with alkyl chain length between C12 and C14, although unmasking of endotoxin is also observed for other alkyl chain lengths in the C8-C16 range. Example 20: Variation of Alkanols (Aliphatic Alcohols) as Modulators for Unmasking Using a Single-Component Modulator This experiment was performed to investigate the effect of various alkanols (aliphatic alcohols) on unmasking in the absence of additional modulator components. The experiment thus investigates the efficiency of endotoxin unmasking using various alkanols (aliphatic alcohols) as single-component modulators. Materials and Methods EndotoxinE. coli O55:B5 (Sigma L2637-5MG) was masked in solutions containing 10 mM sodium citrate and 0.05 wt % Polysorbate 20 for 3 days at room temperature. In order to unmask the samples, samples (1 mL) were mixed with 100 μL of the particular alkanol (i.e. aliphatic alcohol). The alkanols used in the single-component modulator systems were solubilized in EtOH. Concentrations are shown in Table 20a (below). TABLE 20aVariation of alkanols (aliphatic alcohols)UnmaskingApproachAlkanols (size)Concentration [mM]1Dodecanol (C12)50 mM2Tridecanol (C13)50 mM3Tetradecanol (C14)50 mM After addition of unmasking agents, the samples were incubated for 30 minutes and diluted 1:10 as well as 1:100 in depyrogenated water. Endotoxin was detected in both dilutions and the stated recovery reflects the mean recovery of both dilutions. The masking control reflects the non-treated sample after masking, i.e. the solution is not unmasked. The EndoLISA® assay was used for endotoxin detection. Results Table 20b (below) shows the percent recovery, measured using the EndoLISA® assay (Hyglos), of endotoxin recovered from the above masking system by various unmasking approaches employing different alkanols (aliphatic alcohols) in different unmasking approaches using single-modulator systems as specified above in Table 20a. TABLE 20bUnmasking using different alkanols (EndoLISA ®)E. coliO55:B5 (gel)Endotoxin[EU/mL]Positive Control111Recovery [%]Masking Control0Unmasking approach(alkanol size)1 (C12)562 (C13)413 (C14)22.6 The results indicate that a single-component modulator consisting of dodecanol (unmasking approach 1) was most efficient in unmasking ofE. coli O55:B5 (56% recovery), whereas single-component modulators consisting of tridecanol (unmasking approach 2) or tetradecanol (unmasking approach 3) resulted in less recovery ofE. coli O55:B5 (41% and 22.6%, respectively). As expected, the masking controls showed no endotoxin recovery. In summary, the data demonstrate that the most efficient alkanol (aliphatic alcohol) for unmasking ofE. coli O55:B5, when used as a single-component modulator system, is dodecanol, followed by tridecanol and tetradecanol. | 156,337 |
11860159 | DETAILED DESCRIPTION Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. As summarized above, the present disclosure provides systems for use in preparing a labelled biomolecule reagent. In further describing embodiments of the disclosure, systems having an input manager for receiving a labelled biomolecule reagent request and an output manager for providing biomolecule and label storage identifiers are first described in greater detail. Next, a reagent preparatory apparatus for preparing the labelled biomolecule reagent from an activated biomolecule and an activated label are described. Methods for communicating and receiving a labelled biomolecule reagent request and preparing the subject labelled biomolecule reagents are also provided. Systems for Use in Preparing a Labelled Biomolecule Reagent Aspects of the present disclosure include systems for use in preparing a labelled biomolecule reagent. Systems according to certain embodiments include an input manager for receiving a request for a labelled biomolecule reagent, a memory for storing a dataset having a plurality of storage identifiers that correspond to the one or more components of the labelled biomolecule reagent request (e.g., biomolecule, label, etc.), a processing module communicatively coupled to the memory and configured to identify a storage identifier from the dataset that corresponds to the components of the labelled biomolecule reagent request and an output manager for providing the identified storage identifiers. As described in greater detail below, the term “labelled biomolecule” reagent refers to a biological macromolecule coupled (e.g., through a covalent bond) to a detectable marker. The biological macromolecule may be a biopolymer. A “biopolymer” is a polymer of one or more types of repeating units. Biopolymers are typically found in biological systems and particularly include polysaccharides (such as carbohydrates), and peptides (which term is used to include polypeptides, and proteins whether or not attached to a polysaccharide) and polynucleotides as well as their analogs such as those compounds composed of or containing amino acid analogs or non-amino acid groups, or nucleotide analogs or non-nucleotide groups. This includes polynucleotides in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone, and nucleic acids (or synthetic or naturally occurring analogs) in which one or more of the conventional bases has been replaced with a group (natural or synthetic) capable of participating in Watson-Crick type hydrogen bonding interactions. Polynucleotides include single or multiple stranded configurations, where one or more of the strands may or may not be completely aligned with another. Specifically, a “biopolymer” includes DNA (including cDNA), RNA and oligonucleotides, regardless of the source. As such, biomolecules may include polysaccharides, nucleic acids and polypeptides. For example, the nucleic acid may be an oligonucleotide, truncated or full-length DNA or RNA. In embodiments, oligonucleotides, truncated and full-length DNA or RNA are comprised of 10 nucleotide monomers or more, such as 15 or more, such as 25 or more, such as 50 or more, such as 100 or more, such as 250 or more and including 500 nucleotide monomers or more. For example, oligonucleotides, truncated and full-length DNA or RNA of interest may range in length from 10 nucleotides to 108nucleotides, such as from 102nucleotides to 107nucleotides, including from 103nucleotides to 106nucleotides. In embodiments, biopolymers are not single nucleotides or short chain oligonucleotides (e.g., less than 10 nucleotides). By “full length” is meant that the DNA or RNA is a nucleic acid polymer having 70% or more of its complete sequence (such as found in nature), such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more and including 100% of the full length sequence of the DNA or RNA (such as found in nature) Polypeptides may be, in certain instances, truncated or full length proteins, enzyme or antibodies. In embodiments, polypeptides, truncated and full-length proteins, enzymes or antibodies are comprised of 10 amino acid monomers or more, such as 15 or more, such as 25 or more, such as 50 or more, such as 100 or more, such as 250 or more and including 500 amino acid monomers or more. For example, polypeptides, truncated and full-length proteins, enzymes or antibodies of interest may range in length from 10 amino acids to 108amino acids, such as from 102amino acids to 107amino acids, including from 103amino acids to 106amino acids. In embodiments, biopolymers are not single amino acids or short chain polypeptides (e.g., less than 10 amino acids). By “full length” is meant that the protein, enzyme or antibody is a polypeptide polymer having 70% or more of its complete sequence (such as found in nature), such as 75% or more, such as 80% or more, such as 85% or more, such as 90% or more, such as 95% or more, such as 97% or more, such as 99% or more and including 100% of the full length sequence of the protein, enzyme or antibody (such as found in nature) In embodiments of the present disclosure, labels are detectable moieties or markers that are detectible based on, for example, fluorescence emission, absorbance, fluorescence polarization, fluorescence lifetime, fluorescence wavelength, absorbance maxima, absorbance wavelength, Stokes shift, light scatter, mass, molecular mass, redox, acoustic, raman, magnetism, radio frequency, enzymatic reactions (including chemiluminescence and electro-chemiluminescence) or combinations thereof. For example, the label may be a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag (e.g., isotopically pure rare earth element), magnetic particle, microparticle as well as a nanoparticle. Systems include an input manager for receiving a labelled biomolecule reagent request. The labelled biomolecule reagent request may include one or more components. In some instances, the labelled biomolecule reagent request includes a single component and is a labelled biomolecule request (i.e., a request for a biomolecule covalently bonded to a label through a reactive linker). In other instances, the labelled biomolecule reagent request includes two or more components. For example, the labelled biomolecule reagent request includes a biomolecule request and a label request. In certain embodiments, the biomolecule request is an activated biomolecule request that includes a biomolecule and a reactive linker and the label request is an activated label request that includes a label and a reactive linker. The phrases “labelled biomolecule request”, “biomolecule request” and “label request” are used herein to refer to information or data associated with a particular labelled biomolecule, biomolecule or label, respectively. The request may include a string of one or more characters (e.g., alphanumeric characters), symbols, images or other graphical representation(s) associated with a particular labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker. In some instances, the request is a “shorthand” designation of the labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker. For example, the request may include an accession number or an abbreviated probe sequence. The request may also include descriptive information, such as chemical structure or reactivity. Information or data, in certain embodiments, may be any suitable identifier of the labelled biomolecule, biomolecule or label and may include, but is not limited to, the name, monomer sequence, sequence identification number, ascension number or biological source of the biomolecule as well as the name, chemical structure, Chemical Abstracts Service (CAS) registry number or marker class (e.g., fluorescence, magnetic) of the label. In some embodiments, the biomolecule is a biological probe for an analyte of interest and the biomolecule request includes information or data pertaining to a specific binding domain that binds to the analyte of interest. Specific binding domains of interest include, but are not limited to, antibody binding agents, proteins, peptides, haptens, nucleic acids, etc. The term “antibody binding agent” as used herein includes polyclonal or monoclonal antibodies or fragments that are sufficient to bind to an analyte of interest. The antibody fragments can be, for example, monomeric Fab fragments, monomeric Fab′ fragments, or dimeric F(ab)′2 fragments. Also within the scope of the term “antibody binding agent” are molecules produced by antibody engineering, such as single-chain antibody molecules (scFv) or humanized or chimeric antibodies produced from monoclonal antibodies by replacement of the constant regions of the heavy and light chains to produce chimeric antibodies or replacement of both the constant regions and the framework portions of the variable regions to produce humanized antibodies. In some instances, the biomolecule is a polypeptide and the biomolecule request may include information such as polypeptide name, protein name, enzyme name, antibody name or the name of protein, enzyme or antibody fragments thereof, polypeptides derived from specific biological fluids (e.g., blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen), polypeptides derived from specific species (e.g., mouse monoclonal antibodies) as well as amino acid sequence identification number. In other instances, the biomolecule is a nucleic acid and the biomolecule request may include information such as oligonucleotide name, oligonucleotides identified by gene name, oligonucleotides identified by accession number, oligonucleotides of genes from specific species (e.g., mouse, human), oligonucleotides of genes associated with specific tissues (e.g., liver, brain, cardiac), oligonucleotides of genes associate with specific physiological functions (e.g., apoptosis, stress response), oligonucleotides of genes associated with specific disease states (e.g., cancer, cardiovascular disease) as well as nucleotide sequence. As discussed above, labels may include detectable moieties or markers that are detectible based on, for example, fluorescence emission, absorbance, fluorescence polarization, fluorescence lifetime, fluorescence wavelength, absorbance wavelength, Stokes shift, light scatter, mass, molecular mass, redox, acoustic, raman, magnetism, radio frequency, enzymatic reactions (including chemiluminescence and electro-chemiluminescence) or combinations thereof. For example, the label may be a fluorophore, chromophore, enzyme, redox label, radio label, acoustic label, Raman (SERS) tag, mass tag, isotope tag (e.g., isotopically pure rare earth element), magnetic particle, microparticle as well as a nanoparticle. In certain embodiments, the label is a fluorophore (i.e., a fluorescent label, fluorescent dye, etc.). Fluorophores of interest may include but are not limited to dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.), such as an acridine dye, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenyl methane dyes), chlorophyll containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinon-imine dyes, azine dyes, eurhodin dyes, safranin dyes, indamins, indophenol dyes, fluorine dyes, oxazine dye, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, as well as dyes combining two or more of the aforementioned dyes (e.g., in tandem), polymeric dyes having one or more monomeric dye units and mixtures of two or more of the aforementioned dyes thereof. A large number of dyes are commercially available from a variety of sources, such as, for example, Molecular Probes (Eugene, OR), Dyomics GmbH (Jena, Germany), Sigma-Aldrich (St. Louis, MO), Sirigen, Inc. (Santa Barbara, CA) and Exciton (Dayton, OH). For example, the fluorophore may include 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives such as acridine, acridine orange, acridine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, peridinin-chlorophyll protein, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine and derivatives such as cyanosine, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7; 4′,6-diaminidino-2-phenylindole (DAPI); 5′, 5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; IR144; IR1446; Green Fluorescent Protein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™; Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthene; dye-conjugated polymers (i.e., polymer-attached dyes) such as fluorescein isothiocyanate-dextran as well as dyes combining two or more dyes (e.g., in tandem), polymeric dyes having one or more monomeric dye units and mixtures of two or more of the aforementioned dyes or combinations thereof. In some instances, the fluorophore (i.e., dye) is a fluorescent polymeric dye. Fluorescent polymeric dyes that find use in the subject methods and systems are varied. In some instances of the method, the polymeric dye includes a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure which includes a backbone of alternating unsaturated bonds (e.g., double and/or triple bonds) and saturated (e.g., single bonds) bonds, where π-electrons can move from one bond to the other. As such, the conjugated backbone may impart an extended linear structure on the polymeric dye, with limited bond angles between repeat units of the polymer. For example, proteins and nucleic acids, although also polymeric, in some cases do not form extended-rod structures but rather fold into higher-order three-dimensional shapes. In addition, CPs may form “rigid-rod” polymer backbones and experience a limited twist (e.g., torsion) angle between monomer repeat units along the polymer backbone chain. In some instances, the polymeric dye includes a CP that has a rigid rod structure. As summarized above, the structural characteristics of the polymeric dyes can have an effect on the fluorescence properties of the molecules. Any convenient polymeric dye may be utilized in the subject methods and systems. In some instances, a polymeric dye is a multichromophore that has a structure capable of harvesting light to amplify the fluorescent output of a fluorophore. In some instances, the polymeric dye is capable of harvesting light and efficiently converting it to emitted light at a longer wavelength. In some cases, the polymeric dye has a light-harvesting multichromophore system that can efficiently transfer energy to nearby luminescent species (e.g., a “signaling chromophore”). Mechanisms for energy transfer include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer) and the like. In some instances, these energy transfer mechanisms are relatively short range; that is, close proximity of the light harvesting multichromophore system to the signaling chromophore provides for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the signaling chromophore occurs when the number of individual chromophores in the light harvesting multichromophore system is large; that is, the emission from the signaling chromophore is more intense when the incident light (the “excitation light”) is at a wavelength which is absorbed by the light harvesting multichromophore system than when the signaling chromophore is directly excited by the pump light. The multichromophore may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the length of the polymer chain, the backbone contains a large number of conjugated segments in close proximity. Thus, conjugated polymers are efficient for light harvesting and enable optical amplification via energy transfer. In some instances the polymer may be used as a direct fluorescent reporter, for example fluorescent polymers having high extinction coefficients, high brightness, etc. In some instances, the polymer may be used as a strong chromophore where the color or optical density is used as an indicator. Polymeric dyes of interest include, but are not limited to, those dyes described by Gaylord et al. in US Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986, 20130190193 and 20160025735 the disclosures of which are herein incorporated by reference in their entirety; and Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010,39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32), pp 12600-12607, the disclosures of which are herein incorporated by reference in their entirety. In some embodiments, the polymeric dye includes a conjugated polymer including a plurality of first optically active units forming a conjugated system, having a first absorption wavelength (e.g., as described herein) at which the first optically active units absorbs light to form an excited state. The conjugated polymer (CP) may be polycationic, polyanionic and/or a charge-neutral conjugated polymer. The CPs may be water soluble for use in biological samples. Any convenient substituent groups may be included in the polymeric dyes to provide for increased water-solubility, such as a hydrophilic substituent group, e.g., a hydrophilic polymer, or a charged substituent group, e.g., groups that are positively or negatively charged in an aqueous solution, e.g., under physiological conditions. Any convenient water-soluble groups (WSGs) may be utilized in the subject light harvesting multichromophores. The term “water-soluble group” refers to a functional group that is well solvated in aqueous environments and that imparts improved water solubility to the molecules to which it is attached. In some embodiments, a WSG increases the solubility of the multichromophore in a predominantly aqueous solution (e.g., as described herein), as compared to a multichromophore which lacks the WSG. The water soluble groups may be any convenient hydrophilic group that is well solvated in aqueous environments. In some cases, the hydrophilic water soluble group is charged, e.g., positively or negatively charged or zwitterionic. In certain cases, the hydrophilic water soluble group is a neutral hydrophilic group. In some embodiments, the WSG is a hydrophilic polymer, e.g., a polyethylene glycol, a cellulose, a chitosan, or a derivative thereof. As used herein, the terms “polyethylene oxide”, “PEO”, “polyethylene glycol” and “PEG” are used interchangeably and refer to a polymer including a chain described by the formula —(CH2—CH2—O—)n— or a derivative thereof. In some embodiments, “n” is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 5 to 15, or 10 to 15. It is understood that the PEG polymer may be of any convenient length and may include a variety of terminal groups, including but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal groups. Functionalized PEGs that may be adapted for use in the subject multichromophores include those PEGs described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165. Water soluble groups of interest include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, ester, polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, ammonium, guanidinium, polyamine and sulfonium, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including, polyethers, —COOM′, —SO3M′, —PO3M′, —NR3+, Y′, (CH2CH2O)pR and mixtures thereof, where Y′ can be any halogen, sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, each R can be independently H or an alkyl (such as methyl) and M′ can be a cationic counterion or hydrogen, —(CH2CH2O)yyCH2CH2XRyy, —(CH2CH2O)yyCH2CH2X—, —X(CH2CH2O)yyCH2CH2—, glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and RZZand RYYare independently selected from H and C1-3 alkyl. The polymeric dye may have any convenient length. In some cases, the particular number of monomeric repeat units or segments of the polymeric dye may fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units or segments, or such as 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In certain instances, the number of monomeric repeat units or segments of the polymeric dye is within the range of 2 to 1000 units or segments, such as from 2 to 750 units or segments, such as from 2 to 500 units or segments, such as from 2 to 250 units or segment, such as from 2 to 150 units or segment, such as from 2 to 100 units or segments, such as from 2 to 75 units or segments, such as from 2 to 50 units or segments and including from 2 to 25 units or segments. The polymeric dyes may be of any convenient molecular weight (MW). In some cases, the MW of the polymeric dye may be expressed as an average molecular weight. In some instances, the polymeric dye has an average molecular weight of from 500 to 500,000, such as from 1,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000 or even an average molecular weight of from 50,000 to 100,000. In certain embodiments, the polymeric dye has an average molecular weight of 70,000. In certain instances, the polymeric dye includes the following structure: wherein CP1, CP2, CP3and CP4are independently a conjugated polymer segment or an oligomeric structure, wherein one or more of CP1, CP2, CP3and CP4are bandgap-modifying n-conjugated repeat units. In some embodiments, the conjugated polymer is a polyfluorene conjugated polymer, a polyphenylene vinylene conjugated polymer, a polyphenylene ether conjugated polymer, a polyphenylene polymer, among other types of conjugated polymers. In some instances, the polymeric dye includes the following structure: wherein each R1is independently a solubilizing group or a linker-dye; L1and L2are optional linkers; each R2is independently H or an aryl substituent; each A1and A2is independently H, an aryl substituent or a fluorophore; G1and G2are each independently selected from the group consisting of a terminal group, a rrconjugated segment, a linker and a linked specific binding member; each n and each m are independently 0 or an integer from 1 to 10,000; and p is an integer from 1 to 100,000. Solubilizing groups of interest include, but is not limited to a water-soluble functional group such as a hydrophilic polymer (e.g., polyalkylene oxide, cellulose, chitosan, etc.), as well as alkyl, aryl and heterocycle groups further substituted with a hydrophilic group such as a polyalkylene oxide (e.g., polyethylglycol including a PEG of 2-20 units), an ammonium, a sulphonium, a phosphonium, as well has a charged (positively, negatively or zwitterionic) hydrophilic water soluble group and the like. In some cases, the polymeric dye includes, as part of the polymeric backbone, a conjugated segment having one of the following structures: where each R3is independently an optionally substituted water-soluble functional group such as a hydrophilic polymer (e.g., polyalkylene oxide, cellulose, chitosan, etc.) or an alkyl or aryl group further substituted with a hydrophilic group such as a polyalkylene oxide (e.g., polyethylglycol including a PEG of 2-20 units), an ammonium, a sulphonium, a phosphonium, as well has a charged (positively, negatively or zwitterionic) hydrophilic water soluble group; Ar is an optionally substituted aryl or heteroaryl group; and n is 1 to 10000. In certain embodiments, R3is an optionally substituted alkyl group. In certain embodiments, R3is an optionally substituted aryl group. In some cases, R3is substituted with a polyethyleneglycol, a dye, a chemoselective functional group or a specific binding moiety. In some cases, Ar is substituted with a polyethyleneglycol, a dye, a chemoselective functional group or a specific binding moiety. In some instances, the polymeric dye includes the following structure: wherein each R1is a solubilizing group or a linker-dye group; each R2is independently H or an aryl substituent; L1and L2are optional linkers; each A1and A3are independently H, a fluorophore, a functional group or a specific binding moiety (e.g., an antibody); and n and m are each independently 0 to 10000, wherein n+m>1. The polymeric dye may have one or more desirable spectroscopic properties, such as a particular absorption maximum wavelength, a particular emission maximum wavelength, extinction coefficient, quantum yield, and the like (see e.g., Chattopadhyay et al., “Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments.” Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polymeric dye has an absorption curve between 280 and 850 nm. In certain embodiments, the polymeric dye has an absorption maximum in the range 280 and 850 nm. In some embodiments, the polymeric dye absorbs incident light having a wavelength in the range between 280 and 850 nm, where specific examples of absorption maxima of interest include, but are not limited to: 348 nm, 355 nm, 405 nm, 407 nm, 445 nm, 488 nm, 640 nm and 652 nm. In some instances, the polymeric dye has an absorption maximum wavelength in a range selected from the group consisting of 280-310 nm, 305-325 nm, 320-350 nm, 340-375 nm, 370-425 nm, 400-450 nm, 440-500 nm, 475-550 nm, 525-625 nm, 625-675 nm and 650-750 nm. In certain embodiments, the polymeric dye has an absorption maximum wavelength of 348 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 355 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 405 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 407 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 445 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 488 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 640 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 652 nm. In some embodiments, the polymeric dye has an emission maximum wavelength ranging from 400 to 850 nm, such as 415 to 800 nm, where specific examples of emission maxima of interest include, but are not limited to: 395 nm, 421 nm, 445 nm, 448 nm, 452 nm, 478 nm, 480 nm, 485 nm, 491 nm, 496 nm, 500 nm, 510 nm, 515 nm, 519 nm, 520 nm, 563 nm, 570 nm, 578 nm, 602 nm, 612 nm, 650 nm, 661 nm, 667 nm, 668 nm, 678 nm, 695 nm, 702 nm, 711 nm, 719 nm, 737 nm, 785 nm, 786 nm, 805 nm. In some instances, the polymeric dye has an emission maximum wavelength in a range selected from the group consisting of 380-400 nm, 410-430 nm, 470-490 nm, 490-510 nm, 500-520 nm, 560-580 nm, 570-595 nm, 590-610 nm, 610-650 nm, 640-660 nm, 650-700 nm, 700-720 nm, 710-750 nm, 740-780 nm and 775-795 nm. In certain embodiments, the polymeric dye has an emission maximum of 395 nm. In some instances, the polymeric dye has an emission maximum wavelength of 421 nm. In some instances, the polymeric dye has an emission maximum wavelength of 478 nm. In some instances, the polymeric dye has an emission maximum wavelength of 480 nm. In some instances, the polymeric dye has an emission maximum wavelength of 485 nm. In some instances, the polymeric dye has an emission maximum wavelength of 496 nm. In some instances, the polymeric dye has an emission maximum wavelength of 510 nm. In some cases, the polymeric dye has an emission maximum wavelength of 570 nm. In certain embodiments, the polymeric dye has an emission maximum wavelength of 602 nm. In some instances, the polymeric dye has an emission maximum wavelength of 650 nm. In certain cases, the polymeric dye has an emission maximum wavelength of 711 nm. In some instances, the polymeric dye has an emission maximum wavelength of 737 nm. In some instances, the polymeric dye has an emission maximum wavelength of 750 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 786 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 421 nm±5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm±5 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 570 nm±5 nm. In some instances, the polymeric dye has an emission maximum wavelength of 602 nm±5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 650 nm±5 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 711 nm±5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 786 nm±5 nm. In certain embodiments, the polymeric dye has an emission maximum selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm and 786 nm. In some instances, the polymeric dye has an extinction coefficient of 1×106cm−1M−1or more, such as 2×106cm−1M−1or more, 2.5×106cm−1M−1or more, 3×106cm−1M−1or more, 4×106cm−1M−1or more, 5×106cm−1M−1or more, 6×106cm−1M−1or more, 7×106cm−1M−1or more, or 8×106cm−1M−1or more. In certain embodiments, the polymeric dye has a quantum yield of 0.05 or more, such as 0.1 or more, 0.15 or more, 0.2 or more, 0.25 or more, 0.3 or more, 0.35 or more, 0.4 or more, 0.45 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 0.99 or more and including 0.999 or more. For example, the quantum yield of polymeric dyes of interest may range from 0.05 to 1, such as from 0.1 to 0.95, such as from 0.15 to 0.9, such as from 0.2 to 0.85, such as from 0.25 to 0.75, such as from 0.3 to 0.7 and including a quantum yield of from 0.4 to 0.6. In certain cases, the polymeric dye has a quantum yield of 0.1 or more. In certain cases, the polymeric dye has a quantum yield of 0.3 or more. In certain cases, the polymeric dye has a quantum yield of 0.5 or more. In certain cases, the polymeric dye has a quantum yield of 0.6 or more. In certain cases, the polymeric dye has a quantum yield of 0.7 or more. In certain cases, the polymeric dye has a quantum yield of 0.8 or more. In certain cases, the polymeric dye has a quantum yield of 0.9 or more. In certain cases, the polymeric dye has a quantum yield of 0.95 or more. In some embodiments, the polymeric dye has an extinction coefficient of 1×106or more and a quantum yield of 0.3 or more. In some embodiments, the polymeric dye has an extinction coefficient of 2×106or more and a quantum yield of 0.5 or more. The labelled biomolecule reagent is prepared by coupling an activated biomolecule to an activated label. The term “activated” is used herein to refer to a biomolecule or label having a reactive linker or a reactive moiety that, when carried out under appropriate conditions, reacts with a second reactive linker or second reactive moiety to form a chemical linkage, such as for example, an ionic bond (charge-charge interaction), a non-covalent bond (e.g., dipole-dipole or charge-dipole) or a covalent bond. In some embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce an ionic bond. In other embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce a non-covalent bond. In yet other embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce a covalent bond. In certain embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce a covalent bond. Any convenient protocol for forming a covalent bond between the reactive linker of the activated biomolecule and the reactive linker of the activated label may be employed, including but not limited to addition reactions, elimination reactions, substitution reactions, pericyclic reactions, photochemical reactions, redox reactions, radical reactions, reactions through a carbene intermediate, metathesis reaction, among other types of bond-forming reactions. In some embodiments, the activated biomolecule may be conjugated to the activated label through reactive linking chemistry such as where reactive linker pairs include, but is not limited to: maleimide/thiol; thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol; N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester), or pentafluorophenyl-ester (PFP ester)/amine; bissuccinimidylester/amine; imidoesters/amines; hydrazine or amine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl or amine; carbohydrate-periodate/hydrazine or amine; diazirine/aryl azide chemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide; carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol and amine/BMPH (N-[β-Maleimidopropionic acid]hydrazide·TFA)/thiol; azide/triarylphosphine; nitrone/cyclooctyne; azide/tetrazine and formylbenzamide/hydrazino-nicotinamide. In certain embodiments, the reactive linker of the activated biomolecule and the reactive linker of the activated label undergo a cycloaddition reaction, such as a [1+2]-cycloaddition, a [2+2]-cycloaddition, a [3+2]-cycloaddition, a [2+4]-cycloaddition, a [4+6]-cycloaddition, or cheleotropic reactions, including linkers that undergo a 1,3-dipolar cycloaddition (e.g., azide-alkyne Huisgen cycloaddition), a Diels-Alder reaction, an inverse electron demand Diels Alder cycloaddition, an ene reaction or a [2+2] photochemical cycloaddition reaction. In certain embodiments, the biomolecule request and the label request include information or data pertaining to the reactive linker of the activated biomolecule and the activated label. For example, the biomolecule request and the label request may include information or data pertaining to the name of the reactive linker, a chemical structure, a structural description of the reactive linker or the reactive linker CAS number. In certain embodiments, the biomolecule request and the label request includes the name of reactive linker pairs, such as where the reactive linker pairs is may be selected from maleimide/thiol; thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol; N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester), or pentafluorophenyl-ester (PFP ester)/amine; bissuccinimidylester/amine; imidoesters/amines; hydrazine or amine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl or amine; carbohydrate-periodate/hydrazine or amine; diazirine/aryl azide chemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide; carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol and amine/BMPH (N-[β-Maleimidopropionic acid]hydrazide·TFA)/thiol; azide/triarylphosphine; nitrone/cyclooctyne; azide/tetrazine and formylbenzamide/hydrazino-nicotinamide; a diene/a dienophile; and a 1,3-dipole/a dipolarophile. The input manager is configured to receive the request for the labelled biomolecule. To receive the labelled biomolecule reagent request, the input manager is operatively coupled to a graphical user interface where one or more labelled biomolecule reagents requests are entered. In certain instances, the labelled biomolecule reagent request is entered on an internet website menu interface (e.g., at a remote location) and communicated to the input manager, over the internet or a local area network. In some embodiments, the input manager is configured receive a plurality of labelled biomolecule reagent requests. For example, the input manager may be configured to receive 2 or more labelled biomolecule reagent requests, such as 5 or more, such as 10 or more and including 25 or more labelled biomolecule reagent requests. Where the request for a labelled biomolecule reagent includes only a single component and is a labelled biomolecule request, the input manager may be configured to receive 2 or more labelled biomolecule requests, such as 5 or more, such as 10 or more and including 25 or more labelled biomolecule requests. Where the labelled biomolecule reagent request includes two components, such as a biomolecule request and a label request, the input manager may be configured to receive 2 or more biomolecule requests, such as 5 or more, such as 10 or more and including 25 or more biomolecule requests and configured to receive 2 or more label requests, such as 5 or more, such as 10 or more and including 25 or more label requests. In some instances, the input manager is configured to receive a labelled biomolecule reagent request that includes a single biomolecule request and single label request. In other instances, the input manager is configured to receive a labelled biomolecule reagent request that includes a single biomolecule request and a plurality of different label requests. In yet other instances, the input manager is configured to receive a labelled biomolecule reagent request that includes a plurality of different biomolecule requests and a single label request. In still other instances, the input manager is configured to receive a labelled biomolecule reagent request that includes a plurality of different biomolecule requests and a plurality of different label requests. The input manager is configured to receive labelled biomolecule requests from a single user or a plurality of different users, such as 2 or more different users, such as 5 or more different users, such as 10 or more different users, such as 25 or more different users and including 100 or more different users. In embodiments, the input manager is also configured to receive a quantity request corresponding to the desired amount of requested labelled biomolecule reagent. The quantity request may be entered by typing a numerical and a unit (e.g., μg, μmoles, μM, etc.) value into a text box, selecting a check box corresponding to the appropriate numerical and unit values or selecting a numerical value from a first drop-down menu and a unit value from a second drop-down menu. In some embodiments, the input manager is operatively coupled to one or more searchable databases (e.g., catalog) of labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers. In certain instances, the input manager includes a database of labelled biomolecules. In other instances, the input manager includes a database of activated biomolecules and activated labels. In yet other instances, the input manager includes a database of biomolecules, labels and reactive linkers. All or part of each database of labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers may be displayed on the graphical user interface, such as in a list, drop-down menu or other configuration (e.g., tiles). For example, the graphical user interface may display a list of each labelled biomolecule, activated biomolecule, biomolecule, activated label, label and reactive linkers simultaneously (i.e., on a single screen) or may contain drop-down menus for each component of the labelled biomolecule reagent request. In other embodiments, the labelled biomolecule reagent request is provided by inputting information into appropriate text fields, selecting check boxes, selecting one or more items from a drop-down menu, or by using a combination thereof. In one example, the graphical user interface includes a drop-down menu to input a labelled biomolecule reagent request by selecting one or more labelled biomolecules from the drop-down menu. In another example, the graphical user interface includes a first drop-down menu to input a biomolecule request and a second drop-down menu to input a label request by selecting one or more biomolecules and one or more labels from the first and second drop-down menus. In yet another example, the graphical user interface includes a first drop-down menu to input a biomolecule request, a second drop-down menu to input a label request and a third drop-down menu to input a reactive linker request by selecting one or more biomolecules, one or more labels and one or more reactive linkers from the drop-down menus. In still another example, the graphical user interface includes a first drop down menu to input an activated biomolecule request and a second drop-down menu to input an activated label request by selecting one or more activated biomolecules and one or more activated linkers from the first and second drop-down menus. In another example, the graphical user interface includes a list of labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers that are available in the database. For example, the graphical user interface may display a list of each labelled biomolecule, activated biomolecule, biomolecule, activated label, label and reactive linkers simultaneously on one or more screens or may contain drop-down menus for each component of the labelled biomolecule reagent request. In some instances, a list of all available labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers displayed on a single page. In other instances, the list of all available labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers displayed on a plurality of pages, such as 2 or more pages, such as 3 or more pages, such as 5 or more pages, such as 10 or more pages and including 25 or more pages. In yet other instances, the list of all available labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers are each displayed in separate drop-down menus on a single page. FIG.4depicts a graphical user interface for communicating a request for a labelled biomolecule reagent according to certain embodiments. To communicate the labelled biomolecule reagent request, a user inputs a biomolecule request and a label request onto Request form400. The label request is inputted by selecting a detectable marker (e.g., a fluorophore) from drop down menu401A and the biomolecule request is inputted by selecting a biomolecule (e.g., an antibody probe) from drop-down menu401B. Request form400also includes a text box for entering the quantity request402corresponding to the desired amount of labelled biomolecule reagent in micrograms. In certain embodiments, the input manager includes a search engine for searching for, adding or modifying labelled biomolecule reagent requests and for responding to user queries (e.g., inputted into the graphical user interface locally or from a remote location over the internet or local area network). In some instances, each persistent object in the system memory has an associated table in a system database and object attributes are mapped to table columns. In a further aspect, each object has an object relational mapping file which binds that object to the table in the database. Objects are also associated with each other and this association is mapped as the relation between the tables. Objects are also associated with each other by many different relationships, such as one-to-one, one-to-many, many-to-one and many-to-many. Search criteria provided in user queries may include descriptions of attributes or properties associated with an object or by values corresponding to those attributes. Relationships may also be used as search criteria. Basic search criteria can depend upon an object's attributes and advanced search criteria can depend upon association of the object with other objects, e.g., by searching properties of related objects. In certain embodiments, search engines of interest include a finder framework, which will construct a plurality of searchable conditions (e.g., all possible queryable conditions). When a user specifies an entity or object to search for, the framework generates all possible search conditions for that object and then gives the result as per the conditions selected by the user. Using the search engine, a user of the system can search for available labelled biomolecules, biomolecules, activated biomolecules, labels, activated labels and reactive linkers. The search engine is also configured for searching for pending or completed labelled biomolecule reagent requests. In addition, a user can use the search engine to inquire and find labelled biomolecules, biomolecules, activated biomolecules, labels, activated labels and reactive linkers that may be of interest. For example, a user can search for a particular biomolecule that functions as a specific antigen probe or a label that is detectable by fluorescence of a predetermined wavelength of light. Search conditions may be different for different objects and in one instance, a generic finder framework gives a generic solution for such searching. In certain embodiments, the search engine can build queries, save queries, modify queries, and/or update queries used to identify labelled biomolecules, biomolecules, activated biomolecules, labels, activated labels or reactive linkers. In some instances, the search results can be shared, compared or modified. In certain instances, systems are configured to set a maximum of search results that fit a search criteria to be displayed on the graphical user interface. In some embodiments, search results are displayed on a Webpage which includes capabilities for allowing possible actions. Such capabilities can include, but are not limited to, links, buttons, drop down menus, fields for receiving information from a user, and the like. In certain aspects, the system further includes a result formatter for formatting search results (e.g., to build appropriate user interfaces such as Web pages, to specify links, provide a way to associate actions (e.g., “delete,” “edit,” etc.) with images, text, hyperlinks and/or other displays. The system may also display the search criteria for an object under search on the web page. In one aspect, the system takes input data from the finder framework and creates a web page dynamically showing the search criteria for that object. In another aspect, the finder framework creates all possible queryable conditions for the object under search. These conditions are displayed on search web page as different fields. A user can select or specify value(s) for these field(s) and execute a search. The fields that are to be displayed have their labels in localized form. Fields may be in the form of a “select” box, or a text box or other area for inputting text. For example, a user may desire to search for a biomolecule. Biomolecules in the searchable database include queryable conditions such as compound name or sequence number (e.g., accession number). In one embodiment, the search engine supports searching for each of the labelled biomolecules, biomolecules, activated biomolecules, labels, activated labels and reactive linkers in the database. In some instances, the system provides a generic finder framework to create all queryable conditions for an object under search. Such conditions will generally depend upon the properties of the object and its relationship(s) with other objects. In other embodiments, the finder framework retrieves localized field names for these conditions and their order and stores these in the system memory (e.g., in an objectdefinition.xml file). In one example, fields are displayed on a search page in the order in which they are stored in a file as a set of search parameters for which a user can select or enter values. The search parameters may be in the form of a list of objects and the parameters may relate to attribute categories. For example, in response to a user searching for a labelled biomolecule, the system may display the queryable conditions: “name of labelled biomolecule,” “keywords used for search,” “created by,” “modified by,” “modification date,” “annotation” and the like. The finder framework can return the queryable conditions in the form of a collection, which can be displayed on a search page, which lists or represents the various search fields corresponding to the attribute categories in a localized form. A user may enter values for these fields and perform, e.g., selecting one or more of a labelled biomolecule, biomolecule, activated biomolecule, label, activated label and reactive linker having a specific name, structure, registry number, etc., providing specific keywords, identifying a desired domain, creator, modification date, annotation, and the like. The system then displays a list of labelled biomolecules, biomolecules, activated biomolecules, labels, activated labels or reactive linkers that satisfy the search conditions. In certain embodiments, the system displays information regarding the criteria used to perform the search. In certain embodiments, the input manager includes a labelled biomolecule design platform which is configured to provide a recommendation for choosing one or more biomolecules, activated biomolecules, labels, activated labels or reactive linkers. In some instances, the design platform is configured to provide a recommendation for choosing one or more biomolecules, activated biomolecules, labels, activated labels or reactive linkers based on user input of one or more parameters of the desired labelled biomolecule. For example, parameters of the desired labelled biomolecule which may be inputted by the user into the design platform may include, but are not limited to, desired physical properties of the labelled biomolecule (e.g., molecular mass, melting point, purity, etc.); desired chemical properties of the labelled biomolecule (e.g., chemical structure, structural similarity to a second labelled biomolecule, ionizability, solvation, hydrolysis, chemical reactivity, enzymatic reactivity, binding affinity, etc.); spectroscopic properties (e.g., absorbance wavelength range, absorbance maxima, emission wavelength range, emission maxima, Stokes shift, quantum yield, molar extinction coefficient, etc.) In other instances, the design platform is configured to provide a recommendation for choosing one or more biomolecules, activated biomolecules, labels, activated labels or reactive linkers based on the application of the labelled biomolecule. For example, the design platform may be configured to provide a recommendation for choosing each component of the labelled biomolecule based on instruments that will be used (e.g., flow cytometer, fluorescence spectrometer, etc.), instrument configuration, as well as experimental parameters (e.g., target abundance such as antigen density on a cell). The graphical user interface may include one or more text input fields or drop-down menus for inputting data used by the design platform to provide a recommendation for choosing one or more biomolecules, activated biomolecules, labels, activated labels or reactive linkers. The labelled biomolecule design platform may be configured to provide a recommendation for a plurality of different biomolecules, activated biomolecules, labels, activated labels or reactive linkers based on information (e.g., properties of the labelled biomolecule or expected application of the labelled biomolecule) inputted by the user. For example, the design platform may be configured to recommend 2 or more different biomolecules, activated biomolecules, labels, activated labels or reactive linkers based on information inputted by the user, such as 3 or more, such as 4 or more, such as 5 or more, such as 10 or more and including 25 or more biomolecules, activated biomolecules, labels, activated labels or reactive linkers. In certain embodiments, the labelled biomolecule design platform is configured to provide a recommendation as to the combination of biomolecule, label, activated label or reactive linker that is best suited for a particular application (e.g., configuration of a flow cytometer). For example, the design platform may be configured such that a user enters a list of one or more biomolecules and one or more labels as well as application information (e.g., instrument configuration, target abundance, etc.) and the design platform outputs combinations a recommendation of biomolecules, labels, activated labels and reactive linkers best suited for the stated application. In certain embodiments, the recommendation for a labelled biomolecule, biomolecule, activated biomolecule, label, activated label or reactive linker is displayed on a display (e.g., an electronic display) or may be printed with a printer, such as onto a human (paper) readable medium or in a machine readable format (e.g., as a barcode). In other embodiments, the recommendation for a labelled biomolecule, biomolecule, activated biomolecule, label, activated label or reactive linker may be communicated to the input manager and the recommended labelled biomolecule may be prepared as described above. Systems of the present disclosure also include a memory for storing a dataset having a plurality of storage identifiers that correspond with the components the of the label biomolecule reagent request. The term “memory” is used herein in its conventional sense to refer to a device that stores information for subsequent retrieval by a processor, and may include magnetic or optical devices (such as a hard disk, floppy disk, CD, or DVD), or solid state memory devices (such as volatile or non-volatile RAM). A memory or memory unit may have more than one physical memory device of the same or different types (for example, a memory may have multiple memory devices such as multiple hard drives or multiple solid state memory devices or some combination of hard drives and solid state memory devices). The memory may be a computer readable medium or permanent memory. In embodiments, the memory may include one or more datasets having a plurality of storage identifiers that correspond to each labelled biomolecule, biomolecule, label, activated biomolecule, activated label and reactive linker in the system database. The datasets stored in the memory include storage identifiers that correspond with each labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker. The storage identifiers may be presented in the dataset as a string of one or more characters (e.g., alphanumeric characters), symbols, images or other graphical representation(s) associated with a particular labelled biomolecule, biomolecule, label, activated biomolecule, activated label or linker. In some instances, the storage identifier is abbreviated designation of the labelled biomolecule, biomolecule, label, activated biomolecule, activated label or linker. For example, the storage identifier may include references to accession number, sequence identification number, identifiable probe sequence, CAS registry number or may be a custom identification code. The number of storage identifiers in each dataset stored in memory may vary, depending on the type of storage identifiers. For example, the dataset stored in memory having a plurality of labelled biomolecule storage identifiers may include 10 or more labelled biomolecule storage identifiers, such as 25 or more, such as 50 or more, such as 100 or more identifiers, such 250 or more, such as 500 or more and including 1000 or more labelled biomolecule storage identifiers. The dataset stored in memory having a plurality of biomolecules or activated biomolecules may include 25 or more biomolecule or activated biomolecule storage identifiers, such as 50 or more, such as 100 or more, such as 250 or more, such as 500 or more and including 1000 or more biomolecule or activated biomolecule storage identifiers. The dataset stored in memory having a plurality of labels or activated labels may include 5 or more label or activated label storage identifiers, such as 10 or more, such as 15 or more, such as 25 or more and including 50 or more label or activated label storage identifiers. In certain embodiments, the dataset stored in memory having a plurality of reactive linkers includes 2 or more reactive linker storage identifiers, such as 3 or more, such as 5 or more, such as 10 or more and including 15 or more reactive linker storage identifiers. The memory is in operative communication with a processing module that identifies one or more storage identifiers from the dataset that corresponds to the request received by the input manager. In some embodiments, the request for a labelled biomolecule reagent is a labelled biomolecule request and the processing module identifies a labelled biomolecule storage identifier from a dataset in the memory having a plurality of labelled biomolecules storage identifiers. In other embodiments, the request for a labelled biomolecule reagent includes a biomolecule request and a label request and the processing module identifies: 1) a biomolecule storage identifier from a first dataset in the memory having a plurality of biomolecule storage identifiers; and 2) a label storage identifier from a second dataset in the memory having a plurality of label storage identifiers. In still other embodiments, the request for a labelled biomolecule reagent includes a biomolecule request, a label request and a reactive linker request and the processing module identifies: 1) a biomolecule storage identifier from a first dataset in the memory having a plurality of biomolecule storage identifiers; 2) a label storage identifier from a second dataset in the memory having a plurality of label storage identifiers; and 3) a reactive linker storage identifier from a third dataset in the memory having a plurality of reactive linker storage identifiers. When a particular storage identifier that corresponds to a labelled biomolecule request, biomolecule request, label request, activated biomolecule request, activated label request or reactive linker request are not available (i.e., cannot be identified by the processing module from any dataset in the memory), the memory may include algorithm for providing a recommendation for an alternative labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker. The recommendation may be based on similarities in chemical structure, reactivity, probe target, binding affinity, target abundance, target density, label cross-talk, size, price, etc. as the requested labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker. The memory may be configured to provide a recommendation for one or more alternatives, such as 2 or more alternatives, such as 3 or more alternatives and including 5 or more alternatives, depending on the similarity between the requested component and available labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linkers. The processing module may include a commercially available processor such as a processor made by Intel Corporation, a SPARC® processor made by Sun Microsystems, or it may be one of other processors that are or will become available. The processor executes the operating system, which may be, for example, a WINDOWS®-type operating system from the Microsoft Corporation; a Unix® or Linux-type operating system or a future operating system; or some combination thereof. The operating system interfaces with firmware and hardware in a well-known manner, and facilitates the processor in coordinating and executing the functions of various computer programs that may be written in a variety of programming languages, such as Java, Perl, C++, other high level or low level languages, as well as combinations thereof, as is known in the art. The operating system, typically in cooperation with the processor, coordinates and executes functions of the other components of the computer. The operating system also provides scheduling, input-output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques. Processing modules of the subject systems include both hardware and software components, where the hardware components may take the form of one or more platforms, e.g., in the form of servers, such that the functional elements, i.e., those elements of the system that carry out specific tasks (such as managing input and output of information, processing information, etc.) of the system may be carried out by the execution of software applications on and across the one or more computer platforms represented of the system. The one or more platforms present in the subject systems may be any type of known computer platform or a type to be developed in the future, although they typically will be of a class of computer commonly referred to as servers. However, they may also be a main-frame computer, a work station, or other computer type. They may be connected via any known or future type of cabling or other communication system including wireless systems, either networked or otherwise. They may be co-located or they may be physically separated. Various operating systems may be employed on any of the computer platforms, possibly depending on the type and/or make of computer platform chosen. Appropriate operating systems include WINDOWS NT®, Sun Solaris, Linux, OS/400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, and others. Other development products, such as the Java™2 platform from Sun Microsystems, Inc. may be employed in processors of the subject systems to provide suites of applications programming interfaces (API's) that, among other things, enhance the implementation of scalable and secure components. Various other software development approaches or architectures may be used to implement the functional elements of system and their interconnection, as will be appreciated by those of ordinary skill in the art. Systems of the present disclosure also include an output manager that provides the identified storage identifiers from the processing module. In some embodiments, the output manager includes an electronic display and the identified storage identifiers are outputted onto the electronic display. One or more storage identifiers may be outputted onto the electronic display simultaneously, such as 2 or more, such as 3 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 100 or more and including 500 or more storage identifiers. The output manager may display the storage identifiers of the labelled biomolecule reagent requests from a single user or from a plurality of users, such as from 2 or more users, such as 5 or more users, such as 10 or more users, such as 25 or more users and including 100 or more users. The output manager may be configured to organize the displayed storage identifiers, as desired, such as grouping the storage identifiers according to each request for a labelled biomolecule, by user or by type of storage identifier (e.g., labelled biomolecule storage identifier, biomolecule storage identifier, label storage identifier, reactive linker storage identifier). In other embodiments, the output manager includes a printer and the identified storage identifiers are printed onto a human (paper) readable medium or as in a machine readable format (e.g., as a barcode). In certain embodiments, the output manager communicates the storage identifiers assembled by the processing module, e.g., one or more labelled biomolecule storage identifiers, biomolecule storage identifiers, label storage identifiers, reactive linker storage identifiers in an electronic format to a user, such as over a local area network or over the Internet. The electronic communication of data by the output manager may be implemented according to a convenient protocol, including but not limited to, SQL, HTML or XML documents, email or other files, or data in other forms. The data may also include Internet URL addresses so that a user may retrieve additional SQL, HTML, XML, or other documents or data from remote sources. Systems of the present disclosure for inputting a labelled biomolecule reagent request, storing a plurality of storage identifiers that correspond with the components the of the label biomolecule reagent request, identifying one or more storage identifiers and for outputting the identified storage identifiers include a computer. In certain embodiments, a general-purpose computer can be configured to a functional arrangement for the methods and programs disclosed herein. The hardware architecture of such a computer is well known by a person skilled in the art, and can comprise hardware components including one or more processors (CPU), a random-access memory (RAM), a read-only memory (ROM), an internal or external data storage medium (e.g., hard disk drive). A computer system can also comprise one or more graphic boards for processing and outputting graphical information to display means. The above components can be suitably interconnected via a bus inside the computer. The computer can further comprise suitable interfaces for communicating with general-purpose external components such as a monitor, keyboard, mouse, network, etc. In some embodiments, the computer can be capable of parallel processing or can be part of a network configured for parallel or distributive computing to increase the processing power for the present methods and programs. In some embodiments, the program code read out from the storage medium can be written into memory provided in an expanded board inserted in the computer, or an expanded unit connected to the computer, and a CPU or the like provided in the expanded board or expanded unit can actually perform a part or all of the operations according to the instructions of the program code, so as to accomplish the functions described below. In other embodiments, the method can be performed using a cloud computing system. In these embodiments, the data files and the programming can be exported to a cloud computer that runs the program and returns an output to the user. A system can, in certain embodiments, include a computer that includes: a) a central processing unit; b) a main non-volatile storage drive, which can include one or more hard drives, for storing software and data, where the storage drive is controlled by disk controller; c) a system memory, e.g., high speed random-access memory (RAM), for storing system control programs, data, and application programs, including programs and data loaded from non-volatile storage drive; system memory can also include read-only memory (ROM); d) a user interface, including one or more input or output devices, such as a mouse, a keypad, and a display; e) an optional network interface card for connecting to any wired or wireless communication network, e.g., a printer; and f) an internal bus for interconnecting the aforementioned elements of the system. The memory of a computer system can be any device that can store information for retrieval by a processor, and can include magnetic or optical devices, or solid state memory devices (such as volatile or non-volatile RAM). A memory or memory unit can have more than one physical memory device of the same or different types (for example, a memory can have multiple memory devices such as multiple drives, cards, or multiple solid state memory devices or some combination of the same). With respect to computer readable media, “permanent memory” refers to memory that is permanent. Permanent memory is not erased by termination of the electrical supply to a computer or processor. Computer hard-drive ROM (i.e., ROM not used as virtual memory), CD-ROM, floppy disk and DVD are all examples of permanent memory. Random Access Memory (RAM) is an example of non-permanent (i.e., volatile) memory. A file in permanent memory can be editable and re-writable. Operation of the computer is controlled primarily by an operating system, which is executed by the central processing unit. The operating system can be stored in a system memory. In some embodiments, the operating system includes a file system. In addition to an operating system, one possible implementation of the system memory includes a variety of programming files and data files for implementing the method described below. In certain cases, the programming can contain a program, where the program can be composed of various modules, and a user interface module that permits a user to manually select or change the inputs to or the parameters used by the program. The data files can include various inputs for the program. In certain embodiments, instructions in accordance with the method described herein can be coded onto a computer-readable medium in the form of “programming,” where the term “computer readable medium” as used herein refers to any storage or transmission medium that participates in providing instructions and/or data to a computer for execution and/or processing. Examples of storage media include a floppy disk, hard disk, optical disk, magneto-optical disk, CD-ROM, CD-R, magnetic tape, non-volatile memory card, ROM, DVD-ROM, Blue-ray disk, solid state disk, and network attached storage (NAS), whether or not such devices are internal or external to the computer. A file containing information can be “stored” on computer readable medium, where “storing” means recording information such that it is accessible and retrievable at a later date by a computer. The computer-implemented method described herein can be executed using programs that can be written in one or more of any number of computer programming languages. Such languages include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), as well as any many others. In any embodiment, data can be forwarded to a “remote location,” where “remote location,” means a location other than the location at which the program is executed. For example, a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. As such, when one item is indicated as being “remote” from another, what is meant is that the two items can be in the same room but separated, or at least in different rooms or different buildings, and can be at least one mile, ten miles, or at least one hundred miles apart. “Communicating” information references transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network). “Forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and includes, at least in the case of data, physically transporting a medium carrying the data or communicating the data. Examples of communicating media include radio or infra-red transmission channels as well as a network connection to another computer or networked device, and the internet or including email transmissions and information recorded on websites and the like. Some embodiments include implementation on a single computer, or across a network of computers, or across networks of networks of computers, for example, across a network cloud, across a local area network, on hand-held computer devices, etc. In certain embodiments, one or more of the steps described herein are implemented on a computer program(s). Such computer programs execute one or more of the steps described herein. In some embodiments, implementations of the subject method include various data structures, categories, and modifiers described herein, encoded on computer-readable medium(s) and transmissible over communications network(s). Software, web, internet, cloud, or other storage and computer network implementations of the present invention could be accomplished with standard programming techniques to conduct the various assigning, calculating, identifying, scoring, accessing, generating or discarding steps. FIG.5depicts a computer system500of the present disclosure according to certain embodiments. The computer system includes user interface501that includes a keyboard501a, a mouse501band monitor501cfor inputting a labelled biomolecule reagent request. User interface501is operatively coupled to a memory502that includes operating system502a, system files502band datasets that include a plurality of storage identifiers that correspond to the components of the labelled biomolecule reagent request: 1) labelled biomolecule request502d;2) biomolecule request502e;3) label request502f;4) activated biomolecule request502g;5) activated label request502h; and 6) reactive linker request502i. Memory502also includes a database502jthat includes a searchable inventory listing of labelled biomolecules502k, biomolecules502l, labels502mand reactive linkers502n. The memory and user interface are operatively coupled to a processor503through connection504that includes a storage drive506that is controlled by disk controller505. As described above, the processor identifies one or more storage identifiers from the dataset that corresponds to the request received by the input manager. To output the identified storage identifiers, systems of interest according to this embodiment include a network interface controller507which outputs the storage identifiers. Network interface controller507may be interfaced with an electronic display to visually display the identified storage identifiers or may be interfaced with a printer for presenting the identified storage identifiers onto a human (paper) readable medium or as in a machine readable format (e.g., as a barcode). In certain instances, network interface controller507communicates the storage identifiers in an electronic format, such as over a local area network or over the internet and may be implemented according to any electronic format, including but not limited to, SQL, HTML or XML documents, email or other files, or data in other forms. FIG.6illustrates a flow diagram600for receiving, processing and outputting a request for a labelled biomolecule reagent according to certain embodiments. Receiving and processing601the request starts with inputting the one or more components of the labelled biomolecule reagent request (602). As discussed above, the labelled biomolecule reagent request may include one or more of 1) a labelled biomolecule request; and 2) a biomolecule request and a label request. In some instances, the biomolecule request is an activated biomolecule request where biomolecule is coupled to a reactive linker. In other instances, the label request is an activated label request where the label is coupled to a reactive linker. After the systems has received the labelled biomolecule reagent request, a processor determines the components of the request (i.e., labelled biomolecule request; or biomolecule request and label request) and the system searches (603) the memory for storage identifiers that correspond to that particular request. When the appropriate dataset is retrieved, the processing module identifies one or more storage identifiers that correspond with the components of the labelled biomolecule reagent request (604). If more than one labelled biomolecule reagent request is inputted by a single user, the system may repeat the above until all storage identifiers from the user's requests are located and identified by the processor (605). Systems are configured to output (606) the identified storage identifiers once the labelled biomolecule reagent request from the user has been processed. The output manager may display the storage identifiers on an electronic display or print the storage identifiers (607). The storage identifiers may also be communicated electronically (608), such as to a reagent preparatory apparatus or over the internet to a third party manufacturer. In some embodiments, systems include a reagent preparatory apparatus for preparing the labelled biomolecule reagent that corresponds to the requested labelled biomolecule received by the input manager. The reagent preparatory apparatus is operatively coupled to the output manager and is configured to receive the identified storage identifiers (e.g., labelled biomolecule storage identifier, biomolecule storage identifier, label storage identifier, reactive linker storage identifier) and produce the labelled biomolecule reagent according to the received storage identifiers. In these embodiments, the reagent preparatory apparatus may be in communication with the output manager locally, such as through a cable or local area network or may be in a remote location and connected to the output manager through a wide-area network or through the internet. To facilitate connectivity between the reagent preparatory apparatus and the output manager, systems may include any suitable connectivity protocols, such as a cables, transmitters, relay stations, network servers, network interface cards, Ethernet modems, telephone network connections as well as satellite network connections. In certain embodiments, the reagent preparatory apparatus includes a graphical user interface where the storage identifiers from the output manager are manually inputted into an input manager operatively coupled to the graphical user interface of the reagent preparatory apparatus. In certain embodiments, the reagent preparatory apparatus is fully automated. By “fully automated” is meant that the reagent preparatory apparatus receives the identified storage identifiers from the output manager and prepares, formulates and packages the labelled biomolecule reagent with little to no human intervention or manual input into the subject systems. In certain embodiments, the subject systems are configured to prepare, purify and package the labelled biomolecule reagent from an activated biomolecule and activated label without any human intervention. The reagent preparatory apparatus includes a sampling device that provides an activated biomolecule and an activated label to a contacting apparatus. The sampling device may be any convenient device in fluid communication with each source of activated biomolecule and activated label, such as for example, a high throughput sample changer having a plurality of reagent vials containing activated biomolecules and activated labels. The sampling device may also include microfluidic channels, syringes, needles, pipets, aspirators, among other sampling devices. The contacting apparatus may be any suitable apparatus which allows for an activated biomolecule to be contacted with an activated label. For example, in some embodiments, the contacting apparatus is a sample chamber (e.g., enclosed, sealed, air-tight, open, plate, etc.). In other embodiments, the contacting apparatus is a microtube. In other embodiments, the contacting apparatus is a test tube. In yet other embodiments, the contacting apparatus is a glass flask (e.g., beaker, volumetric flask, Erlenmeyer flask, etc.). In still other embodiments, the contacting apparatus is a 96-well plate. In certain embodiments, the subject systems may further include a packaging unit configured to seal the produced labelled biomolecule reagent in the contacting apparatus (e.g., microtube, test tube, etc.). In other embodiments, the produced labelled biomolecule reagent is first characterized and further purified, diluted, concentrated or re-formulated before sealing in a container and packaged with the packaging unit. The contacting apparatus may further include an agitator for mixing the combined activated biomolecule and activated label. The agitator may be any convenient agitator sufficient for mixing the subject compositions, including but not limited to vortexers, sonicators, shakers (e.g., manual, mechanical, or electrically powered shakers), rockers, oscillating plates, magnetic stirrers, static mixers, rotators, blenders, mixers, tumblers, orbital shakers, bubbles, microfluidic flow, among other agitating protocols. In some embodiments, the reagent preparatory apparatus also includes a source of activated biomolecules and activated labels. The source may include a plurality of activated biomolecules and activated labels. In some instances, the reagent preparatory apparatus includes a source containing 5 or more different types of activated biomolecules, such as 10 or more, such as 100 or more, such as 250 or more, such as 500 or more and including 1000 or more different types of activated biomolecules. For example, the reagent preparatory apparatus may include a source containing 5 or more different types of activated antibody probes or activated oligonucleotide probes, such as 10 or more, such as 100 or more, such as 250 or more, such as 500 or more and including 1000 or more different types of activated antibody probes or activated oligonucleotide probes. In some embodiments, the reagent preparatory apparatus includes a source containing 5 or more different types of activated labels, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more and including 100 or more different types of activated labels. For example, the reagent preparatory apparatus may include a source containing 5 or more different types of activated fluorophores, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more and including 100 or more different types of activated fluorophores. The source of activated biomolecules and activated labels may be any suitable reservoir that is capable of storing and providing one or more type of activated biomolecule and activated label to the contacting apparatus. In one example, the source is a single high throughput reservoir that stores a plurality of different types of activated biomolecules and activated labels in separate, partitioned reagent chambers. In another example, the source of activated biomolecules and activated labels is a plurality of individual vials of each activated biomolecule and each activated label. In yet another example, the source of activated biomolecules and activated labels is a reservoir with pre-measured aliquots of each activated biomolecule and each activated label. For example, the reservoir may include pre-measured aliquots of each activated biomolecule and each activated label sufficient to prepare one or more labelled biomolecules, such as 2 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 100 or more, such as 500 or more and including 1000 or more labelled biomolecules. Depending on the particular design of reservoir containing the activated biomolecules and activated labels, the reagent preparatory apparatus may further include one or more inlets for delivering the activated biomolecules and activated labels to the contacting apparatus. The reagent preparatory apparatus may also include one or more reagent purifiers. Reagent purification protocols of interest may include, but is not limited to size exclusion chromatography, ion exchange chromatography, filtration (e.g., membrane filters, size cut-off filtration), liquid-liquid extraction, passive dialysis, active dialysis, centrifugation, precipitation, among other purification protocols. The reagent preparatory apparatus may also include a reagent analyzer. In certain embodiments, the sample analyzer may be mass cytometry, mass spectrometry (e.g., TOF mass spectrometry, inductively coupled plasma mass spectrometry), absorbance spectroscopy, fluorescence spectroscopy, volumetric analysis, conductivity analysis, nuclear magnetic resonance spectroscopy, infrared spectroscopy, UV-vis spectroscopy, colorimetry, elemental analysis, liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry systems. For example, the apparatus may include analytical separation device such as a liquid chromatograph (LC), including a high performance liquid chromatograph (HPLC), fast protein liquid chromatography (FPLC) a micro- or nano-liquid chromatograph or an ultra high pressure liquid chromatograph (UHPLC) device, a capillary electrophoresis (CE), or a capillary electrophoresis chromatograph (CEC) apparatus. However, any manual or automated injection or dispensing pump system may be used. For instance, the subject sample may be applied to the LC-MS system by employing a nano- or micropump in certain embodiments. Mass spectrometer systems may be any convenient mass spectrometry system, which in general contains an ion source for ionizing a sample, a mass analyzer for separating ions, and a detector that detects the ions. In certain cases, the mass spectrometer may be a so-called “tandem” mass spectrometer that is capable of isolating precursor ions, fragmenting the precursor ions, and analyzing the fragmented precursor ions. The ion source may rely on any type of ionization method, including but not limited to electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron impact (EI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI) or inductively coupled plasma (ICP) ionization, for example, or any combination thereof (to provide a so-called “multimode” ionization source). In one embodiment, the precursor ions may be made by EI, ESI or MALDI, and a selected precursor ion may be fragmented by collision or using photons to produce product ions that are subsequently analyzed. Likewise, any of a variety of different mass analyzers may be employed, including time of flight (TOF), Fourier transform ion cyclotron resonance (FTICR), ion trap, quadrupole or double focusing magnetic electric sector mass analyzers, or any hybrid thereof. In one embodiment, the mass analyzer may be a sector, transmission quadrupole, or time-of-flight mass analyzer. The reagent preparatory apparatus may also be configured to formulate the labelled biomolecule reagent with one or more excipients, such as a buffer, preservative, drying agent, etc. In certain embodiments, the reagent preparatory apparatus is configured to formulate the labelled biomolecule reagent with one or more buffers. Example buffers may include but are not limited to PBS (phosphate) buffer, acetate buffer, N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer, 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS) buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, citrate buffer, tris(hydroxymethyl)methylamine (Tris) buffer, N-tris(hydroxymethyl)methylglycine (Tricine) buffer, 3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid (TAPSO) buffer, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES) buffer, 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES) buffer, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer, dimethylarsinic acid (Cacodylate) buffer, saline sodium citrate (SSC) buffer, 2(R)-2-(methylamino)succinic acid (succinic acid) buffer, potassium phosphate buffer, N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, among other types of buffered solutions. The reagent preparatory apparatus may also include a packing unit for packaging the labelled biomolecule reagent. In certain embodiments, the packaging unit may package the prepared labelled biomolecule reagent and prepare the labelled biomolecule reagent for shipping, such as by mail. In certain instances, the prepared labelled biomolecule reagent is dispensed into a container and sealed. In other instances, the labelled biomolecule reagent is dispensed into a container, sealed and further packaged such as in a pouch, bag, tube, vial, microtube or bottle. Where desired, the packaging may be sterile. In certain embodiments, systems of interest include an on-demand standalone labelled biomolecule reagent dispensing station configured to: 1) receive one or more requests for a labelled biomolecule reagent; 2) prepare the requested labelled biomolecule reagent and 3) deliver the prepared labelled biomolecule reagent to the requestor (e.g., customer). For example, the standalone reagent dispensing station may be a self-vending machine that is configured to receive one or more labelled biomolecule reagent requests from a customer, prepare the requested labelled biomolecule and dispense the prepared labelled biomolecule to the customer on demand. Depending on the number of labelled biomolecule reagent requests and the amount of each labelled biomolecule reagents requested, standalone reagent dispensing stations of interest may prepare and dispense the labelled biomolecule to the requestor on demand in 10 seconds or more after input of the labelled biomolecule request, such as in 15 seconds or more, such as in 30 seconds or more, such as in 1 minute or more, such as in 5 minutes or more, such as in 10 minutes or more, such as in 15 minutes or more, such as in 30 minutes or more and including in 60 minutes or more, such as in 1.5 hours or more, such as in 2 hours or more, such as in 2.5 hours or more, such as in 3 hours or more, such as in 4 hours or more, such as in 5 hours or more, such as in 6 hours or more, such as in 8 hours or more, such as in 10 hours or more, such as in 12 hours or more, such as in 16 hours or more, such as in 18 hours or more and including in 24 hours or more. In some instances, the standalone reagent dispensing station is configured to prepare and dispense the labelled biomolecule to the requestor on demand in a duration that ranges from 5 seconds to 60 seconds, such as from 10 seconds to 50 seconds and including from 15 seconds to 45 seconds. In other instances, the standalone reagent dispensing station is configured to prepare and dispense the labelled biomolecule to the requestor on demand in a duration that ranges from 1 minute to 60 minutes, such as from 2 minutes to 55 minutes, such as from 5 minutes to 50 minutes, such as from 15 minutes to 45 minutes and including from 20 minutes to 40 minutes, for example preparing and dispensing the labelled biomolecule to the requestor in 30 minutes. In still other instances, the standalone reagent dispensing station is configured to prepare and dispense the labelled biomolecule to the requestor on demand in a duration that ranges from 0.5 hours to 24 hours, such as from 1 hour to 20 hours, such as from 1.5 hours to 18 hours, such as from 2 hours to 16 hours, such as from 2.5 hours to 12 hours, such as from 3 hours to 10 hours, such as from 3.5 hours to 8 hours and including from 4 hours to 6 hours. In these embodiments, the subject standalone reagent dispensing stations may include the components for receiving a labelled biomolecule reagent request and preparing the requested labelled biomolecule reagent, as described above. For instance, the standalone labelled biomolecule reagent dispensing station may include an input module for receiving a request for a labelled biomolecule; a reagent preparatory apparatus; and a dispensing module for outputting a packaged labelled biomolecule. In these embodiments, the input module may include an input manager for receiving a request for a labelled biomolecule, a memory for storing a dataset having a plurality of storage identifiers that correspond to the one or more components of the labelled biomolecule reagent request (e.g., biomolecule, label, etc.), a processing module communicatively coupled to the memory and configured to identify a storage identifier from the dataset that corresponds to the components of the labelled biomolecule reagent request and an output manager for providing the identified storage identifiers. The standalone station also includes, as described above, a graphical user interface as well as user input devices for communicating the labelled biomolecule request to the input manager of the standalone dispensing station. In embodiments, the output manager is communicatively coupled to the reagent preparatory apparatus in the standalone reagent dispensing station which is configured with one or more sources of biomolecules, labels, reactive linkers, activated biomolecules and activated labels and a contacting station for coupling an activated biomolecule and an activated label to produce the requested labelled biomolecule. In certain embodiments, the standalone reagent dispensing station includes a plurality of pre-synthesized labelled biomolecules and the standalone reagent dispensing station is configured to aliquot an amount of the pre-synthesized labelled biomolecule reagent into a container and dispense the labelled biomolecule reagent to the requestor. The standalone labelled biomolecule reagent dispensing station also includes a dispensing module that is configured to provide a packaged labelled biomolecule reagent. In embodiments, the dispensing module may include a packaging unit for packaging the prepared labelled biomolecule reagent. In certain instances, the prepared labelled biomolecule reagent is dispensed into a container and sealed. In other instances, the labelled biomolecule reagent is dispensed into a container, sealed and further packaged such as in a pouch, bag, tube, vial, microtube or bottle. Where desired, the packaging may be sterile. In certain embodiments, the standalone reagent dispensing station is fully automated, where a labelled biomolecule request is received and and the station prepares, purifies and packages the labelled biomolecule reagent with little to no human intervention or manual input into the subject systems apart from the labelled biomolecule request. Methods for Preparing a Labelled Biomolecule Reagent Aspects of the present disclosure also include methods for preparing a labelled biomolecule reagent. Methods according to certain embodiments include receiving a request for a labelled biomolecule reagent and preparing a labelled biomolecule. In other embodiments, methods include receiving a request for a labelled biomolecule reagent with one or more input managers as described above, identifying a storage identifier that corresponds with the labelled biomolecule reagent request; outputting the one or more identified storage identifiers and preparing the labelled biomolecule from the identified storage identifiers. As discussed above, the labelled biomolecule reagent is a biological macromolecule that is coupled (e.g., covalently bonded) to a detectable marker. In some embodiments, methods include preparing a polypeptide coupled to a detectable marker, a nucleic acid coupled to a detectable marker, a polysaccharide coupled to a detectable marker, or a combination thereof. In one example, the biomolecule is an oligonucleotide, truncated or full-length DNA or RNA. In another example, the biomolecule is a polypeptide, protein, enzyme or antibody. In certain instances, the biomolecule is a biological probe having a specific binding domain sufficient to bind an analyte of interest. Specific binding domains of interest include, but are not limited to, antibody binding agents, proteins, peptides, haptens, nucleic acids, etc. The term “antibody binding agent” as used herein includes polyclonal or monoclonal antibodies or fragments that are sufficient to bind to an analyte of interest. The antibody fragments can be, for example, monomeric Fab fragments, monomeric Fab′ fragments, or dimeric F(ab)′2 fragments, as well as molecules produced by antibody engineering, such as single-chain antibody molecules (scFv) or humanized or chimeric antibodies produced from monoclonal antibodies by replacement of the constant regions of the heavy and light chains to produce chimeric antibodies or replacement of both the constant regions and the framework portions of the variable regions to produce humanized antibodies. Labels of interest include detectable markers that are detectible based on, for example, fluorescence emission, fluorescence polarization, fluorescence lifetime, fluorescence wavelength, absorbance maxima, absorbance wavelength, Stokes shift, light scatter, mass, molecular mass, redox, acoustic, raman, magnetism, radio frequency, enzymatic reactions (including chemiluminescence and electro-chemiluminescence) or combinations thereof. Labels of interest may include, but are not limited to fluorophores, chromophores, enzymes, redox labels, radiolabels, acoustic labels, Raman (SERS) tag, mass tag, isotope tag (e.g., isotopically pure rare earth element), magnetic particles, microparticles and nanoparticles. Methods include receiving a request for a labelled biomolecule reagent. In embodiments of the present disclosure, the labelled biomolecule reagent request includes one or more of: 1) a labelled biomolecule request; and 2) a biomolecule request and a label request. In some instances, the biomolecule request is an activated biomolecule request where biomolecule is coupled to a reactive linker. In other instances, the label request is an activated label request where the label is coupled to a reactive linker. The labelled biomolecule reagent request may be received by any convenient communication protocol including, but not limited to, receiving the labelled biomolecule reagent request over the telephone, by facsimile, electronic mail or postal mail. In certain embodiments, the labelled biomolecule reagent request is communicated by inputting the labelled biomolecule reagent request into a graphical user interface on a computer, such as through an internet website. One or more labelled biomolecule reagent requests may be received (simultaneously or sequentially), such as receiving 2 or more labelled biomolecule reagent requests, such as 5 or more, such as 10 or more and including receiving 25 or more labelled biomolecule reagent requests. Where the request for a labelled biomolecule reagent includes only a single component and is a labelled biomolecule request, methods may include receiving 2 or more labelled biomolecule requests, such as 5 or more, such as 10 or more and including 25 or more labelled biomolecule requests. Where the labelled biomolecule reagent request includes two components, such as a biomolecule request and a label request, methods may include receiving 2 or more biomolecule requests, such as 5 or more, such as 10 or more and including 25 or more biomolecule requests and configured to receive 2 or more label requests, such as 5 or more, such as 10 or more and including 25 or more label requests. In some instances, methods including receiving a labelled biomolecule reagent request that includes a single biomolecule request and single label request. In other instances, methods include receiving a labelled biomolecule reagent request that includes a single biomolecule request and a plurality of different label requests. In yet other instances, the methods include receiving a labelled biomolecule reagent request that includes a plurality of different biomolecule requests and a single label request. In still other instances, methods include receiving a labelled biomolecule reagent request that includes a plurality of different biomolecule requests and a plurality of different label requests. The labelled biomolecule reagent requests may be received from a single user or a plurality of users, such as from 2 or more users, such as from 5 or more users, such as from 10 or more users, such as from 25 or more users and including receiving labelled biomolecule requests from 100 or more users. In certain embodiments, methods include receiving a request for a labelled biomolecule reagent and inputting the request into a graphical user interface of an input manager (as described above) entered through. In other embodiments, the user making the labelled biomolecule reagent request inputs the request directly into the graphical user interface. The labelled biomolecule request, in these embodiments, may be entered into the graphical user interface and communicated to the input manager as a string of one or more characters (e.g., alphanumeric characters), symbols, images or other graphical representation(s) of the labelled biomolecule. In some instances, the request is a “shorthand” designation or other suitable identifier of the labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker. For example, the request may include biomolecule name, label name, ascension number, sequence identification number, abbreviated probe sequence, chemical structure or Chemical Abstracts Service (CAS) registry number. As described above, after the labelled biomolecule request is received by the input manager, a processing module of the subject systems identifies one or more storage identifiers from a dataset stored in memory that corresponds to the components of the received labelled biomolecule reagent request (e.g., a labelled biomolecule storage identifier, a biomolecule storage identifier, a label storage identifier, a reactive linker storage identifier, etc.) The storage identifiers that correspond to each component of the received labelled biomolecule reagent request is outputted by an output manager. In some instances, each labelled biomolecule storage identifier is displayed on a monitor. In other instances, the storage identifiers is outputted by printing in a machine (e.g., as a barcode) or human readable format. Where the labelled biomolecule reagent is prepared by a computer controlled reagent preparatory apparatus (as described in greater detail below), the output manager is operatively coupled to the reagent preparatory apparatus and each storage identifier may electronically communicated to the reagent preparatory apparatus, such as through an internet protocol, including but not limited to SQL, HTML or XML documents, email or other files, or data in other forms. Depending on the number of labelled biomolecule requests received, one or more storage identifiers may be simultaneously outputted by the output manager, such as 2 or more, such as 3 or more, such as 3 or more, such as 5 or more, such as 10 or more, such as 25 or more, such as 100 or more and including outputting 500 or more storage identifiers. Each set of outputted storage identifiers may correspond with the labelled biomolecule requests from a single user or from a plurality of users. In certain embodiments, the output manager organizes (e.g., groups together) storage identifiers based on a predetermined criteria before displaying or printing the storage identifiers. In one example, the output manager groups together all of the storage identifiers from a particular user. In another example, the output manager groups together all of the same labelled biomolecule storage identifiers. In yet another example, the output manager organizes the storage identifiers based on name or type of biomolecule (e.g., antibody, oligonucleotide). In still another example, the output manager organizes the storage identifiers based on the name or type of label (e.g., fluorescein, coumarin). In some embodiments, methods include preparing a labelled biomolecule reagent according to the received request and/or the outputted storage identifiers. In some embodiments, preparing the labelled biomolecule reagent includes selecting an activated biomolecule and an activated label from a storage having a plurality of activated biomolecules and a plurality of activated labels. Each labelled biomolecule reagent may be prepared manually by one or more individuals, such as in a laboratory or may be prepared with a computer-controlled reagent preparatory apparatus (e.g., a high throughput preparatory system) as described above. In some instances, where the outputted storage identifier is a labelled biomolecule storage identifier, methods include retrieving the labelled biomolecule from a storage that corresponds to the outputted labelled biomolecule storage identifier. In these instances, methods may further include purifying the labelled biomolecule from the storage or adding one or more additional reagents (e.g., buffers, antioxidants, etc.) as desired. In other instances, the retrieved labelled biomolecule may be packaged and shipped to the user without further purification or additions to the composition. In other embodiments, the labelled biomolecule is prepared by contacting an activated biomolecule that corresponds with the outputted biomolecule storage identifier with an activated label that corresponds with the outputted label storage identifier. Any convenient reaction protocol may be employed to mix the activated biomolecule with the activated label, so long as reaction is sufficient to form a covalent bond between the reactive linker of the activated biomolecule and the reactive linker of the activated label. Mixing, in certain embodiments, may include stirring the mixture with a magnetic stir bar or manually stirring the mixture as well as vortexing of agitating the mixture either manually (i.e., by hand) or mechanically (i.e., by a mechanically or electrically powered shaking device). The activated biomolecule and activated label are contacted for a duration sufficient to couple the activated biomolecule to the activated label, such as for 1 minute or longer, such as for 5 minutes or longer, such as for 10 minutes or longer and including for 30 minutes or longer. As discussed above, the activated biomolecule and activated label each include a reactive linker which when carried out under appropriate conditions, react together to form chemical linkage, such as for example, an ionic bond (charge-charge interaction), a non-covalent bond (e.g., dipole-dipole or charge-dipole) or a covalent bond. In some embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce an ionic bond. In other embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce a non-covalent bond. In yet other embodiments, the reactive linker or moiety of the activated biomolecule reacts with the reactive linker or moiety of the activated label to produce a covalent bond. In certain embodiments, the reactive linker of the activated biomolecule and the reactive linker of the activated label react to produce a covalent bond. Any convenient protocol for forming a covalent bond between the reactive linker of the activated biomolecule and the reactive linker of the activated label may be employed, including but not limited to addition reactions, elimination reactions, substitution reactions, pericyclic reactions, photochemical reactions, redox reactions, radical reactions, reactions through a carbene intermediate, metathesis reaction, among other types of bond-forming reactions. In some embodiments, the activated biomolecule may be conjugated to the activated label through reactive linking chemistry such as where reactive linker pairs include, but is not limited to: maleimide/thiol; thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol; N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester), or pentafluorophenyl-ester (PFP ester)/amine; bissuccinimidylester/amine; imidoesters/amines; hydrazine or amine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl or amine; carbohydrate-periodate/hydrazine or amine; diazirine/aryl azide chemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide; carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol and amine/BMPH (N-[β-Maleimidopropionic acid]hydrazide·TFA)/thiol; azide/triarylphosphine; nitrone/cyclooctyne; azide/tetrazine and formylbenzamide/hydrazino-nicotinamide. After contacting the activated biomolecule and activated label for a duration sufficient to form a chemical linkage (e.g., covalent bond) between each respective reactive linker, the labelled biomolecule may be further purified, such as by microextraction, gel electrophoresis, liquid-liquid extraction, centrifugation, precipitation, passive or active dialysis, or solid phase chromatography, including but not limited to ion exchange chromatography, liquid chromatography employing a reverse phase stationary column, size exclusion chromatography, high performance liquid chromatography and preparatory thin layer chromatography, ultrafiltration (membrane filters with size cut offs), among other purification protocols. Methods may also include analysis of the prepared labelled biomolecule reagent. By analyzed is meant characterizing the chemical composition of the labelled biomolecule reagent, including but not limited to the amount and types of compounds in the prepared reagent composition as well as any impurities present. Analysis of the prepared labelled biomolecule reagent may be conducted using any convenient protocol, such as for example by physical measurements (e.g., mass analysis, density analysis, volumetric analysis, etc.) mass spectrometry (e.g., TOF mass spectrometry, inductively coupled plasma mass spectrometry), mass cytometry, absorbance spectroscopy, fluorescence spectroscopy, conductivity analysis, infrared spectroscopy, UV-vis spectroscopy, colorimetry, elemental analysis and nuclear magnetic resonance spectroscopy. In some instances, analysis of the labelled biomolecule is conducted by mass spectrometry. In some instances, analysis of the labelled biomolecule is conducted by fluorescence spectroscopy. In some instances, analysis of the labelled biomolecule is conducted by gas chromatography. In some instances, analysis of the labelled biomolecule is conducted by liquid chromatography. In some instances, analysis of the labelled biomolecule is conducted by elemental analysis. In certain embodiments, analysis of the labelled biomolecule reagent is conducted by gas chromatography-mass spectrometry. In other embodiments, analysis of the labelled biomolecule reagent is conducted by liquid chromatography-mass spectrometry. For example, the apparatus may include analytical separation device such as a liquid chromatograph (LC), including a high performance liquid chromatograph (HPLC), fast protein liquid chromatography (FPLC) a micro- or nano-liquid chromatograph or an ultra high pressure liquid chromatograph (UHPLC) device, a capillary electrophoresis (CE), or a capillary electrophoresis chromatograph (CEC) apparatus. However, any manual or automated injection or dispensing pump system may be used. For instance, the subject sample may be applied to the LC-MS system by employing a nano- or micropump in certain embodiments. Mass spectrometer systems may be any convenient mass spectrometry system, which in general contains an ion source for ionizing a sample, a mass analyzer for separating ions, and a detector that detects the ions. In certain cases, the mass spectrometer may be a so-called “tandem” mass spectrometer that is capable of isolating precursor ions, fragmenting the precursor ions, and analyzing the fragmented precursor ions. The ion source may rely on any type of ionization method, including but not limited to electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron impact (EI), atmospheric pressure photoionization (APPI), matrix-assisted laser desorption ionization (MALDI) or inductively coupled plasma (ICP) ionization, for example, or any combination thereof (to provide a so-called “multimode” ionization source). In one embodiment, the precursor ions may be made by EI, ESI or MALDI, and a selected precursor ion may be fragmented by collision or using photons to produce product ions that are subsequently analyzed. Likewise, any of a variety of different mass analyzers may be employed, including time of flight (TOF), Fourier transform ion cyclotron resonance (FTICR), ion trap, quadrupole or double focusing magnetic electric sector mass analyzers, or any hybrid thereof. In one embodiment, the mass analyzer may be a sector, transmission quadrupole, or time-of-flight mass analyzer. After preparation (as well as purification and analysis, where desired) of the labelled biomolecule reagent, each prepared labelled biomolecule reagent may be loaded into a container for packaging and delivery in accordance with the labelled biomolecule request (i.e., transported to the user originating the labelled biomolecule request). In certain embodiments, the labelled biomolecule reagent is prepared and delivered to the user in the container used to contact the activated biomolecule with the activated label. For example, the labelled biomolecule reagent may be packaged and delivered in the microtube used to contact the activated biomolecule with the activated label. Methods may also include delivering the packaged labelled biomolecule reagent to the requestor, such as by mail. The prepared labelled biomolecule reagent may be packaged with other components, such as for using or storing the labelled biomolecule reagent, including but not limited to buffers, syringes, needles, micropipets, glass slides, desiccants, etc. In addition, the packaged labelled biomolecule reagent may further include instructions for storing and using the labelled biomolecule reagent. The instructions may be recorded on a suitable recording medium, such as printed on paper or plastic, etc. The instructions may be present as a package insert, such as in the labeling of the container. In other embodiments, the instructions may be present as electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, SD card, USB drive etc. In yet other embodiments, the actual instructions are not present in the package, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a paper or plastic insert having a web address where the instructions can be viewed and/or from which the instructions can be downloaded. Methods for Requesting and Receiving a Labelled Biomolecule Reagent Aspects of the present disclosure also include methods for requesting and receiving a labelled biomolecule reagent. Methods according to certain embodiments include communicating a request for a labelled biomolecule reagent, the labelled biomolecule request including one or more of: 1) a labelled biomolecule request; and 2) a biomolecule request and a label request and receiving a labelled biomolecule reagent that includes a biomolecule covalently bonded to a label. In practicing the subject methods, the labelled biomolecule request may be communicated by any convenient communication protocol including, but not limited to, communicating the labelled biomolecule request over the telephone, by facsimile, electronic mail or postal mail. In certain embodiments, the labelled biomolecule request is communicated by inputting the labelled biomolecule reagent request into a graphical user interface on a computer, such as on an internet website. One or more labelled biomolecule reagent requests may be communicated, such as communicating 2 or more labelled biomolecule reagent requests, such as 5 or more, such as 10 or more and including communicating 25 or more labelled biomolecule reagent requests. In some embodiments, methods include communicating a labelled biomolecule reagent request that includes a single biomolecule request and a single label request. In other embodiments, the labelled biomolecule reagent request includes a single biomolecule request and a plurality of label requests. In yet other embodiments, the labelled biomolecule reagent request includes a plurality of biomolecule requests and a single label request. In still other embodiments, the labelled biomolecule request includes a plurality of biomolecule requests and a plurality of label requests. In certain embodiments, the labelled biomolecule reagent request includes one or more labelled biomolecule requests. In certain embodiments, the labelled biomolecule reagent request is communicated by inputting the request on a graphical user interface, such as on an internet website. The graphical user interface may display all or part of a database (e.g., catalog) of labelled biomolecules, activated biomolecules, biomolecules, activated labels, labels and reactive linkers. Each category from the database may be displayed as a list, drop-down menu or other configuration. The labelled biomolecule reagent request may be entered by inputting information or data associated with the biomolecule and the label into appropriate text fields or by selecting check boxes or selecting one or more items from a drop-down menu, or by using a combination thereof. In one example, a labelled biomolecule reagent request is inputted into the graphical user interface by selecting a labelled biomolecule from a drop-down menu. In another example, a labelled biomolecule reagent request is inputted into the graphical user interface by selecting one or more biomolecules from a first drop-down menu and one or more labels from a second drop-down menu. In yet another example, a labelled biomolecule reagent request is inputted into the graphical user interface by selecting one or more biomolecules from a first drop-down menu, one or more labels from a second drop-down menu and one or more reactive linkers from a third drop-down menu. To input a labelled biomolecule reagent request, information or data associated with a particular labelled biomolecule, biomolecule or label is entered onto the graphical user interface. The information or data entered may be a string of one or more characters (e.g., alphanumeric characters), symbols, images or other graphical representation(s) of the labelled biomolecule. In some instances, a “shorthand” designation or other suitable identifier of the labelled biomolecule, biomolecule, label, activated biomolecule, activated label or reactive linker are entered. For example, biomolecule name, label name, ascension number, sequence identification number, abbreviated probe sequence, chemical structure or Chemical Abstracts Service (CAS) registry number may be entered. In some embodiments, the labelled biomolecule reagent includes a polypeptide and the request may include information such as polypeptide name, protein name, enzyme name, antibody name or the name of protein, enzyme or antibody fragments thereof, polypeptides derived from specific biological fluids (e.g., blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen), polypeptides derived from specific species (e.g., mouse monoclonal antibodies) as well as amino acid sequence identification number. In certain embodiments, the labelled biomolecule reagent includes a biological probe and the request includes information or data associated with a specific binding domain. In other embodiments, the labelled biomolecule reagent includes a nucleic acid and the request may include information such as oligonucleotide name, oligonucleotides identified by gene name, oligonucleotides identified by accession number, oligonucleotides of genes from specific species (e.g., mouse, human), oligonucleotides of genes associated with specific tissues (e.g., liver, brain, cardiac), oligonucleotides of genes associate with specific physiological functions (e.g., apoptosis, stress response), oligonucleotides of genes associated with specific disease states (e.g., cancer, cardiovascular disease) as well as nucleotide sequence identification number. In certain embodiments, methods for requesting a labelled biomolecule further include completing a questionnaire or survey related to the labelled biomolecule request. In these embodiments, the requestor of the labelled biomolecule is prompted with a series of questions, or in the form of a questionnaire or survey related to the labelled biomolecule request. For example, the questionnaire or survey may include one question related to the labelled biomolecule request, such as 2 or more questions, such as 3 or more questions, such as 4 or more questions and including 5 or more questions related to the labelled biomolecule request. The content of questionnaire or survey may vary depending on the information that is desired. For instance, questions in the questionnaire or survey may include, but are not limited to, requests to provide the contents of a requestor's reagent inventory, the types of experiments being conducted with the labelled biomolecule as well as the timing of the use of the labelled biomolecule reagent. The questionnaire may also include one or more open text fields for inputting. For example, the questionnaire may be an open text feedback form. In some embodiments, methods include prompting the requestor to complete the series of questions or survey before the labelled biomolecule request is communicated (e.g., inputted into the graphical user interface). In other embodiments, methods include prompting the requestor to complete the series of questions or survey after the labelled biomolecule request is completed. In still other embodiments, the requestor may be prompted with questions related to the labelled biomolecule request concurrently with communicating the labelled biomolecule request. For instance, methods may include prompting the requestor with a question about the specific use (e.g., experiments being conducted) of the labelled biomolecule when communicating the labelled biomolecule request. As described above, the completed series of questions or survey may be used by the design platform to provide a recommendation for one or more labelled biomolecule, biomolecule, activated biomolecule, label, activated label or reactive linker. For example, the answers to the questions or survey may be used by the design platform to recommend a labelled biomolecule, biomolecule, activated biomolecule, label, activated label or reactive linker that is best suited for use with a particular analytical instrument (e.g., flow cytometer, fluorescence spectrometer) or that is best suited for the intended application of the labelled biomolecule. The design platform, in certain embodiments, is configured to use the answers to the completed series of questions or surveys to provide a recommendation for a labelled biomolecule, biomolecule, activated biomolecule, label, activated label or reactive linker based on the target density (e.g., antigen density on a cell) The answers to the series of questions or survey may be communicated using the same or different protocol as used to communicate the labelled biomolecule request (e.g., telephone, facsimile, email, graphical user interface at a standalone station, graphical user interface through the internet). For example, where the labelled biomolecule is request is communicated through a graphical user interface through the internet, answers to the series of questions may also be inputted through the graphical user interface, such as with drop down menus or text fields. Methods according to embodiments of the present disclosure also include receiving the labelled biomolecule reagent. The labelled biomolecule reagent may be received loaded in a container and may be packaged with one or more ancillary components, such as for using or storing the subject composition. In certain embodiments, the labelled biomolecule reagent is received with buffers, syringes, needles, micropipets, glass slides, desiccants, etc. The packaged labelled biomolecule reagent may also be received with instructions for storing and using the labelled biomolecule reagent, such as instructions printed on paper, plastic or on a computer readable medium (e.g., CD-ROM, SD-card, USB drive) or as an insert providing instructions for retrieving instructions for storing and using the subject compositions from a remote source, such as on the internet. Storage Containing a Plurality of Activated Biomolecules and a Plurality of Activated Labels Aspects of the disclosure also include a storage containing a plurality of activated biomolecules and a plurality of activated labels. As discussed in detail above, the subject labelled biomolecule reagents are prepared by contacting an activated biomolecule with an activated label. In some embodiments, the activated biomolecules in the storage are polypeptides, nucleic acids, polypeptides or a combination thereof that are coupled to a reactive linker. In certain instances, the activated biomolecules in the storage are biological probes coupled to a reactive linker where the probe includes a specific binding domain for an analyte of interest, such as antibody binding agents, proteins, peptides, haptens, nucleic acids, etc. Activated labels are marker compounds that may be detectible based on, for example, fluorescence emission, absorbance, fluorescence polarization, fluorescence lifetime, fluorescence wavelength, absorbance maxima, absorbance wavelength, Stokes shift, light scatter, mass, molecular mass, redox, acoustic, raman, magnetism, radio frequency, enzymatic reactions (including chemiluminescence and electro-chemiluminescence) or combinations thereof. For example, the label may be a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag (e.g., isotopically pure rare earth element), magnetic particle, microparticle as well as a nanoparticle. In certain embodiments, activated labels in storage are fluorophores coupled to a reactive linker. Fluorophores of interest may include, but are not limited to, dyes suitable for use in analytical applications (e.g., flow cytometry, imaging, etc.), such as an acridine dye, anthraquinone dyes, arylmethane dyes, diarylmethane dyes (e.g., diphenyl methane dyes), chlorophyll containing dyes, triarylmethane dyes (e.g., triphenylmethane dyes), azo dyes, diazonium dyes, nitro dyes, nitroso dyes, phthalocyanine dyes, cyanine dyes, asymmetric cyanine dyes, quinon-imine dyes, azine dyes, eurhodin dyes, safranin dyes, indamins, indophenol dyes, fluorine dyes, oxazine dye, oxazone dyes, thiazine dyes, thiazole dyes, xanthene dyes, fluorene dyes, pyronin dyes, fluorine dyes, rhodamine dyes, phenanthridine dyes, as well as dyes combining two or more dyes (e.g., in tandem) as well as polymeric dyes having one or more monomeric dye units, as well as mixtures of two or more dyes thereof. For example, the fluorophore may be 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine and derivatives such as acridine, acridine orange, acrindine yellow, acridine red, and acridine isothiocyanate; allophycocyanin, phycoerythrin, peridinin-chlorophyll protein, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS); N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant Yellow; coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine and derivatives such as cyanosine, Cy3, Cy5, Cy5.5, and Cy7; 4′,6-diaminidino-2-phenylindole (DAPI); 5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin; diethylaminocoumarin; diethylenetriamine pentaacetate; 4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid; 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein isothiocyanate (FITC), fluorescein chlorotriazinyl, naphthofluorescein, and QFITC (XRITC); fluorescamine; IR144; IR1446; Green Fluorescent Protein (GFP); Reef Coral Fluorescent Protein (RCFP); Lissamine™; Lissamine rhodamine, Lucifer yellow; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Nile Red; Oregon Green; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 4,7-dichlororhodamine lissamine, rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives; xanthene; dye-conjugated polymers (i.e., polymer-attached dyes) such as fluorescein isothiocyanate-dextran as well as dyes combining two or more of the aforementioned dyes (e.g., in tandem), polymeric dyes having one or more monomeric dye units and mixtures of two or more of the aforementioned dyes thereof. In some instances, the fluorophore (i.e., dye) is a fluorescent polymeric dye. Fluorescent polymeric dyes that find use in the subject methods and systems are varied. In some instances of the method, the polymeric dye includes a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure which includes a backbone of alternating unsaturated bonds (e.g., double and/or triple bonds) and saturated (e.g., single bonds) bonds, where 7-electrons can move from one bond to the other. As such, the conjugated backbone may impart an extended linear structure on the polymeric dye, with limited bond angles between repeat units of the polymer. For example, proteins and nucleic acids, although also polymeric, in some cases do not form extended-rod structures but rather fold into higher-order three-dimensional shapes. In addition, CPs may form “rigid-rod” polymer backbones and experience a limited twist (e.g., torsion) angle between monomer repeat units along the polymer backbone chain. In some instances, the polymeric dye includes a CP that has a rigid rod structure. As summarized above, the structural characteristics of the polymeric dyes can have an effect on the fluorescence properties of the molecules. Any convenient polymeric dye may be utilized in the subject methods and systems. In some instances, a polymeric dye is a multichromophore that has a structure capable of harvesting light to amplify the fluorescent output of a fluorophore. In some instances, the polymeric dye is capable of harvesting light and efficiently converting it to emitted light at a longer wavelength. In some cases, the polymeric dye has a light-harvesting multichromophore system that can efficiently transfer energy to nearby luminescent species (e.g., a “signaling chromophore”). Mechanisms for energy transfer include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer) and the like. In some instances, these energy transfer mechanisms are relatively short range; that is, close proximity of the light harvesting multichromophore system to the signaling chromophore provides for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the signaling chromophore occurs when the number of individual chromophores in the light harvesting multichromophore system is large; that is, the emission from the signaling chromophore is more intense when the incident light (the “excitation light”) is at a wavelength which is absorbed by the light harvesting multichromophore system than when the signaling chromophore is directly excited by the pump light. The multichromophore may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the length of the polymer chain, the backbone contains a large number of conjugated segments in close proximity. Thus, conjugated polymers are efficient for light harvesting and enable optical amplification via energy transfer. In some instances the polymer may be used as a direct fluorescent reporter, for example fluorescent polymers having high extinction coefficients, high brightness, etc. In some instances, the polymer may be used as an strong chromophore where the color or optical density is used as an indicator. Polymeric dyes of interest include, but are not limited to, those dyes described by Gaylord et al. in US Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986 and 20130190193 the disclosures of which are herein incorporated by reference in their entirety; and Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp 6417-6418; Feng et al., Chem. Soc. Rev., 2010,39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133 (32), pp 12600-12607, the disclosures of which are herein incorporated by reference in their entirety. In some embodiments, the polymeric dye includes a conjugated polymer including a plurality of first optically active units forming a conjugated system, having a first absorption wavelength (e.g., as described herein) at which the first optically active units absorbs light to form an excited state. The conjugated polymer (CP) may be polycationic, polyanionic and/or a charge-neutral conjugated polymer. The CPs may be water soluble for use in biological samples. Any convenient substituent groups may be included in the polymeric dyes to provide for increased water-solubility, such as a hydrophilic substituent group, e.g., a hydrophilic polymer, or a charged substituent group, e.g., groups that are positively or negatively charged in an aqueous solution, e.g., under physiological conditions. Any convenient water-soluble groups (WSGs) may be utilized in the subject light harvesting multichromophores. The term “water-soluble group” refers to a functional group that is well solvated in aqueous environments and that imparts improved water solubility to the molecules to which it is attached. In some embodiments, a WSG increases the solubility of the multichromophore in a predominantly aqueous solution (e.g., as described herein), as compared to a multichromophore which lacks the WSG. The water soluble groups may be any convenient hydrophilic group that is well solvated in aqueous environments. In some cases, the hydrophilic water soluble group is charged, e.g., positively or negatively charged or zwitterionic. In certain cases, the hydrophilic water soluble group is a neutral hydrophilic group. In some embodiments, the WSG is a hydrophilic polymer, e.g., a polyethylene glycol, a cellulose, a chitosan, or a derivative thereof. As used herein, the terms “polyethylene oxide”, “PEO”, “polyethylene glycol” and “PEG” are used interchangeably and refer to a polymer including a chain described by the formula —(CH2—CH2—O—)n— or a derivative thereof. In some embodiments, “n” is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 5 to 15, or 10 to 15. It is understood that the PEG polymer may be of any convenient length and may include a variety of terminal groups, including but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal groups. Functionalized PEGs that may be adapted for use in the subject multichromophores include those PEGs described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165. Water soluble groups of interest include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, ester, polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, ammonium, guanidinium, polyamine and sulfonium, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including, polyethers, —COOM′, —SO3M′, —PO3M′, —NR3+, Y′, (CH2CH2O)pR and mixtures thereof, where Y′ can be any halogen, sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, each R can be independently H or an alkyl (such as methyl) and M′ can be a cationic counterion or hydrogen, —(CH2CH2O)yyCH2CH2XRyy, —(CH2CH2O)yyCH2CH2X—, —X(CH2CH2O)yyCH2CH2—, glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and RZZand RYYare independently selected from H and C1-3 alkyl. The polymeric dye may have any convenient length. In some cases, the particular number of monomeric repeat units or segments of the polymeric dye may fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units or segments, or such as 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In certain instances, the number of monomeric repeat units or segments of the polymeric dye is within the range of 2 to 1000 units or segments, such as from 2 to 750 units or segments, such as from 2 to 500 units or segments, such as from 2 to 250 units or segment, such as from 2 to 150 units or segment, such as from 2 to 100 units or segments, such as from 2 to 75 units or segments, such as from 2 to 50 units or segments and including from 2 to 25 units or segments. The polymeric dyes may be of any convenient molecular weight (MW). In some cases, the MW of the polymeric dye may be expressed as an average molecular weight. In some instances, the polymeric dye has an average molecular weight of from 500 to 500,000, such as from 1,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000 or even an average molecular weight of from 50,000 to 100,000. In certain embodiments, the polymeric dye has an average molecular weight of 70,000. The polymeric dye may have one or more desirable spectroscopic properties, such as a particular absorption maximum wavelength, a particular emission maximum wavelength, extinction coefficient, quantum yield, and the like. In some embodiments, the polymeric dye has an absorption curve between 280 and 850 nm. In certain embodiments, the polymeric dye has an absorption maximum in the range 280 and 850 nm. In some embodiments, the polymeric dye absorbs incident light having a wavelength in the range between 280 and 850 nm, where specific examples of absorption maxima of interest include, but are not limited to: 348 nm, 355 nm, 405 nm, 407 nm, 445 nm, 488 nm, 640 nm and 652 nm. In some instances, the polymeric dye has an absorption maximum wavelength in a range selected from the group consisting of 280-310 nm, 305-325 nm, 320-350 nm, 340-375 nm, 370-425 nm, 400-450 nm, 440-500 nm, 475-550 nm, 525-625 nm, 625-675 nm and 650-750 nm. In certain embodiments, the polymeric dye has an absorption maximum wavelength of 348 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 355 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 405 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 407 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 445 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 488 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 640 nm. In some instances, the polymeric dye has an absorption maximum wavelength of 652 nm. In some embodiments, the polymeric dye has an emission maximum wavelength ranging from 400 to 850 nm, such as 415 to 800 nm, where specific examples of emission maxima of interest include, but are not limited to: 395 nm, 421 nm, 445 nm, 448 nm, 452 nm, 478 nm, 480 nm, 485 nm, 491 nm, 496 nm, 500 nm, 510 nm, 515 nm, 519 nm, 520 nm, 563 nm, 570 nm, 578 nm, 602 nm, 612 nm, 650 nm, 661 nm, 667 nm, 668 nm, 678 nm, 695 nm, 702 nm, 711 nm, 719 nm, 737 nm, 785 nm, 786 nm, 805 nm. In some instances, the polymeric dye has an emission maximum wavelength in a range selected from the group consisting of 380-400 nm, 410-430 nm, 470-490 nm, 490-510 nm, 500-520 nm, 560-580 nm, 570-595 nm, 590-610 nm, 610-650 nm, 640-660 nm, 650-700 nm, 700-720 nm, 710-750 nm, 740-780 nm and 775-795 nm. In certain embodiments, the polymeric dye has an emission maximum of 395 nm. In some instances, the polymeric dye has an emission maximum wavelength of 421 nm. In some instances, the polymeric dye has an emission maximum wavelength of 478 nm. In some instances, the polymeric dye has an emission maximum wavelength of 480 nm. In some instances, the polymeric dye has an emission maximum wavelength of 485 nm. In some instances, the polymeric dye has an emission maximum wavelength of 496 nm. In some instances, the polymeric dye has an emission maximum wavelength of 510 nm. In some cases, the polymeric dye has an emission maximum wavelength of 570 nm. In certain embodiments, the polymeric dye has an emission maximum wavelength of 602 nm. In some instances, the polymeric dye has an emission maximum wavelength of 650 nm. In certain cases, the polymeric dye has an emission maximum wavelength of 711 nm. In some instances, the polymeric dye has an emission maximum wavelength of 737 nm. In some instances, the polymeric dye has an emission maximum wavelength of 750 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 786 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 421 nm±5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm±5 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 570 nm±5 nm. In some instances, the polymeric dye has an emission maximum wavelength of 602 nm±5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 650 nm±5 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 711 nm±5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 786 nm±5 nm. In certain embodiments, the polymeric dye has an emission maximum selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm and 786 nm. In some instances, the polymeric dye has an extinction coefficient of 1×106cm−1M−1or more, such as 2×106cm−1M−1or more, 2.5×106cm−1M−1or more, 3×106cm−1M−1or more, 4×106cm−1M−1or more, 5×106cm−1M−1or more, 6×106cm−1M−1or more, 7×106cm−1M−1or more, or 8×106cm−1M−1or more. In certain embodiments, the polymeric dye has a quantum yield of 0.05 or more, such as 0.1 or more, 0.15 or more, 0.2 or more, 0.25 or more, 0.3 or more, 0.35 or more, 0.4 or more, 0.45 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, 0.9 or more, 0.95 or more, 0.99 or more and including 0.999 or more. For example, the quantum yield of polymeric dyes of interest may range from 0.05 to 1, such as from 0.1 to 0.95, such as from 0.15 to 0.9, such as from 0.2 to 0.85, such as from 0.25 to 0.75, such as from 0.3 to 0.7 and including a quantum yield of from 0.4 to 0.6. In certain cases, the polymeric dye has a quantum yield of 0.1 or more. In certain cases, the polymeric dye has a quantum yield of 0.3 or more. In certain cases, the polymeric dye has a quantum yield of 0.5 or more. In certain cases, the polymeric dye has a quantum yield of 0.6 or more. In certain cases, the polymeric dye has a quantum yield of 0.7 or more. In certain cases, the polymeric dye has a quantum yield of 0.8 or more. In certain cases, the polymeric dye has a quantum yield of 0.9 or more. In certain cases, the polymeric dye has a quantum yield of 0.95 or more. In some embodiments, the polymeric dye has an extinction coefficient of 1×106or more and a quantum yield of 0.3 or more. In some embodiments, the polymeric dye has an extinction coefficient of 2×106or more and a quantum yield of 0.5 or more. In embodiments, the activated biomolecules and activated labels for preparing the labelled biomolecule reagent in accordance with the labelled biomolecule reagent request are obtained from the storage. The storage may have 10 or more different activated biomolecules, such as 25 or more, such as 50 or more, such as 100 or more, such as 250 or more, such as 500 or more and including 1000 or more activated biomolecules. In one example, the storage includes 10 or more different activated oligonucleotides, such as 25 or more, such as 50 or more, such as 100 or more, such as 250 or more, such as 500 or more and including 1000 or more activated oligonucleotides. In another example the storage includes 10 or more different activated polypeptides, such as 25 or more, such as 50 or more, such as 100 or more, such as 250 or more, such as 500 or more and including 1000 or more activated polypeptides. The storage may also include 10 or more different activated labels, such as 15 or more, such as 20 or more, such as 30 or more, such as 40 or more and including 50 or more different activated labels. Each of the plurality of activated biomolecules and activated labels may be present in the storage in any suitable container capable of storing and providing the activated biomolecule or activated label when desired. In some embodiments, the plurality of different activated biomolecules and plurality of different activated labels are stored in a single reservoir partitioned into separate reagent chambers. In other embodiments, each of the plurality of different activated biomolecules and plurality of different activated labels are stored in individual containers (e.g., bottles, jugs, etc.) In yet other embodiments, each of the plurality of different activated biomolecules and plurality of different activated labels are stored in a plurality of vials, where each vial includes pre-measured aliquots of each activated biomolecule and each activated label. Each container in the storage may also include a label identifying the components of the activated biomolecule or activated label (e.g., name, structure, CAS registry number, ascension number, probe sequence, etc. of the biomolecule, label and reactive linker) The label may also include one or more machine readable components such as a Quick Response (QR) code or a bar code. In some embodiments, the storage also includes a database of available activated biomolecules and activated labels. The database may be a printed catalog in paper or electronic form or may be a searchable electronic database, such as searchable by keyword, chemistry structure, ascension number, monomer sequence (e.g., amino acid or nucleotide sequence) or by CAS chemical registry number. Utility The subject systems and methods find use in preparing complex biological reagents (e.g., biological macromolecules coupled to detectable markers)—a process that is generally time consuming, financially inefficient and extraordinarily labor intensive when conducted on a large scale. The present disclosure provides a fast, efficient and highly scalable process for delivering high quality and performance specific products across a wide range of biomolecule and detectable label portfolios. The systems and methods described herein also provide a unique and new way to request and provide customized biological reagents. In addition being able to choose pre-synthesized reagents from an extensive database (e.g., an online database), the subject systems and methods provide for user customization, where the user can create any desired labelled biomolecule on-demand. By simply choosing a biological macromolecule and a detectable marker on an easy-to-use graphical interface, a user can request any labelled biomolecule, which are used in a variety of different research applications and in medical diagnosis. The present disclosure also provides access to large portfolios of complex biological reagents that are not possible when prepared by small scale synthesis. The subject systems and methods are scalable facilitating the preparation, on-demand, of thousands of different combinations of biomolecules and detectable markers. In certain embodiments, the subject systems provide fully automated protocols so that the preparation of customized detectable biomolecule probes requires little, if any human input. The present disclosure also finds use in applications where cell analysis from a biological sample may be desired for research, laboratory testing or for use in therapy. In some embodiments, the subject methods and systems may facilitate analysis of cells obtained from fluidic or tissue samples such as specimens for diseases such as cancer. Methods and systems of the present disclosure also allow for analyzing cells from a biological sample (e.g., organ, tissue, tissue fragment, fluid) with enhanced efficiency and low cost as compared to using probe compositions synthesized de novo. Notwithstanding the appended clauses, the disclosure set forth herein is also defined by the following clauses:1. A system for use in preparing a labelled biomolecule reagent, the system comprising:an input manager for receiving a request for a labelled biomolecule reagent;a memory for storing a dataset comprising a plurality of labelled biomolecule storage identifiers;a processing module communicatively coupled to the memory and configured to identify one or more labelled biomolecule storage identifiers from the dataset that corresponds to the components of the labelled biomolecule reagent request;an output manager for providing the identified labelled biomolecule storage identifiers.2. The system of clause 1, wherein the request for a labelled biomolecule reagent comprises a biomolecule request and a label request.3. The system of clause 2, wherein the memory comprises a first dataset comprising a plurality of biomolecule storage identifiers for a plurality of activated biomolecules and a second dataset comprising a plurality of label storage identifiers for a plurality of activated labels.4. The system of any one of clauses 1 to 3, wherein the output manager is operatively coupled to a communication component configured to display the identified labelled biomolecule storage identifiers.5. The system of clause 4, wherein the communication component is an electronic display.6. The system of clause 4, wherein the communication component is a printer.7. The system of any one of clauses 1 to 6, wherein the input manager is operatively coupled to a graphical user interface.8. The system of any one of clauses 1 to 7, wherein the graphical user interface comprises an internet website menu interface.9. The system of any one of clauses 1 to 8, wherein the input manager is configured to receive a plurality of labelled biomolecule requests.10. The system of clause 9, wherein the input manager is configured to simultaneously receive a plurality of biomolecule requests and label requests.11. The system of clause 9, wherein the input manager is configured to receive a plurality of biomolecule requests and label requests from the same user.12. The system of clause 9, wherein the input manager is configured to receive a plurality of biomolecule requests and label requests from a plurality of users.13. The system of any one of clauses 1 to 12, wherein the memory comprises algorithm for providing a recommendation for an alternative biomolecule when a biomolecule storage identifier that corresponds to the biomolecule request is not available.14. The system of any one of clauses 1 to 13, wherein the memory comprises algorithm for providing a recommendation for an alternative label when a label storage identifier that corresponds to the label request is not available.15. The system of any one of clauses 1 to 14, further comprising a reagent preparatory apparatus for preparing the labelled biomolecule reagent, wherein the reagent preparatory apparatus is operatively coupled to the output manager and is configured to:receive the identified biomolecule storage identifier and label storage identifier; andproduce a labelled biomolecule reagent corresponding to the received biomolecule storage identifier and the label storage identifier.16. The system of clause 15, wherein the reagent preparatory apparatus comprises a sampling device configured to provide an activated biomolecule and an activated label to a contacting apparatus.17. The system of clause 16, further comprising a contacting apparatus configured for contacting the activated biomolecule with the activated label to produce the labelled biomolecule reagent.18. The system of any one of clauses 16 to 17, further comprising a labelled biomolecule reagent analyzer.19. The system of clause 18, wherein the analyzer comprises a purification component for purifying the labelled biomolecule reagent.20. The system of clause 19, wherein the purification component comprises liquid chromatography.21. The system of any one of clauses 16 to 20, further comprising a solvent chamber configured to provide one or more solvents to the contacting apparatus.22. The system of any one of clauses 16 to 21, wherein the contacting apparatus is a microtube.23. The system of any one of clauses 15 to 22, wherein the system comprises a reagent packaging unit configured to seal the produced labelled biomolecule reagent in the container.24. The system of any one of clauses 1 to 23, wherein the biomolecule is a compound selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.25. The system of clause 24, wherein the nucleic acid is an oligonucleotide, DNA or RNA.26. The system of clause 25, wherein the biomolecule is an oligonucleotide.27. The system of clause 24, wherein the polypeptide is a protein, enzyme or antibody.28. The system of clause 27, wherein the biomolecule is an antibody.29. The system of any one of clauses 1 to 28, wherein the label is a compound selected from the group consisting of a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.30. The system of any one of clauses 1 to 29, wherein the memory comprises 25 or more biomolecule storage identifiers.31. The system of clause 30, wherein the memory comprises 25 or more antibody storage identifiers.32. The system of any one of clauses 1 to 31, wherein the memory comprises 10 or more label storage identifiers.33. The system of clause 32, wherein the memory comprises 25 or more fluorophore storage identifiers.34. The system of any one of clauses 1 to 33, wherein activated biomolecule and activated label each independently comprise a covalently coupled reactive linker.35. A method comprising:communicating a request for a labelled biomolecule reagent, the request comprising one or more of:a labelled biomolecule request; anda biomolecule request and a label request; andreceiving one or more labelled biomolecule reagents, each labelled biomolecule reagent comprising a biomolecule covalently coupled to a label through a linker.36. The method of clause 35, further comprising selecting a labelled biomolecule reagent from a first dataset comprising a plurality of labelled biomolecule storage identifiers.37. The method of any one of clauses 35 to 36, further comprising selecting:a biomolecule from a second dataset comprising a plurality of biomolecule storage identifiers; anda label from a third dataset comprising a plurality of label storage identifiers.38. The method of any one of clauses 35 to 37, wherein communicating the request comprises inputting the labelled biomolecule reagent request into a graphical user interface operatively coupled to an input manager of a system configured to receive the labelled biomolecule reagent request.39. The method of clause 38, wherein the graphical user interface comprises an internet website menu interface.40. The method of any one of clauses 35 to 39, wherein communicating the labelled biomolecule reagent request comprises providing the labelled biomolecule reagent request by mail, electronic mail or over the telephone.41. The method of any one of clauses 35 to 40, wherein the method comprises communicating a request for a plurality of labelled biomolecule reagents.42. The method of clause 41, wherein the request for a plurality of labelled biomolecule reagents comprises a plurality of biomolecule requests and a plurality of label requests.43. The method of clause 41, wherein the request for a plurality of labelled biomolecule reagents comprises a single biomolecule request and plurality of label requests.44. The method of clause 41, wherein the request for a plurality of labelled biomolecule reagents comprises a plurality of biomolecule requests and a single label request.45. The method of any one of clauses 35 to 44, wherein the received labelled biomolecule reagent is sealed in a container.46. The method of any one of clauses 35 to 44, wherein the biomolecule is a compound selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.47. The method of clause 46, wherein the nucleic acid is an oligonucleotide, DNA or RNA.48. The method of clause 46, wherein the biomolecule is an oligonucleotide.49. The method of clause 46, wherein the polypeptide is a protein, an enzyme or an antibody.50. The method of clause 46, wherein the biomolecule is an antibody.51. The method of any one of clauses 35 to 50, wherein the label is a compound selected from the group consisting of fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.52. The method of clause 51, wherein the label is a fluorophore.53. A method comprising:communicating a request for a labelled biomolecule reagent to a system comprising:an input manager that receives a labelled biomolecule reagent request;a memory for storing a dataset comprising a plurality of labelled biomolecule storage identifiers;a processing module communicatively coupled to the memory and configured to identify one or more labelled biomolecule storage identifiers from the dataset that corresponds to the components of the labelled biomolecule reagent request;an output manager for providing the identified labelled biomolecule storage identifiers; andreceiving a labelled biomolecule reagent comprising a biomolecule covalently coupled to a label.54. The method of clause 53, wherein the request for a labelled biomolecule reagent comprises a biomolecule request and a label request.55. The method of any one of clauses 53 to 54, wherein the memory comprises a first dataset comprising a plurality of biomolecule storage identifiers for a plurality of activated biomolecules and a second dataset comprising a plurality of label storage identifiers for a plurality of activated labels.56. The method of any one of clauses 53 to 55, wherein communicating the request for a labelled biomolecule reagent comprises inputting one or more of: a labelled biomolecule request, a biomolecule request and a label request into a graphical user interface operatively coupled to the input manager.57. The method of clause 56, wherein the graphical user interface comprises an internet website menu interface.58. The method of any one of clauses 53 to 57, wherein the method comprises communicating a request for a plurality of labelled biomolecule reagents.59. The method of clause 58, wherein the request for the plurality of labelled biomolecule reagents comprises a plurality of biomolecule requests and a plurality of label requests.60. The method of clause 58, wherein the request for the plurality of labelled biomolecule reagents comprises a single biomolecule request and plurality of label requests.61. The method of clause 58, wherein the request for the plurality of labelled biomolecule reagents comprises a plurality of biomolecule requests and a single label request.62. The method of any one of clauses 53 to 61, wherein the received labelled biomolecule reagent is sealed in a container.63. The method of any one of clauses 53 to 62, wherein the biomolecule is a compound selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.64. The method of clause 63, wherein the nucleic acid is an oligonucleotide, DNA or RNA.65. The method of clause 64, wherein the biomolecule is an oligonucleotide.66. The method of clause 63, wherein the polypeptide is a protein, an enzyme or an antibody.67. The method of clause 66, wherein the biomolecule is an antibody.68. The method of any one of clauses 53 to 67, wherein the label is a compound selected from the group consisting of a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.69. The method of clause 68, wherein the label is a fluorophore.70. A method comprising:receiving a request for a labelled biomolecule reagent, the request comprising one or more of:a labelled biomolecule request; anda biomolecule request and a label request;preparing a labelled biomolecule reagent corresponding to the labelled biomolecule reagent request by contacting an activated biomolecule with an activated label to produce the labelled biomolecule reagent, wherein the preparing comprising selecting an activated biomolecule and an activated label from a storage comprising a plurality of activated biomolecules and a plurality of activated labels.71. The method of clause 70, wherein the method comprises receiving a request for a plurality of labelled biomolecule reagents.72. The method of clause 71, wherein the request for a plurality of labelled biomolecule reagents comprises a plurality of biomolecule requests and a plurality of label requests.73. The method of clause 71, wherein the request for a plurality of labelled biomolecule reagents comprises a single biomolecule request and plurality of label requests.74. The method of clause 71, wherein the request for a plurality of labelled biomolecule reagents comprises a plurality of biomolecule requests and a single label request.75. The method of any one of clauses 70 to 74, wherein contacting comprises manually combining the activated biomolecule with the activated label in a contacting apparatus.76. The method of clause 75, wherein the contacting apparatus is a microtube.77. The method of any one of clauses 70 to 76, wherein the activated biomolecule and the activated label are contacted in a contacting apparatus of a reagent preparatory apparatus by a computer-controlled sampling device.78. The method of any one of clauses 70 to 77, further comprising purifying the labelled biomolecule reagent.79. The method of any one of clauses 70 to 78, further comprising transporting the labelled biomolecule reagent to a remote location.80. The method of any one of clauses 70 to 79, wherein the request for a labelled biomolecule reagent is received through an internet website.81. The method of any one of clauses 70 to 80, wherein the request for a labelled biomolecule reagent is received over the telephone.82. The method of any one of clauses 70 to 81, wherein the request for a labelled biomolecule reagent is received through the mail.83. The method of any one of clauses 70 to 82, wherein the request for a labelled biomolecule reagent is received through electronic mail.84. The method of any one of clauses 70 to 83, further comprising providing a recommendation for an alternative labelled biomolecule when the labelled biomolecule corresponding to the request is not available.85. The method of any one of clauses 70 to 84, further comprising providing a recommendation for an alternative biomolecule when the biomolecule that corresponds to the biomolecule request is not available.86. The method of any one of clauses 70 to 85, further comprising providing a recommendation for an alternative label when the label that corresponds to the label request is not available.87. The method of any one of clauses 70 to 86, wherein the biomolecule is a compound selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.88. The method of clause 87, wherein the nucleic acid is an oligonucleotide, DNA or RNA.89. The method of clause 88, wherein the biomolecule is an oligonucleotide.90. The method of clause 87, wherein the polypeptide is a protein, an enzyme or an antibody.91. The method of clause 90, wherein the biomolecule is an antibody.92. The method of any one of clauses 70 to 91, wherein the label is a compound selected from the group consisting of a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.93. The method of clause 92, wherein the label is a fluorophore.94. A method comprising:receiving a request for a labelled biomolecule reagent with a system comprising:an input manager that receives a labelled biomolecule reagent request;a memory for storing a dataset comprising a plurality of labelled biomolecule storage identifiers;a processing module communicatively coupled to the memory and configured to identify one or more labelled biomolecule storage identifiers from the dataset that corresponds to the components of the labelled biomolecule reagent request;an output manager;identifying the labelled biomolecule storage identifiers that corresponds with the labelled biomolecule reagent request;outputting the identified labelled biomolecule reagent storage identifier. 95. The method of clause 94, wherein the request for a labelled biomolecule reagent comprises a biomolecule request and a label request.96. The method of any one of clauses 94 to 95, further comprising displaying the outputted labelled biomolecule reagent storage identifier onto an electronic display.97. The method of any one of clauses 94 to 96, further comprising printing the outputted labelled biomolecule reagent storage identifier.98. The method of any one of clauses 94 to 97, wherein the method comprises receiving a plurality of requests for labelled biomolecule reagents.99. The method of clause 98, wherein the plurality of requests are received from the same user.100. The method of clause 98, wherein the plurality of requests are received from different users.101. The method of clause 98, wherein the request for the labelled biomolecule reagents comprises a plurality of biomolecule requests and a plurality of label requests.102. The method of clause 98, wherein the request for the labelled biomolecule reagents comprises a single biomolecule request and plurality of label requests.103. The method of any one of clauses 94 to 102, wherein the request for the labelled biomolecule reagents comprises a plurality of biomolecule requests and a single label request.104. The method of clause 103, further comprising contacting an activated biomolecule associated with biomolecule storage identifier with an activated label associated with the label storage identifier to produce the labelled biomolecule reagent.105. The method of clause 104, wherein contacting comprises manually combining the activated biomolecule with the activated label in a contacting apparatus.106. The method of clause 105, wherein the contacting apparatus is a microtube.107. The method of clause 105, wherein the activated biomolecule and the activated label are contacted in a contacting apparatus of a reagent preparatory apparatus by a computer controlled sampling device.108. The method of clause 105, further comprising purifying the labelled biomolecule reagent.109. The method of any one of clauses 94 to 108, further comprising transporting the labelled biomolecule reagent to a remote location.110. The method of any one of clauses 94 to 109, wherein the request for a labelled biomolecule reagent is received through an internet website.111. The method of any one of clauses 94 to 110, wherein the request for a labelled biomolecule reagent is received over the telephone and inputted into the input manager.112. The method of any one of clauses 94 to 111, wherein the request for a labelled biomolecule reagent is received through the mail and inputted into the input manager.113. The method of clause 112, wherein the request for a labelled biomolecule reagent is received through electronic mail and inputted into the input manager.114. The method of any one of clauses 94 to 113, further comprising providing a recommendation for an alternative labelled biomolecule from a database when a labelled biomolecule storage identifier that corresponds to the labelled biomolecule request is not available.115. The method of any one of clauses 94 to 114, further comprising providing a recommendation for an alternative biomolecule from a database when a biomolecule storage identifier that corresponds to the biomolecule request is not available.116. The method of any one of clauses 94 to 115, further comprising providing a recommendation for an alternative label from a database when a label storage identifier that corresponds to the label request is not available.117. The method of any one of clauses 94 to 116, wherein the biomolecule is a compound selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.118. The method of clause 117, wherein the biomolecule is a nucleic acid.119. The method of clause 118, wherein the nucleic acid is an oligonucleotide, DNA or RNA.120. The method of clause 119, wherein the biomolecule is an oligonucleotide.121. The method of clause 117, wherein the polypeptide is a protein, an enzyme or an antibody.122. The method of clause 121, wherein the biomolecule is an antibody.123. The method of any one of clauses 94 to 122, wherein the label is a compound selected from the group consisting of a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.124. The method of clause 123, wherein the label is a fluorophore.125. A system comprising:a plurality of activated biomolecules;a plurality of activated labels; anda reagent preparatory apparatus for preparing a labelled biomolecule reagent, wherein the reagent preparatory apparatus is configured to:receive an identified biomolecule storage identifier and label storage identifier; andproduce a labelled biomolecule reagent corresponding to the received biomolecule storage identifier and the label storage identifier.126. The system of clause 125, wherein the reagent preparatory apparatus comprises a sampling device configured to provide an activated biomolecule and an activated label to a contacting apparatus.127. The system of clause 126, wherein the reagent preparatory apparatus comprises a contacting apparatus configured for contacting the activated biomolecule with the activated label to produce the labelled biomolecule reagent.128. The system of clause 127, further comprising a labelled biomolecule reagent analyzer.129. The system of clause 128, wherein the analyzer comprises a purification component for purifying the labelled biomolecule reagent.130. The system of any one of clauses 125 to 126, wherein the system comprises a reagent packaging unit configured to seal the produced labelled biomolecule reagent in a container.131. The system of any one of clauses 125 to 130, wherein the reagent preparatory apparatus is operatively coupled to a system for receiving a labelled biomolecule reagent request, the system comprising:an input manager for receiving a biomolecule request and a label request for a labelled biomolecule reagent;a memory for storing a first dataset comprising a plurality of biomolecule storage identifiers for a plurality of activated biomolecules and a second dataset comprising a plurality of label storage identifiers for a plurality of activated labels;a processing module communicatively coupled to the memory and configured to identify a biomolecule storage identifier and a label storage identifier from the first dataset and second dataset that correspond to the biomolecule request and label request;an output manager for providing the identified biomolecule storage identifier and label storage identifier.132. The system of any one of clauses 125 to 131, wherein the system comprises 1000 or more different activated biomolecules.133. The system of any one of clauses 125 to 132, wherein the biomolecule is selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.134. The system of clause 133, wherein the biomolecule is an oligonucleotide.135. The system of clause 134, wherein the system comprises 1000 or more different types of oligonucleotides.136. The system of clause 133, wherein the biomolecule is an antibody.137. The system of clause 136, wherein the system comprises 1000 or more different types of antibodies.138. The system of any one of clauses 125 to 137, wherein each activated biomolecule comprises a reactive linker.139. The system of any one of clauses 125 to 138, wherein the system comprises 100 or more different activated labels.140. The system of clause 139, wherein the label is selected from the group consisting of a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.141. The system of any one of clauses 125 to 140, wherein activated label comprises a reactive linker.142. A storage comprising:a plurality of activated biomolecules; anda plurality of activated labels.143. The storage of clause 142, wherein the storage comprises 1000 or more different activated biomolecules.144. The storage of any one of clauses 142 to 143, wherein the biomolecule is selected from the group consisting of a polypeptide, a nucleic acid and a polysaccharide.145. The storage of clause 144, wherein the biomolecule is an oligonucleotide.146. The storage of clause 145, wherein the storage comprises 1000 or more different types of oligonucleotides.147. The storage of clause 144, wherein the biomolecule is an antibody.148. The storage of clause 147, wherein the storage comprises 1000 or more different types of antibodies.149. The storage of any one of clauses 142 to 148, wherein each activated biomolecule comprises a reactive linker.150. The storage of any one of clauses 142 to 149, wherein the storage comprises 100 or more different activated labels.151. The storage of clause 150, wherein the label of the activated labels is selected from the group consisting of a fluorophore, chromophore, enzyme, redox label, radiolabels, acoustic label, Raman (SERS) tag, mass tag, isotope tag, magnetic particle, microparticle and nanoparticle.152. A labelled biomolecule reagent dispensing system comprising:an input module for receiving a request for a labelled biomolecule;a reagent preparatory apparatus; anda dispensing module for outputting a packaged labelled biomolecule.153. The labelled biomolecule reagent dispensing system of clause 152, wherein the input module comprises:a graphical user interface for communicating a labelled biomolecule request to an input manager;an input manager for receiving a request for a labelled biomolecule;a memory for storing a dataset having a plurality of storage identifiers that correspond to the one or more components of the labelled biomolecule reagent request;a processing module communicatively coupled to the memory and configured to identify a storage identifier from the dataset that corresponds to the components of the labelled biomolecule reagent request; andan output manager for providing the identified storage identifiers.154. The labelled biomolecule reagent dispensing system of any one of clauses 152 to 153, wherein the reagent preparatory apparatus comprises one or more of a source of a labelled biomolecule, a source of a biomolecule, a source of a label, a source of a reactive linker, a source of an activated biomolecule and a source of an activated label.155. The labelled biomolecule reagent dispensing system of any one of clauses 152 to 154, wherein the reagent preparatory apparatus comprises:a sampling device configured to provide an activated biomolecule and an activated label to a contacting apparatus.156. The labelled biomolecule reagent dispensing system of clause 155, further comprising a contacting apparatus configured for contacting the activated biomolecule with the activated label to produce the labelled biomolecule reagent.157. The labelled biomolecule reagent dispensing system of any one of clauses 155 to 156, further comprising a labelled biomolecule reagent analyzer.158. The labelled biomolecule reagent dispensing system of clause 157, wherein the analyzer comprises a purification component for purifying the labelled biomolecule reagent.159. The labelled biomolecule reagent dispensing system of clause 158, wherein the purification component comprises liquid chromatography.160. The labelled biomolecule reagent dispensing system of any one of clauses 153 to 159, further comprising a solvent chamber configured to provide one or more solvents to the contacting apparatus.161. The labelled biomolecule reagent dispensing system according to any one of clauses 152 to 160, wherein the dispensing module comprises a reagent packaging unit configured to seal the produced labelled biomolecule reagent in the container.162. The labelled biomolecule reagent dispensing system according to clause 161, wherein the container is selected from the group consisting of a pouch, bag, tube, vial, microtube or bottle.163. The labelled biomolecule reagent dispensing system according to clause 161, wherein the packaging unit is further configured to dispense the sealed container with labelled biomolecule in a second container.164. The labelled biomolecule reagent dispensing system according to clause 163, wherein the second container is selected from the group consisting of a pouch, bag, tube, vial, microtube or bottle. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. | 186,734 |
11860160 | DETAILED DESCRIPTION OF THE INVENTION In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. Where applicable in the following description, definitions of specific terms and phrases are provided to clarify their specific use in the context of the invention. Example 1: Paper-Based Vertical Flow Immunoassay (VFI) for Diagnosis of Bio-Threat Pathogens During the last century, in parallel to progress made in basic and applied microbiology, there has been concerning advances made toward the weaponization of biological agents and biotoxins (Jansen et al., 2014; L W et al., 1997). Currently, there are nearly seventy biological agents listed by the U.S. Department of Health and Human Services as potential threats to public health and national security. Exposure to these agents can cause a variety of human and animal diseases (Higgins et al., 1999; Lakoff, 2008).Burkholderia pseudomallei, a soil and water dwelling bacterium that causes the deadly disease melioidosis in human and animals, is considered a high priority biological threat because of its potential to be weaponized and ability to produce fatal diseases. Recently, the total number of melioidosis cases worldwide has been estimated to be 165,000 of which 89,000 people die (Limmathurotsakul et al., 2016). Melioidosis can be very challenging to diagnose and treat, as current diagnostic method often lack the required sensitivity. Melioidosis has broad clinical manifestations that range from mild infection of the skin and soft tissues to organ abscesses (Wiersinga et al., 2012), and is one of the most common sources of community-acquired sepsis in many tropical and subtropical regions of southeast Asia and northern Australia (Limmathurotsakul et al., 2016). Infections vary from mild disease to overwhelming septicemia within 24 to 48 hours after symptom onset, and untreated cases of melioidosis have a fatality rate as high as 90% (Limmathurotsakul et al., 2010b). Early diagnosis and treatment are key factors in improving disease management and lowering the mortality rate of infected patients (Currie et al., 2010). Despite several advances in immunological and molecular approaches to detectB. pseudomallei(Sirisinha et al., 2000), culturing ofB. pseudomalleifrom clinical samples (e.g. blood, urine, pus, sputum, etc.) remains the gold-standard for melioidosis diagnosis. However, laboratory culture is very time-consuming and often challenging due to the low levels of bacteria present in many clinical samples. Culture and other molecular diagnostic approaches, including PCR, require sophisticated equipment and highly-skilled personnel that are often unavailable in resource-restricted regions. These difficulties can lead to the infection being misdiagnosed and grossly underreported in these areas (Limmathurotsakul et al., 2010b). Moreover, recent statistical studies have found that the culture method is not a reliable gold standard for diagnosis of melioidosis infection due to low sensitivity (Limmathurotsakul et al., 2010a). For these reasons, there is a crucial need for a simple, rapid, point-of-care (POC) diagnostic tool that can quickly and accurately detect bio-threat agents such asB. pseudomalleifrom clinical samples. With the advancement of nanotechnology and bio-sensing technology, paper materials have been utilized for biomedical applications (Hu et al., 2014; Parolo and Arben Merkogi, 2013; Warren et al., 2014; Yetisen et al., 2013). Paper based devices have been reported to detect proteins (Li et al., 2010), nucleic acids (He et al., 2012), and cellular activities (Liu et al., 2009). A majority of the reported paper devices are designed in a lateral flow immunoassay (LFI) format where the fluid flow is parallel to the surface of the paper. This format typically consists of several paper segments joined together with a backing layer (Yetisen et al., 2013) and the testing fluid is transported through these segments by the capillary force of the paper. The main benefits of LFI include low cost, rapid results, flexibility and ease of use. However, in order to achieve an adequate flow rate, the pore size of the paper materials are limited to several micrometres and above, which could hinder the biomolecule capturing and thus the assay's sensitivity. In addition, sample volume constraint and the difficulties of multiplexing are other hurdles that confront LFI development. Several LFI prototypes for multiplexing detection have been reported using a paper device with branched geometry (Li et al., 2011; Mu et al., 2014), multiple testing lines (Rivas et al., 2015), and microarray (Helene and Svahn, 2014; Lafleur et al., 2012; Safenkova et al., 2016). Overall, the number of integrated assays is still limited and they require careful design of the membrane geometry and microarray layout to prevent cross-contamination so that the upstream reactions do not affect the downstream reactions. One approach to improving upon the LFI platform is the use of vertical flow-through devices. This alternative format is similar in that it is a membrane based immunoassay, however, fluids are applied vertically to the surface of the paper rather than parallel (Desmet et al., 2011; Oh et al., 2013; Pauli et al., 2015). One design simply replaces the conventional lateral flow segments in a stacking manner, and allowing the liquid to diffuse from the bottom to the top layers (Eltzov and Marks, 2017; Herendarcikove et al., 2017; Park and Park, 2017). Another method is to push the assay reagents through one single membrane in steps, and allow for the targets to react with the reagents on the membrane. A vertical flow allergen microarray assay was built with to detect IgE activities in human serum samples with allergen components (Chinnasamy et al., 2014; Reuterswsrd et al., 2015). Similarly, an immunofiltration assay for simultaneous detection of HIV p24 and hepatitis B virus antigens was recently reported (Cretich et al., 2015). Another vertical flow platform was developed using 96-well vacuum plate to draw reagents through a nitrocellulose membrane to detect IgM specific to the lipopolysaccharides (LPS) derived fromSalmonella typhi(Ramachandran et al., 2013). Recent efforts have been made to investigate the possibilities of coupling the vertical flow assay with more advanced readout technologies, such as the surface-enhanced Raman spectroscopy (SERS), toward higher sensitivity (Berger et al., 2016; Clarke et al., 2017). This example presents a paper-based vertical flow immunoassay (VFI) device for the detection of bio-threat pathogens.B. pseudomallei, the causative agent of melioidosis, was used as the model bacterium. Previous studies have validated the capsular polysaccharide (CPS), a polymer of 1, 3-linked 2-O-acetyl-6-deoxy-b-D-manno-heptopyranose residues, as a diagnostic biomarker forB. pseudomallei(AuCoin, 2012; Nuti et al., 2011). It has also been shown that CPS can be successfully detected via LFI (Houghton et al., 2014; Robertson et al., 2015). It is clear that some melioidosis patients have low levels of CPS that are undetectable by the CPS LFI. In this example, VFI parameters were optimized in order to develop a POC assay with improved sensitivity and both simplex and multiplex detection capabilities of biothreat pathogens. There are a number of important parameters that needed to be considered in order to optimize the VFI platform of the disclosed device. The VFI membrane area is large, but the sensing spots with surface reagents can be reduced in size by depositing small volumes of reagents. This allows for multiplexed detection by incorporating different assays on the same membrane creating a spatially independent spot microarray. Previous work reported that increasing the sample volume could improve the sensitivity of flow-through immuno-sensors (Pauli et al., 2015). However, the effect of sample flow rate has not been studied with the vertical flow devices. Flow rate is an important factor as it impacts the time needed to perform such assays, and point-of-care devices by definition are rapid. In the VFI platform of the disclosed device, this example demonstrates that increasing the sample flow rate directly increases the sample volume per unit area, thus improving the VFI-based assay's sensitivity. Lastly, the flow path of VFI device was dramatically reduced to only the thickness of the membrane compared to a flow path of several centimetres in the LFIs. This allowed for the utilization of membranes with submicron pore sizes. Results demonstrate that use of nanopore membranes can significantly improve the sensitivity of the VFI's assay. Analysis of paper-based immuno-biosensor:FIG.1Aillustrates the principle of a paper-based immuno-biosensor in the vertical flow format, as used in the disclosed vertical flow detection device1. The porous structures11of membrane3were approximated as bundled tubes to help estimate the transport process, as shown inFIG.1B. In a porous material24with pore radius d, a liquid flows through the membrane3with a flow23velocity of {right arrow over (μ)}, and a sensing length of Ls, which is the detectable thickness9of the membrane3. The sensing area (also the imaging surface) is the top surface Av, e.g., first surface5, which is a circular shape with radius Rm; a second surface is on the bottom (no observable) side of the membrane separated by membrane thickness9.FIG.1Cillustrates an internal surface of the porous structure11is coated with capture antibody, e.g., capture agent19, with a surface concentration of γ. The diffusivity of the target is D, and the antigen-antibody pair has an association constant of kon, and mass transfer coefficient kc. There are two critical dimensionless numbers in the VFI system (Schlappi et al., 2016; Squires et al., 2008; Zimmermann et al., 2005). The first one is the Damköhler numbers (Da), which characterizes the relation between adsorption rate and transport rate. Da=adsorptionratetransportrate=kon·γkc(1) The second one is the Péclet number (Pe), which can be used to compare convection rate and diffusion rate. Pe=convectionratediffusionrate=u/LsD/d2(2) To capture target antigen13with low concentration, two conditions are desired: (1) Efficient capture assay (Da>>1), in which the rate of the antigen13binding to the capture antibody19is faster than the rate of antigen13molecules transport to the pore wall of, e.g., porous structure11. High flow speed23increases the transport rate Kc, decreases Da, and reduces the capture efficiency. However, this can be counterbalanced by using an assay with fast binding kinetics. In this example, the focus was on low concentration antigen13detection with high capture antibody density, thus assuming the capture is fast enough to ensure Da>>1. (2) Non-diffusion-limited assay (Pe<1), allows for all delivered antigens13to diffuse to the pore wall before they are convected through the sensing area. Previous simulation showed that keeping Pe<1 ensures >90% capture efficiency (Schlappi et al., 2016). Setting Pe<1 in Equation (2) provides the following constraint on the volumetric flow rate (Q): Q<ΦDLsAvd2(3) Here ϕ is the porosity of the membrane3. According to Equation (3), decreasing the membrane3pore size is an effective way to increase the maximum flow23rate Q to allow more antigen13to be detected by the sensor. Based on the theoretical analyses, it was determined a VFI design for device1with high flow23speed and small membrane3pore size could improve the assay sensitivity. MATERIALS AND METHODS: Construction of the VFI platform: As shown inFIG.2A, the VFI platform of device1comprises a 13 mm diameter nitrocellulose membrane3(the actual flow through area is 10 mm diameter) encapsulated in a stainless steel filter holder29(Swinny Filter Holder 13 mm, Millipore, MA, USA) together with supporting and sealing components, e.g., support21and holder29. A polytetrafluoroethylene (PTFE) gasket25and o-ring77are placed below and on top of the paper membrane3respectively to seal the liquid pathway and prevent leakage. A syringe6and pump31(FIG.30B) (New Era Pump Systems, Inc., NY, USA) can push the samples and reagents vertically through the paper membrane3at a controlled flow23rate. In previous vertical flow systems, where a commercial stainless steel membrane support was used, large signal variation was reported in membranes with pore size larger than 0.1 μm (Chinnasamy et al., 2014). It was suspected the variation came in part from the flexibility of the stainless steel support. Therefore, the support21is fabricated with silicon, which has a higher Young's modulus than stainless steel, using deep etching method. The silicon grid mechanically supported the membrane3against the flow23with less deformation, thus reducing the signal variation. Accordingly, any of the devices provided herein may use a support formed of a material having a higher Young's modulus than that of stainless steel, including a silicon grid. The VFI in device1can be made using nitrocellulose membrane3because of its high protein-binding capability (Lu et al., 2010) and availability in a range of small pore sizes. Four nitrocellulose membranes, Amersham Protran 0.1 μm NC, 0.2 μm NC, 0.45 μm NC, and Whatman AE98 (pore size 5 μm) (GE Healthcare Life Sciences, PA, USA), are tested and compared. The membranes are cut into 13 mm diameter disks using a CO2laser (VersaLaser 2.30, Universal Laser Systems, AZ, USA). The capture antibody microarray is dispensed onto the paper disks using a micro solenoid robotic dispenser (AD1520 micro-dispenser with BioJet Elite, Biodot, CA, USA) with a droplet volume of 1 nanolitre creating a circular spot with 220 μm diameter. Burkholderia pseudomalleidetection microarray: Detection of theB. pseudomalleiis based on a sandwich immunoassay targeting the CPS, a capsular antigen13that is shed by the bacterium during infection (Nuti et al., 2011). Capture antibody19is immobilized on the paper membrane3as an array of capture agent spots79using the Biodot micro-dispenser.FIG.2Bshows the layout of theB. pseudomalleidetection microarray that comprises 120 replicated detection spots79,3negative control spots79, and19positive control spots79. In the detection spots79, CPS specific mAb 4C4 (10 mg/mL) is deposited with one 1 nL droplet per spot79. In positive control spots79, goat anti-mouse IgM+IgG+IgA (1 mg/mL) (SouthernBiotech, Birmingham, AL, USA) is deposited with three 1 nL droplets per spot79. In negative control spots79, 1×PBS is deposited with one 1 nL droplet per spot79. The dispensed membrane3is stored at room temperature in an aluminium pouch with silica desiccant until use. The detailed processes of bacterial isolation and culture, biomarker discovery, and monoclonal antibody affinity characterization were introduced in a previous report (Nuti et al., 2011). VFI operation workflow: CPS is a linear polymer composed of repeating saccharide units (Perry et al., 1995). As a result, the repeating epitope binding sites allow for the same antibody to be used for capture and detection of CPS. In the VFI workflow shown inFIG.2C, CPS-specific mAb 4C4 was immobilized on the paper membrane3as the capture antibody array and 4C4-labeled gold nanoparticle12(4C4-GNP) was used as the detection agent. The 4C4-GNP12stock solution was prepared through passive absorption of mAb 4C4 to 40 nm diameter colloidal gold. After washing to remove free antibody, the 4C4-GNP12solution was concentrated to an OD=9 at 540 nm, which equals to a concentration of 1.5 nM. As the CPS and 4C4-GNP12were pushed through the membrane3, the antigen13-antibody10complex bound to the capture agent19antibody spot79array, forming the sandwich assay. After the experiment, the membrane3was scanned with a regular table-top scanner to extract the colorimetric signals generated by the gold nanoparticles12as shown in the “Top View” inFIG.2C. The VFI test procedure can be completed in less than 30 min. Detailed experimental procedure included flowing 1 mL of 1×PBS (phosphate buffered saline, Gibco, MA, USA) through the membrane3for equilibration of the membrane3. The membrane3was then treated by flowing blocking buffer (10 mM borate buffer containing 2.5% Triton X-100, pH=8) to block the membrane3and prevent non-specific binding. CPS spiked assay buffer solution (0.1 M PB buffer containing 0.1% Triton X-100 and 0.1% BSA, pH=7.2) were pushed using syringe6flow device (e.g., pump31) through the membrane3at a controlled flow23rate and duration. Two reaction schemes, namely sequential and premixing protocol are tested. Sequential protocol: The CPS spiked assay buffer solution was pushed through the membrane3to allow the CPS to bind to the capture antibody agent19on the membrane3. The 4C4-GNP12solution was then pushed through the membrane3. During the second step, the 4C4-GNP12bound to the CPS that was already captured and bound to the capture antibody agent19on the membrane3. Premixing protocol: The CPS spiked assay buffer solution was pre-mixed with the 4C4-GNP12for 10 min. This premixing step provides additional time for the 4C4-GNP12to bind the free CPS in solution. This mixture was then pushed by syringe3through the membrane3to allow capture of the CPS-4C4-GNP12complex by the capture antibody19on the membrane3. After the sample was processed, the membrane3was washed by flowing 1.5 mL blank assay buffer through the membrane3to remove non-specific or loosely bound proteins and excess 4C4-GNP12. The VFI device1was dismantled and the membrane3was placed on a filter paper (Whatman qualitative filter paper, Grade 1, GE Healthcare Life Sciences, PA, USA) for 5 min as a fast drying step before it was scanned using a table-top scanner. The entire VFI test process was finished in less than 30 min. 1×PBS is from ThermoFisher. Bovine serum albumin (BSA), Triton X-100, PB buffer, boric acid, and sodium tetraborate is from Sigma Aldrich. All the chemicals used are of analytical grade and are applied without any further purification or modification. Image processing and data analysis: After the drying process, the VFI membrane3was scanned with a consumer-grade table-top scanner (CanonScan 9000F II) and Scan IJ Utility (default software for the CanonScan), with 48 bits RGB settings and 2400-dpi resolution exported into an uncompressed TIFF file format. The 48 bits RGB image was then converted to 16 bits grayscale image using the built-in function rgb2gray of Matlab (Mathworks, MA, USA). The resulting image was imported into ImageJ, where the spots79were analysed using a microarray grid to extract the mean grayscale values from the spots79with subtracted local background. Data processing and analysis were performed using Excel 2016 (Microsoft, WA, USA). VFI Reaction Scheme Our initial in silico theoretical analyses indicates a porous membrane based immuno-sensor that incorporates membranes with smaller pore size and high sample flow speed provides better sensitivity than traditional LFI. The following experimental results verify the theoretical findings and characterize the effects of the membrane pore size and sample flow speed on the limit-of-detection (LOD) of the VFI with aB. pseudomalleiassay. Initially the experiments that are performed compared the two VFI reaction schemes, i.e. sequential and premixing. The sequential approach relied on the capture of CPS by the capture antibody agent19microarray of spots79, followed by binding of the 4C4-GNP12to the captured CPS as the 4C4-GNP12solution was pushed by syringe6through the membrane3. The premixing approach gave the antigen13(CPS) and detection antibody10particle (4C4-GNP12) additional time for interaction and binding before being pushed through and captured by the capture antibody agent19microarray of spots79. Both approaches used a 0.2 μm pore size nitrocellulose membrane3, 1 ng/mL CPS spiked into 1 mL assay buffer solution, and a10 μL of the OD=9 (540 nm) 4C4-GNP12. As shown inFIG.3B, the premixing protocol yielded detection spots79(the centre array) with stronger signals than the sequential protocol. The relative intensity was increased by 6.5 times as shown inFIG.3A. In the example, the improved sensitivity of the premixing approach is believed to be attributable to the increase in binding time between CPS and 4C4-GNP12and better efficiency of the liquid reaction when compared to the surface reaction. All subsequent experiments were performed using the premixing method. VFI with membrane of 5 μm pore size: After selecting the reaction scheme, experiments were performed using a nitrocellulose membrane3of 5 μm pore size (Whatman AE98, GE Healthcare). This membrane3has a similar pore size to the nitrocellulose membrane3used in the CPS LFI prototype (10 μm pore size, Whatman FF120 hp, GE Healthcare) but without the backing layer (Houghton et al., 2014). The premixed sample solution was pushed by syringe6through the membrane3at a volumetric flow23rate of 1.5 mL/min for 10 min, a flow23rate comparable to that of an LFI. Purified CPS was tested on the VFI to determine the LOD under this condition. Dilutions of CPS (1, 0.5, 0.2, 0.1, 0.04, 0.02, and 0.004 ng/mL) were prepared in the assay buffer and applied to the VFI membrane3. 15 μL of the 4C4-GNP12solution was added to the sample and mixed for 10 min before the flow-through step. The LOD was defined as the concentration that generated a signal that was greater than 3 times the standard deviation (SD) above the background signal. As shown inFIG.4, the LOD was determined to be at or slightly lower than 0.1 ng/mL. Effect of flow speed for membrane of 5 μm pore size: Next, the effect of flow23speed on the sensitivity of the assay was studied. It was suspected that a faster flow23speed would deliver more antigen13to the membrane3sensor, which could improve the sensitivity. The VFI system was tested using 1 ng/mL CPS spiked in assay buffer with different flow23speeds using a constant assay time of 10 min with the AE98 membrane3. The volume of the 4C4-GNP12stock solution added to the CPS sample was adjusted to keep the final concentration of GNP12constant. According to the results shown inFIG.5, as the flow23speed (equals to volume flow23rate divided by the surface area81of the membrane3) increased, signals from both the detection and positive control spots79were increased. Knowing that increasing the sample flow23speed could improve the sensitivity, the LOD of the VFI device1was tested again with a higher flow23rate of 5 mL/min. Dilutions of purified CPS (1, 0.5, 0.2, 0.1, 0.04, 0.02, and 0.004 ng/mL) were prepared in the assay buffer and applied to the VFI membrane3. 50 μL 4C4-GNP12solution was added to the CPS sample and mixed for 10 min. As shown inFIG.6, the LOD was determined to be at or slightly lower than 0.04 ng/mL. VFI membrane pore size effect: Four nitrocellulose membranes3with different pore sizes (0.1 μm, 0.2 μm, 0.45 μm, and 5 μm) were tested to determine the effect of membrane3pore size on the sensitivity of the CPS VFI. 0.1 μm is the smallest pore size of commercially available nitrocellulose membrane. One filter paper (Whatman qualitative filter paper, Grade 1, effective pore size 11 μm) was also tested, but failed in the capture antibody immobilization step. 1 ng/mL CPS spiked in assay buffer solution was used throughout as the testing sample. Samples were processed with a flow23rate of 1.5 mL/min and 10 min assay time. Results from using different nitrocellulose membranes3are shown inFIG.7. A decrease in the pore size of the nitrocellulose membrane resulted in enhancement of the signals for the detection spots79. The signal from the 0.1 μm membrane3was twice as strong as that observed for the 5 μm membrane3. Effect of flow speed for membrane of 0.1 μm pore size: In the example, the effect of flow23speed was tested using the 0.1 μm membrane, which showed the best signal among the different pore size membranes3. Assay buffer solution spiked with 1 ng/mL CPS was pushed through the membrane3with flow23rates of 0.5, 1, 1.5, 2, 3, 4, 5 mL/min for a constant time of 10 min. According to the results shown inFIG.8, increased signal intensity at higher flow23rate was observed. An increase in sensitivity with higher flow23rate was also observed with the 5 μm pore size membrane3, but overall signal was stronger with the 0.1 μm pore membrane3. There was no significant change in the background levels with the change in pore size of the membrane3. LOD of optimized VFI system: The effects of the two critical factors, i.e. membrane pore size and sample flow speed, were proven individually through experiments with spiked samples and showed good agreement with the theoretical model. Using the knowledge gained from the flow23rate and membrane3pore size studies, the best performing flow23rate and membrane3conditions were integrated to determine the LOD of the VFI device1at optimal conditions, i.e., smallest membrane3pore size and highest flow23rate. The 0.1 μm pore size membrane3was selected and applied the sample at the highest flow23rate of 5 mL/min. Dilutions of purified CPS (1, 0.5, 0.2, 0.1, 0.04, 0.02, and 0.004 ng/mL) were prepared in the assay buffer and mixed with 50 μL 4C4-GNP12solution for 10 min and then passed through the VFI membrane3using syringe6. The scanned images of the membranes3are shown inFIG.8, together with the signals extracted from each membrane3. The LOD was determined to be at or slightly lower than 0.02 ng/mL. VFI for multiplexing detection of bio-threat pathogens—proof of concept: With the ability to accommodate a microarray of spots79utilizing different capture antibody agents19, VFI is inherently suitable for multiplex biomarker detection. For biothreat detection, a proof-of-concept experiment was performed using the VFI device1platform to detect two targets simultaneously—CPS and PGA (a biomarker for the bacteriumBacillus anthracis, which is the causative agent of anthrax) (Gao et al., 2015). The design of the multiplexing VFI membrane3is shown inFIG.10A. The detection microarray was divided into two parts. Half was coated with mAb 4C4 targeting CPS, and the other half was coated with mAb 8B10 targeting PGA. Goat anti-mouse IgM+IgG+IgA and 1×PBS were also dispensed onto the same membrane3as a positive and negative control. Four samples with different antigen13contents (CPS negative/PGA negative, 1 ng/mL CPS/PGA negative, CPS negative/1 ng/mL PGA, and 1 ng/mL CPS/1 ng/mL PGA) were processed with the VFI membrane3of 0.1 μm pore size at a flow rate of 1 mL/min and 10 min assay time.FIG.10Bshows the scanned images of the four membranes3.FIG.10Cshows the signal intensities from the four membranes3. Detection spots79showed positive signals only when the corresponding antigen13was present in the sample. This experiment demonstrated that the VFI device1platform was able to detect multiple biothreat agents simultaneously. Unlike LFI, the detection spots79were spatially separate and reactions were independent from each other in the VFI device1, making the VFI especially suitable for large-scale multiplexing detection. However, there is still chance for cross-reactivity or non-specific binding to occur (Juncker et al., 2014), which may require further characterization of the premixing condition and buffer solution. This experiment demonstrates that the VFI platform was able to detect multiple bio-threat agents simultaneously. Unlike LFI, the detection spots are spatially separate and reactions are independent from each other in the VFI system, making the VFI devices provided herein especially suitable for larger-scale multiplexing detection. VFI fluidic simulation: To investigate the flow uniformity across the membrane3, fluid simulation was performed using FEM software COMSOL Multiphysics 5.0 (CONSOL, Inc., Los Angeles, CA, USA). The simulation was started with the stainless steel holder29chamber, without any paper membrane3or Si grid support21. Due to the nature of the fluid dynamics, the velocity was significantly higher in the central part of the holder29chamber, as shown inFIG.11A. Upon implementation of the support21(modelled as non-deformable solid structure with multiple openings) and the paper membrane3(modelled as a porous thin film) into the model, the liquid flow profile was changed significantly. As shown inFIG.11B, the support21and the membrane3evened out the fluid flow23across the flow chamber.FIG.11Cshows a zoom-in view of the flow23magnitude profile across the membrane3. The velocity is higher on top of the hollow part of the Si grid support21than that on top of the solid part. This variation of velocity translates into the variation of pressure with the paper membrane3, as shown inFIGS.11D and11E. However, the pressure on the top surface5of the paper membrane3, which is the most critical part as the imaging plane, the pressure variance is within 1%. Therefore, the flow23and pressure across the membrane3can be considered uniform. DISCUSSION: In this example, the detection of biothreat pathogens is based on a sandwich immunoassay that has been integrated into a nano-porous nitrocellulose membrane3, which generates colorimetric signals as readout. The VFI device1technology provides a simple, miniaturized, and rapid (sample-to-answer time under 30 min) platform for bio-threat pathogen detection. When testing the effect of the flow rate in a flow-through device1, a common experimentation scheme is to keep the sample volume constant and change the flow rate (Zimmermann et al., 2005). This is effective in pursuing an assay with high processing speed with limited sample volume. However, in real world applications, especially when dealing with abundant sample such as urine or environment water, sample volume is no longer a constraint. Therefore the experiments in this example were performed with a constant assay time of 10 min and varied the flow23rate, and equivalently the sample volume, to compare the VFI's detection sensitivity. The 10 min assay time was selected to match the time needed for a LFI test. The effect of flow23speed was tested with two types of membrane3(with pore size of 5 μm and 0.1 μm). In both cases, the signals from the detection spots79were enhanced as the flow23rate increased. The porous structures23of the membrane3were modelled as bundled nanotubes, and this relation between flow23speed and signal intensity can be explained with the classical model for flow through surface immunosensors (Squires et al., 2008). As the flow23rate increased, the thickness of the depletion zone, in which the target antigen13can be captured by the surface antibody capture agent19, was reduced. However, the increased flow23rate also delivered more samples to the membrane3, which counter-balanced the loss of binding due to the reduced depletion zone thickness. Due to the design of the disclosed VFI device1, only the sample that was pushed through the regions with immobilized capture antibody agent1could be detected. The rest of the sample that went through the other regions was wasted. Therefore, it was the sample volume per unit area rather than the total sample volume that determined the amount of sample being delivered to the detection region. The higher the sample volume per unit area, the larger the amount of target antigens13that could be captured by the capture antibody agent19. Table 1 summarizes the key features of some previous immuno-filtration devices. The disclosed VsI device1has a much higher sample volume per unit area compared with previous work. As a result, the sensitivity of the VFI was improved. TABLE 1Key feature comparison between the presented VFI and previousreported immune filtration devices. Because of the high samplevolume per unit area in the disclosed VFI device 1,a better sensitivity was achieved.SampleMembraneSamplevolume persizevolumeunit areaReference(mm2)(μL)(μL/mm2)SensitivityVFI78.550000636.940.02 ng/mL(Hárendarc̆íková et al.,70.85200028.23NA2017)(Pauli et al., 2015)63.59500078.631.0 ng/mL(Berger et al., 2016)25100.5110 μg/mL(Clarke et al., 2017)63.591201.8953.1 μg/mL(Cretich et al., 2015)132.67150011.310.42 ng/mL(Chinnasamy et al.,132.6710007.541 ng/mL2014)(Oh et al., 2013)0.201681.530.01 μg/mL(Reuterswärd et al.,132.671000075.381.9 μg/mL2015) The average CV across the membrane3was calculated to be 10%. It is comparable to previously developed system (Chinnasamy et al., 2014). Fluidic simulation was conducted to investigate the flow23profile inside the filter paper holder and confirmed the uniformity across membrane3, as shown inFIG.11. Notably, any high CV values (CV>0.85) when using a Si support21were not observed, even with the larger-pore membranes3, which was reported in previous literature (Chinnasamy et al., 2014). It is possible that the Si grid support21that was fabricated in the example has a higher Young's modulus than the commercially available stainless steel grid. It was less susceptible to deformation, and thereby preventing inhomogeneous flow23from happening. As predicted based on the theoretical model and later demonstrated through the experiments of the example, the advantage of VFI arises primarily from two factors—the samples volume per unit area and the membrane3pore size. Traditional LFI is relying on capillary force to transport the liquid through a long membrane (˜40 mm), which posts limitations on the membrane3pore size. A common range of the LFI membrane pore size is 3˜12 μm (Posthuma-Trumpie et al., 2009). However, VFI has a short membrane3flow23path (˜130 μm) that allows for membranes3with submicron pore size to be used. Smaller pore size offers a higher protein loading capacity that creates more binding-sites inside the porous structures11. The smaller pore size also reduces the required diffusion distance for an antigen13to be captured by the antibody capture agent19on the membrane surface. It has been shown that reducing the membrane pore size in the LFI system can also be an effective way to increase the assay sensitivity (Henderson and Stewart, 2002). Based on these reasons, the nitrocellulose membrane3with 0.1 μm pore size was selected in the example as the optimized substrate for the CPS assay. However, 0.2 μm and 0.45 μm options remain open for applications where the target antigen13is bigger than CPS. One of the major hurdles preventing getting even better sensitivity was the low 4C4-GNP12concentration. The current VFI requires a large total sample volume to achieve high sample volume per unit area. Using a large sample volume in turn would require a sizable amount of labelled nanoparticles12to increase the binding efficiency. To solve this problem, one potential solution is to reduce the total volume of the sample while maintaining the high sample volume per unit. One approach to this would be developing a miniaturized VFI device1with smaller surface area81. This example describes the development and optimization of a prototype of the disclosed paper-based Vertical Flow Immunoassay (VFI) device1for the rapid diagnosis of bio-threat pathogens.Burkholderia pseudomallei, the causative agent of melioidosis, was used as a model bacterium target and a sandwich assay was developed to detect the capsular polysaccharide (CPS) as the diagnostic biomarker. The VFI device1incorporates at least 120 detection spots79that can potentially be distinct from each other, an assay time of less than 30 min, and gold nanoparticle12mediated colorimetric readout that is amendable to regular table-top scanner. The effects of sample flow23rate and membrane3pore size on the performance of the disclosed VFI device1were investigated. The results of the example agreed well with the theoretical analysis showing that high flow23speed through a nanopore membrane3was the key to achieving higher sensitivity. A deep-etched silicon grid support21was used instead of the commercial stainless steel support, which helped prevent large signal variation with membranes3of larger pore size. Two reaction schemes were compared—the premixing method where the sample and antibody-labelled nanoparticle12were mixed in advance proved to be more effective. A limit-of-detection (LOD) of 0.02 ng/mL was demonstrated with purified CPS. The multiplexing biothreat detection with CPS and PGA using the VFI device1platform was also demonstrated. The disclosed VFI device1offers a valuable approach to detect and reduce the bio-threat agents in a variety of resource-limited or clinical conditions. See, e.g., Chen et al. “Paper-based Vertical Flow Immunoassay (VFI) for detection of bio-threat pathogens” Talanta 191(1): 81-88 (January 2019—available online Aug. 17, 2018) and supporting supplementary materials, all of which is hereby incorporated by reference. Example 2: Design Rules for Vertical Paper-Based Immuno-Diagnostic System (VPI-DS) This example addresses uniformity and PGA array printing, Purified and conjugated mAb for PGA, and development of mAbs for LPS, LcrV and F1, determine the effect of pH and ionic strength by LFI and tests of LcrV and F1 LFI. VPI assay using the disclosed device1was also evaluated for CPS and automatic image analysis software. TSignal variation across membranes: Several approaches were taken to solve the issue of the nitrocellulose membrane3wrinkle after the screening experiment.FIGS.12A-12Cdepict images of membranes3exhibiting these issues to varying extents. Issue: In most of the processed samples, a dark circular stain14can be seen on the nitrocellulose membrane3after the screening experiment. It is due to the wrinkle of the membrane3after liquid runs through, and the wrinkle generates uneven paper surface, which then imaged as the dark stain14. The stain14is problematic because it might change the gray-scale intensity of the detection spots79and the background and lead to false detection results. FIGS.13A-13Cdepict several failed approaches to solve this issue, including: 1) Use the Si grid support21instead of the stainless steel grid as the membrane3support. We tried to take advantage of the high stiffness of Si to maintain the shape of the membrane3better (FIG.13A). 2) Tube regulator33. We elongated the vertical tubing35connected to the outlet37of the screening chamber39to guarantee the liquid runs through the membrane3vertically (FIG.13B). 3) Customized O-ring77. We fabricated our own o-ring77with a flat surface to apply more uniform force on the membrane3(FIG.13C). FIG.14is a schematic diagram illustrating the proposed reasoning behind the wrinkle: As we assemble the screening chamber39, the force (clamping and twisting force) applied on the membrane3is higher at the edge of the membrane3, which forms the membrane3into a dome shape. As the liquid runs through, higher pressure is applied at the center, and thus generates the wrinkle. FIGS.15A and15Billustrate a solution to the membrane3wrinkle and staining issue. Final solution: pre-wet the membrane3with PBS even before assembling the screening chamber. So far, we have solved the problem with the air bubbles and the membrane3wrinkle. The quality is largely improved (as can be seen in the comparison ofFIG.15BtoFIG.15A). Signal uniformity study: One of the concerns with the VFI is that the variation of the liquid flow23across the membrane3might introduce signal non-uniformity. We have presented in silico results of the pressure applied on the membrane3, which appears to be consistent and relatively uniform across the membrane3. Analysis is performed with some of the samples to study the signal uniformity. We use the 10 min sample from the drying time experiment as an example. Even though variation can be seen among the many detection spots79in the array, no clear pattern can be observed as has been reported in previous publications. The variation appears random. With the current device1, the signal variation is about 15%, which is a significant improvement compared to values reported in the literature. The dispensing process, which is mostly determined by the accuracy of the dispenser itself, has more than 10% variation according to the vendor specifications. The systems and methods provided herein facilitate signal detection and sensitivity that is much improved over lateral flow systems. For example,FIG.16Aillustrates a membrane with different capture agents (e.g., antibodies in this example) on the surface, schematically modelled as hollow tubes to reflect the cross-sectional area of flow associated with each capture antibody.FIG.16Bis a close-up of a cross section, illustrating four different capture antibodies: one control and three different capture, with a corresponding colorimetric read-out.FIG.16Cillustrates a model based on flow speed (μ), sensing length (Ls), membrane pore size (d) and diffusivity (D). The diffusion time (tdif) and resident time (tres), are calculated as: tdif=d2/(4D) tres=Ls/μ In this manner, the nanopores of the vertical flow systems (VFI) provided herein allows for better target capture under high flow speed. In addition, the VFI has a short flow path, on the order of 100 μm or less, compared to typically 40 mm or more in lateral flow systems (LFI). PGA assay dispensing and VFI testing: Other than the CPS assay, we started some of the early work with the PGA assay. The sandwich for the PGA is provided below. The capture antibody (8B10) agent19and detection antibody10(8B10-GNP) are used in this assay, as shown inFIG.17. With the same recipe, we are able to dispense the 8B10 capture antibody (8B10) agent19onto the nitrocellulose membrane3using Biodot dispenser, as shown inFIGS.18A and18B. Conceptual experiments test the 8B10 sandwich assay at a concentration of 5 ng/mL. The results are shown inFIGS.18C and18D. The sandwich assay works on both the 0.2 μm and 0.45 μm membrane3. The results of 5 ng/mL CPS and 5 ng/mL CPS are provided side by side, the PGA assay actually shows even stronger signal. We expect this assay to be more sensitive. Development and purification of mAbs: Production and purification ofBacillus anthracisPGA mAbs: TheB. anthracispoly gamma D-glutamic acid (PGA) specific mAb, 8B10, is purified using recombinant Protein A affinity chromatography. The purified mAb was also conjugated to 40 nm colloidal gold. Both the unlabeled and labeled mAb along with purified PGA are provided as part of the second antibody-antigen pair to be optimized in the vertical flow assay format. Production and purification of FranciscellatularensisLPS mAbs: The hybridoma cell line producing theF. tularensisLPS reactive mAb, 1A4, is grown in tissue culture Integra flasks. One round of purification using recombinant Protein A affinity chromatography provides a yield of 40 mg of antibody. However, it has been found that the cell line is unstable under high-density Integra culture conditions. In response, a new set of mice are immunized withF. tularensisLPS in order to isolate a larger library of high affinity monoclonal antibodies to LPS. Serum titers from 6 weeks post-immunization indicated multiple mice have high levels of LPS specific antibodies. After additional boosting with Ft LPS, splenocytes is isolated for production of hybridomas. Production, purification and characterization ofY. pestisLcrV and F1 mAbs: LcrV monoclonal antibodies: SPR analysis was performed on all eight purified LcrV monoclonal antibodies to determine mAb binding kinetics and affinity to purified recombinant LcrV (Table 2, below). The association rate constant (ka) and disassociation rate constant (kd) was determined for each mAb and used to calculate the equilibrium dissociation constant or “affinity” (KD=kd/ka). Alternatively, affinity was also calculated using a steady state equilibrium model (SSKD). A tenfold higher association rate (ka) was observed for mAb 8F10 when compared to all the other LcrV monoclonal antibodies. Monoclonal antibody 6F10 exhibited the slowest dissociation rate amongst the eight antibodies tested. Overall, mAb 8F10 performed the best in the SPR analysis and had the highest binding affinity. LcrV mAbs were used in an antigen13capture ELISA format to determine the best antibody pairs for LFI production. All eight LcrV mAbs were HRP-conjugated and used to detect rLcrV, which was captured by unlabeled LcrV antibody in in an ELISA format. The limit of detection (LOD in ng/ml) for each antibody pair was calculated (Table 3, below). F1 monoclonal antibodies: A library of twelve monoclonal antibodies F1 antibodies were generated and purified using recombinant Protein A affinity chromatography. Screening strategy for the 12 purified F1 mAbs was changed (from the LcrV strategy) to conserve purified antigens13and ELISA workload. All twelve F1 mAbs were gold labeled and wet tested in prototype LFIs and top eight mAb candidates were selected for further screening with SPR analysis and antigen13capture ELISAs. No significant differences in binding kinetics or affinity were observed between the F1 mAbs with the exception mAb 11C7, which had a ten-fold higher association rate when compared to the other antibodies (Table 4, below). All eight F1 mAbs were HRP-conjugated and tested in an antigen13capture ELISA format using recombinant F1. The limit of detection for each antibody pair was determined using a cutoff of 5 times the background level (Table 5). Purification of biomarkers: Biomarkers (LPS, PGA, F1) are purchased commercially. Effects of pH and ionic strength by LFI (CPS): Initial experiments comparing effects of pH and ionic conditions on mAb binding reactivity showed that a direct comparison could not be made between surface plasmon resonance (SPR) and lateral flow immunoassay (LFI) results. It was decided that this line of testing would proceed using only the LFI format, as this immunoassay format is most similar to the vertical flow assay being developed. In order to investigate the effect of ionic conditions on background and sensitivity of the 4C4 lateral flow immunoassay, LFIs were performed using phosphate buffered saline (pH 7.4) at different ionic strengths with or without surfactant P20. Significant background was observed at NaCl salt concentration below 100 mM (ionic concentration: 0.125 mol/L). Increase in salt concentration up to 300 mM (ionic concentration: 0.325 mol/L) did not produce any significant change in LFI performance (Table 1, below). These results indicate that using PBS with an ionic concentration above 0.325 mol/L might reduce false positive results in our LFIs. To complete the testing of ionic conditions, we plan to test 4C4 LFIs at higher ionic conditions and possibly test buffers that may be more compatible with the vertical flow assay format of the disclosed device1. Development ofY. pestisLcrV and F1 LFI prototypes: Since the majority of LcrV mAbs performed well in the antigen13capture ELISA, all LcrV mAbs were gold conjugated and sprayed as test lines onto nitrocellulose for evaluation as the detector and/or capture antibody in the LFI platform. Initial testing of all LcrV LFI prototypes has been completed and there are several promising candidates with a visual limit of detection between 1-10 ng/ml (FIG.19). The eight F1 antibodies with highest reactivity in ELISA were selected for similar testing in LFI format (FIG.20). Several prototypes have a visual limit of detection at 1-10 ng/ml with recombinant F1. Initial testing was conducted using rLcrV or rF1 spiked into buffer. Current efforts are now focused on optimizing the LFI prototypes for use with human serum as the matrix. As shown inFIG.19, lateral flow immunoassays (LFIs) (A) 8F7:6F10, (B) 8F10:2B2, (C) 8F10:6E5, (D) 8F10:6F10 were tested with varying concentrations of rLcrV: from left to right: 1 ug/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 500 pg/mL and negative control. Images were recorded after 20 min. As shown inFIG.20, lateral flow immunoassays (LFIs) (A) 4E5:3F2, (B) 10D9:3F2, (C) 11C7:3F2, (D) 11C7:15C4 were tested with varying concentrations of recombinant F1: from left to right: 1 ug/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 500 pg/mL and negative control. Images were recorded after 20 min. VPI CPS Optimization: Different parameters are tested individually first to get a working range. Then a DOE screening by DSD is conducted to select critical parameters, then a full factorial experiment will be done for the critical parameters for final optimization. Membrane material comparison and optimization:FIG.21shows examples of membranes3used for the optimization and comparison for this example. One of the advantages of the vertical flow23system in the disclosed device1is that the liquid transport does not rely on the porous structure11of the nitrocellulose membrane3, which allows for membrane3materials with much smaller pore size. Such membranes3with small pore sizes have much higher loading capacity to bind more capture antibody agent19. The diffusion range is also largely reduced to facilitate the antibody-antigen binding. We first examined the two types of vertical flow nitrocellulose membranes3with 0.2 μm and 0.45 μm pore size, the typical lateral flow membrane (10 μm pore size), and the filter paper with SEM (the results with2K magnification can be seen above). One can imagine that with such finer porous structure11the detection efficiency can be significantly improved. We then tested six different materials in the VFI system of the disclosed device1with the CPS assay. The 8 μm and 12 μm nitrocellulose membranes3were selected to resemble the lateral flow membrane as they the closest provided by the same vendor. MaterialNCMNCMNCMNCMNCMFilterPore size0.10.20.4581211(μm) The results with 1 mL of CPS at the concentration of 5 ng/mL at the screening speed of 1 mL/min can be seen inFIG.22. In all of the nitrocellulose membrane3samples, the positive control spots79(top and bottom rows ofFIG.22) can be clearly seen, and the detection spots79(the center array) have various intensities. However, on the filter paper, the dispensed microarray was smeared, and no signal can be observed from either the detections or the control spots. Now we look at the gray scale intensity we acquired from the images (FIGS.23A and23B). Please note that we are using 16 bit gray sclae scanning, so the range of the values should be between 1 to 65535. We can see that as the pore size goes smaller, the signals get enhanced. Especially for the detection spots79, the signal from the 0.1 um membrane3is 16 times stronger than the 12 um membrane3. The results strongly support our theory that by choosing a nitrocellulose material with much smaller pore size, the detection sensitivty can be significantly improved. Drying time study: One of the many advantages of the VFI system of the disclosed device1is that it allows for processing of large volume of samples (mL level) which is impossible for lateral flow testing strip (L level) at high speed. However, it still requires a certain amount of drying time before scanning the membranes3for image analysis and signal extraction. We studied the effect of the drying time (10-30 mins), which is the time between taking the membranes3out of the liquid and scanning the image. The scanned images are shown inFIG.24. The gray scale intensity of the detection spots79can be seen inFIGS.25A and25B. As the drying time increases, the absolute intensity (raw intensity) of the detection spots79and background both go up. It can be explained that as the excessive water in the membrane3evaporates, the reflective index of the membrane3changes, and the membrane3appears lighter in color. In contrast, the relative intensity (detection spots79subtract the background) does not change much as the drying time goes up. Therefore, we can select the shortest drying time of 10 mins and keep the entire testing time as short as possible. Noted is that the drying time can be further reduced by using a drying pad91(FIG.31A), or similar active drying mechanism. Detection antibody10study: As an important part of the sandwich assay, the binding between the detection Ab10and the captured antigen13largely determines the intensity of the final signal. Since the CPS is a multivalent target, as the concentration of the detection Ab10increases, more nanoparticle12binds to the antigen13. Hence the signal is enhanced. However, as it reaches and surpasses the saturation point, further increases of the detection Ab10concentration only increases the background. Therefore, it is important to characterize and find the optimum concentration of the detection antibody10. Here, we use 1 mL CPS 1 ng/mL spiked in buffer solution as the sample solution, and test different amount of detection Ab10from 5 μL to 60 μL. The results can be seen inFIG.26. As shown inFIGS.27A and27B, the detection Ab10concentration increases, the intensity of the detection spots79and the positive control spots79are both enhanced. The values of the detection spots79and positive control spots79show the same rule that as the concentration of the detection Ab10increases, both signals get improved. Noted is that the difference between 40 μL ad 60 μL is much smaller than that between 20 μL and 40 μL. It indicates that the sandwich assay might reach the saturation point with about 40-60 μL detection antibody10. Sample volume and capture antibody agent19concentration study: The capture antibody captures the antigen13as the screening sample go through the membrane3. As one can expect, the more capture antibody agent19dispensed on the membrane3, the more antigen13be captured. It also has a saturation point, beyond this point adding more capture antibody agent19does not further increase the amount of antigen13captured. We can certainly dispense capture antibody agent19solution with different concentration, but the dispenser has its limitations on the viscosity of the dispensing liquid. Therefore, we choose to dispense multiple droplets of the capture antibody agent19solution to increase the amount of capture antibody agent19on the membrane3. The capability of processing samples on mL level is another advantage of the VFI system of the disclosed device1. The sample volume determines the actual amount of antigen13passing through the detection spots79. Increasing the sample volume compensates for a decrease of the target concentration, and thus improves the system sensitivity of the disclosed device1. We designed the experiments shown inFIG.28to test these two factors at the same time. The microarray (on the left) has 6 rows of detection spots79. Each row was dispensed with different number of droplets (1-6 droplets, the concentration of the capture antibody agent19solution is 10 mg/mL), so that the amount of capture antibody agent19was different among the rows. We prepared six screening samples of different volume (1 mL to 10 mL), but with the same CPS concentration of 0.04 ng/mL. The samples were screened at the flow23speed of 1 mL/min. The results can be seen inFIG.29. As the sample volume increases, the signal intensity increases. 10 mL sample show significantly higher signal than the 1 mL sample. However, within the same sample, the result does not vary much among the spots79of different capture antibody agent19concentration. It indicates that even single droplet of the 10 mg/mL 4C4 capture antibody agent19is more than enough to capture all the antigen13passing through. With the single parameter studies above, it helps to identify the important factors and the range of interest. 3.5 mm membrane3system development for the disclosed device1: We now identified the sample per unit area is a dominant factor in the VFI system. There are two methods to increase it 1) increase the total sample volume, and 2) reduce the size of the membrane3. The first method is relatively straightforward and does not require any modification of the experiment apparatus. It is especially suitable for bio samples typically with large volume such as urine. However, it does require a much longer time to process the sample. We also tried some conceptual work to reduce the size of the membrane3, as shown inFIGS.30A-30C. Compared with the standard 13 mm membrane3, we fabricated a smaller version with diameter of only 3.5 mm. It is small enough to fit into the space inside a needle41(FIG.30A). We also fabricated smaller version of the gasket25, the Si grid support21, o-ring77(FIG.30C), and an adaptor43to assemble them into the needle chamber45(FIG.30B). Theoretically, it can be operated without the syringe6pump31and be highly portable. FIGS.31A,31B and31Cdepict experiment setup and gray scale intensity results for testing of the 3.5 mm membrane3setup with the CPS (5 ng/mL) assay at the flow23speed of 0.5 mL/min. The results plotted inFIG.31Cshow significantly stronger signal (up to 20 times) compared with that from the standard 13 mm disk membrane3. Such smaller embodiments of the disclosed device1can be especially useful for applications in which the sample volume is small and requires ultra-high sensitivity. Completed the design of a new VFI device1that involves six design factors: The most important main effects driving the relative grayscale intensity were the antigen13concentration and detection antibody10concentration. There were also two significant two-way interaction effects involving the antigen13concentration, along with a significant quadratic effect involving the antigen13concentration. Additional design parameters are membrane3pore size, buffer pH and buffer ionic strength. Examples of relevant design parameters are: 1. Membrane3pore size (0.1-0.45 μm); 2. Antigen13Concentration (0.5-1.5 ng/mL); 3. Detection Ab10Concentration (note that the range has not been determined yet; the current saturation point is 60 μL); 4. Flow23Rate (0.5-1.5 mL/min); 5. Buffer pH (6.5-8.5); 6. Ionic Strength (0.1-0.5). The goal is to identify ideal values of all of these design factors that will produce the largest relative grayscale intensity with the disclosed device1. Based on the evidence for curvature in the response surface, a definitive screening design (DSD) is again appropriate. A DSD can accommodate six design factors in a relatively small amount of runs (17 total) and will enable an estimate of second-order effects without additional runs. The 17 run design table with randomized run orders is shown below: AntigenDetectionBufferMembraneConcen-AbFlowBufferIonicThicknesstrationConcentrationRatepHStrength10−1−1−1−1−12−1−11−11131−11−1−10411−11−1−15111−11−1601111170000008−1−1−11−11911−1−101101−1−1011111011−1112−11−111013−1110−1−114−110−1−11151−1011−116−10−1−11−117−1−1110−1 Automatic image analysis software: To minimize the impact of the observer, an objective observation and analysis software system is needed. The software will analyze the image of the membranes3to locate the test area and measure the values for each of the test spots79. The programming environment is LabVIEW. The first step is to locate the pattern and orient to a known position. The second step is to analyze the spots79in the pattern to determine their intensity. Presently, the software can read the file. In the coarse adjustment, it will rotate the image to orient the image, as shown inFIG.32. Once located, the software will need to do a fine adjustment to be sure the image is straight. Having oriented the image, the software can detect the pattern and evaluate each spot79dot. Currently it provides the average for the center 25 pixels of each spot79dot, as shown inFIG.33. At the current state of the program, automatic orientation is successful in 80% of the test cases. We are examining and testing methods for improving this process. Presently a user is required to tune the analysis process. This part can be automated using known methods and procedures. Risk and Issues: Risk 1—The variation of the biodot dispenser might generate systematic variation. Mitigation strategy: Use industrial level, or piezo-actuated microdispenser. Resolution: The droplet size we are working with (1 nL) is usually the cut off size of micro-solenoid dispenser. The variation of the droplet is larger than the normal value of 10%. Using an industrial level microdispenser could increase the stability of the entire VFI system in the disclosed device1. Risk 2—mAb 1A4 cell line instability. Mitigation strategy: While the 1A4 cell line is still producing, instability and slow growth prompted the decision to make more hybridomas producing Ft reactive mAbs. Resolution: Ten mice were immunized with Ft LPS and are currently being monitored and boosted for maximum immune response. Splenectomy and fusions will be performed to produce a larger library of highly reactive Ft mAbs. Other factors to examine include:B. pseudomalleiantibody-antigen binding determined by LFI at different ionic conditions. Production, purification and characterization of a library of highly reactiveY. pestisLcrV and F1 specific mAbs. Developed and testedY. pestisLcrV and F1 LFI prototypes. Determined limit of detection with early-stage prototypes using recombinant protein spiked into buffer. Purification of 1A4 (Ft LPS specific) mAb. New murine immunizations and generation of titers specific toF. tularensisLPS. Opportunities include: Re-circulate the sample with a peristaltic pump31, as shown inFIGS.34A and34B. An alternative way is to increase the sample per unit area without requiring a larger sample volume. In this manner, additional opportunity for capture is available, for bi-directional flow47. Incorporate some active drying step to further reduce the membrane3drying time to below 5 mins. With the VFI system of the disclosed device1, we are able to use membranes3with pore size as small as 0.1 μm, which is 100 times smaller than the membranes used for LFI. With such a small diffusion range, the antigen13are easier to be captured by capture agent19when transiting the membrane3pore. The sample per unit area is the most dominant factor in the current sandwich assay. We achieved the largest signal enhancement by reducing the size of the membrane3. Testing of the 4C4 LFIs with chase buffers of different ionic conditions emphasized the importance of optimal ionic conditions for antibody-antigen binding. TABLE 14C4 LFI prototype was tested with buffer alone or 1 ng/ml CPS inphosphate buffered saline (pH 7.4) at different ionic strengths with orwithout surfactant P20 in the chase buffer. Optical Density wasobtained using ESE Reader after 20 min. Results represent themean values of two independent experiments.IonicSignal −NaClStrengthCPSBack-Back-(mM)(mol/L)(1 ng/ml)groundgroundPBI chase buffer500.075170222−521000.1258538471370.162475421500.175410412000.2255821372500.275450453000.32538038PBS + 0.05% Surfactant P20 chase buffer500.075145184−391000.12511145661370.1628821671500.175830832000.225132221102500.27511401143000.32515013137 TABLE 2Binding kinetics and affinity of LcrV mAbs determined by SPR analysis.Readings represent mean values of two independent experiments.kaKD (M)RmaxChi2SSKDRmaxChi2Clone(1/Ms)kd (1/s)kd/ka(RU)(RU2)(M)(RU)(RU2)2B23.6E+047.3E−052.1E−09771.412.3E−07780.384E81.1E+047.2E−046.5E−089410.765.1E−07952.355D32.3E+044.0E−043.4E−08856.083.7E−07871.366E51.5E+041.9E−051.3E−091054.345.1E−071001.396F101.7E+044.9E−063.3E−101293.074.1E−071272.088F31.4E+041.4E−049.8E−09804.173.0E−07760.388F71.1E+042.0E−042.0E−08545.612.8E−07440.258F101.2E+055.6E−055.5E−10491.096.9E−08440.15 TABLE 3The limit of detection (LOD) of LcrV mAb pairs determined by antigen 13capture ELISA. LOD was calculated as the concentration of rLcrV (ng/ml) yieldingsignal at five times background (OD = 450 nm). The readings represent mean valuesof two independent ELISAs with each concentration performed in triplicate.Detection mAb (HRP-conjugated)2B24E55D36E56F108F38F78F10Capture2B24.2110.1626.875.945.779.32ND40.08mAb4E510.1315.7047.8722.8717.342.202.541.765D313.0322.92105.6624.3519.443.552.511.926E524.0127.8537.2133.9521.743.060.840.616F107.6215.6739.5610.7211.412.050.840.658F30.752.153.891.120.8920.3612.066.278F70.812.349.5632.1652.38125.6731.777.698F101.072.685.644.752.3351.1812.6914.44 TABLE 4Binding kinetics and affinity of F1 mAbs were determined by SPR analysis.Readings represent mean values of two independent experiments.kaKD (M)RmaxChi2SSKDRmaxChi2Clone(1/Ms)kd (1/s)kd/ka(RU)(RU2)(M)(RU)(RU2)3F21.7E+044.8E−042.8E−08693.233.4E−07670.254E53.4E+044.9E−041.4E−08963.092.1E−07920.734F121.5E+041.4E−039.4E−08738.943.6E−07691.795E101.6E+045.7E−053.7E−091204.212.4E−07910.4110D99.2E+035.2E−065.2E−10703.135.7E−07670.2411B81.7E+045.3E−053.1E−091033.893.5E−071010.6711C72.0E+056.3E−043.2E−09737.691.1E−07520.7715C41.8E+042.1E−041.1E−089317.752.5E−07733.21 TABLE 5The limit of detection (LOD) of F1 mAb pairs determined by antigen 13capture ELISA. LOD was calculated as the concentration of rF1 (ng/ml) yieldingsignal at five times background (OD = 450 nm). The readings represent mean valuesof two independent ELISAs with each concentration performed in triplicate.Detection mAb (HRP-conjugated)3F24E54F125E1010D911B811C715C4Capture3F22.891.085.971.292.831.520.661.11mAb4E52.772.252.497.861.9015.822.751.904F1210.301.485.502.181.933.440.781.645E107.765.023.112.903.354.672.702.6310D92.541.202.203.194.761.710.601.3411B82.394.012.073.003.161.600.622.0911C715.212.332.735.972.771.581.551.9215C44.324.883.105.502.552.530.863.61 Example 3: High Flow Sensitive Vertical Flow Diagnostics Devices1and Method of Use There are many scenarios requiring biomarker(s) measurement on site with limited resources in a point-of-care setting, such as for cardiovascular disease and cancer prognosis monitoring, as well as infectious disease diagnostics and biothreat detection. In many cases, these biomarkers need to be detected at low concentration (e.g. <1 ng/ml). However, current paper-based lateral flow Immunoassay (LFI), the most popular POC testing format, is not sensitive enough for this requirement. One example is the capsule polysaccharide (CPS), a biomarker for melioidosis, a tier 1 biothreat agent listed by the U.S. government. The current LFI has a limit of detection (LOD) of 1 ng/ml in clinics. At this sensitivity, still a significant portion of the infected patients are not diagnosed by the assay. Simple POC assay with higher sensitivity is critically needed. Previously, different ways of improving the lateral flow immunoassay sensitivity have been reported. They are mainly divided into two groups. One exploits kinetics of the sensor, i.e. the effects of paper pore size, geometry and sample volume. It is well known that smaller pore size with a slower flow in LFI results in a better sensitivity. Furthermore, Parolo et al. (2013) reported geometry to concentrate sample for better sensitivity in LFI; Pauli et al. (2015) reported a variation of LFI in a syringe vertical flow format to have LOD on-demand by passing different volume of sample through the paper; Oh et al. (2013) reported reduced paper membrane size and larger volume increased sensitivity. However, no effect of sample flow speed has been mentioned. In another work by Chinnasamy, et al. (2014) for multiplexed detection of biomarkers in a vertical flow format, fast fluid flow was reported to reduce background due to shear force, and an optimal flow speed of ˜ 1.5 ml/min (i.e. 0.33 mm/sec) was reported. Another manner is to improve sensitivity is by novel detection mechanisms. Most LFI assays use gold nanoparticle as labels for simple colorimetric detection. Other detection schemes, such as dual gold nanoparticle, silver enhancement, or linked enzyme for chemiluminescent detection, can improve sensitivity, but at the cost of increased complexity. In this application, in contrary to the well-known effect of slower flow for higher sensitivity in LFI, we achieve a benefit of fast flow23for higher sensitivity in the disclosed vertical flow device1. In ligand-receptor binding biosensors such as immunoassay, the sensitivity is limited by the amount of sample passing through the sensor, and the efficiency of the capturing agent on the sensor. Because POC tests are preferred to be done in a short period of time, a higher flow speed to deliver more sample within a POC time frame (e.g. 10 min), and a smaller paper membrane pore size for better capture efficiency, will improve the assay sensitivity. Any of the devices1and methods provided herein may have:1. The flow23speed higher than that in traditional assay (˜3.3 mm/sec), but smaller than what would detach the capturing of the target and label by shear (dependent on specific binding kinetics and strength, and may be about >33 mm/sec). Such shear can help to remove non-specific binding to reduce background, further improving signal and sensitivity. Accordingly, any of the devices and methods provided herein may relate to a flow speed or flux that is less than 33 mm/sec, but greater than 3.3 mm/sec, or between about 33 mm/sec and 10 mm/sec, or between about 33 mm/sec and 20 mm/sec, or any sub-ranges thereof; 2. The paper membrane3pore size is equal or smaller than traditional LFI for better capturing efficiency (<15 um). Generally, the disclosed vertical flow device1comprises: 1. A porous membrane3that is loaded with the capturing agent19and control reagents for the diagnostic assay; 2. A porous membrane support21that can mechanically support the membrane3against the liquid flow23to avoid damage. The support can be Si wafer or steel piece with a grid with multiple pores to allow fluid flowing through, or a porous membrane3or combination or others; 3. A gasket25to press down the paper membrane3and prevent liquid leakage. 4. A holder29that can hold the gasket25/membrane3/support21assembly and press the gasket25against the membrane3on the support25so that fluid flows through the membrane3and support21only within the inner gasket25area; 5. A POC pump31(e.g. syringe6that can be hand-actuated or connected to a powered pump31) that generates pressure difference across the membrane3/support21to drive the fluid flow23at any desired flow23rate through the membrane3vertically. The pump31can be substituted by manual pushing depending on the flow23rate needed for different applications; 6. One or multiple spots79of capturing agent19immobilized on the top surface5of the membrane3and/or the interior of the membrane3, such as along the pore surface. Using the micro-dispensing technology, an array of capture antibody agent19spots79can be dispensed on the membrane3. Each spot can detect one type of target. This microarray design also minimizes the cross contamination between different assays; 7. A sample solution that may or may not contain the target biomarkers that can be captured by the capture spots79on the membrane3; 8. A detection solution containing a labeling agent that can link to the target biomarkers specifically and generate detectable signals. 9. In one embodiment, the sample solution and detection solution flow23through the membrane3sequentially; In another embodiment, the sample and detection solutions are mixed and then flowed23through the membrane3as a complex; 10. A detection system49that detects the label on the membrane3surface5, such as gold particle12based colorimetric detection by a table top scanner. Both faster flow23and smaller pore size will increase the working pressure to flow sample through the paper membrane3. The devices1and methods provided herein may function under relatively high pressure (˜<500 bar, such as in HPLC). For manual generation of high pressure, a syringe6with small piston area can be used (e.g. piston diameter <5 mm). For the holder29to be able to withstand the high pressure, it is also good to miniaturize the size of the chamber39hosting the gasket25/membrane3/support21. For limited sample volume, shrinking the membrane3area81will be able to reach the desired sample volume per unit area required for the high flow23operation. For limited sample volume, two supports21sandwiching the membrane3with gaskets25to prevent fluid leak will allow sample to be pushed through the membrane3sensor back and forward with bi-directional flow47to increase effective sample volume. The support21and gasket25can be integrated on the holder29for a simpler device1and operation thereof. To reduce the membrane3area, wax can be printed and melt on the membrane3to block the undesired membrane3area. According to the Department of Health and Human Services, about 70 agents (pathogens and toxins) can potentially pose severe threats to human and animal health upon exposure. Other CBRN threats are also major risks for security and accidents. The exposure often occurs in resource-limited situations, such as rural districts or even in battlefields. Therefore, there is a clear need to develop rapid, point-of-care (POC), multiplexed, and simple-to-use diagnostic devices that can work independently under these settings. Paper, as an extremely cheap and widely available material, has been well pursued to perform such tasks. The commonly seen lateral-flow format has dominated rapid diagnostics over the last decades because of its low cost and low complexity. However, it is difficult to perform multiplex detection with the dipstick design because assay performance can decrease as the number of target antigens13increase. In addition, the sample volume is limited because it is relying only on the capillary force of the paper to transport the liquid. Most importantly, the sensitivity is inadequate for certain infections where biomarkers accumulate below the limit-of-detection (LOD). These limitations can often lower the accuracy of the test, and thus degrade its clinical significance. Provided herein is a novel vertical flow paper-based immunoassay (VFI) device1platform that can be easily adapted for different diagnostic applications. As illustrated, the testing membrane3that contains the assay reagents is inserted into a syringe6pump31needle41chamber45using an adapter43. A mechanical grid support21is placed underneath the membrane3to support the membrane3against the liquid flow23. An o-ring77is placed on top of the membrane3for suppression and preventing liquid leakage. The testing sample is stored in the syringe and moved through the membrane3vertically either by manual pushing or with a syringe6pump31. The device1can be disassembled after the screening test to retrieve the membrane3for imaging and other subsequent analyses. The disclosed devices1and methods are compatible with different detection methodologies other than colorimetric detection. For example, electrochemical detection of biomarkers can be performed with printed electrodes on the membrane3substrate. Other schemes such as optical (e.g. SERS) or magnetic detections can also be implemented. A vertical flow diagnostic device1may comprise any one or more of: The porous membrane3: 1. Can be nitrocellulose membrane3, PVDF membrane3, and filter paper as the membrane3, etc.; 2. Has a high tolerance for the pore size selection (conventional ranges between 0.01 μm to 20 μm). Smaller pore size largely reduces the diffusion range, facilitates the capture reaction involving capture agent19, and increases the sensitivity; 3. Has a thickness9from 10 μm to 1000 μm, but preferably 10-30 μm to have sufficient sites for target capture, and also minimize flow23resistance. The membrane support21: 1. Can be Si, stainless steel, etc; 2. Has large enough thickness to support the membrane3, such as >10 μm; 3. Has pore size of 1 μm to 1000 μm; 5. Multiple pores within the gasket25area with distributions that gives desired flow23rate profile within the gasket25area, such as uniform flow23across the membrane3. The gasket25: 1. Can be elastic material such as o-ring; 2. Can also be microfabricated material with structures that defines the inner gasket25area; Three configurations are exemplified below, including for different application scenarios. Configuration 1: The fluid flow23can be controlled at a predefined optimal rate so that during certain realistic time frame (e.g. 10 mins for point-of-care application), the labeling signal is maximized. Manual pushing is used to push the fluid through the membrane3. The piston area of the syringe6is reduced to allow maximum pressure available to reach the optimal flow23rate. The non-specific binding from clinical sample can also be reduced by the fluid flow23. The o-ring77opening is relatively large, making it suitable for high multiplexed detection with large sample volume. Configuration 2: The inner gasket25area is controlled to be smaller than the membrane3area in configuration 1 (˜96 mm2, ˜3.5 mm diameter) down to similar to a single membrane3support pore size (e.g. ˜0.00008 mm2, ˜10 um diameter for 0.1-μm-pore membrane3). It is suitable for applications that need very good sensitivity, low requirement for multiplexing, and small sample volume. Configuration 3: The membrane3is sandwiched between two membrane supports21. Knife edges can be fabricated on the support21to integrate the gasket25with the support21. This sandwich configuration allows bi-directional fluid flow47to enable recycling through the membrane3with optimal flow rate. It can be used for applications that need ultra-high sensitivity with limited sample volume. Example 4: Development of a Vertical Flow Paper-Based Immunoassay (VFI) Method for Multiplexing Detection of Tier I Bio-Threat Agents More than seventy biological agents and toxins have been determined to pose severe threats to both human and animal health. Exposure to these agents often occurs in austere settings such as battlefields and rural areas where resources are limited. Therefore, it is imperative to develop point-of-care diagnostic tools that are sensitive, cost-effective and simple-to-use that are amenable to multiplexing. We have developed and characterized a vertical flow paper-based immunoassay (VFI) microfiltration device1that performs multiplexed detection of Tier I bio-threats. The device1platform is based on microbial antigen13capture that generates colorimetric signals for direct visualization in less than 10 min. Burkholderia pseudomallei(Tier I agent) is the causative agent of melioidosis, a devastating bacterial infection. A sandwich immunoassay was constructed to detect theB. pseudomalleicapsular polysaccharide (CPS) in the vertical flow format. A CPS-specific monoclonal antibody (mAb 4C4) was immobilized on a nitrocellulose membrane3(pore size <1 m) and served as the capture antibody agent19. A micro-dispenser was used to spot mAb 4C4 in an spot79array format on the nitrocellulose membrane3. Gold nanoparticles12(GNP) linked with mAb 4C4 served as the detection antibody10, which produces colorimetric signals following binding to CPS. The VFI was run by pre-mixing detection antibody10with buffer solution spiked with CPS then the sample was passed through the membrane3vertically with a syringe6pump31. After a washing and a drying step, the membrane3was scanned with a standard tabletop scanner and analyzed using an automated imaging analysis software. To characterize the VFI system of the disclosed device1, a design of experiment (DOE) screening analysis was created in JMP Pro13. Seven VFI parameters were studied, including six continuous factors—flow23rate, assay time, GNP12amount, premixing time, buffer pH, buffer ionic strength, and one categorical factor-membrane3type. Results and Conclusion: Traditional paper-based lateral flow assays may have limited sensitivity and multiplexing capabilities due to the small sample volume and the need of relatively large membrane pore size (>10 μm), which is inefficient for target capture. The disclosed VFI device1provides a good solution to these problems by implementing active fluid pumping. According to the DOE results, flow23rate and assay time were the two most important factors affecting the average signal intensity, followed by membrane3type, pH, and premixing time. There was a 2-factor interaction between flow23rate and assay time, indicating that the sample volume per unit area might be the key to further improving the sensitivity of the disclosed VFI device1. As for the signal variation, GNP12amount and membrane3type were the dominating factors, followed by flow23rate. The screening design identified key factors that will be studied for further VFI device1optimization. Under these optimum experimental conditions, the current VFI's limit-of-detection (LOD) for the CPS assay is 4 pg/mL (10 times lower than a previous lateral flow device). We also demonstrate multiplexing detection of CPS and PGA (a biomarker forB. anthracis, the causative agent of anthrax). The VFI system of the disclosed device1may be characterized for detection of a variety of biothreats, and validated for multiplexing capabilities and improvement of performance through miniaturization. Example 5: Design Guidelines and System Specifications Parameter 1—Membrane3materials and pore size: Design rules: Membrane3material with high protein binding capability is preferred. Smaller the membrane3pore size, better the detection sensitivity. Due to the extremely small flow23path (˜130 um), nanopore membrane3can be used in the VFI system of the disclosed device1. Suggested value: Nitrocellulose membrane3with 0.1 um pore size shows the best sensitivity with the assays for melioidosis, anthrax, and plague. Parameter 2—Capture antibody agent19: Design rules: The higher the density of the capture antibody agent19on the membrane3, the more binding sites are available to capture the target antigens13. The concentration of capture antibody agent19should be pushed to saturation based on the loading capacity of the membrane3material. Suggested value: One droplet (with a volume of 1 nanoliter) capture antibody agent19solution (10 mg/mL) is deposited on the membrane3as the detection spot79. Parameter 3—pH: Design rules: In the tested range (pH 6.4˜8.4) in buffer, pH can be significant. Suggested value: assay dependent. Parameter 4—Ionic strength: Design rules: In the range we tested (50˜450 mM), value below 150 mM gave high background, no significant effect of the ionic strength was observed for values between 150˜450 mM in the VFI system. Suggested value: 150˜450 mM. Parameter 5—Matrix Type Design rules: Three types of most commonly used human body fluid serum, plasma, urine can be used in the disclosed VFI device1. Serum and plasma have narrow pH and ionic strength range, and variations from these two factors are usually not significant. Human urine sample, which is known for big variations of pH and ionic strength, (up to 10-fold difference), which requires more in depth characterization in the VFI system of the disclosed device1. A pre-filtration through 0.2 um Polyethersulfone (PES) membrane is preferred to remove any micro-particles and cellular components that might clog the VFI membrane3. Suggested value: Serum, plasma or urine. Parameter 6—VFI device1reaction scheme: Design rules: Premixing scheme: mix the sample with the detection antibody10labeled GNP12before it is flowed through the VFI membrane3; Sequential scheme: flow the sample and the detection antibody10labeled solution through the membrane3in sequential steps. Suggested value: Our result shows premixing is preferred, but may be assay dependent. Parameter 7—Detection antibody10labeled AuNP12: Design rules: For assay that uses the same antibody for both detection and capture, such as the CPS assay, increasing the concentration of the detection antibody12labeled AuNP12does not improve detection sensitivity significantly. For assay that uses different antibody for detection and capture, such as the LcrV assay, increasing the concentration of the detection antibody10labeled AuNP12could significantly improve the detection sensitivity. More experiments are currently being performed to find the optimum concentration of the antibody labeled AuNP12in the LcrV assay that give the best contrast between the detection spot79and the background. Gold nanostars versus standard spheroid NP12are also under evaluation as the branched NP have higher scattering coefficients and tunable wavelength for possibly increasing reflectance when used in CCD based camera detection. Suggested value: Assay dependent. Parameter 8—Unit flow rate: Design rules: Unit flow rate (mm/s) is defined as the sample volumetric flow rate (mL/min or cm3/min) divided by the surface area81(mm2) of the VFI membrane3. The higher the unit flow rate is, the more samples can be delivered to the sensing area81in a constant time, and the better detection sensitivity can be achieved. The unit flow rate is limited by the pump31—higher the flow23rate, higher the required pressure, which could either stall the pump31or break the VFI housing (e.g., holder29). Suggested value: Based on the pump31we are currently using (New Era syringe pump NE1000), flow23rates can readily be 1.5 mm/s. Parameter 9—VFI test time: Design rules: Premixing time: it is the time the sample is mixed with the antibody labeled AuNP12solution. Longer the premixing time is, better the antigen13can be labeled with the AuNP12. VFI running time: it is the time the sample (mixed with the labelled AuNP12) being pushed through the VFI membrane3. Given a constant flow23rate, longer the running time is, the better the detection sensitivity. Suggested value: The current setting is 10 min premixing time and 10 min running time. The 10 min running time was selected to match the time needed for a lateral flow test, but could be modified based on the need of the users. Parameter 10—Membrane support21grid: Design rules: The rigidity of the membrane support21determines the deformation of the membrane3during the flow through step, which could impact the signal uniformity across the membrane3. The more rigid the support21can be, the less non-uniform the signal is across the membrane3. Suggested value: Deep etched Si support21is used in the current VFI setup for the disclosed device1. Parameter 11—Membrane3size and multiplexity: Design rules: The surface area81of the VFI membrane3determines the total number of spots79and thus the multiplexity of the test can be integrated with the disclosed VFI device1. Spot79size is a limiting factor of the multiplexity, which is dependent on the droplet size of the micro-dispenser. With our current micro-solenoid value dispenser, the smallest droplet volume is 1 nanoliter. However, with more advanced piezo-dispenser, the droplet size can be reduced to picoliter level, so that the spot79size can be made much smaller. Suggested value: With our current dispenser, we could have 7 spots/mm2; but other dispenser showed 11 spots/mm2. Parameter 12—Sample volume: Design rules: Sample volume is decided by the unit flow rate and the surface area81of the membrane3. VFI accommodates a much larger range of samples volumes: currently the standard VFI processes samples 1 mL to 50 mL and above, and the mini VFI processes samples from 500 μL to 5 mL. Adding an extra dilution step, the lower bound can be further extended to 100 μL without losing much sensitivity. Such wide range of sample volume gives the disclosed VFI device1a lot of flexibility in dealing with different types of samples. Suggested value: Standard VFI device1processes samples 1 mL to 50 mL and above, and the mini VFI device1processes samples from 100 μL to 5 mL. Parameter 13—Device1Integration and software: Design rules: Currently the membrane3and support21for the disclosed device1are designed into Luer-lock devices to fit most standard syringes6. In the case of nucleic acid configuration, the system of the disclosed device1will also integrate a sample preparation module coupled with the VFI device1. Data analysis software for imaging with smartphone device51camera53. Suggested value: Plastic syringes and Luer-lock connector. Example 6: Vertical Flow23by a Passive Capillary Driven System55 Other systems are available to ensure appropriate vertical flow23occurs across the membrane3. For example, a passive/capillary pumped vertical flow device1is compatible with large sample volume/area. A similar sample volume/area is achieved compared to an active pumped device1(e.g., a pump31powered by an external source of energy, such as electricity). Replacement of an active pump31with a passive flow23makes the process simpler. The device1in this example is a vertical flow device1for singleplex or multiplex biomarker detection. The difference between this device1and previously described vertical flow device1is that the reagent/fluid of this device1is pumped by the passive capillary force of an absorbent material57, in contrast to an active mechanical pump31. FIG.35provides an illustration of an exemplary embodiment of the passive capillary driven vertical flow system55. Relevant aspects include: (1) There is a detection membrane3, similar to the detection membrane3in other vertical flow device1, where capturing agent(s)19can be immobilized on the membrane3; (2) The detection area81has an area of “A”. This area81can be defined by a gasket25mechanism, or by printing a layer of liquid impermeable material (the liquid blocking material58) on the membrane3, such as wax printing or by a masking tape. (3) There is a “sample tube/holder”59on top of the detection membrane3to hold the sample. The sample will be pre-filtered before going into the detection membrane3. The pre-filter56can be external, or together with the sample tube/holder59. (4) There is an absorbent material57, acting as a capillary pump to pull the sample through the detection membrane3over time so that: (a) the sample volume going through the detection area81is higher than ˜200 μL per mm2of detection membrane3, i.e. total volume of 200 mm*A; (b) where the capillary action is completed in a point-of-care time frame, e.g. 10-30 min, which means that the fluid flow23speed is higher than ˜0.2 mm/sec; (c) where the absorbent material57that accommodates all the sample volume fans out relative uniformly, with “A” as the center. In practical use, usually “A” has an area of 100 to 0.01 mm2, “A” can have a circular shape. “A” could have a line shape, but the absorbent material57also fans out beyond the outline of “A” uniformly. Example 7: Cost-Effective Methods for Expedient Dosimetry to Support Diagnosis of Radiation Injury A mass-casualty nuclear disaster, such as detonation of a terrorist dirty bomb or a nuclear power plant incident, requires an effective and fast medical response in order to treat and save thousands of lives. First responders must precisely assess the absorbed radiation dose in order to distinguish those who need immediate medical intervention from those who are candidates for delayed treatment. Although there is no bio dosimetry method approved by the U.S. Food and Drug Administration (FDA) yet, the dicentric chromosome assay (DCA) is currently considered the “gold-standard”. This assay is very specific to ionizing radiation and low background levels of dicentric chromosomes allow it to be highly sensitive. However, like all cytogenetics-based assays, the DCA is labor intensive and takes a long time to estimate the dose, an important limitation for radiation dose assessment in an emergency scenario. The development of gene expression profiles, especially in peripheral blood lymphocytes, has been suggested as an alternate approach to radiation bio dosimetry. Exposure of human cells to environmental stresses, including IR, is known to activate multiple signal transduction pathways, and rapidly results in complex patterns of gene expression change. In contrast to DCA or the micronucleus assay, gene expression does not require cell division and can be analyzed quickly with advanced molecular assays (Lacombe et al., 2018). However, in order to process and analyze such large numbers of samples and return results in a few hours, technology development requires automation and miniaturization to provide a point-of-care device integrated in a high-throughput platform. With the advancement of nanotechnology, paper materials have been utilized for biomedical applications and show promising capacities and advantages. A majority of the reported paper devices are designed in a lateral flow immunoassay (LFI) format where the fluid flow is parallel to the surface of the paper. The main benefits of LFI include low cost, rapid results, flexibility and ease of use. However, in order to achieve an adequate flow rate, the pore size of the paper materials is limited to several micrometers and above, which hinders biomolecule capturing and thus the assay sensitivity. In addition, sample volume constraints and the difficulties of multiplexing are other hurdles that confront LFI development. One approach to improving upon the LFI platform has been the development of flow-through devices. This alternative format is similar to LFI in that it is a membrane-based immunoassay, however, fluids are applied vertically to the surface of the membrane rather than parallel (Chen et al. 2018). As such, vertical flow immunoassays provide better sensitivity, are faster, and avoid the hook effect. Provided herein is a cost-effective, rapid flow-through paper-based device1for nucleic acid detection—offering an accurate, reliable, miniaturized and automated high-throughput bio dosimetry platform that will have a higher specificity and sensitivity than the existing DCA assay. Technical Objectives: The assessment of gene expression profiles, especially from human circulating cells in biofluids such as blood, is a promising approach to standard but labor- and cost-intensive cytogenetic assays for radiation bio dosimetry. Provided herein is a Vertical Flow Paper-based device1Platform (VFP) (FIG.36) for assessing expression level of radiation dosimetry genes from PBMCs after irradiation. In this manner, the devices and methods can accurately measure the absorbed dose of irradiation: Detection of 4 different genes (3 radiation-responsive genes and 1 housekeeping gene) using VFP approach. The device is to detect three radiation-responsive genes on the same membrane3and measure and calculate their expression level between non-irradiated and irradiated samples by using housekeeping gene also detected on the same membrane3for normalization (total of four genes on a single membrane3). Results indicate that two genes from cell lines are able to be amplified and detected on VFP membrane3simultaneously. Here, we use whole blood as a biological sample to demonstrate assay feasibility on human biofluid. Blood samples are irradiated and RNA extracted and quantified. PCR primers are designed for a panel of genes, with target(s) amplified using standard PCR and amplicons assessed by using agarose gel. The three radiation-responsive genes and the housekeeping gene with the highest signal are selected to be passed and detected on VFP membrane3. In order to assess the ability of the disclosed VFP device1to detect and quantify gene expression change after irradiation, gene expression using VFP is analyzed and compared with real-time qPCR approach. Relevant aspects include: Whole blood sample collection and sample processing (cell culture, irradiation, RNA extraction, RNA quantification, etc.); development of PCR primers for a panel of radiation-responsive genes and housekeeping genes (primer design, PCR experiment optimization, product assessment); detection using VFP and comparison with real-time qPCR (confirmation of range, accuracy, and throughput along with specificity, multiplexing, signal sensitivity, etc.) Other technical aspects include isothermal amplification using recombinase polymerase amplification (RPA)—We develop an isothermal amplification method using recombinase polymerase amplification (RPA) while simultaneously developing a VFP for diagnosis of radiation injury. This approach facilitates a faster sample preparation by avoiding standard amplification and, hence, will be easier to integrate into a fully automated sample preparation platform which gets combined with the disclosed VFP device1, for maturation into phase II prototype. RPA is developed using whole blood, using the same primers described in technical objective1. The quality of the amplicons is assessed using agarose gel or ELISA prior to testing on the VFP. These aspects include: whole blood samples collection/processing (cell culture, irradiation, RNA extraction, RNA quantification, etc.); RPA development (experimental optimization, primer design, product assessment); detection using the disclosed VFP device1(confirmation of range, accuracy, and throughput along with specificity, multiplexing, signal sensitivity); Optimization of isothermal amplification for VFP (time, temperature, etc.) To accurately measure an absorbed dose of irradiation in a manner that is readily scalable, aspects to consider include: a) PCR primers (Primer design) for the designated target gene panel and b) PCR product quality assessment method; detector selection to achieve best signal to noise ratio; quantification strategy for reporting the results; Sample Type, Collection and storage; Sample processing. The system is assessed to demonstrate binding affinity in terms of best Signal to Noise ratio. Methods: Sample collection and processing—Whole blood samples, pooled from different individuals and mixed gender, is ordered from a certified company (BioChemed). Blood collected with heparin or sodium citrate is preferred since chelating agents sometimes used as anticoagulants (e.g. EDTA) could interfere with magnesium and thus reduce efficiency of PCR. Upon reception, blood is aliquoted in 1.5 mL microcentrifuge tube (˜1 mL) and exposed to 0, 2, 4 and 6 Gy X-rays using the X-RAD 320 (Precision X-Ray Inc., North Branford, CT). Irradiation is performed at 320 kVp and 12.5 mA with a 2 mm Al filter. The source-to-axis distance is 42 cm and dose-rate 3Gy/min. The beam is calibrated using a UNIDOS E PTW T10010 electrometer and TN30013 ionization chamber, with measurement done in air, for a 15 cm×15 cm field size. After irradiation, blood samples can be diluted 1:1 with RPMI 1640 medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen) and incubated in 6-well plates for 24 hours at 37° C. in a humidified incubator with 5% CO2. After 24 hours, RNA is extracted using QIAamp® RNA Blood Mini Handbook (Qiagen) following manufacturer's recommendations. RNA quality is tested and quantified by using Agilent 2100 Bioanalyzer System (Agilent) and Epoch™ Microplate Spectrophotometer (Biotek) and then stored at −80° C. until use. Standard amplification—In order to maximize success rate, PCR primers from several radiation-responsive and housekeeping genes are designed. BAX, CDKN1A, DDB2, FDXR, GADD45A, and HIST1H3D are selected as radiation-responsive genes. These genes have been identified by numerous studies as radiation dosimetry biomarkers candidates in PBMCs and have been recently reviewed in several meta-analysis (Lacombe et al., 2018; Lu et al. Sci Rep. 2014). CDR2, MRPS5 and MRPS18A genes are selected as housekeeping genes. These genes have been published and are currently used for normalization in a validated and approved diagnostics' radiation blood test (DxTerity Diagnostic, Lucas et al., 2014). PCR primers are designed by using PRIMER-Blast (NCBI). Whether some genes have several transcript variants, primers pairs targeting all these variants are preferentially selected. Primers crosslinking with others genes are excluded to ensure specificity. Amplicons whose size is above 400 base pairs (bp) are selected in order to be detectable by agarose gel. In order to be detected by immunoassay using VFP, for each primer pair, forward primer are modified on its5′ end by adding FITC group and reverse primers are modified on its5′ end by adding a different chemical group for each genes (Cy3, DIG, DNP, etc.). RNA is first converted into cDNA by using RNA to cDNA EcoDry™ Premix (Oligo dT) kit (Takara). This kit is a convenient dry master mix that allows efficient and accurate first-strand cDNA synthesis. As such, reconstitution is simple by adding PCR-grade water along with your RNA to master mix. This system thus facilitates potential integration into automated point-of-care platform for sample preparation. Second, standard amplification by PCR is performed using High Yield PCR EcoDry™ Premix. Similarly to the RT-PCR kit, this kit contains a lyophilized master mix which are reconstituted with PCR-grade water, cDNA and the designed primers. Amplification efficiency and amplicon size are assessed by conventional 2% agarose gel with ethidium bromide before VFP analysis. In order to assess VFP quantification efficiency, amplicon are also amplified by real-time qPCR. The same primers, but without any modifications on their 5′ end, are used to perform qPCR. qPCR are performed on Stratagene Mx3005p (Agilent) by using RT2SYBR Green ROX qPCR Mastermix (Qiagen) following manufacturer's recommendations. Relative fold-change are calculated by the ΔΔCT method. Data are normalized to CDR2 and/or MRPS5 and/or MRPS18A genes expression levels in order to define the best individual or panel housekeeping genes. One analysis is performed based on linear regression. The R2 value for a linear fit (fold change vs. radiation dose) are calculated for each gene and genes with R2>0.9 (or the three genes with the highest R2 value) are selected as behaving linearly. The three radiation-responsive genes and the housekeeping gene with the highest signal on agarose gel and radiation-responsive gene with the best linearity is selected for multiplex VFP analysis. Gene expression levels are normalized with housekeeping genes and fold changes are calculated and compared between VFP and qPCR. The three radiation-responsive genes and the housekeeping gene with the highest signal on agarose gel and the highest fold change ratio between non-irradiated and irradiated samples detected with qPCR are selected for multiplex VFP analysis. Construction of the VFP device1and data analysis—The disclosed VFP device1comprises either a 13 mm or 3 mm diameter nitrocellulose membrane3encapsulated in a stainless steel filter holder29(Swinny Filter Holder 13 mm XX3001200, Millipore, MA, USA). A polytetrafluoroethylene (PTFE) gasket25and o-ring77is placed below and on top of the paper membrane3respectively to seal the liquid flow23pathway. The gasket25and o-ring77is purchased as a package together with the Swinny filter holder77from Millipore. A syringe6pump31(NE-1000 automatic single syringe pump, New Era Pump Systems, Inc., NY, USA) is used to push the samples and reagents vertically through the paper membrane3at a controlled flow23rate. We fabricate the support21with silicon using deep etching method instead of using commercially available stainless steel support. The silicon grid of the support21mechanically supports the membrane3against the flow23during the flow through processes. A VFP device1may be built using nitrocellulose membrane3because of its high protein-binding capability and availability in a range of small pore sizes. Four nitrocellulose membranes3, Amersham Protran 0.1 μm NC, 0.2 μm NC, 0.45 μm NC, and Whatman AE98 (pore size 5 μm) (GE Healthcare Life Sciences, PA, USA), could be tested and compared. The four types of membrane3all consist of 100% pure nitrocellulose with different pore sizes. The membranes3are cut into shape using a CO2laser (VersaLaser 2.30, Universal Laser Systems, AZ, USA). The capture antibody agent19microarray of spots79is dispensed onto the paper disks using a micro solenoid non-contact robotic dispenser (AD1520 micro-dispenser with BioJet Elite, Biodot, CA, USA) with a droplet volume of 1 nanoliter, which creates circular spots79of 220 μm in diameter. Specific capture agent19antibodies (against one extremity of the amplicon labelled specifically with a different compound for each gene) are first immobilized on the paper membrane3. All amplicon also containing a FITC modified5′ end, thus a FITC-labelled gold nanoparticle12(FITC-GNP) are used as unique detection agent for all the target. Two experimental procedures, i.e. premixing and sequential, can be tested and compared. PCR products are spiked in buffer solution (0.1 M PB buffer containing 0.1% Triton X-100 and 0.1% BSA, pH=7.2) containing the FITC-GNP12for 10 min. In the meantime, the membrane3are treated with blocking buffer (10 mM borate buffer containing 2.5% Triton X-100, pH=8). The sample mixture is pushed through the membrane3to allow capture of the PCR products-FITC-GNP12complex by the capture agent19antibodies on the membrane3. After the sample is processed, the membrane3is washed by flowing 1.5 mL blank assay buffer through the membrane3to remove non-specific or loosely bound amplicon and excess FITC-GNP12. The VFP device1is dismantled and the membrane3placed on a filter paper (Whatman qualitative filter paper, Grade 1, GE Healthcare Life Sciences) for 5 min as a drying step. Subsequently, the VFP membrane3is scanned with a consumer-grade table-top scanner (CanonScan 9000 F II) and Scan IJ Utility (default software for the CanonScan), with 48 bits RGB settings and 2400-dpi resolution exported into an uncompressed TIFF file format. The 48 bits RGB image will be then converted to 16 bits grayscale image using the built-in function rgb2gray of Matlab (Mathworks, MA, USA). The resulting image is imported into ImageJ, where the spots79are analyzed using a microarray grid to extract the mean grayscale values from the spots79with subtracted local background. The limit-of-detection (LOD) is defined as the concentration that generated a signal that is greater than 3 standard deviations (SD) above the background signal. Data processing and analysis is performed. Specificity of the VFP membrane3is assessed by flowing the individual amplicons though a membrane3printed with the four capture agent19antibodies. Indeed, the membrane3is first coated with the four specific capture agent19antibodies. Then, samples containing only one amplicon is flowed through the membrane3. This experiment is repeated four times, for each amplicon. Signal is detected for each capture agent19antibodies. Signal for the targeted antibodies is considered significant and specific if signal is at least two times higher than the background (determined as signal generated by spot79printed with PBS). Radiation-responsive genes expression levels are normalized with the selected housekeeping gene. Similarly to qPCR data processing, resulting fold changes are plotted into a linear fit curve in function of radiation dose in order to compare VFP and qPCR results and assess VFP power as biodosimetry gene expression assay. Isothermal nucleic acid amplification-Isothermal nucleic acid amplification can be based on Recombinase Polymerase Amplification (RPA) which represents a hugely versatile alternative to polymerase chain reaction (PCR) for the development of fast, portable, nucleic acid detection assays. RPA is developed from the same whole blood samples as previously described. RNA is extracted according to the same protocol as previously described. RPA amplification can be entirely performed by using TwistAmp kit (TwistDX) following manufacturer's recommendation. This kit has been developed to amplify DNA, but we add reverse transcriptase (AMV reverse transcriptase, Sigma-Aldrich, efficient at 42° C.) to the reaction mix in order to amplify RNA. The same PCR primers designed for standard PCR are used for the reaction. RPA products can be assessed by agarose gel and finally detected with VFP. First, reaction is performed at 30 min at 42° C. If efficient, different timing points are assessed to decrease to up to 10 min if possible. VFP being very sensitive, we expect that even a short time of amplification can be sufficient to detect signal. However, although manufacturer claims that RPA can work with PCR primers, RPA may require longer primers amplifying shorter sequence. If we cannot detect any amplicon, new primers are designed specifically oriented for RPA. For such an approach, agarose gel technique may not be ideal to detect RPA product (shorter than PCR products) and as a result we can use sandwich ELISA to assess RPA efficiency. We are able to detect two different genes (CDKN1A and HIST1H3D) on the same VFP membrane3with a high specificity suggesting the possibility of using a multiplex approach for several targets. By quantifying colorimetric signal on VFP membrane3, we also illustrate an ability to detect difference in gene expression levels between non-irradiated and irradiated samples. We also detect the surface capsular polysaccharide (CPS) ofBurkholderia pseudomalleipathogen. We optimize parameters and critical factors of VFP. We thus show that increasing the flow23speed (up to 1.06 mm/s) and reducing the membrane3pore size (down to 0.1 μm) can improve the sensitivity by at least 5 times. The VFP's limit-of-detection for CPS spiked in buffer solution is determined to be 0.02 ng/mL suggesting that the disclosed VFP device1shows great potential as a point-of-care tool for immuno-detection of molecules in a variety of clinical and resource-restricted conditions (e.g. rural or battlefield environment). This example provides a bio dosimetry device1suitable for rapidly measuring expression levels of a low-density genes that can define radiation exposure, dose and injury in case of a public health emergency. The devices and methods may be validated, including to satisfy agency regulations, with a large population of cancer patients from multiple sites and different treatment modalities. Example 8: Human-Centered Design Augmentation of the Vertical Flow Paper-Based Health Monitoring Platform This example presents a human-centered design of a VFP platform working in microgravity environment for space mission, including integrated sample preparation modules for gene expression based health monitoring. The platform is designed to ensure safe operation in spaceflight, as well as usability to the unique demographics of the end user (astronauts). This addresses the need in the field to develop human-centered designs for the disclosed Vertical Flow Paper-based Platform (VFP) device1with more operability and usability, improved efficiency and safety, decreased errors and reduced learning curve for space health monitoring. The disclosed VFP device1is a mobile point-of-care (POC) uses a miniaturized “syringe-like” cartridge that is capable of detecting tens or hundreds of biomarkers in small or large volume of bodily fluids in a short period of time (˜10 min) (e.g., as shown and described above with reference toFIGS.2A-2C). The device1platform is based on antigen13capture by specific antibody pairs with colorimetric signals from nanoparticle12labels that can be detected by direct eye visualization or by a standard camera53of a smartphone51as a reader. The disclosed VFP device1platform has demonstrated ˜25× improved sensitivity comparing with standard lateral flow assay for antigen13biomarker detection. The TRISH VFP project is expanding the capability of the disclosed device1platform by detecting gene expression biomarkers for astronaut health monitoring in space flight (e.g. biodosimetry or infection). The current VFP platform of the disclosed device1is designed for POC detection of antigen13biomarkers on earth, but has not been optimized for space missions. The expansion of the device1platform for gene expression biomarker detection also requires an additional module for the sample preparation process. For devices1operated during space missions, it is imperative to ensure safety of the mission, including safety of general public, the astronauts, NASA workforce and high value equipment. It is also important to consider the demographics of astronauts. They are highly educated, trained and motivated, but overtasked. There are also numerous factors in a space mission that will decrease the capacity of processing information, memory recall, strength and attention for astronauts during space mission, such as reduced gravity, fatigue due to circadian misalignment, decreased visual acuity with headward fluid shifts, acoustic sensitivity and other stresses of long-duration deep space exploration. The special environment (e.g. ionizing radiation) and limited resources in space mission (mass, power, volume etc.) are additional factors that need to be considered for device used during spaceflight. To address the above-mentioned conditions, we design a user-centered VFP platform with automated sample preparation for nucleic acid biomarker detection for space missions. The nucleic acid VFP platform has a modular plug-and-play design to minimize the need of astronaut intervention.FIG.37shows a process400flow diagram of operation of the platform. It mainly comprises three modules, i.e. Block403: Sample Prep1—white blood cell sorting, Block405: Sample Prep2—RNA amplification (including cell lysis, mRNA reverse transcription, cDNA isothermal amplification), and Block407: the final VFP assay and detection. Block403, Sample Prep1—White Blood Cell Sorting Module: Current gene expression biodosimetry biomarkers are detected from lymphocytes in blood because it is a systemic bodily fluid that can be collected with minimal invasiveness. To detect gene expression from white blood cells, red blood cells usually need to be removed to reduce the interference from their large amount of RNA background. Traditionally, this is done by Ficoll-Paque density gradient separation. However, this process requires multiple manual handling and is difficulty to automate. To overcome this issue, a white blood cell sorting microfluidic chip based on deterministic lateral displacement (DLD) is used, where cells with different sizes flow through a micro-post array with columns of the array tilted slightly to the cell stream and those larger than a “critical size” defined by the array are bumped out of (and separated from) the original stream]. We have demonstrated the DLD chip for gene expression applications and integrate it into the VFP platform as the initial step of sample preparation.FIG.38shows layout of a DLD chip40and fluorescent images of white blood cells60being sorted into designated channels62. Block405/Sample Prep2—RNA Amplification Module: The RNA amplification module provides white blood cell lysis, mRNA reverse transcription and isothermal DNA amplification. RNA can be prepared through different processes for detection, by organic extraction, filter based spin basket, magnetic particles or direct lysis. The direct lysis method: (i) has the highest potential for accurate RNA representation, (ii) it can work with small samples, and (iii) it is amenable for simple automation. Direct lysis assay chemistry can be used for VFP gene expression detection and implemented through a microfluidic cartridge. The extracted mRNA needs to be reverse transcripted to cDNA and amplified for VFP detection. Traditionally, cDNAs are amplified through a cyclic heating/cooling process by polymerase chain reaction (PCR). However, several isothermal amplification processes have been reported recently for simple automation and POC application, such as loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA) and nucleic acid sequence based amplification (NASBA) etc. A recombinase polymerase amplification (RPA) process can be adopted that overcomes the drawbacks of other isothermal amplifications. It is also possible to integrate the reverse transcription process together with the amplification process. Commercial kits are available for gold nanoparticle12labeled reagents for lateral flow test, which can be adopted directly for VFP assay and detection. A microfluidic cartridge controlled by a microprocess controller is designed to automate the reverse transcription and amplification process for VFP detection. Block407/VFP Assay and Detection Module: This module comprises main features of current antigen13detection VFP platform of any of the devices described herein, including a syringe6cartridge for sample processing, and a reader for detection and data analysis. The main detection membrane3ishoused inside a membrane holder29. The membrane holder29can be attached to the syringe6cartridge so that the amplified DNA sample can be pushed through the membrane3for target capture by capture agent(s)19. Then the membrane holder29can be detached and mounted to a reader for array scanning and data analysis.FIG.36shows a current design of a spring loaded powerless syringe6cartridge for easy fluid handling, with a zoomed in view of the current design of a miniature membrane holder29attached to a smartphone51adapter157for data acquisition. These elements will be reconfigured to interface with upstream sample preparation modules (e.g., blocks403and/or405). Overall Platform Design: Finally, the Sample Prep and VFP modules (blocks403,405, and407) are integrated together through human-centered cartridge design. This serves the human systems integration to ensure operational relevance to optimize space mission success and preserve human health. Design activities are aimed at emphasizing unequivocal value of the product platform to the NASA customer through system and crew factors, i.e. astronaut crew and space medicine team, and for the broader purpose of human spaceflight operations (i.e. clinical space medicine, expedition support). Once aboard the space-based platform (e.g., currently the International Space Station (ISS), but relevant to other non-Earth outposts are orbital habitats), researchers are able to monitor their experimental data daily with planned upgrades to near real-time capability. This high throughput, reconfigurable and automated architecture allows for scalable and affordable use of microgravity.FIG.39shows a process workflow700of a human-centric cartridge design integrated into a modular automated sample preparation and monitoring system. The operational constrains are followed during design, such as the storage temperatures during each stage of space mission, power requirement, telemetry rate etc. Design includes internal electronics for control of fluid flow23and filtering, per specifications. Suitable image analysis software is used in the system, to ensure relevant data gathering and validation. A VFP platform design, including sample preparations for gene expression biomarker detection that can operate in microgravity environment for spaceflight health monitoring using a simple fingerprick, which is integrated into an automated sample preparation, processing, and monitoring system of workflow700including disposable cartridges and appropriate sample port that is ground approved for space mission is thus provided. Example 9: Development of Vertical Flow Paper-Based Immunoassay (VPI) Diagnostic System for Multiplex Pathogens Detection This example provides a Vertical Integrated Flow Assay System Technology (“VERIFAST”) for Multiplex Pathogens Detection. The VERIFAST is a rapid (<30 min) point-of-need diagnostics device1for use at multiple echelons of care and in the field. An initial feasibility study will demonstrate the performance of the proposed biomarkers panel and assay kits for the integrated vertical flow immunoassay platform of the disclosed device1. The next stage of VERIFAST™ will be to define a detailed user requirement that will satisfy FDA devices guidelines. Areas of requirements will include each of the constituent parts of the multiplex biomarkers panel and protocol, including sample preparation, stabilization/transportation, preparation, quantification/detection, and interpretation of the multiplex array images, and their incorporation into a fully integrated automated point-of-need (PON) system. The outcome will be a list of requirements from which the deliverables can be checked and modified appropriately. Key processes to integrate into the system for the VERIFAST system are: Enhancements to the sample preparation for clinical (e.g. blood, urine) and non-clinical samples (e.g. growth media and soil suspension matrices); Optimization of the multiplex signatures and assay chemistries (i.e.5pathogens; enhanced optical properties of nanoparticles12); Upgrade of the optical detection module from the current benchtop scanning system to the compact smartphone51module for point-of-need configuration; Modified and optimized data interpretation software for the proper analysis of multiplex biomarkers from serum and environmental or lab-grown samples. The sample preparation cartridge and instrumentation hardware are designed, built and tested (iteratively) to optimize the process to provide as high a success rate as possible. Following successful attainment of appropriate data protection and securitization of data, these expert systems can be made available through a gateway to an external server. This task is coordinated to ensure compatibility with specified mobile systems. The packaging of system-ready sample preparation cartridges is delivered for subsequent evaluation. A main objective is to deliver a paper-based microfluidic Vertical Flow Integrated Assay Technology platform (VERIFAST), in a portable, rapid multiplex bio-fluid preparation, processing, and ‘sample-to-answer’ analysis system designed for rapid detection (<30 min) and multiplexed identification of biological features in human clinical fluids or environmental samples. The technical aspects include: Bioassay design and integration. We design the workflow integration for the microfluidic device including sample collection, sample volume processing, buffer compositions, sample run time, and result analysis; Evaluate and validate the VERIFAST assay for each of the biomarkers with respect to standard LFI assays. The VERIFAST system is compared to the relevant LFI that has either previously been developed or is in the process of being developed. This will indicate whether the VERIFAST can achieve improved analytical sensitivity over the LFI, especially in the proposed multiplexing configuration; Design, assemble and validate the overall VERIFAST platform. Through an iterative process, the VERIFAST assay is optimized for analytical sensitivity (e.g. limit of detection, LOD); Translate the VERIFAST platform system to pre-clinical studies; Product development and preparation for FDA approval. Product Description: Below is a summary of the specification of the current VFI system of the disclosed device1and its next generation (VERIFAST™) TABLEProduct DescriptionModuleVERIFAST System FeaturesSample CollectionStandard sample collection methodCompatible with urine, serum, plasma;adaptable to soil preparationSample volume200 μL to 5 mL and aboveBio-AssayProtein assay:Burkholderia pseudomallei,Burkholderia mallei,Bacillus anthracis,Francisella tularensisandYersinia pestisTest time30 minVERIFAST hardwareVERIFAST: a fully disposable device withinterface and analysisthe testing membrane and other functionalSoftwarehardware (membrane support, liquid sealingetc.) pre-assembled in a sealed packageTesting kit: including the blocking buffer,washing buffer and dilution buffer (optional)Imaging probe: an optical system thatconnects the VERIFAST to the cellphonecamera with desired magnification and focaldistanceVERIFAST analysis app: an iOS orAndroid based App that can image themembrane with desired specs and runanalysis to examine the signals from eachspot and generate a report automatically forreference FIG.36is an Illustration of the VERIFAST Development for Point-of-Need Concept Relevant for the sample collection module include: The collection device of module must be capable of reliably collecting a range of volumes from at least 200 μL to 5 mL of whole blood; or being adapted to prepare soil suspension for media culture; The collection device of module must be compatible with use in remote environment by a relatively unskilled individual given appropriate training; The collection device of module must be capable of reliably delivering the collected bio-sample to the next sub-systems of the multiplex assay detection of the VERIFAST system platform; The collection device of module must have a suitable method for tracking and transferring the user tracking information to the VERIFAST system platform to ensure custody chain of information. Relevant aspects for the VFI membrane include: The membrane substrate has good mechanical properties for accommodating various flow23rate regimes and it has controlled pore sizes compatible for optimizing all necessary fluidic properties suitable for a miniaturized configuration; the membrane can be preloaded with all required reagents; the membrane can be packaged so as to have a minimum shelf life of 6 months with 3 years being preferable. Relevant aspects for a multiplex assay include: The assay is capable of discriminating, five pathogens biomarkers of biological fluids collected at a point-of-need in variable volumes from small (e.g. <200 μL) to large (>5 mL) quantities; The assay can have an analytical range that is linear across two orders of logarithmic scale, preferably <nanomolar target concentration depending on the input specimen; Analytical performance requirements for the assay (accuracy and precision) are derived from the final multiplex biomarkers algorithm, and are designed to allow reproducible diagnostics prior or near a therapeutic decision point; the assay is capable of being read by human eye or using an optical imaging system readily available on a mobile smartphone device51or the like; the assay is able to start with an input of whole blood or urine and determine the biological exposure of an individual to at least a pathogen from multiple targeted biomarkers whose antibody couplings are specifically producing scattering signals from a nanoparticle12label which can be detected by an imaging sensor (e.g. smartphone51camera53) TABLEInstrument CharacteristicsCycleThe instrument shall perform the assay from biospecimenTimecollection to multiplex analysis in less than 30 minDeviceThe VERIFAST device shall automate the running of theentire sample processing and assay, control all requiredfunctions, perform detection analysis, data collection anddata interpretation without any user intervention postsample and reagents kit loadingDetectionThe device shall be capable of interfacing with a mobileappliance for detecting antibodies reactions directlythrough light scattering imagingRegulatoryThe VERIFAST device shall be compatible with FDAclass II regulationFieldThe device shall be of a portable size and weight, allowingPortablepoint-of-need operations and mobilityNetworkingThe device shall have networking capabilities compatiblewith standard and specialized communications systems andprotocols, per specificationDataThe device shall be capable of performing all dataProcessingprocessing requirements and deliver a diagnostics testwhile functioning in standalone or networked modesPowerThe device shall run without external power or becompatible with battery-pack remote operationsInstallationThe device shall be capable of being transported to newlocations without any setup requirements aside fromconnecting to an electronic appliance like a smartphone (orother equivalent handheld reader if applicable)PatientThe device shall be equipped with suitable patient/sampleTrackingidentification and/or tracking hardware and database (ifapplicable)DutyThe device shall be capable of running 24 h/day with norequired maintenance The VERIFAST platform enables the operator to load a plastic syringe6or equivalent cartridge module containing patient samples individually. The device1will process in four steps, performing the multiplex pathogens assay, recording the biomarkers interactions and performing the analysis required to provide a diagnostic result: 1. Biofluid filtering and pre-processing (e.g. serum and plasma separation); 2. Ab labeling and hybridization reaction with antigens13using multiplex assay; 3. Imaging detection (e.g. nanoparticles12scattering); 4. Numerical processing of biomarkers detection to determine result. Results are recorded in a database on the instrument and provided to the operator in a format to be agreed upon specific requirements and applications, for example in a mobile setting or field operational scenario. The operator can dispose of the processed devices1. The VERIFAST technology platform is summarized as a multiplex pathogen bio-markers panel system which is broken into several key subsystems previously described above with reference to the various embodiments of the disclosed device1. The VERIFAST system platform integrates advances in several interdisciplinary fields to substantially improve and change the capability to perform immunoassay testing with multiple biomarkers of at least five bacterial pathogens in a remote setting by providing a new self-contained integrated paper-based microfluidic vertical flow device1diagnostic platform system. Some singleplex Lateral Flow Immonoassays (LFI) already exist for bacterial detection and are in use for other applications. However, we know of no multiplex Tier 1 Pathogens-based system that are currently used in a fully automated miniaturized point-of-need format suitable for operating by non-expert users and without power requirement, or multiple steps manual preparation beyond the sample loading into a self-contained vertical flow device pre-loaded with all necessary assay reagents, onboard fluidic management, and multiplex detection, either by direct visualization or imaging using standard mobile electronic appliances (e.g. smartphone51camera53). In this example, we build upon the combined knowledge and interdisciplinary knowledge and outline the necessary validation required to ready this integrated technology for use in mobile settings. What distinguishes the VERIFAST platform is that not only is the detection highly sensitive and specific, but also analysis for characterization of multiplex biological signals is performed simultaneously rather than in a series of independent assays. The integrated system approach of this example leads to a fully handheld “sample-to-answer” analysis approach that is based on the integration of existing sub-components, both technological and scientific. This device and methods can focus on current bacterial biomarkers of critical pathogens in biological fluids, although the assays can also be designed for dual use with other non-clinical targets. The detection biomarkers used in this study are validated for specificity by screening them on multiple systems across diverse platform technologies. With the VERIFAST system platform, the ability to assess the biological responses to pathogens exposure or not, and rapidly in a readily available fluids or environmental samples, represents a significant leap forward in effectively protecting the field operators engaged in active missions. FIG.39is an illustration of the assay workflow700for this example, e.g., the VERIFAST Automated Assay Workflow. The solution presented in this example shifts from the current paradigm of clinical laboratories testing capabilities to a comprehensive handheld diagnostics capacity of dual testing of clinical and environmental samples in field forward austere environments with limited resources. Such a transformation is only now possible due to advances in molecular assay technology, the development and validation of highly sensitive and specific biomarkers for bacterial pathogens and the advancement of multiplex vertical flow immunoassay technology. The disclosed VERIFAST system platform integrates these advancements into a medical defense solution to allow for the rapid identification of pathogens in biofluids at their earliest possible exposure time points. The transfer of existing technologies from a research lab to a field forward setting is not a simple endeavor and this translational work is the primary focus of this example. For instance, in a research laboratory the focus is often on assays that evaluate a single specific target, and these assays are often employed one at a time. In contrast, in a point-of-need setting the ability to detect quickly a broad spectrum of targets is the main objective because many of these biological perturbations can exhibit similar symptoms. The key innovation of our approach is to combine these existing technologies for point-of-need, paper-based microfluidic vertical flow immunoassay currently undergoing extensive platform validation and rapid prototyping, with reagents kits that are extensively validated into a robust diagnostic platform to simultaneously test for multiple biological responses to Tier 1 pathogens exposure for field forward application by personnel operating in austere environments. The AuCoin laboratory specializes in biomarker discovery and development of immunoassays for infectious diseases. The team developed multiple platforms including In Vivo Microbial Antigen Discovery or “InMAD” for the identification of shed/secreted microbial antigens13within patient samples (Yuan, Fales, et al. 2012; Yuan, Khoury, 2012). This has led to the development and optimization of multiple assay for direct detection of microbial antigens13(Yuan, Fales, et al. 2012; Wang, et al., 2016; Indrasekara, Meyers, et al., 2014; Liu et al., 2015; Indrasekara, Johnson, et al., 2018). Our assay development workflow includes (i) identifying multiple shed microbial biomarkers in patient samples, (ii) isolating large panels of well-characterized capture agent19mAbs to each target (producing >20 mAbs to each target ensures superior assay performance), (iii) down selecting mAbs and optimizing antigen-capture ELISA for biomarker detection and (iv) transferring the assay to different diagnostic formats (e.g. LFI, VFI, etc.). The program has been focused on developing LFIs for a number of pathogens including, but not limited to all of the Tier I bacterial select agents classified by the Department of Health and Human Services. These pathogens includeBacillus anthracis(anthrax),Burkholderia pseudomallei(melioidosis),Burkholderia mallei(glanders),Francisella tularensis(tularemia) andYersinia pestis(plague). These are the infectious disease that are studied in this current example. The table below summarizes the biomarkers for the five pathogens and current status of each of the singleplex LFI assays. We continue to use the same biomarkers for the VERIFAST system. The results are compared with corresponding LFIs. Currently, the LFI developed forB. pseudomalleiis anticipated to identifyB. malleias well, this is due to the conserved capsular polysaccharide (CPS) produced by each species. TheB. anthracisandB. pseudomalleiLFIs are developed in collaboration with InBios International (Seattle, WA) and have been shown to achieve a limit of detection (LOD) of 1 ng/ml. Both of these assays are in-hand and are currently being evaluated in the pre-clinical setting. LFI forF. tularensisis in progress, and the biomarkers and lead mAbs are available for VFI system evaluation. The biomarkers forY. pestishave been identified and mAbs are currently being purified. Monoclonal antibody production, growth of BSL3 pathogens, purification of bacterial targets, and development of prototype assays is ongoing at AuCoin laboratory. TABLELFI Development:TargetLeadLFIPathogen(Biomarker)AntibodyPrototypeTRL*B. anthracisCapsule (PGA)mAb 8B10Yes6B. pseudomalletCapsule (CPS)mAb 4C4Yes5B. malletCapsule (CPS)mAb 4C4Yes4Y. pestisLcrV and F1MultipleYes4F. tularensisLipopolysaccharidemAb 1A4In progress4(LPS)*Technology Readiness Level FIG.40Ashows a design of the paper based VFI device1(Cheng, et al., 2019) according to this example. The device has a nitrocellulose membrane3based vertical flow immunoassay (VFI) diagnostic device1. The insert ofFIG.40Ais an example membrane3showing positive signals from the microarray of spots79. The prototype ofFIG.40Aincludes a nitrocellulose membrane3(e.g. diameter of 3.5 mm) with spotted antibody capture agent79array, a silicon membrane support21, and an o-ring77sitting inside a polycarbonate housing (e.g., holder29). The VFI device1uses a standard Luer-lock fitting to connect to the sample syringe6. After the sample run is completed, the Luer-lock can be conveniently disconnected with the sample syringe6and re-connected to the smartphone51camera53imaging probe, as shown in the prototyped imaging detection system49module design is shown inFIG.40B. The setup shown inFIG.40Bis an example of an imaging detection system59module used to scan the VFI membrane3with smartphone51camera53. In the process, the membrane3stays inside the VFI device1, which minimizes the users' exposure to the potentially bio-hazard samples. The insert ofFIG.40Ais a membrane3example showing positive signals from the microarray of spots79. The microarray spots79are deposited with capture agent19antibodies targeting different biomarkers of the pathogens panel. The microarray membrane3is fabricated with a BioDot™ micro-solenoid dispenser with droplet size of 1 nanoliter, which yields a spot79size of ˜220 μm on the membrane3. Quality of the VFI membrane3, including spot79size, spot79spatial resolution, and number of multiplexing can be further improved with advanced micro piezodispensing system with in-line camera feedback module. The Si support21is a circular silicon disk with flow through holes (diameter of 150 μm) fabricated using deep RIE methods. It provides mechanical support to the membrane3against the liquid flow23. Silicon support21has a higher Young's modulus than commercially available stainless steel support, and thus less susceptible to deformation under flow23pressure. It significantly improves the signal uniformity across the spot79microarray on the membrane3. As a significant part of the ongoing effort to optimize the vertical flow diagnostic platform, multiple system parameters are investigated both theoretically and experimentally. The disclosed VFI device1platform is detecting the bio-threat pathogens based on sandwich immunoassay. There are two critical dimensionless numbers in the VFI system of the disclosed device1. One is the Damköhler numbers (Da), which characterizes the relation between adsorption rate and transport rate. The second one is the Péclet number (Pe), which is the ratio between convection rate and diffusion rate. To capture target antigen13with low concentration using capture agent(s)19, two conditions are desired: (1) Efficient capture assay (Da>>1), in which the rate of the antigen13binding to the capture agent19antibody is faster than the rate of antigen13molecules transport to the pore wall in membrane3; (2) Non-diffusion-limited assay (Pe<1), allows for all delivered antigens13to diffuse to the membrane3pore wall before they are convected through the sensing area81. Based on these two requirements, the VFI design has high flow23speed and small membrane3pore size to improve the assay sensitivity. The table below summarizes the factors investigated for the VFI system of the disclosed device1to determine their effect on the performance of the VFI system of the disclosed device1. They can be divided into four groups, related to the sample, device, assay, and operation protocol respectively. SampleDeviceSample typeMembrane sizeSample pHMembrane materialSample ionic strengthSupporting strengthSample volumeAssayOperationCapture antibodyUnit flow rateDetection antibodyTest timeColorimetric agentMixing timeDilution factor Some of the factors are found to be very significant, such as the membrane3material pore size and sample flow23rate. Experiment results indicate that nitrocellulose membrane3with smaller nanometer pore size provide the best sensitivity with the current four bio-threat assays implemented on the VFI platform of the disclosed device1. The higher the unit flow rate is, the more sample is delivered to the sensing area81in a constant time, and the better detection sensitivity can be achieved. In contrast, some factors are less important, such as the density of the capture agent19antibody—as long as the spotted region of the membrane3is saturated, adding more capture agent19antibody to the same region does not further improve the sensitivity of the disclosed VFI device1. Performance of the VFI System in Detecting Bio-Threat Pathogens and Multiplex Detection. The table below provides key specifications of a VFI device in comparison with the lateral flow immunoassay system using the same assay: Vertical flow assay (VFI)Lateral flow assay (LFI)SampleAssayLimit ofSampleAssayLimit ofPathogenAntigenvolumetimedetectionvolumetimedetectionB.pseudomalleiCPS5 mL30 min4 pg/mL100 μL15 min40 pg/mLB. malleiB. anthracisPGAY. pestisLcrV0.2 ng/mlF. tularensisLPS Due to the larger flow through area81and integration of active pumping mechanism (e.g., pump31), the VFI device1is capable of processing samples with larger volume, such as urine or environmental water soil suspension samples. The total test time of the disclosed VFI device1is longer than LFI, due to the longer sample preparation steps for larger sample volumes, but still under the 30 min benchmark. The sensitivity of the disclosed VFI device1is about 25 times more sensitive than a similar LFI configuration. We perform experiments using the VFI platform of the disclosed device1to detect two targets simultaneously—CPS and PGA. The design of the multiplexing VFI membrane3is shown inFIGS.10A-10C, with multiplexing VFI with CPS and PGA assay detected on the same membrane3.FIG.10Bshows scanned images of the four membranes3with different samples (CPS−/PGA−, CPS+/PGA−, CPS−/PGA+, CPS+/P). The detection spot79microarray was divided into two parts. Half was coated with mAb 4C4 capture agent19targeting the CPS, and the other half was coated with mAb 8B10 capture agent19targeting the PGA. Four samples with different antigen81contents (CPS negative/PGA negative, 1 ng/mL CPS/PGA negative, CPS negative/1 ng/mL PGA, 1 ng/mL CPS/1 ng/mL PGA) were processed with the VFI membrane3.FIG.10Bshows the scanned images of the four membranes3.FIG.10Cshows the signal intensities from the four membranes3. The detection spots79showed positive signals only when the corresponding antigen81existed in the sample. This experiment demonstrated that the VFI platform of the disclosed device1was able to detect multiple biothreat agents simultaneously. Unlike LFI, the detection spots79were spatially separate and independent from each other in the VFI system of the disclosed device1, making the disclosed VFI device1especially suitable for large-scale multiplexing detection. FIG.41shows the user interface of the current benchtop image analysis software161. The workflow of the software to analyze the miniVFI membrane has three major steps: 1) Identify and rotate the image using a pre-defined template; 2) Locate each spots79in the microarray. After identifying the template, an x/y two-axes coordinate is created in relative to the template. The coordinates of each spot79can be calculated based on the pitch distance and the scanning dpi; 3) Obtain the intensity from each spot79and its background; 4) Generate a diagnostic report (positive, negative, and questionable) for each spot79, which can potentially for one type of biomarker. We compared the results with manual methods using ImageJ. On the average, the difference was up to 15%. Considering that the signal intensity from the membrane has a variation of 10% itself, the results from the software is quite accurate. Note: the software161system has also been adapted to analyze LFI strips. Among plasmonic nanoparticles, surfactant-free branched gold nanoparticles have exhibited exceptional properties as a nanoplatform for a wide variety of applications ranging from surface-enhanced optical sensing and imaging applications to photothermal treatment and photo-immunotherapy for cancer treatments. A surfactant- and capping agent-free route to synthesize gold nanoparticles with multiple sharp branches protruding from a spherical core has been introduced, which is referred to as “gold nanostars” (GNS) (Nuti, et al., 2011). The biocompatible, ligand-free surface chemistry of that requires no ligand exchange or extensive purification protocols, ease of direct surface functionalization, and superior surface area available (compared to spherical gold nanoparticles of the same diameters) for biomolecule loading are few major advantages offered by GNS in comparison to other types of gold nanoparticles (Jiang et al., 2011); Yuan, Weng, et al., 2011; Yuan, Khoury, et al., 2012; Yuan, Fales, et al., 2012; Wang, et al., 2016; Indrasekara, Meyers, et al., 2016; Liu, Ashton, et al., 2015; Indrasekara, Johnson, et al., 2018; Lenshof, et al., 2009; Kim, et al., 2017; Kuo, et al., 2015). In addition, GNS exhibit unique and extraordinary optical properties such as high scattering crosssection, plasmonenhanced absorption and geometry controlled plasmon absorption band tunability in the visible to near infrared spectral range (Nuti, et al., 2011; Gong, et al., 2013). Collectively, both chemical and optical properties of the GNS could provide a versatile platform for enhancing the optical detection performance of the application of the VERIFAST platform. The effectiveness and reliability of nanoparticles in biomedical applications strongly rely on the consistency and reproducibility of physical, chemical, and optical properties of nanoparticles, which is mainly governed by their morphological features. Design parameters have been developed for the purpose-tailored manufacturing GNS in a reliable manner. There are equipped with optimized bottom-up synthesis protocols that improve reproducibility and homogeneity of GNS, and the modulation of their morphology, particularly the branch density, geometrical features of branches to obtain desired optical properties. Through systematic modulation of experimental parameters, GNS can be manufactured with branch densities ranging from 3-30 branches/particle, overall size from 30-200 nm, and optical absorption ranging from 530 nm-900 nm, as shown inFIG.42(Nuti, et al., 2011; Gong, et al., 2013).FIG.42shows transmission electron microscopy (TEM) images and absorption spectra showing the morphological and optical tuning of high quality GNS with high reproducibility using surfactant and capping agent-free seed mediated synthesis approach. Biomarkers Panel Development: Purification and QC (Quality Control) Testing of mAbs Milligram quantities (≥500 mg) of each mAb capture agent19used in the VFI format are purified. The capture agent19mAbs are needed to develop prototype antigen81capture assays, LFIs, and VPIs. Existing hybridoma cell lines are cultured in Integra bioreactors (Integra systems) containing hybridoma growth media. Hybridoma supernatant fluid will be routinely collected and mAbs will be purified by affinity chromatography on protein-A columns. Fresh vials of hybridoma cell lines will be periodically woken up to ward off negative effects that can occur as a result of genetic drift and antibody subclass is routinely monitored. Each lot of capture agent19mAbs will undergo rigorous QC testing that includes enzyme-linked immunosorbent assay (ELISA), Western blot and surface plasmon resonance (SPR−/Biacore×100) assays to confirm capture agent19mAb performance. SPR assay allows for a thorough evaluation of mAb affinity that will include determination of kinetic binding data (on- and off-rates). Briefly, purified bacterial biomarkers are immobilized to a Biacore sensor chip. Samples (two-fold serial dilution of mAb ([333-5.2 nM]) are injected over the sensor surface for 60 s, after which the capture agent19mAb is allowed to passively dissociate. BIA evaluation software is used to determine the Ka (on-rate), Kd (off-rate) and KD(equilibrium dissociation constant or “affinity”). The dissociation constant (KD) is determined using the steady-state model in BIA evaluation software. The detection mAbs are conjugated to horseradish peroxidase (HRP) for ELISA, or GNP12and GNS for LFI and VPI using either passive absorption or functionalized surface chemistry. The conjugated reagents also undergo quantification and QC methods that include validation in ELISA or LFI format. Purification of biomarkers and preparation of bacterial isolates: Milligram quantities (≥20 mgs) of each biomarker (bacterial antigen81) are purified and used. This is required to test the performance of each of the prototype LFIs and VPIs that are developed. Purification ofB. pseudomalleiCPS,B. anthracisPGA andF. tularensisLPS will require growth of the pathogen (in some cases select agent exempt strains may be used). Biomarkers expressed byY. pestiswill be expressed by recombinant methods. In addition, heat or chemically inactivated whole cells and whole cell lysates of each bacterial pathogen will be produced. The work will be performed in a BSL3 laboratory. The recombinant proteins and bacterial preparations are used to determine the LOD (pg/ml and cfu/ml) of the VFI for each biomarker. Prototype singleplex LFI development: Prototype singleplex LFIs are produced and used to compare with corresponding VFIs. The goal for the VFI is to achieve at least a 10-fold improvement in LOD. Prototype LFIs forB. anthracis, B. pseudomalleiandB. malleiare currently in-hand. Prototype LFIs forF. tularensisandY. pestisusing biomarkers LPS, LcrV and F1 are currently being produce for this this project. A BioDot 3050 will be used to fabricate the prototype LFIs. Each component and parameter of the LFI will be optimized based on traditional methodology, including the sample pad, sample volume, chase buffer, conjugate pad, mAb labeling, membrane, test line mAb, absorbent pad etc. Analytical sensitivity/limit of detection (LOD) will be determined for each prototype LFI assay. In addition, purified biomarkers and whole bacterial cells will be spiked into relevant sample matrices (e.g. blood, urine) and LOD will be determined. Performance results are compared to corresponding VPIs. Both LFIs membranes and VFI membranes3are imaged with the same image analysis algorithm. Optical Detection Agent Development To optimize the optical properties of the VFI gold nanoparticles12labeling, the main task will focus on optimizing the fabrication processes. Synthesis of surfactant and capping agent-free GNS by a seed-mediated approach has been described (Yuan, Khoury, et al., 2012). This method of GNS synthesis is very simple in comparison to other nanoparticle syntheses that it only takes about 10 seconds to complete while allowing excellent fine tuning of the morphological features and the optical absorption (localized surface plasmon) position (Yuan, Khoury, et al., 2012; Yuan, Fales, et al., 2012; Wang, et al., 2016; Indrasekara, Meyers, et al., 2014; Liu, Ashton, et al., 2015; Indrasekara, Johnson, et al., 2018). Briefly, rapid sequential addition of AgNO3(shape directing agent) and ascorbic acid (reducing agent) to an acidic mixture of polycrystalline spherical gold nanoparticles (gold seeds) and gold chloride instantaneously yield GNS (FIGS.43A and43B).FIG.43Ais a schematic representation of the GNS synthesis.FIG.43Bare TEM and 3D modeling images (scale bar 50 nm) of GNS formed under varying AgNO3concentrations: (S5: 5 uM, S10: 10 uM, S20: 2 uM, S30: 30 uM). As-synthesized GNS can either be stored at 4° C. until further use or can be coated with polyethylene glycol to improve the colloidal stability and further functionalization purposes. Ability to produce batches of gold nanoparticles12and coupling with Abs: When being used in the field, the disclosed VERIFAST device1processing comprises two major steps. The first step is to flow the sample through the membrane3so that the antigens81can bind to the capture agent19antibody. The second step is to flow a washing buffer through the membrane3to remove excessive nanoparticles12and non-specific loose-bond antigens81. We can use a three-way valve system163(schematic of a proof of concept actuator is shown inFIG.44A) to deliver the two liquids to the membrane3. Compare with the current laboratory practice, in which users change syringe6to switch liquid, using a valve system163offers several benefits. It is easy to operate. It also eliminates the air bubble issues because of the pressure drop when switching syringes6. It makes a liquid closed system to reduce the user's exposure to the potentially hazardous samples. In our current laboratory setup, a syringe6pump31(uses 120V AC power supply) is used to push the liquids through the membrane3of the prototype device1. Considering the limited power supply in the field, we propose to use alternative methods that do not require external power supply to actuate the liquids.FIG.44Bis a prototyped design using a gas syringe4to push liquid through the membrane3. Our preliminary test of a 15-lb gas syringe4has shown the ability to flow reagents within the desired timeframe. A more miniaturized and simplified liquid actuation method will be integrated to the VERIFAST system platform. Si membrane support21fabrication: Si membrane support21is an important component of the VFI device1that facilitate uniform signals across the membrane3. Currently, it is fabricated by semiconductor microfabrication techniques. First, photolithography is used to pattern a resist layer on a double-side-polished Si wafer. Then the resist is thermally harden in an oven or on a hotplate to serve as a mask for deep reactive ion etching (DRIE) of the Si wafer to make anisotropic through-hole arrays in the substrate. Mask design, resist spinning, photolithography and resist baking may be done in a cleanroom. More supports21per Si wafer will lower the unit cost of the support21, but it could also cause the wafer to break because more through-holes are etched in the wafer). Once the optimal design is reached, the support21fabrication will be scaled up for production of the prototyping devices1. Array Printing with Replicates on Miniaturized Membrane:FIGS.45A-45Cillustrate aspects of membrane3array printing for the present example.FIG.45Aillustrates the surface area81of the nitrocellulose membrane3in a VFI device1design (ϕ=3.5 mm; the gray region of the membrane3is covered by the o-ring77). The flow through region on the Si support21with etched through holes is within a circle of 2 mm diameter. FIG.45Bis a design of the multiplex array of spots79. Using micro-solenoid dispenser, the minimum droplet volume is 1 nanoliter, which yields a spot79size of 230 μm. We are able to fit up to 6 detection spots79inside the flow through area81, only one for each antigen81. However, in order to improve the accuracy and reliability of the test, duplicate or even triplicate spots79for each antigen81is desired.FIGS.45C and45Dis a design of the multiplex microarray of spots79for five antigens81with duplicates and triplicates respectively. In order to fit in these extra detection spots79within our miniaturized membrane3, the spot79size and pitch distance both need to be reduced. Based on the spot79size, we can calculate the desired droplet volume is about 450 picoliter. Current micro-solenoid dispenser is not capable of doing this picoliter dispensing. A piezo-dispenser is can achieve such small droplet size and improve the dispensing spatial resolution. With a piezo liquid dispenser, different parameters are tested to achieve the desired spot79size and pitch distance. A protocol is developed for printing multiplexed cAb array on the center of the miniaturized membrane3. A procedure for low volume production of the multiplexed cAb array membrane3is also developed. Briefly, multiple miniaturized membranes are cut from a large membrane3sheet by CO2laser, then the small membranes3are loaded into a rigid membrane holder for array printing. The membrane3holder can be fabricated by CNC machining of polycarbonate or other hard plastics. The rigidity of polycarbonate allows precise alignment of the dispenser to the small membranes3. Sample Preparation and Assay Optimization in Sample Matrices: Sample preparation is a significant part of any assay for real world applications. Sample preparation modules and protocols suitable for PON settings (e.g. in <5 min timeframe) for multiple sample matrices, such as plasma and urine, are desired. The quality of the prepared sample may be characterized to ensure minimal loss of antigen and similar assay analytical sensitivity to benchtop procedures. Plasma extraction cartridge for blood: The selected biomarkers pathogens are bacterial antigens in patient's circulation system for rapid disease diagnosis (Nuti, et al., 2011). Plasma/serum are common samples for pathogens immunoassays. Traditional, plasma/serum are prepared by centrifugation of whole blood and collecting the cell-free supernatant. However, it is not realistic to use bulky, power-demanding centrifugation in a PON setting. A simple and rapid plasma/serum generation method is critically needed. Among blood components such as plasma and serum, the plasma can be faster due to the fact it does not require coagulation of the clotting factors, and is usually the choice for PON applications. Due to the importance of plasma separation from whole blood, a number studies to achieve this have been reported, including using electric force (Jiang et al., 2011), acoustic force (Lenshof, et al., 2009), hydrodynamics (Kim et al., 2017), structured (Kuo, et al., 2015) or membrane filtration (Gong, et al., 2013). Among those techniques, membrane based filtration is the simplest form. In membrane based filtration, a commercial asymmetric polysulfone membrane (e.g. Vivid Plasma Separation Membrane, Pall) can be used to separate plasma from whole blood in as fast as 2 minutes.FIGS.46A and46Billustrates a basic structure of a membrane-based plasma extraction device165(FIG.46A), and a fabricated finger actuated plasma extractor167(FIG.46B). As shown inFIG.46A, the membrane169is sandwiched before a top plate171and a bottom plate173. The top plate171has an inlet175and a chamber177with supporting structures179to accommodate a blood sample. The bottom plate173has capillary structures181to extract plasma from the membrane169. The plasma outlet183is another capillary structure that can pump out the extracted plasma. The plasma separation extractor device (165,167) have been fabricated and tested for biodosimetry. The knowledge gained can be leveraged for this task. The geometry and surface tensions of both top171and bottom173-plates are investigated to achieve fast plasma extraction with minimal sample loss. Currently the membrane169has a limited whole blood capacity of <50 μL/cm2. However, by scaling up the membrane area81, handling of milliliter-scale blood in the field has been reported (Gong, et al., 2013). Further scaling up will be explored using 3D structures, e.g. stacked membranes169. Moreover, cell sedimentation can be used to improve the speed and throughput of plasma extraction (Dimov, et al., 2011; Galligan, et al., 2015). For example, the basic structure ofFIGS.46A and46Bcan be flipped upside down with a superhydrophobic blood chamber177at the bottom to improve the filter capacity by cell sedimentation. About 6.5×better membrane169capacity has been reported in a similar device (Liu, Liao, et al., 2016). Other phenomena, such as pressure/vacuum and aggregation of red blood cells by isotonic dilution are also exploited to achieve the goal of extracting several milliliters of plasma in under ˜5 min. Finally, the quality of the extraction plasma is characterized using spike recovery experiments for all five antigens81and compared to the benchtop procedures. Antigen81loss and change of analytical sensitivity are used to guide the optimization of the devices/protocols. Urine is another common sample matrix for the VFI device and applications utilizing the VFI device. Different from blood sample, urine does not contain large amount of cells that need to be separated, and can be used directly with any of the disclosed VFI devices. However, unlike well-regulated plasma, urine has a large variable composition. The pH can vary between pH 4.5-8.0, osmolality from 50-1300 mOsm/kg, and urine specific gravity (USG) from 1.005-1.030 (Sviridov, et al., 2009). We do show that pH can affect the assay result, and ionic strength of <0.15 can increase the signal background. However, it was also reported that pH variation did not affect much detection of IL-6 in dog urine (Wood, et al., 2011). These effects will be further studied in human urine matrix (see Assay optimization in sample matrices). If needed, sample pH can be tested using pH strips and neutralized by HCl or NaOH. The ionic strength can be increased by adding NaCl. Plasma matrix optimization: It has been reported that plasma or serum can have strong inhibitory effects on antigens in immunoassays (Tate, et al., 2004; de Jager, et al., 2009). Spike and recovery experiments have shown the recovery rate in serum/plasma as low as 1% for certain antigens (Rosenberg-Hanson, et al., 2014). This is also observed in our VFI assay.FIG.47shows 25 ng/ml LcrV spiked into either buffer or serum. Plotted are signals of spiked 25 ng/mL LcrV in buffer and serum showing strong matrix inhibitory effect. The signal from serum sample is significantly less than that of buffer, even with a 4× higher GNP12concentration. However, dilution of plasma can partially alleviate this matrix inhibitory effect, and it is even suggested that slightly diluted sample is preferred than undiluted sample (Rosenberg-Hanson, et al., 2014). On the other hand, we also experienced membrane clogging when flowing undiluted serum sample through the smallest 0.1 μm nitrocellulose membrane. Both observations suggest a dilution of plasma samples for use in a VFI device. Nonetheless, smaller dilution factor is still preferred to avoid significant impact on assay sensitivity. Due to the complexity of the matrix effects, we use Design of Experiment (DOE) for optimizing the VFI device1assays in plasma matrix. The critical parameters are: (1) plasma dilution factors; (2) membrane3pore size; (3) flow23speed; (4) GNP12-dAb10concentration. First, a screening experiment is conducted to determine which of the four factors have a significant effect on the assay performance. The goal of this phase of experimentation is to eliminate design factors that are not having a meaningful impact on the response. The second experiment is a response surface-type design, where the true relationship between the design factors and their interactions is determined, to facilitate factor level optimization. Membrane blocking and suitable dilution buffer is also tested out separately beforehand. Furthermore, we previously dedicated 10 min for sample and gold nanoparticle12-detection antibody10premixing, and the sample running time was another 10 min. In this example, we adopt a new approach to run the sample immediately after a short mixing time. This way, the sample running time is close to 20 min to double the sample passed through the sensor while still allowing liquid antigen-antibody reaction in solution at the same time. Each antigen is optimized individually because dilution of plasma samples is not linear and its effect on different antigens can vary from antigen to antigen. As mentioned previously, human urine varies greatly in terms of pH (4.5-8.0), osmolality (0.05-1.3 Osm/kg), and USG 1.002-1.030 (Sviridov, et al., 2009; Cook, et al., 2000). It also contains a complex mixture of proteins and high amounts of urea, hippuric acid, etc. relative to plasma (Sviridov, et al., 2009). Inhibitory effect by urine matrix was also reported with some interesting observation that higher USG improved recovery of certain cytokine (Wood, et al., 2011). It would be a significant endeavor to fully study the effects of urine matrix on VFI. Accordingly, initial efforts study the effects of pH and ionic strength of urine sample. The pH and ionic strength of purchased urine sample is measured by pH meter (or pH strip) and conductivity meter. Then the pH and ionic strength can be adjusted by titration of acid/based and NaCl, and VFI can be performed for spiked antigens81under different pH, ionic strength conditions. Besides the pH and ionic strength, other relevant parameters such as dilution factor, membrane pore size, flow speed and GNP12-dAb10concentration is also tested for assay optimization using similar DOE strategies to that in plasma matrix study. The USG and osmolality data is requested from the urine supplier and monitored during the project. The results influence the final sample preparation strategy for the VFI assay with the disclosed device1. Demonstration of Specimen Collection & Processing Modules: The experimental approach for the platform optimization will be sequential. Typically, a screening experiment will be conducted to determine which of the selected factors may have a significant effect on the assay performance. Then, a response surface-type design, where the true relationship between the design factors and their interactions can be determined, will be applied to facilitate factor level optimization. For the screening phase, a definitive screening design (DSD) will likely be used, as a DSD serves as an efficient screening design while also providing some attractive correlation properties and the ability to fit an adequate statistical model to find efficiencies in the second phase of experimentation. Depending on the outcome of the screening phase either an augmented DSD or a traditional response surface design such as a central composite design will be used. Software is required to reliably analyze the images acquired from the VFI membranes3. Existing software algorithms (CK Point #2.4) will examine the image, locate the test pattern, orient the image as required, then analyze the dot pattern to determine the values of the signal spots79compared to background values to determine if the target is detected. This software will monitor a selected Cloud location for any incoming images. When they arrive, the program opens the image, analyzes it, then moves it to a secure location for archiving, and reports the results. This configuration will be re-programmed for accommodating its conversion onto a mobile handheld device platform (e.g. smartphone51or equivalent electronic appliance) within the data security and communications protocols and requirements (e.g., as established by the defense community). Application for Electronic Appliance (e.g. Smartphone51) Software can facilitate a reliable and easy-to-use end user interface. An app designed for the military approved smartphones51(e.g. Nett Warrior network with iPhone and Galaxy devices) can obtain the required 48-bit image of the test membrane3using the CCD phone51camera53. The app can then send the image to a military-approved network location to be analyzed by the existing software, or the app can be written so that the analysis would occur directly on the smartphone51, with the results being sent to the military approved network location. The sample size for each of the primary five pathogens categories will be determined based on the table below, which shows the relationship between sample size and 95% confidence interval for a number of estimated sensitivities and specificities. Numberofinfected(non−infected)subjectsEstimated test sensitivity (or specificity)**required*50%60%70%80%90%95%5013.9%13.6%12.7%11.1%8.3%6.0%1009.8%9.6%9.0%7.8%5.9%4.3%1508.0%7.8%7.3%6.4%4.8%3.5%2006.9%6.8%6.4%5.5%4.2%3.0%5004.4%4.3%4.0%3.5%2.6%1.9%10003.1%3.0%2.8%2.5%1.9%1.4%*As defined by the reference standard culture test**95% confidence interval around the estimated sensitivity/specificity (+/− value in table) The goal of field testing is to precisely establish the performance metric of the VERIFAST PON test. The larger the collected samples number, the better precision, or narrower the 95% Cl width, will be obtained for both the sensitivity and specificity of the multiplex test. As an example, if we would achieve a PON test sensitivity of 90%, with respect to the reference standard lab-grown culture test, by collecting 500 subjects then the 95% Cl will be about +/−2.6%. Similarly, a specificity of 70% derived from recruiting1000uninfected samples will have a 95% Cl of +/−2.8%. This metrics should provide a mean to estimate the number of patient or nonhuman primate (NHP) samples per category that will be needed after delivery of the devices to the sponsor for validation studies. Example 10: Detection of Nucleic Acids and Biologics Any of the systems and processes described herein may be used to detect nucleic acids and, more generally, biologics. For example,FIGS.48-50demonstrate the usefulness of the vertical flow detection device to detect a range of biologics, including those corresponding to a biothreat.FIGS.48and49illustrate capture agents connected to the membrane that are antibodies specific to F1 (plague) and LPS. Photographs of the VFI membrane are provided on the right having control spots and detection spots.FIG.50summarizes detection results for four Tier I biothreats. FIG.51is a summary of one process for obtaining nucleic acid from cells. RNA can be extracted from a cell line and, as desired, amplification of a target may occur, including by standard RT and standard PCR. Samples may then be introduced to any of the VF systems described herein. AlthoughFIG.51illustrates a cell culture, the systems and methods provided herein are compatible with a range of sample starting materials, including but not limited to, a tissue biopsy or biological fluid.FIG.52is a schematic illustration of detection of nucleic acid using any of the systems described herein. A capture antibody is connected to the membrane that is specific for a target nucleic acid sequence. A detection antibody can be used to detect the presence of a target nucleic acid sequence bound to the capture antibody. The systems and methods provided herein have high specificity. For example,FIG.53illustrates detection of CDKN1A using vertical flow for a positive sample (CDKN1A amplicon diluted in 1 mL buffer solution) at the nucleotide spot (left most bar) and at the negative spot (right bar). No statistically significant colorimetric signal is detected for the negative sample (1 mL buffer solution). FIG.54summarizes results for detection of CDKN1A and HIST1H3D using a vertical flow device. The system has high specificity for the desired target as illustrated by the negative sample and the samples with only one of the two targets present. The systems provided herein are useful for detection of cells exposed to radiation.FIG.55top panel summarizes an irradiation protocol, where cells are not exposed (0 Gy) or are exposed (5 Gy) to radiation at a dose of 3 Gy/min. RNA is extracted and amplified and the resultant amplicons run on an agarose gel (bottom left panel) or on a vertical flow device. No statistical difference is observed for the agarose gel, whereas as a meaningful difference with radiation is reliably detected by the vertical flow device and method. This indicates that the methods and devices provided herein are highly sensitivity and specificity, making them useful for a range of applications. For example, the number of genes able to be detected by the membrane can be increased. Further quantification is achieved by using, for example, one or more housekeeping genes to facilitate normalization. One example using four genes is: (1) CDKN1A (reverse: biotin); (2) HIST1H3D (reverse: digoxigenin); (3) DDB2 (reverse: dinitophenyl); (4) housekeeping gene MRPS5 (reverse: Cy3). Similarly, multiplexing for protein detection is compatible by use of different capture antibodies, each specific for relevant protein or portions thereof. Examples of protein detection include detection ofB. pseudomalleiby targeting the capsular polysaccharide (CPS). Other intra-cellular protein biomarkers (e.g. lymphocyte proteins FDXR, BAX, DDB2 and ACTN1) can also be interrogated with a vertical flow device of the present invention. The vertical flow devices and systems are also compatible with a sample that is a blood sample. Sample preparation, upstream of introduction to the membrane, can be simplified, such as by using isothermal recombinase polymerase amplification (RPA). The systems are compatible with various form factors, including in a fluidic cartridge and can be integrated into various systems. For example, fluidic pumps such as syringe pumps can be replaced with a small, light, self-powered system that is transportable. General requirements include a total volume of about 2 mL to 5 mL and constant flow in the range of 0.2-0.5 mL/min. Examples can include simple and robust systems that is non-powered and capable of applying a constant load, such as a constant force spring and/or ball slide to reduce friction and flow variability. 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Modeling and optimization of high-sensitivity, low-volume microfluidic-based surface immunoassays. Biomed. Microdevices 7, 99-110. doi:10.1007/s10544-005-1587-y STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS All references throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material; are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference). The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. The specific embodiments provided herein are examples of useful embodiments of the present invention and it will be apparent to one skilled in the art that the present invention may be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps. When a group of substituents is disclosed herein, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated. Whenever a range is given in the specification, for example, a size range, a number range, a pore size range, a porosity range, a thickness range, LOD range, a temperature range, a time range, a flow-rate range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. For example, when composition of matter are claimed, it should be understood that compounds known and available in the art prior to Applicant's invention, including compounds for which an enabling disclosure is provided in the references cited herein, are not intended to be included in the composition of matter claims herein. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. | 194,791 |
11860161 | DETAILED DESCRIPTION OF THE INVENTION Several embodiments described herein are related to the identification, amelioration, and/or treatment of a wide variety of autoimmune or immune related diseases or disorders including, for example, multiple sclerosis, Crohn's disease, SLE, Alzheimer's disease, rheumatoid arthritis, psoriatic arthritis, enterogenic spondyloarthropathies, insulin dependent diabetes mellitus, autoimmune hepatitis, thyroiditis, transplant rejection and celiac disease. Some embodiments concern the use of a PD-L1 leukocyte expression assay to identify the presence, absence, or progression of an autoimmune disease, for example. More embodiments relate to the use of a PD-L1 agonist or ligand to ameliorate or treat an autoimmune disease. Some embodiments concern methods, wherein a PD-L1 ligand is provided to a patient that has been identified as having an autoimmune disease, such as SLE, and the general health or welfare of the patient is improved during the course of treatment. Optionally, the improvement in said patient is monitored or measured before, during, or after administration of said PD-L1 ligand using conventional clinical evaluation or observation, analysis of diagnostic markers for the disease, or by using one or more of the diagnostic techniques described herein. Active SLE is associated with failure of antigen presenting cells to upregulate programmed cell death ligand-1. Antigen presenting cells (APC) maintain peripheral T cell tolerance in part via expression of negative costimulatory molecules such as programmed cell death ligand-1 (PD-L1). APC in peripheral blood, including CD14+/CD11c+ monocytes (Mo) and CD14-/CD11c+ myeloid dendritic cells (mDC), have been implicated in the pathogenesis of SLE. Patients with active disease generally have decreased numbers of Mo in peripheral blood mononuclear cells, and their APC generally fails to upregulate PD-L1 appropriately when cultured ex vivo, as measured by flow cytometry. APC from healthy individuals or SLE patients in remission tend to upregulate PD-L1 surface expression by day one, with peak expression on day two or three, and declining expression through day six, all in the absence of exogenously-added stimuli. Therefore, failure of APC to upregulate PD-L 1 correlates with abnormal T lymphocyte regulation and loss of peripheral tolerance in SLE. Programmed death ligand-1 (PD-L1; also known as B7-H1/CD274), is a B7 family glycoprotein inducibly expressed on many hematopoietic and parenchymal cells in response to inflammatory stimuli. It regulates immune tolerance by binding to the programmed death-1 (PD-1) receptor on lymphocytes, causing suppression of T-effector function, and permissiveness of regulatory T-cell function. PD-L1 may also suppress T-cell activation by signaling through the B7-1 receptor. Although mRNA for PD-L1 can be found in many healthy human tissues, baseline protein expression appears to be limited to cells of monocytic origin. Both myeloid dendritic cells (mDC) and monocytes (Mo) express PD-L1 protein, and anti-PD-L1 antibody increases the stimulatory capacity of mature and immature DCs for T-effector cells. Endogenous or transgene-driven expression of PD-L1 on antigen presenting DCs leads to diminished T-cell reactivity in vitro and in vivo, as demonstrated in murine models of autoimmunity. The importance of PD-L1 in self-tolerance has also been demonstrated in experimental animals in which blockade or absence of the PD-L1:PD-1 pathway results in various forms of autoimmune disease, including a spontaneous lupus-like glomerulonephritis in C57BL/6 mice. The receptor for PD-L1 is shared by a second ligand, PD-L2, (B7-DC/CD273), which can also inhibit T-cell activation, but is less widely expressed and appears to play some non-redundant roles in self-tolerance. DNA polymorphisms in the gene for the shared PD-1 receptor have been linked to SLE susceptibility in some populations of adults and children; however, T-cell expression of PD-1 protein has not been found to differ significantly between SLE patients and controls. In contrast to the PD-1 gene studies, genetic polymorphisms in PD-L1 did not appear to be linked to SLE. However, both immature mDC and Mo from children with SLE failed to up-regulate PD-L1 normally, and this deficiency was associated with increased disease activity, indicating an important role for this negative co-stimulator in the pathogenesis of SLE. As discussed above, PD-L1 on antigen presenting cells binds to PD-1 on T lymphocytes, and regulates their activity. Animals without PD-1 develop an autoimmune disease similar to SLE, with T lymphocytes reacting to self proteins. Some embodiments relate to the discovery that patients with active SLE express almost no PD-L1 on their antigen presenting cells. The same patients, when their disease is in remission, express PD-L1. Accordingly, some embodiments concern methods to identify the presence, absence, or progression of an autoimmune disease, such as SLE, in a subject that has been identified as having an autoimmune disease or a subject identified as being at risk for developing an autoimmune disease, wherein the presence, absence, or amount of PD-L1 in a biological sample from said subject is analyzed, detected, or determined. In some embodiments, such assays are performed by staining peripheral blood lymphocytes obtained from a subject with a fluorescence-labeled antibody specific for PD-L1, along with antibodies to cell surface markers for monocytes and dendritic cells (CD11c and CD14). Optionally, the frequency of cells expressing PD-L1 or the amount of PD-L1 on a subject's peripheral blood lymphocytes in the sample is detected using flow cytometry, ELISA, or other immunological detection techniques. Thus, some embodiments include methods to identify the presence, absence, or likelihood to acquire an autoimmune disease, such as SLE, wherein a molecule that specifically binds to PD-L1, such as an antibody, binding partner for PD-L1, or a binding fragment thereof (e.g., an identifiable ligand for PD-L1), is contacted with a biological sample obtained from a patient (e.g., blood) or a component isolated therefrom (e.g., a peripheral blood lymphocytes) for a time sufficient to create a PD-L1/binding partner complex and the presence, absence, or amount of said PD-L1/antibody or binding partner complex is measured or detected, which then indicates the presence, absence, or likelihood to acquire the autoimmune disease. The assays described above may be used to assess the efficacy of a treatment regimen or the progression of a treatment protocol or the progression of an autoimmune disease. Other embodiments relate to methods to identify individuals that are at risk for developing an autoimmune disease, or individuals that are at risk for relapse of a preexisting autoimmune disease. The identifiable ligand for PD-L1 or PD-L1 binding partner may be an antibody (e.g., a monoclonal antibody or a polyclonal antibody, which may be humanized or modified) or a fragment of an antibody that binds to a PD-L1 antigen. Polyclonal and monoclonal antibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., (1988). Antigen-binding fragments of such antibodies, which may be produced using conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab, F(ab′), and F(ab′)2fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided. The monoclonal antibodies may be chimeric antibodies, e.g., humanized versions of murine monoclonal antibodies. Such humanized antibodies may be prepared by known techniques. In some embodiments, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature332:323, 1988), Liu et al. (PNAS84:3439, 1987), Larrick et al. (Bio/Technology7:934, 1989), and Winter and Harris (TIPS14:139, May, 1993). The identifiable PD-L1 ligand or binding partner for PD-L1 may also be a peptide or peptidomimetic that binds to PD-L1. Peptides and peptidomimetics that bind to PD-L1 can be identified using computer modeling of the binding regions of antibodies that interact with PD-L1 and identifying peptides and peptidomimetics with similar structures. Peptides and peptidomimetics that bind to PD-L1 can also be identified by screening detectably labeled PD-L1 against libraries of peptides and peptidomimetics and determining the presence of detectably labeled PD-L1/binding partner complexes. Alternatively, detectably labeled peptides and peptidomimetics can be screened against PD-L1 and the presence of detectably labled binding partner/PD-L1 complexes can be identified. Preferably, the ligands for PD-L1 (e.g., an antibody, a PD-L1 antigen-binding fragment thereof or PD-L1 ligand) is detectably labeled. The label may be colorimetric, fluorescent, a radioisotope, or a metal. More embodiments relate to the use of PD-L1 or a nucleic acid encoding PD-L1 as a therapeutic to regulate T lymphocytes in patients suffering from an autoimmune disease, such as SLE. In some embodiments, for example, PD-L1 is provided to or administered to a patient that has been identified as having an autoimmune disease, such as SLE, and the presence, absence, or progression of the autoimmune disease or a marker thereof is measured or detected using clinical evaluation or diagnostic assay. In more embodiments, a nucleic acid encoding PD-L1 (e.g., DNA or RNA), preferably a nucleic acid that has been codon optimized for expression in humans is provided or administered to a patient that has been identified as having an autoimmune disease, such as SLE, and the presence, absence, or progression of the autoimmune disease or a marker thereof is measured or detected using clinical evaluation or diagnostic assay. Some additional embodiments relate to methods of inducing or increasing PD-L1 expression in patients. In some embodiments, for example, caspase inhibitors can be administered to a patient with autoimmune disease in order to induce or increase PD-L1 expression in the patient, thereby treating or preventing the autoimmune disease or ameliorating the symptoms of the autoimmune disease. Such inducement or increase can be achieved by administering to the patient an amount of at least one caspase inhibitor in an amount sufficient to induce or cause an increase in the expression of PD-L1 by a patient's cells. In some embodiments, the caspase inhibitors can be combined with one or more therapeutic capable of treating or preventing the autoimmune disease or ameliorating the symptoms of the autoimmune disease. In certain embodiments, the caspase inhibitors can be specific caspase inhibitors, pan caspase inhibitors, poly-caspase inhibitors or a combination thereof. In some embodiments, the caspase inhibitors are, for example, at least one of Z-WEHD-fmk, Z-VDVAD-fmk, Z-DEVD-fmk, Z-YVAD-fmk, Z-VEID-fmk, Z-IETD-fmk Z-LEHD-fmk, Z-AEVD-fmk, Z-LEED-fmk, Z-VAD-fmk and OPH. However, the current embodiments are not limited to such examples and encompass any pharmaceutically acceptable caspase inhibitor. More embodiments relate to methods of inducing or increasing PD-L1 expression by the cells of a patient by removing cells from the patient and exposing the cells to at least one caspase inhibitor ex vivo in order to induce or increase the expression of PD-L1 on the cells, washing the cells and then administering the cells to a patient. In some embodiments, an additional therapeutic can be combined with at least one caspase inhibitor that is either administered to the patient in vivo or exposed to cells of the patient ex vivo to induce or increase PD-L1 expression by the cells. Examples of such additional therapeutics include therapeutics targeting HLA molecules, CD18, CD2, CD4, CD28, Fc-gamma 3 receptor, Fc gamma receptor 2a, CTLA4, or TGF-b. EXAMPLE 1 PD-L1 Protein Levels Downmodulated in Control Mo Using Specific siRNA In this model, siRNA technology was used to diminish mRNA for PD-LI. Normal PBMC were obtained from healthy volunteers and frozen in liquid nitrogen. At the time of experiments, cells were thawed, washed, and diluted to 1-2×106cells/ml in culture medium consisting of RPMI 1640 supplemented with L-glutamine (CellGro), 10% heat-inactivated AB human serum, 1% penicillin/streptomycin (CellGro), and 0.1% beta-mercaptoethanol. Cells were plated in round-bottom 96-well plates (Corning Costar) and incubated at 37° C. in a humidified cell chamber with 5% CO2. After a four-hour rest period, total PBMC were incubated with Mo nucleofection buffer (Amaxa) plus specific anti-human PD-L1 siRNA or control siRNA (sc-39699, Santa Cruz Biotechnology, Inc.) and nucleofected as per the manufacturer's protocol (Amaxa). PBMC were returned to the incubator and cell surface PD-L1 levels determined by flow cytometry 24 hours later. Cells were surface-stained using various fluorochrome- or biotin-conjugated monoclonal antibodies, including: anti-CD3, anti-PD-L1 (eBioscience), anti-CD11c, and anti-CD14, (Pharmingen/BD Biosciences), with isotype-matched, fluorochrome-/biotin-labeled irrelevant monoclonal antibodies as controls. Some cells were cultured for longer time periods to assay the level of intracellular proteins (cytokines and Foxp3). To determine intracellular caspase activity, selected cultures were incubated with a cell-permeant fluorochrome-derivative of the appropriate caspase inhibitor for each caspase under investigation. Prior to staining for intracellular cytokines, cells were restimulated for four hours with 1 ug/ml phorbol myristate acetate (PMA) plus 14 uM ionomycin, in the presence of GolgiStop (BD Biosciences) to prevent cytokine secretion. Cells were permeabilized with the appropriate proprietary buffers according to the manufacturer's protocols (BD Biosciences, eBioscience, or BioLegend), and intracellular cytokine production was assayed using fluorochrome-conjugated monoclonal antibodies to interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-17 (IL-17) (Pharmingen/BD Biosciences). Prior to staining for Foxp3 (BioLegend), cells were permeabilized, but not restimulated. All samples were blocked using 0.5% human serum and anti-FcR antibody (Miltenyi) during staining. Dead cells were excluded from the analysis using a dead cell marker dye. After staining, PBMC were fixed using 2% paraformaldehyde in PBS after preliminary experiments indicated no effect of cell fixation on expression levels of PD-L1 or other surface markers. Flow cytometry was performed using an LSR II cytometer (Becton Dickinson), and the data were analyzed using FlowJo software (Macintosh Version 6.3). Mo and immature myeloid DC (mDC) were identified using surface markers characteristic of each cell type. Results were compared between populations using Student's t-test, and significance assigned where p<0.05. In the siRNA studies it was confirmed that the distribution of leukocyte subtypes in PBMC were not altered by this experimental manipulation (Table I), and that the number of cells expressing PD-L1 also remained unchanged in these cultures (Table II). However, expression of PD-L1 protein on Mo and mDC was reduced by 24 hours (Table III), while the levels PD-L1 of on other PBMC subsets remained unchanged. TABLE ILeukocyte subsets in nucleofected PBMC from two healthy controls.Ancestry% Live of% CD14+% CD1c+% CD11c+% CD3+Subset Value Type ForLymphsof Liveof Liveof Liveof LiveMbr No Stain Control9900.020.030Mbr IgG Contral Control901.33.92958NOP511(2) d1 PDL1 High734.22.72552Control siRNANOP511(2) d1 PDL1 Low PDL1765.43.22954siRNANOP511(2) d1 PDL1 High PDL1745.53.53354siRNANOP505A(2) d1 PDL1 High661.32.32759Control siRNANOP505A(2) d1 PDL1 Low692.33.32756PDL1 siRNANOP505A(2) d1 PDL1 High631.33.42956PDL1 siRNANOP505A(2) d1 PDL1 GFP752.52.72757vector no zap PBMC from two healthy individuals were subjected to nucleofection using control siRNA or two different concentrations of PD-L1 siRNA and results compared with those in nonnucleofected control cultures to confirm that specific leukocyte subsets were not preferentially destroyed by this manipulation. Numbers represent percent of each cell type remaining in culture at the end of day one. TABLE IIPercent of each leukocyte subset expressing PD-L1 after nucleofection.Ancestry% PDL1+ of% PDL1+ of% PDL1+ of% PDL1+ of% PDL1+ ofSubset Value Type ForLiveCD14+CD1c+CD11c+CD3+Mbr No Stain0*00*ControlMbr IgG Control0.80.41.7260.8ControlNOP511(2) d1 PDL1 High7.2978.7154Control siRNANOP511(2) d1 PDL1 Low6.7917.5162.1PDL1 siRNANOP511(2) d1 PDL1 High7.2917.8172.7PDL1 siRNANOP505A(2) d1 PDL13.2757.67.32.2High Control siRNANOP505A(2) d1 PDL1 Low3.7642.78.92.4PDL1 siRNANOP505A(2) d1 PDL15.1677.2114.6High PDL1 siRNANOP505A(2) d1 PDL1 GFP36198.11.9vector no zap PBMC from two healthy individuals were subjected to nucleofection using control siRNA or two different concentrations of PD-L1 siRNA and results compared with those in nonnucleofected control cultures to confirm that the percent of cells in each specific leukocyte subset was not altered by this manipulation. Numbers represent percent of each cell type expressing PD-L1 in culture at the end of day one. TABLE IIIPD-L1 expression in nucleofected leukocyte subsets.AncestryPDL% MF1PDL1 MF1 ofPDL1 MF1 ofPDL1 MF1 ofSubset Value Type Forof CD14+CD1c+CD11c+CD3+Mbr No Stain Control*298113*Mbr IgG Control Control151240325172NOP511(2) d1 PDL1 High135787752398557Control siRNANOP511(2) d1 PDL1 Low PDL176996211741410siRNANOP511(2) d1 PDL1 High PDL170136901608299siRNANOP505A(2) d1 PDL1 High5405635864322Control siRNANOP505A(2) d1 PDL1 Low PDL13108255599332siRNANOP505A(2) d1 PDL1 High PDL13512420675434siRNANOP505A(2) d1 PDL1 GFP3582535640308vector no zap PBMC from two healthy individuals were subjected to nucleofection using control siRNA or two different concentrations of PD-L1 siRNA and results were compared with those in nonnucleofected control cultures to confirm that PD-L1 was specifically downmodulated in CD14+ Mo and mDC, while other cell types were unaffected. Numbers represent mean fluorescence intensity (MFI) of PD-L1 in of each cell type under various treatment conditions at the end of day one. Symbols represent **p<5×10−5, #p<0.033, and *p<5×10−4. In order to allow interaction of APC with autologous T cells, a culture of each PBMC sample was incubated for another four days. At the end of this time, PBMC were assessed for intracellular cytokine production in T lymphocytes as well as for expression levels of the regulatory T cell marker, Foxp3. Although Mo treated with siRNA expressed significantly lower amounts of surface PD-L1, production of IFN-γ, TNF-α, and IL-17, and the level of intracellular Foxp3 expression remained unchanged in T cells. The T cells examined in these studies were autologous cells, incubated and treated concurrently with the APC, thus (1) limiting the number and magnitude of the T lymphocyte response as compared to an allogeneic reaction, and (2) allowing the possibility that T cells in these cultures may have been affected by the nucleofection process itself, even though preliminary studies gave no indication of adverse effects on T cell survival or function. These experiments showed that the distribution of leukocyte subtypes in PBMC were not altered by this experimental manipulation (Table I), and that that the number of cells expressing PD-L1 also remained unchanged in these cultures (Table II). Neither of these parameters was altered, however, expression of PD-L1 protein on Mo and mDC were reduced by 24 hours (Table III andFIG.1), while the levels PD-L1 of on other PBMC subsets remained unchanged. EXAMPLE 2 PD-L1 Protein Levels Were Increased in APC by Inhibition of Caspase Activity PBMC were cultured as above and a duplicate well of each sample treated with 50 uM OPH. PD-L1 upregulation on APC was confirmed in these cultures, and an aliquot of each sample was incubated further in order to allow interaction of APC with autologous T cells in culture. Three days later, the cells were fixed and permeabilized, and the T lymphocytes were assayed for intracellular expression of the regulatory T cell protein, Foxp3. It was found that OPH increased PD-L1 expression on the surface of both Mo and mDC at day one. Identification of endogenous Treg in the PBMC cultures using Foxp3 protein revealed not only fewer Treg in lupus PBMC, but also less Foxp3 protein per cell, indicating that development and/or survival of Treg was abnormal in SLE. Although cultures treated with OPH expressed significantly higher amounts of PD-L1 on the surface of APC, the level of Foxp3 expression in both healthy control and SLE cultures remained unchanged, both with respect to Foxp3 MFI and to the number of CD4+cells expressing Foxp3. These experiments showed that diminished PD-L1 levels on lupus APC directly affect Treg development. EXAMPLE 3 PD-L1 Signaling was Inhibited Using Anti-PD-1 Antibodies Soluble anti-PD-1 antibody was utilized to block PD-L1 signaling in autologous PBMC cultures, and the effect on T cell cytokine production was evaluated after five days of incubation. It was found that soluble anti-PD-1 antibody at this concentration did not significantly affect intracellular production of IFN-γ, TNF-α, or IL-17 in T cells (seeFIG.8), but this may have also been due in part to the fact that the T cells were minimally stimulated, as they were responding to autologous APC. These experiments did demonstrate the ability to detect small changes in T lymphocyte cytokine production among total PBMC. PBMC from eight healthy individuals were incubated with autologous APC in the presence of soluble control IgG or anti-PD-1 antibody to assess the effect of blocking PD-L1 signaling on T cell cytokine production. After five days, CD4 and CD8 T cells were identified and assayed for the presence or absence of intracellular cytokines. These experiments showed that soluble anti-PD-1 antibody at this concentration did not significantly affect intracellular production of IFN-γ, TNF-α, or IL-17 in T cells. EXAMPLE 4 Caspase Activity Levels Were Measured in APC From Children With and Without SLE Cells were cultured as above, and PBMC were stained with Annexin V and propidium iodide (PI) (both from Becton Dickinson) to identify both dead and dying cells. After apoptotic cells were omitted from our analyses, the intracellular levels of caspases in living leukocytes were quantitated using fluorochrome derivatives of known caspase inhibitors which only bind at the active site (Immunochemistry Techologies, Inc.), thus revealing both caspase identity and activity level simultaneously in each individual cell. Using PBMC from five controls and two children with SLE, the mean fluorescence intensity (MFI) for each active caspase was measured, and results between controls and SLE compared using a 2-tailed t-test. Significance was assigned where p<0.05. Surprisingly, for all of the eight caspases tested, it was found that enzyme activity levels in non-apoptotic cells exhibited the pattern: Mo>mDC>other PBMC, indicating an important role for caspases in normal Mo function (seeFIG.9). However, when comparing control Mo to SLE Mo, it was found that among individual caspases tested, only the activity of caspase-13 was significantly different between the two groups. The activity levels of caspases 1, 2, and 9 also appeared to be higher among SLE Mo. These experiments showed the baseline expression of caspases and their activity, level in human cells. EXAMPLE 5 Individual Caspases Were Inhibited and APC Tested for Expression of PD-L1 This study examined the direct contribution of each caspase to the regulation of PD-L1 expression in vitro. Amino acids 70-180 of PD-L1 are shown inFIG.10, with potential caspase cleavage sites marked by the four arrows. Caspase numbers are listed above each site in order of likelihood of cleavage. In order to determine which caspase (or caspases) is responsible for downmodulation of PD-L1 during active lupus, the effects of specific caspase inhibitors on PD-L1 expression in human APC were tested (Table IV). Control and SLE PBMC were incubated for one day in the presence of the individual caspase inhibitors at doses ranging from 10 uM to 50 uM (R&D Systems), with the appropriate level of DMSO as the carrier control (0.5% final concentration). Treated cells were assayed for cell death and apoptosis using Annexin V and PI (example shown inFIG.4), as well as stained for cell-surface expression of PD-L1 on APC. TABLE IVSpecific caspase inhibitors and their targets.InhibitorCaspase targetZ-WEHD-fmk1Z-VDVAD-fmk2Z-DEVD-fmk3 (and 7)Z-YVAD-fmk4Z-VEID-fmk6Z-IETD-fmk8Z-LEHD-fmk9Z-AEVD-fmk10Z-LEED-fmk13Z-VAD-fmk or OPHPolycaspase inhibitor The inhibitors enter living cells and bind irreversibly at the caspase active sites, preventing further proteolytic activity by the enzyme. After incubation with caspase inhibitors, PBMC were gated by forward and side scatter and analyzed for percent cell death and apoptosis using Annexin V and PI (seeFIG.10). It was found that treatment of PBMC with the specific caspase inhibitors was less potent for reducing cell death and apoptosis than the poly-caspase inhibitors, OPH and Z-VAD-fmk. It was also found that treatment of PBMC with low doses of the caspase-specific inhibitors (10-25 uM) did not affect PD-L1 expression by APC. However, higher doses of some caspase-specific inhibitors (50 uM), did alter PD-L1 levels, both in Mo and mDC (example shown inFIG.11). Normal PBMC were incubated in medium with or without various caspase inhibitors, and PD-L1 protein levels assessed at day one. This graph shows the PD-L1 quantitation results for one set of control mDC treated with various inhibitors. Notably, using Method 1 above, it was observed that an elevation of active caspases 1, 2, 9, and 13 in SLE cells, with statistical significance demonstrated for caspase 13. In this set of experiments to determine the effect of these caspases on PD-L1 expression, we found that caspase-13 seemed to figure prominently in PD-L1 regulation (see Chart 6, under Z-LEED-fmk), confirming the usefulness of this multifaceted approach. Although the caspase-3 inhibitor (Z-DEVD-fmk) also greatly increased PD-L1 levels, caspase-3 acts as a master regulator of the caspase cascade and therefore it is not yet clear whether this enzyme acts directly on PD-L1 or via another caspase. Accordingly, these experiments demonstrate the direct contribution of each caspase to the regulation of PD-LI expression in vitro. EXAMPLE 6 Downregulation of PD-L1 Protein Expression in Normal Mo by Induction of Apoptosis Healthy human PBMC were cultured and half of each sample were exposed to pro-apoptotic conditions—in this case, to withdrawal of serum from the culture medium. After 24 hours, cells were surface stained as above to identify Mo and to measure PD-L1 protein expression. It was found that among normal PBMC cultured in the absence of serum, PD-LI protein levels dropped dramatically by 24 hours, as did the number of Mo expressing PD-L1 (Chart 7). The average PD-LI MFI on Mo was reduced by more than half, while the percent of Mo expressing this negative costimulator dropped by one third. This PD-LI profile observed in the context of serum withdrawal was remarkably similar to that obtained using cells from patients with active SLE, indicating that the loss of PD-L1 in lupus APC is indeed due to heightened caspase activity in these cells. The effect of various caspase inhibitors on PD-L1 expression in normal APC was also tested, as healthy cells can be made “lupus-like” by subjecting them to pro-apoptotic conditions (seeFIG.12). Normal PBMC were cultured in standard medium containing serum (med), medium without serum (serum-) to increase apoptosis, or in medium containing 50 uM of poly-caspase inhibitor (OPH) to decrease apoptosis. This system was used to test the effects of other pro-apoptotic conditions on caspase activation and PD-L1 expression, to determine if different apoptotic signals regulate PD-L1 differently. Such conditions include: UV irradiation, Fas:FasL signaling, and heat shock, as well as testing the direct effects of SLE serum on APC, as it has very recently been demonstrated that incubation of healthy leukocytes with lupus serum induces “classical” caspase-dependent apoptosis. These experiments provided evidence that the upregulation of caspase activity in normal cells should also lead to the downmodulation of PD-L1. EXAMPLE 7 Direct Cleavage of Human PD-L1 Protein in Vitro As shown above, there exist significant differences in caspase activity between control and SLE APC, as well the PD-L1-enhancing effects of polycaspase inhibitors on human PBMC. These findings indicate that PD-L1 is directly cleaved by one or more active caspases. Purified PD-L1 and control protein targets are incubated with individual caspases in the appropriate buffers under the conditions specified by the manufacturer. The resultant peptide products arc fractionated by SDS-PAGE. Protein fragments are identified by size and gel-purified for further identification. Any molecules of interest are sequenced to confirm or refute potential PD-L1 caspase cleavage sites. Caspases of interest identified in these experiments are combined with other caspases in PD-L 1 cleavage experiments, to determine whether a sequential or concurrent proteolysis of PD-L1 may occur during downmodulation of PD-L1 in living cells. We found that several caspases are capable of cleaving PD-L1 in vitro. EXAMPLE 8 Elevated Caspase Activity Inhibits Programmed Cell Death Ligand-1 Expression in Human Leukocytes and is Associated with Active SLE As discussed above, APC from patients with active SLE are deficient in PD-L1, but regain the ability to express this protein during disease remissions. Using flow cytometric analysis, the levels of endogenous caspase activity were measured in live APC from children with and without active SLE, and the effect of caspase inhibitors on expression of PD-L1 was tested. Active SLE was associated with excessive leukocyte apoptosis, which was inversely correlated with PD-L1 protein levels on APC. Treatment with caspase inhibitors not only reduced leukocyte apoptosis, but also significantly increased expression of PD-L1 on both Mo and mDC. Although PD-L1 levels were elevated by caspase inhibitors, protein expression of CD80/86 was not increased, suggesting an overall decrease in the positive costimulatory capacity of these cells. Caspase inhibitors also increased PD-L1 levels on control and remission APC, suggesting a normal role for these proteases in regulation of this negative costimulatory molecule, and indicating that PD-L1 or its upstream signaling pathways are direct targets of caspases. This indicates that excessive leukocyte caspase activity in active SLE is linked to decreased PD-L1 protein expression on professional APC. EXAMPLE 9 Peripheral venous blood from volunteers was collected into heparin- or citrate-containing tubes (Vacutainer, Becton Dickinson) after informed consent was obtained. Clinical and laboratory data were collected for each sample at the time of blood draw (Table V). TABLE VSLEAge atDiseaseLow C3Lympho-I.V.Subjectdrawduration&/or C4peniaSLEI.V.cyclo-Daily oralDaily oralDaily oralWeekly oral#Gender(years)(years)at drawat drawstatusbsteroidscphosphamideMMF (mg)HCQpred (mg)MTX (mg)1F15.5>5dna−stable−−−+−−2F12.5<1−−stable2 mo prior−−+20−3F15.9<1+−stable−−−−≤10−4F18.71-3+−stable3 mo prior3 mo prior−+−−5F7.7<1na−stable−−−+60−8.11-3−−stable1 mo prior−1000+15−6F16.13-5nanastable−−−+≤10−17.3>5−−stable−−−−−−7F13.11-3−+flare−−−+≤10−15.43-5−+stable>12 mo prior−>6 mo prior+≤10−8F15.73-5+−flare−−−+−1517.5>5−−stable−−−+−209F16.01-3++flare−−−+≤102518.93-5−+stable−−−+≤102.521.2>5+−stable−−−+≤10−10F10.7<1+−stable−−−+−−11.61-3+−flare−−−+−−11F11.2<1++flare1 mo prior1 mo prior−+30−12F12.1<1++flare−−−+≤10−13M15.01-3−−flare−−−+≤101514F15.6>5++flare−−−+≤10−15F17.21-3++flare−−−+20−16F6.41-3+−flare−−−−−−17F9.9<1+−flare−−−−−−18F15.4<1na−flare−−−−−−19F16.6<1na−flare−−−−−− PBMC were isolated by density centrifugation over a Ficoll-Paque gradient (Amersham), frozen in heat-inactivated AB human serum (Valley Biomedical) with 7% DMSO (Sigma), and stored in liquid nitrogen until use. In preliminary studies, PBMC samples were split into frozen and fresh aliquots and tested to confirm a lack of effect of freeze-thaw on our experimental outcomes. PBMC were thawed, washed, and diluted to 1-2×106cells/ml in culture medium consisting of RPMI 1640 with L-glutamine (CellGro), 10% heat-inactivated NB human serum (Valley Biomedical), 1% penicillin/streptomycin (CellGro), and 0.1% beta-mercaptoethanol. Cells were plated in round-bottom 96-well plates (Coming Costar) and incubated at 37° C. in a humidified cell chamber with 5% CO2. Some wells were treated with pan-/poly-caspase inhibitors at the time of plating: 50 uM Q- Val-Asp-(non-o-methylated)—OPh (OPH) or Z- Val-Ala-Asp-(beta-o-methyl)-fluroromethylketone (Z-VAD) (both from R&D Systems), or DMSO as the carrier control (0.5% final concentration). At the timepoints indicated, cells were surface-stained using fluorochrome- or biotin-conjugated monoclonal antibodies: anti-CD1c, (Miltenyi), anti-CD3, anti-PD-L1 (eBioscience), anti-CD11b, anti-CD11c, anti-CD14, anti-CD-86, anti-PD-L2 (Pharmingen/BD Biosciences), anti-CD45RO, anti-CD80, anti-CD83, and/or anti-HLA-DR (BioLegend), with isotype-matched, fluorochrome/biotin-labeled irrelevant monoclonal antibodies as controls. All samples were blocked using 0.5% human serum and anti-FeR antibody (Miltenyi) during staining. Cells were fixed using 2% paraformaldehyde in PBS after preliminary experiments indicated no effect of cell fixation on expression levels of PD-L1 and other surface markers. To assess apoptosis, some cultures were stained in parallel with Annexin V and propidium iodide (PI) (both from Becton Dickinson) as per the manufacturer's instructions. Flow cytometry was performed using a FACSCalibur or LSR II cytometer (Becton Dickinson), and data were analyzed using FlowJo software (Macintosh Version 6.3). Populations were compared using a 2-tailed t-test, and significance assigned where p<0.05. A total of 26 PBMC samples were collected from 19 SLE patients ranging in age from 6-21 years old (mean=14.3+/−3.7); 13 of these samples were obtained from patients with active (recurrent or newly diagnosed) SLE and designated “flare” samples; 13 were obtained from patients with inactive SLE and designated “remission” samples; (Table I). Control PBMC were obtained from 17 healthy volunteers ranging in age from 6-23 years old (mean=16.5+/−5.3); age was not significantly different between the SLE and control groups. Female subjects comprised 18/19 of the SLE patients and 14/17 of the controls. After one day of culture in the absence of exogenously added stimuli, PD-L1 was expressed on a proportion of CD3−cells from healthy subjects, but there was near-complete absence of PD-L1 on PBMC from patients with active SLE (FIG.1A). CD3−PBMC from patients in lupus remission had regained the ability to express PD-L1. To further characterize these CD3−PD-L1+ cells, levels of several APC surface markers in control PBMC were and it was found that the PD-L1+ cells were of myeloid lineage by staining for CD14 (FIG.1B). These CD14loand CD14hipopulations expressed CD11c, CDIIb, CD45RO, and HLA-DR, and corresponded to CD 1c+/−CD80/CD86himDC and CD1c−CD80/CD86loMo populations, respectively, demonstrating that PD-L1 was primarily expressed on professional APC (FIG.1C). Similar to prior findings there was not found a significant amount of CD83 on these cells, supporting the idea that the mOC in these cultures were phenotypically immature. APC profiles in PBMC from patients in SLE flare or remission were similar to those of controls. To assess levels of PD-L1 on immature mOC and Mo, PBMC were cultured as above and APC identified by double-staining for CD11c and CD14 (FIG.2A). In comparison to APC from healthy controls, it was found that both immature mOC and Mo from children with active SLE failed to upregulate PD-L1, while APC from children in lupus remission expressed normal or increased amounts of this negative costimulator (FIG.2A). These findings were reproducible using immature mDC and Mo from multiple individuals (FIG.2B). Compared to control APC, mean PD-L1 expression was more than three-fold lower in immature mOC and Mo from children in SLE flare, but nearly two-fold higher in Mo from children in SLE remission, indicating that this negative costimulator may play a role in inhibiting the autoreactive immune response. In support of this concept, serial samples drawn from four patients at different times revealed inverse correlation of PD-L1 expression with SLE disease activity (FIG.2C), with lower levels during lupus flares and higher levels during remissions. Not only were PD-L1 levels significantly lower in SLE flare, but these PBMC also had a lower percentage of APC expressing PD-L1. Two- to three-fold fewer mDC and nearly half as many Mo were PD-L1+in SLE flare samples as compared to APC from healthy controls and patients in SLE remission (FIG.2D). To rule out the possibility that APe from patients with active SLE were merely delayed in upregulation of PD-L1, expression of this protein was measured over a five-day time period (FIG.3). Normal APC expressed little PD-L1 at initiation of culture, but levels rapidly increased over time, with peak PD-L1 expression in both mDC and Mo by day three (FIGS.3A and3C). These findings are in agreement with previous work which showed that purified normal human Mo expressed very little PD-1 or PD-L2 upon initial isolation, but spontaneously upregulated PD-L1 after 24 h of culture. As was observed in short-term cultures, it was found that APC from patients in SLE remission expressed PD-L1 at or above normal levels; the kinetics of PD-L1 induction in these cells were similar to those of control cells (FIGS.3A and3C). In contrast to control APC, immature mDC and Mo from children with active SLE expressed abnormally low levels of PD-L1 throughout the timecourse, refuting the idea that the low PD-L1 observed in day one cultures was merely due to delayed expression. Although mean PD-L1 expression was consistently lower in active SLE throughout the timecourse, this was not merely due to a lower proportion of APC expressing PD-L1. The total number of mDC and Mo was quantified at each timepoint, as were the number of cells expressing PD-L1, and it was found that the total numbers of mDC and Mo were not significantly different between samples over time (FIGS.3Band D). In active SLE, slightly fewer mDC expressed PD-L1 over the entire culture period (FIG.3B), but the proportion of Mo expressing PD-L1 was similar to that of controls after day one (FIG.3D). The finding that immature mDC and Mo failed to upregulate PD-L 1 in active SLE has significant implications for pathologic conversion of APC to an immunogenic state. Immature mDC ingest apoptotic bodies and cross-present Ags to cytotoxic T cells and lack of P-1 signaling in vivo results in DC-mediated CD8+T cell priming rather than tolerization. Therefore, in the absence of PD-L1, autoantigen presentation by lupus mDC may result in T cell activation, rather than tolerogenesis. In addition to implications for augmented T effector activity, PD-L1 deficiency may also lead to abnormal T regulatory cell (Treg) function and/or development. Prior work revealed that PD-L1 was necessary for the suppressive activity of classic CD4+CD25+Treg in an animal model of GVHD, and that costimulation of naive CD4+T cells with an anti-CD3 antibody plus PD-L1-Ig fusion protein resulted in formation of Trl regulatory cells. It was observed that PBMC from children in SLE flare had the highest level of apoptosis (FIG.4A), and in all cultures, apoptosis was reduced by the addition of OPH, a potent pan-caspase inhibitor (FIG.4B). It was found that OPH also significantly increased PD-L1 expression in Mo of all three groups compared to untreated (FIG.4C), and doubled the mean percentage of Mo expressing PD-L1 in SLE flare (from 45% to 97%). With respect to immature mDC, OPH increased PD-L1 expression in all three groups two- to three-fold (p<0.02), and more than doubled the mean percentage of mDC expressing PD-L1 in all three groups (P<0.013). This concentration of OPH was not sufficient to completely normalize the excessive apoptosis (FIG.4B) nor the deficient Mo PD-L1 expression in SLE flare (FIG.4C), revealing an inverse correlation between apoptosis and PD-L1 (FIG.4D). This reciprocal relationship between caspase activity and Mo PD-L1 expression also held true for all PBMC treated with another caspase inhibitor (Z-VAD). APC was examined for expression of CD80 and CD86 in the presence and absence of OPH to determine the potential consequences of decreased PD-L1 in active SLE. In PBMC from healthy controls, CD80/86+ APC were clearly PD-L1+in the absence of exogenous stimuli, and upregulated PD-L 1 further after treatment with OPH (FIG.7). In contrast, untreated APC from children with active lupus were markedly PD-L1-deficient without any apparent deficiency in expression of CD80/86; suggesting a high level of positive costimulatory capacity in these cells. In the presence of OPH, PD-L1 levels in SLE flare APC approached those of untreated control cells (FIG.7), indicating that the abnormal balance of costimulatory signaling in lupus APC could be ameliorated by inhibition of caspases. Insults which promote apoptosis, such as drugs, infection, or UV irradiation, may inhibit APC from expressing PD-L1 due to activation of caspases. PD-L1-deficient APC could then play a role in triggering lupus-like symptoms by presenting apoptosis-related antigens in an inflammatory context, providing a final common pathway for breakdown of peripheral tolerance in SLE. Pristane, which causes a lupus-like syndrome when injected into normal mice, and chlorpromazine, which causes a lupus-like syndrome in humans, activate caspases and trigger apoptosis in leukocytes. Infliximab, which can cause a lupus-like syndrome in susceptible individuals, was recently shown to promote caspase activation and apoptosis in human macrophages. The above experiments showed that the failure of APC to upregulate PD-L1 contributes to abnormal T lymphocyte regulation and loss of peripheral tolerance in SLE. EXAMPLE 10 Paediatric donors with and without SLE were recruited under a research protocol. Peripheral venous blood was collected into heparin- or citrate-containing tubes (Vacutainer, Becton Dickinson, NJ, USA) after written informed consent was obtained from the child and/or parent/guardian. Blood samples were centrifuged and plasma aliquots. Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation over a Ficoll-Paque gradient (Amersham, Uppsala, Sweden), frozen in heat-inactivated AB human serum (Valley Biomedical, Winchester, MA., USA) with 7% DMSO (Sigma, St. Louis, MO., USA), and stored in liquid nitrogen until use. In preliminary experiments, PBMC samples from four unique donors were split into frozen and fresh aliquots, and evaluated by flow cytometry to confirm, a lack of effect of freeze-thaw on expression levels of PD-L1 (P≥0.7). Clinical and laboratory data were collected for each individual, and all but one of the lupus patients fulfilled the current ACR classification criteria for SLE. As this was a retrospective study, the European Consensus Lupus Activity Measurement (ECLAM) was calculated for all patient samples where information was available (n=24); ECLAM scores ranged from 0 to 6.5, with a mean±S.D. of 2.5±2.0. As no patient had documentation of seizures, psychosis, cerebrovascular accident, cranial nerve disorder, visual disturbance, myositis, pleurisy, pericarditis, intestinal vasculitis or peritonitis at the time of blood draw, we used a modified scoring system to group patients with respect to disease activity, consisting of these remaining categories: mucocutaneous disease (rash, alopecia, mucosal ulcers and finger nodules), arthritis, haematuria, thrombocytopenia and hypocomplementaemia. In addition, we used lymphopenia, rather than leucopenia, as a sensitive measure of active paediatric SLE. Several samples were chosen at random and also assayed for PBMC apoptosis and/or plasma levels of IFN-α, as these markers are strongly linked to SLE disease activity. PBMC apoptosis was considered to be abnormally high if outside the bounds of the 99.95% CI of control cells (PBMC from seven healthy children tested, data not shown) and plasma IFN-α levels were considered to be abnormal if ≥5 times the upper limit of normal (six healthy children tested). As the clinical assessments were gleaned from chart notes written by a panel of different physicians, the objective laboratory data were weighted more heavily in the final determination, with each abnormal laboratory value assigned 2 points, and each abnormal clinical finding assigned 1 point. A total disease activity score of ≥4 points was felt to represent active disease, and called ‘flare’, while a score of <4 was felt to represent inactive disease, and called ‘remission’. This modified scoring system has the limitation that it has not been formally validated; however, there are no validated disease activity scoring systems for paediatric SLE. Moreover, when this modified scale was used to categorize patients into flare and remission groups, the mean ECLAM scores and anti-dsDNA antibody levels were found to be significantly different between the two groups (Table I), suggesting the potential utility of this approach. PBMC were thawed, washed and diluted to 1-2×106cells/ml in culture medium consisting of RPMI 1640 supplemented with L-glutamine (CellGro, Herndon, VA, USA), 10% heat-inactivated AB human serum, 1% penicillin/streptomycin (CellGro) and 0.1% β-mercaptoethanol. Cells were plated in round-bottom 96-well plates (Corning Costar, Corning, NY., USA) and incubated at 37° C. in a humidified cell chamber with 5% CO2. At the time points indicated, PBMC were surface-stained using various fluorochrome- or biotin-conjugated mAbs, including: anti-CD1c, (Miltenyi, Auburn, CA., USA), anti-CD3, anti-PD-L1 (eBioscience, San Diego, CA, USA), anti-CD11b, anti-CD11c, anti-CD14, anti-CD-86, anti-PD-L2 (Pharmingen/BD Biosciences), anti-CD45RO, anti-CD80, anti-CD83 and/or anti-HLA-DR (BioLegend, San Diego, CA, USA), with isotypematched, fluorochrome-/biotin-labelled irrelevant mAbs as controls. All samples were blocked using 0.5% human serum and anti-FcR antibody (Miltenyi) during staining. After staining, PBMC were fixed using 2% paraformaldehyde in PBS after preliminary experiments indicated no effect of cell fixation on expression levels of PD-L1 or other surface markers (data not shown). Some cultures were stained in parallel with Annexin V and propidium iodide (PI) as per the manufacturer's instructions (both from Becton Dickinson) and apoptosis assessed by enumerating the percent of Annexin V-positive PBMC per culture. Flow cytometry was performed using an LSR II cytometer (Becton Dickinson), and the data were analysed using Flow Jo software (Tree Star, Inc., Ashland, OR, USA). Populations were compared using a two-tailed t-test and significance assigned where P<0.05. Due to the fact that some patients had more than one blood draw and were therefore overrepresented in the data set, statistical analyses were repeated using multivariate logistic generalized estimating equations (GEEs), to account for multiple observations in some individuals. Results of GEE analyses confirmed P<0.05 between populations as identified by t-test. A total of 26 PBMC samples were collected from 19 unique SLE patients ranging in age from 6 to 21 yrs (mean±S.D.=14.3±3.7). Clinical and laboratory data for these blood draws are summarized in Table I. Overall, 12 samples were obtained from patients with active (recurrent or newly diagnosed) SLE and categorized as ‘flare’ samples, while 14 were categorized as ‘remission’ samples, as outlined above. Patient age was not significantly different between the SLE flare (13.8±3.1) and remission (14.7±4.2) groups, and there were no statistically significant differences between the groups with respect to medication usage. Control PBMC were collected from 15 healthy volunteers ranging in age from 6 to 23 yrs (15.7±5.2); patient age and gender composition were not significantly different between the control and SLE groups. Females comprised 18/19 of the SLE patients and 12/15 of the controls. To test the hypothesis that PD-L1 expression is abnormal on lupus APC, primary human PBMC were cultured for 1 day in the absence of exogenously added stimuli and PD-L1 levels measured using four-color multiparametric flow cytometry. Consistent with prior findings in normal human leucocytes, we observed virtually no PD-L1 protein on CD3+cells, but PD-L1 was expressed on a proportion of CD3−cells from a healthy subject (FIG.1A). In contrast, there was near-complete absence of PD-L1 on PBMC from a patient with active SLE. Surprisingly, CD3−cells from the same patient during lupus remission had regained the ability to express normal levels of PD-L1. This pattern was reproducible using PBMC from multiple individuals (see below). To characterize the CD3−cells expressing PD-L1, we assessed levels of several surface markers on normal PBMC and found that the PD-L1+cells naturally segregated into CD14-low/negative)(CD14lo) and CD14-high (CD14hi) populations (FIG.1B), demonstrating that PD-L1 was primarily expressed by APC of myeloid lineage, consistent with published data. Examination of the CD14loand CD14hiAPC subsets for CD11c, CD11b, CD1c, CD45RO and HLA-DR revealed expression patterns consistent with immature mDC and Mo, respectively (FIG.1C). Similar to a prior report, we did not observe a significant amount of CD83 on these cells, supporting the idea that the mDC in these cultures were phenotypically immature. To confirm abnormal PD-L1 levels on lupus APC, PBMC were cultured as above and immature mDC and Mo identified by co-staining for CD14 and CD11c (FIG.13A). As noted, we found that both immature mDC and Mo from children with active SLE failed to up-regulate PD-L1, while APC from children in lupus remission expressed normal or increased levels of this negative costimulator (FIG.13B). As the CD14loCD11c+populations in PBMC may have been comprised of a heterogenous mix of differentiating Mo and early mDC, we used the cell surface marker CD1c (BDCA-1) to specifically identify Type I mDC. Gating for CD14loCD11c+CD1c+cells revealed PD-L1 expression consistent with that of the CD14loCD11c+population as a whole, confirming the utility of this method for measuring PD-L1 levels on immature mDC (FIG.13C). These findings were reproducible and statistically significant for immature mDC and Mo from multiple individuals (FIG.13D-F). Compared with control APC, mean PD-L1 expression was more than 3-fold lower on immature mDC and Mo from children in SLE flare, but nearly 2-fold higher on Mo during SLE remission (FIG.13D). To correct for potential inter-experiment variation, the PD-L1 MFI for each set of APC was normalized to background levels, using the PD-L1 MFI of the CD14−CD11c−cells as the denominator for each sample. However, mDC and Mo from patients in SLE flare remained significantly PD-L1-deficient as compared with both normal and remission APC (FIG.13E). Not only were PD-L1 protein levels lower on SLE flare APC, but there were also lower percentages of cells expressing PD-L1 (FIG.13F). Compared with controls, PD-L1 was expressed on nearly 70% fewer SLE flare mDC and nearly 50% fewer Mo. In contrast, the percentages of PD-L1+APC in lupus remission samples were not significantly different than in controls, consistent with the idea that this negative costimulator may play a role in inhibiting the autoreactive immune response. In support of this concept, serial samples drawn from four patients at different times revealed an inverse correlation between Mo PD-L1 expression and disease activity, with lower levels during SLE flares and higher levels during remissions (FIG.13G). To rule out the possibility that APC from patients with active SLE were merely delayed in up-regulation of PD-L1, we measured expression of this protein over a 5 day culture period. Normal APC expressed little PD-L1 at initiation of culture, but levels rapidly increased over time, with peak PD-L1 expression in both immature mDC and Mo by days 1-2, and return to baseline by day 5. In contrast, immature mDC and Mo from children with active SLE expressed abnormally low levels of PD-L1 throughout the time course, refuting the idea that the low PD-L1 observed in day 1 cultures was merely due to delayed surface expression of this protein. As in short-term cultures, APC from children in SLE remission exhibited normal or elevated levels of PD-L1, suggesting a potential functional association between PD-L1 expression and disease activity. In contrast to PD-L1, staining of control PBMC for the related negative co-stimulator, PD-L2, revealed a nearly negligible level of protein expression that did not change over 4 days of culture. These findings are in agreement with previous work that showed that purified Mo from healthy adult volunteers expressed virtually no PD-L1 or PD-L2 upon initial isolation, and spontaneously up-regulated only PD-L1 after 24 h of culture. To determine whether the defect in lupus flare APC was specific to PD-L1, we measured the level of positive co-stimulatory molecules (a combination of CD80 plus CD86) on PBMC from children with and without SLE. We found that although immature mDC and Mo were clearly PD-L1-deficient during SLE flare, they retained the ability to express CD80/CD86 (FIG.14), congruent with prior studies that revealed normal or elevated levels of these proteins on mDC and Mo from patients with SLE. Taken together, these observations suggest that the inability of lupus APC to express PD-L1 cannot be attributed to a global decrease in costimulatory molecule expression during SLE flare, and that loss of the negative PD-L1 signal is not associated with or compensated for by a decrease in positive co-stimulatory signals. Consistent with a prior report, we also observed that although Mo populations were fairly homogenous with respect to expression of CD80/CD86, immature mDC segregated into CD80/CD86loand CD80/CD86hi-expressing groups, suggesting differing abilities for T-cell stimulation (FIG.14). Moreover, in control and SLE remission PBMC, the majority of PD-L1 protein was expressed by CD80/CD86himDC, suggesting that T-cell stimulation by these most potent APC is normally held in check by this negative regulator. Surprisingly, the CD80/CD86hisubset of mDC was markedly lacking in SLE flare, although the reasons for this are currently unclear. The finding of decreased PD-L1 protein during active SLE has significant implications for conversion of APC to a pathological state. Although immature mDC and Mo from children with active SLE failed to up-regulate PD-L1, both cell types retained the ability to express several other markers, including CD80/CD86, at the APC surface. As CD80/CD86-mediated T-effector signaling is normally countered by PD-L1, lupus APC could potentially have an abnormally high capacity for positive T-cell co-stimulation during SLE flare. A hyperstimulatory role for lupus APC is supported by data showing that mDC and Mo from patients with SLE have an increased ability to activate allogenic T-cells. Not only do DCs depend upon PD-L1 signaling to diminish T-cell stimulation, but negative co-stimulation by PD-L1 is more effective in immature DCs than in mature DCs, suggesting a mechanism for the immunogenic presentation of autoantigens in SLE. Immature mDC ingest apoptotic bodies and cross-present Ags to cytotoxic T-cells, and lack of PD-1 signaling in vivo results in DC-mediated CD8+T-cell priming rather than tolerization. Therefore, our data may provide a partial explanation for the self-reactivity observed in lupus patients, whereby PD-L1-deficient immature mDC present apoptosis-related antigens in a pro-inflammatory context. While examining CD80/CD86 expression, it was also noted that the CD80/CD86hisubgroup of mDC was diminished during SLE flare. This is intriguing, as SLE PBMC proliferate poorly in autologous mixed leucocyte reactions (aMLRs), and it has recently been suggested that CD80/CD86himDC are integral for T-cell proliferation during aMLRs. The reason behind the loss of these cells in active SLE is unclear, however, and may be related to increased apoptosis or to tissue sequestration—it has been reported that patients with active Class III and IV lupus nephritis have significantly fewer circulating mDC along with a concomitant increase of immature mDC in renal tissues. It would be interesting to determine whether these renal mDC retain the ability to express PD-L1. In addition to potentially stimulating autoreactive T effector cells, PD-L1-deficient APC may promote abnormal function and/or development of regulatory T lymphocytes (Treg). It has been demonstrated that PD-L1 signaling is necessary for the suppressive activity of classic CD4+CD25+Treg in an animal model of GVHD, and that anti-CD3-stimulated naïve CD4b T cells could be induced to become Trl-type regulatory cells if co-stimulated with PD-L1-Ig. Although decreased Treg number and function have been reported in human SLE it remains to be determined whether PD-L1 plays any role in Treg-related deficiencies. The decreased PD-L1 levels observed on APC from patients with active SLE were not likely a result of medication effects, as the use of immunosuppressive agents was comparable between flare and remission groups (Table I). Three of the children with active SLE and low PD-L1 had been newly diagnosed and had never received any immunosuppression. Additionally, all four of the subjects who provided serial samples (FIG.2G) were on minimally varying medication regimens at the time of their blood draws. Similarly, a prior study of SLE patients revealed no correlation between the use of immunosuppressive agents in vivo and changes in cell surface markers on peripheral blood DC, as well as no significant effect of chloroquine, steroids, 6-mercaptopurine or mycophenolate mofetil on markers of Mo differentiation and maturation in vitro. In addition to cytokine dysregulation, SLE leucocytes undergo apoptosis at an increased rate, and we did note an inverse correlation between PD-L1 expression and PBMC apoptosis (Table I). Following this lead, we have preliminary data demonstrating that in vitro treatment of PBMC with polycaspase inhibitors not only reduced leucocyte apoptosis, but increased the expression of PD-L1 on mDC and Mo in all cultures (data not shown). These findings suggest a role for caspase activity in the normal regulation of PD-L1 and provide a potential explanation for the loss of this negative co-stimulator on APC from patients with active SLE. In support of this idea, it has been reported that caspase-3 is directly responsible for the decreased CD3−-chain expression on the surface of SLE T cells. Our findings complement what is already known regarding PD-L1 expression in human disease; levels of PD-L1 are increased on circulating APC from patients with chronic HIV, hepatitis B or hepatitis C infection, and decreased on DC from patients with multiple sclerosis. As preliminary studies in our laboratory have also indicated abnormally low levels of PD-L1 on APC from patients with some other types of active autoimmune disease, we propose that diminished expression of PD-L1 on circulating APC may be a hallmark of active multi-organ autoimmunity, while elevated levels of PD-L1 on circulating APC may be indicative of chronic infection. If verified in larger samples, this distinction may be medically useful, as it is often unclear whether clinical deterioration in SLE patients represents disease flare or infection. In summary, our findings link active SLE with the inability of peripheral blood APC to express PD-L1, suggesting that PD-L1 may be functionally important in the maintenance of immune tolerance in SLE. Lack of this protein on the surface of immature mDC also suggests a mechanism for the propensity of the immune system to target apoptosis-associated molecules in SLE, as immature mDC typically ingest and present these self-antigens. Given the inverse correlation between PD-L1 and SLE disease activity, future investigations may reveal a role for PD-L1 fusion proteins or other molecules capable of ligating PD-1 in the treatment of SLE or other autoimmune diseases. Larger studies may determine whether intermittent measurements of PD-L1 on circulating APC could provide an additional tool for monitoring the clinical course of SLE. The above experiments showed that both immature mDC and Mo from children with SLE failed to up-regulate PD-L1 normally, and that this deficiency was associated with increased disease activity. As used herein, the term “patient” refers to the recipient of a therapeutic treatment and includes all organisms within the kingdom animalia. In preferred embodiments, the animal is within the family of mammals, such as humans, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer and primates. The most preferred animal is human. As used herein, the terms “treat” “treating” and “treatment” include “prevent” “preventing” and “prevention” respectively. As used herein, the term “autoimmune disease” includes “immune-related disease,” “autoimmune disorder,” “immunologic disorder” and “immune-related disorder.” As used herein, the term “isolated” refers to materials, such as cells or antibodies, which are removed from at least some of the components that normally accompany or interact with the materials in a naturally occurring environment such that they have been altered, “by the hand of man” from their natural state to a level of isolation or purity that does not naturally occur. In some other embodiments, the treatments described herein may be administered alone or in combination with another therapeutic compound. Any therapeutic compound used in treatment of the target autoimmune disease can be used. Many different modes and methods of administration of the therapeutic molecules are contemplated. In some embodiments, delivery routes include, for example, intravenous, intraperitoneal, inhalation, intramuscular, subcutaneous, nasal and oral administration or any other delivery route available in the art. Depending on the particular administration route, the dosage form may be, for example, solid, semisolid, liquid, vapor or aerosol preparation. The dosage form may include, for example, those additives, lubricants, stabilizers, buffers, coatings, and excipients available in the art of pharmaceutical formulations. In some embodiments, gene therapy is utilized to deliver therapeutic molecules to the patient. Many pharmaceutical formulations are contemplated. In some embodiments, the pharmaceutical formulations can be prepared by conventional methods using the following pharmaceutically acceptable vehicles or the like: excipients such as solvents (e.g., water, physiological saline), bulking agents and filling agents (e.g., lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogenphosphate, soft silicic acid anhydride and calcium carbonate); auxiliaries such as solubilizing agents (e.g., ethanol and polysolvates), binding agents (e.g., starch, polyvinyl pyrrolidine, hydroxypropyl cellulose, ethylcellulose, carboxymethyl cellulose and gum arabic), disintegrating agents (e.g., starch and carboxymethyl cellulose calcium), lubricating agents (e.g., magnesium stearate, talc and hydrogenated oil), stabilizing agents (e.g., lactose, mannitol, maltose, polysolvates, macrogol, and polyoxyethylene hydrogenated castor oil), isotonic agents, wetting agents, lubricating agents, dispersing agents, buffering agents and solubilizing agents; and additives such as antioxidants, preservatives, flavoring and aromatizing agents, analgesic agents, stabilizing agents, coloring agents and sweetening agents. If necessary, glycerol, dimethyacetamide, 70% sodium lactate, surfactants and alkaline substances (e.g., ethylenediamine, ethanol amine, sodium carbonate, arginine, meglumine and trisaminomethane) can also be added to various pharmaceutical formulations. In the context of some embodiments, the dosage form can be that for oral administration. Oral dosage compositions for small intestinal delivery include, for example, solid capsules as well as liquid compositions which contain aqueous buffering agents that prevent the expanded Tregcell population or other ingredients from being significantly inactivated by gastric fluids in the stomach, thereby allowing the expanded Tregcell population to reach the small intestines. Examples of such aqueous buffering agents which can be employed in the present invention include, for example, bicarbonate buffer at a pH of from about 5.5 to about 8.7. Tablets can also be made gastroresistent by the addition of, e.g., cellulose acetate phthalate or cellulose acetate terephthalate. EQUIVALENTS The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The foregoing description details certain preferred embodiments of the invention and describes the best mode contemplated by the inventors. It will be appreciated, however, that no matter how detailed the foregoing may appear in text, the invention may be practiced in many ways and the invention should be construed in accordance with the appended claims and any equivalents thereof. | 66,843 |
11860162 | EXAMPLES Example 1: Selection of the SLE Patients and Test Subjects Selection of the patient groups to be tested: Blood samples were analysed from 129 SLE patients, 100 patients with systemic sclerosis (SSc, PSS), 75 patients with rheumatoid arthritis (RA), 537 patients with early RA (period of disease less than 6 months) and 75 patients with ankylosing spondylitis (SPA)/Bekhterev's disease (SPA). 343 blood samples from the Bavarian Red Cross (RFC) were used as control group. An informed consent of the Ethics Commission of the clinical partners and of the biobank of the BRC was received from all test subjects. TABLE 1Patient samples and clinical data (test cohort. I)2. Screen1. ScreenSSc(PSS)Early RASLERAHealthySLETotalSubtype(<6 months)SPAHealthyNumber1297512310010053782343Age39 +/−56.6 +/−41.3 +/−39.8 +/−56.9 +/−Limited56.8 +/−43.7 +/−47.7 +/−(years)1213.21111.913.4n − 5014.310.111.7% female86.17286.28387Diffuse62.215.958.3n = 32% ANA77.5N.D.N.D.10095OverlapN.D.N.D.N.D.n = 9SLAM7.7 +/−7.7 +/−5.15.1SLICC1.45 +/−1.45 +/−1.81.8ANA %ENA-43748positive %U1-RNP(%1313of ENA-4pos.)SM (% of88ENA-4pos.)SS-A/Ro523535(% of ENA-4 pos.)SS0B/Ro601010(% of ENA-4 pos.)Kidney26.434involvement% Example 2: Antigen Production Five cDNA libraries that had been produced from different human, tissues (foetal brain, intestine, lung, liver and T-cells) were used for the production, of the recombinant antigens. All cDNAs were expressed inE. coliunder the transcriptional control of the lactose-inducible promoter. The resultant proteins carry, at their amino terminus, an additional sequence for a hexahistidine purification tag (His6 tag), Target, antigens which were not present, in the cDNA library were produced by chemical synthesis (Life Technologies) and cloned into the expression vector pQE30-NST, which already codes an amino-terminal His6 tag. Following recombinant expression of the proteins, these were isolated in denaturising conditions and purified by means of metal affinity chromatography (IMAC). The proteins were lyophilised and stored set −20° C. until further use (http://www.lifesciences.sourceboioscience.com). Example 3: Production of the BBAs The production of BBAs was adapted to a microtitre plate format, such that 384 coupling reactions could be assessed in parallel using automated pipette systems (Starlet, Hamilton Robotics, Evo Freedom 130, Tecan), For the use of automated pipette systems, the individual bead regions were transferred into coupling pates (96 well Greiner) and the antigens were transferred into 2D barcode vessels (Thermo Scientific). For each coupling reaction, 0.6 to 2.5 million beads and, depending on the antigen, 1 to 100 μg protein were used. All washing and pipetting steps of the coupling reaction were carried out in coupling plates which were fixed on magnets. The beads were washed twice with 100 μl LxAP buffer (100 roM NaH2PO4, pH 6.2) and then, received in 120 μl LxAP buffer. For the activation, 15 μl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; 50 mg/ml) and 15 μl N-hydroxysulfosuccinimide (sulfo-NHS; 50 mg/ml) were added by pipette to form a bead suspension, and these suspensions were then incubated for 20 minutes in the shaker (RT, 900 rpm, protected against light). The beads—were then washed 3× with 150 μl LxKPT buffer and then the protein solution was added. Following an incubation period of two hours in the shaker (RT, 300 rpm, protected against light), the beads were then washed three times with 150 μl LxKPT buffer. To block free binding points, 100 ul LxCBSP buffer (PBS, 1% BSA, 0.05% ProClin300) were added, and these, mixtures were then incubated for 20 min in the shaker (RT, 900 rpm, protected against, light). This was followed by incubation over night at 4-8° C. The BBA was produced by the combination of beads coupled to antigens and was stored at 4-8° C., protected against light, until use. Example 4: Quality Control of the BBAs In order to check the immobilisation of the proteins at the respective bead regions, a coupling control, was carried out. Here, different amounts of beads were used (250, 500 and 750 beads per bead region). For a reaction mixture, 500 beads for example per bead region were diluted in LxCBS buffer (PBS, 1% BSA) and transferred into an assay plate (96 well half area microplate, Greiner). Before each washing step, the assay plate with the beads was placed for 2 minutes on a magnet, and the supernatant was then removed. After three washing steps, the beads were incorporated with 100 μl LxWPT buffer (PBS, 0.05% Tween-20), and 10 μl/ml penta-his antibodies (Qiagen) or LxCBS buffer (PBS, 1% BSA) were added by pipette. Following incubation for 45 minutes in the shaker (RT, 900 rpm, protected against light), the supernatant was removed and the beads were washed in two steps, 5 μl/ml goat, anti-mouse IgG-PE (Phycoerythrin) or goat anti-human IgG-PE (Dianova) were then added as secondary antibody to the reaction mixture and incubated for 30 minutes. Following two washing steps, 100 μl of carrier liquid (Luminex) was added to the beads. The fluorescence signal of the beads was detected with the aid of the FlexMAPSD instrument. Here, the bead count on the one hand and the median of the fluorescence intensity (MFI value) on the other hand were measured. Example 5: Application of BBAs For application, BBAs were incubated with sera and all IgG-based autoantibodies bonded to antigens were detected with the aid of a secondary antibody. In order to enable a high throughput of measurements, the application of BBAs was adapted to a microtiter plate format so that either an 8-channel (Starlet, Hamilton Robotics) or a 96-channel (Evo Freedom. 150, Tecan) automated pipetting system could be used. The sera to be examined were transferred into 2D barcode vessels and then diluted 1:100 with assay buffer (PBS, 0.5% BSA, 10%E. colilysate, 50% low-cross buffer (Candor Technologies)). In order to neutralise human antibodies directed againstE. coli, a pre-incubation of the sera dilutions was performed for 20 min. In this time, 500 beads per bead region were distributed in the assay plate. 50 μl of diluted serum were added to the beads in the coupling plate, and the reaction mixtures were incubated for 18-22 h in the shaker (4-8° C., 900 rpm, protected against light). After three washing steps in each case with 100 μl LxWPT buffer, 5 μl/ml of the detection antibody goat anti-human IgG-PE (Dianova) were added to the reaction mixtures and incubated for 1 h in the shaker (RT, 900 rpm). The beads were then washed three times with 100 pi LxWPT and incorporated in 100 μl carrier liquid (Luminex). The fluorescence signal of the beads was detected with the aid of the FiexMAF3D instrument, Here, the bead count on the one hand and the MFI value (median fluorescence intensity) on the other hand were measured. Example 6: Biostatistical Analysis The biostatistical analysis comprised univariate and multivariate methods for describing the statistical properties of individual antigens and of groups of antigens. In order to discover interesting candidates for panels, the key property was a good separation between the groups of samples based on the MFI values. In order to find antigen candidates for panel generation, univariate testing, receiver operating characteristic (ROC) analyses, correlation profiles, powered partial least squares discriminant analysis (PPLS-DA) and random forests were used as methods. Biostatistical analyses were subject to expert assessment, in order to define final antigen panels. Before the statistical analysis, the MFI values were log 2-transformed in order to reduce the skew in the distributions. If more than 20% of the values ‘were missing, antigens were excluded from the analysis. Missing values—were replaced by median imputation. A quant lie normalisation was carried out under consideration of the reference sera in order to normalise, per BBA set, all measured samples on individual plates. Besides descriptive standardisation for MFI values, non-parametric tests were also carried out with the aid of the two-sided Mann-Whitney-U test in order to uncover differences in the median values of the groups. The test level for multiple testing was corrected in accordance with the Bonferroni-Holm procedure. In addition, the Benjamin-Hochberg procedure inclusive of the determination of the False Discovery Rate (FDR, q-value) was applied. In addition, fold-change and effect size were determined. In order to assess the classification quality, an ROC analysis was carried out, within the scope of which sensitivity, specificity and the area under the ROC curve (AUC) were calculated, in each case inclusive of the 95% confidence interval on the basis of the bootstrap method. Boxplots and volcano plots were used, for graphical representation. A scoring system was implemented on the basis of the univariate results. By means of the application of a PPLS-DA, it was attempted to maximise the correlation between the component of the response matrix. A linear discriminant analysis with the latent component as predictors was used for the final classification, A random forest was applied, in which binary decision trees are combined. The decision trees were formed on the basis of a number of bootstrap samples of a training sample and by random selection of a subgroup of explaining variables at each node. The number of input variables, which was selected randomly with each division step, was determined as the square root of the total number of variables, and the number of trees in the random forest was set to 1000. A cross validation with 500 times throughput was implemented for both multi-variant approaches. Example 7: Autoantibodies/Antigen Reactivities Differentiate SLE from Healthy Controls, Rheumatoid Arthritis and Other Autoimmune Diseases In a first screening the antigen reactivities of 129 SLE patients, 75 RA patients, and 134 healthy controls categorized in accordance with age and sex were differentially tested. For this purpose, the autoantibody reactivities of these blood samples were tested on 5857 antigens coupled to Luminex beads. In order to identify antigens with which the group of all SLE patients can be distinguished from different control croups consisting of healthy samples and patients with RA, univariate statistical tests were carried out, The result, of the statistical test is illustrated as a volcano plot for all 5857 antigens. In the volcano plot, the x-axis shows the relative change of the antigen reactivity in SLE patients compared with healthy controls (FIG.1) and RA patients (FIG.2). The y-axis presents the p-value of the statistical tests.FIGS.1and2show that specific autoantibody reactivities were found which are increased in the group of ail SLE and which can distinguish both from healthy donors and from RA patients. Example 8: Autoantibodies/Antigen Reactivities Differentiate SLE from Healthy Controls, Early Rheumatoid Arthritis and Other Autoimmune Diseases In a second screening with 6088 antigens, the antigens which differentiate between healthy controls and donors with rheumatoid arthritis were tested on patients with early rheumatoid arthritis, SSc and SPA., This is of importance in particular since patients with collagenoses and mixed collagenoses have an overlapping autoantibody profile and therefore are difficult to diagnose, particularly in the early phase FIG.3shows a volcano plot of the antigen reactivities of SLE patients against a combined group of patients with various autoimmune diseases, such as SSc, SPA, early rheumatoid arthritis, and SPA. Following univariate statistical evaluation, a threshold value of p<0.05 and a 1.5 times modified reactivity compared with the control group were applied. A final list of antigen reactivities over both screens was established (Table 2). In order to analyse the frequency of the newly identified antigens in comparison with, known antigens, a threshold value of 3 standard deviations (SD) above the mean value of the healthy samples was defined. Astonishingly, at least 4 additional antigens were identified of which the frequency in SLE patients lies above 15%. These include TMPO (19%) (SEQ ID No. 13), HNRNPA1 (26%) (SEQ ID No. 5), XRCC5 (15%) (SEQ ID No. 22) and MVP (15%) (SEQ ID No. 7). FIG.4shows the frequency of 23 antigens in comparison to the healthy controls. Table 2 summarises the identified antigen reactivities and different group comparisons. TABLE 2List of all antigen reactivitiesStatistical TestSEQGeneGeneGenePanelSLEENA-4 negL. Nephr.SLEID No.IDSymbolNameGroupSLEL. Nephr.Clustervs HVvs SLEvs control11629DBTdihydrolipoamide1xxSLEbranched chainvstransacylase E2AID21737DLATdihydrolipoamide S-1xSLEacetyltransferasevsAID37430EZRezrin1xxxSLEvsAID43017HIST1H2BDhistone cluster 1,1xxSLEH2bdvsAID53178HNRNPA1heterogeneous1xxSLEnuclearvsribonucleoprotein A1AID63181HNRNPA2B1heterogeneous1xxSLEnuclearvsribonucleoproteinAIDA2/B179961MVPmajor vault protein1xxxxSLEvsAID86175RPLP0ribosomal protein,1xxxSLElarge, P0vsAID96176RPLP1ribosomal protein,1xxxxSLElarge, P1vsAID106181RPLP2ribosomal protein,1xxxSLElarge, P2vsAID1130011SH3KBP1SH3-domain kinase1xxSLEbinding protein 1vsAID126625SNRNP70small nuclear1xSLEribonucleoproteinvs70 kDa (U1)AID136628SNRPBsmall nuclear1xxxSLEribonucleoproteinvspolypeptides B andAIDB1146638SNRPNsmall nuclear1xSLEribonucleoproteinvspolypeptide NAID156672SP100SP100 nuclear1xxSLEantigenvsAID166710SPTBspectrin, beta,1xxSLEerythrocyticvsAID176741SSBSjogren syndrome1xxSLEantigen Bvs(autoantigen La)AID187112TMPOthymopoietin1xxxSLEvsAID196737TRIM21tripartite motif-1xxxSLEcontaining 21vsAID206738TROVE2TROVE domain family,1xxSLEmember 2vsRA217431VIMvimentin1xxSLEvsAID227520XRCC5X-ray repair1xxSLEcomplementingvsdefective repair inAIDChinese hamstercells 5 (double-strand-breakre joining)237764ZNF217zinc finger protein1xxSLE217vsAID2464763ZNF574zinc finger protein1xxSLE574vsAID25148741ANKRD35ankyrin repeat2xxSLEdomain 35vsHV2684779ARD1BARD1 homolog B2xxSLE(S. cerevisiae)vsAID27672BRCA1breast cancer 1,2xxSLEearly onsetvsHV28134359C5orf37chromosome 5 open2xxxSLEreading frame 37vsHV299478CABP1calcium binding2xxSLEprotein 1vsHV3090557CCDC74Acoiled-coil domain2xxSLEcontaining 74AvsHV319973CCScopper chaperone for2xxxxSLEsuperoxide dismutasevsAID321410CRYABcrystallin, alpha B2xxSLEvsHV3355802DCP1ADCP1 decapping2xxSLEenzyme homolog Avs(S. cerevisiae)HV3479147FKRPfukutin related2xSLEproteinvsHV3526128KIAA1279KIAA12792xxSLEvsHV3657608KIAA1462KIAA14622xxSLEvsHV371939LGTNligatin2xxSLEvsHV3884298LLPHLLP homolog, long-2xxSLEterm synapticvsfacilitationHV(Aplysia)3911253MAN1B1mannosidase, alpha,2xxSLEclass 1B, member 1vsHV4084930MASTLmicrotubule2xxSLEassociatedvsserine/threonineHVkinase-like4154531MIER2mesorm induction2xxxxSLEearly response 1,vsfamily member 2RA424594MUTmethylmalonyl2xxSLECoenzyme A mutasevsHV43399687myO18Amyosin XVIIIA2xxSLEvsHV448883NAE1NEDD8 activating2xxSLEenzyme E1 andvssubunit 1HV4510458BAIAP2BAI1-associated2xxSLEprotein 2vsHV464869NPM1nucleophosim2xxSLE(nucleolarvsphosphoprotein B23,HVnumatrin)475223PGAM1phosphoglycerate2xxSLEmutase 1 (brain)vsHV4811040PIM2pim-2 oncogene2xxSLEvsHV4954517PUS7pseudouridylate2xxSLEsynthase 7 homologvs(S. cerevisiae)HV506605SMARCE1SWI/snf related,2xxSLEmatrix associated,vsactin dependentAIDregulator ofchromatin,subfamily e, member15123635SSBP2single-stranded DNA2xxxSLEbinding protein 2vsHV5283660TLN2talin 22xxSLEvsHV5351673TPPP3tubulin2xxSLEpolymerization-vspromoting proteinHVfamily member 3547265TTC1tetratricopeptide2xxSLErepeat domain 1vsHV55124930ANKRD13Bankyrin repeat3xSLEdomain 13BvsHV56160AP2A1adaptor-related3xSLEprotein complex 2,vsalpha 1 subunitHV5753335BCL11AB-cell CLL/lymphoma3xx11A (zinc fingerprotein)5879959CEP76centrosomal protein3x76 kDA591153CIRBPcold inducible RNA3xSLEbinding proteinvsHV6051084CRYL1crystallin, lambda 13x6155827DCAF6DDB1 and CUL43xxxSLEassociated factor 6vsAID626993DYNLT1dynein, light chain,3xSLETctex-type 1vsHV63283991FAM100Bfamily with sequence3xSLEsimilarity 100,vsmember BHV649815GIT2G protein-coupled3xSLEreceptor kinasevsinteracting ArfGAP 2HV6584706GPT2glutamic pyruvate3xtransaminase(alanineaminotransferase) 2663059HCLS1hematopoieti cell-3xxSLEspecific Lynvssubstrate 1AID673329HSPD1heat shock 60 kDa3xprotein 1(chaperonin)683490IGFBP7insulin-like growth3xSLEfactor bindingvsprotein 7HV6923392KIAA0368KIAA03683x7084695LOXL3lysyl oxidase-like3x3714133MAP2microtubule-3xSLEassociated proteinvs2HV726837MED22mediator complex3xsubunit 227329079MED4mediator complex3xxsubunit 47410933MORF4L1mortality factor 43xlike 17564963MRPS11mitochondrial3xxSLEribosomal proteinvsHV7681565NDEL1nudE nuclear3xdistribution gene Ehomolog(A. nidulans)-like 17757447NDRG2NDRG family member3xSLE2vsHV784744NEFHneurofilament, heavy3xpolypeptide79153478PLEKHG4Bpleckstrin homology3xdomain containing,family G (withRhoGef domain)member 4B [homosapiena(human)]8011054OGFRopioid growth factor3xxSLEreceptorvsAID8156122PCDHB14protocadherin beta3xSLE14vsHV822923PDIA3protein disulfide3xSLEisomerase family A,vsmember 3HV8323646PLD3phospholipase D3xSLEfamily, member 3vsHV8423759PPIL2peptdylprolyl3xxisomerase(cyclophilin)-like2855557PRIM1primase, DNA,3xSLEpolypeptide 1vs(49 kDa)HV865682PSMA1proteasome (prosome,3xSLEmacropain) subunit,vsalpha type, 1HV875802PTPRSprotein tyrosine3xSLEphosphatase,vsreceptor type, SHV8881890QTRT1queuine tRNA-3xSLEribosyltransferase 1vsHV89116362RBP7retinol binding3xSLEprotein 7, cellularvsHV9010287RGS19regulator of G-3xxprotein signaling 199183642RP3-402G11.5selenoprotein O3xSLEvsHV926389SDHAsuccinate3xxSLEdehydrogenasevscomplex, subunit A,AIDflavoprotein (Fp)9354437SEMA5Bsema domain, seven3xthrombospondinrepeats (type 1 andtype 1-like),transmembranedomain (TM) andshort cytoplasmicdomain,(semaphorin) 5B9459343SENP2SUMO1/sentrin/SMT33xSLEspecific peptidasevs2HV956629SNRPB2small nuclear3xSLEribonucleoproteinvspolypeptide B″AID9627131SNX5sorting nexin 53xSLEvsHV979021SOCS3suppressor of3xxSLEcytokine signalingvs3HV983925STMN1stathmin 13xSLEvsHV9981551STMN4stathmin-like 43xSLEvsHV10027097TAFSLTAF5-like RNA3xSLEpolymerase II,vsp300/CBP-associatedHVfactor (PCAF)-associated factor,65 kDa10179521TCEAL4transcription3xSLEelongation factor Avs(SII)-like 4HV10210040TOM1L1target of mybl3xSLE(chicken)-like 1vsHV10322974TPX2TPX2, microtubule-3xSLEassociated, homologvs(Xenopus laevis)HV10451567TTRAPTRAF and TNF3xreceptor associatedprotein1058615USO1US01 homolog,3xxvescicle dockingprotein (yeast)10610869USP19ubiquitin specific3xSLEpeptidase 19vsRA10729761USP25ubiquitin specific3xpeptidase 25108375690WASH5PWAS protein family3xxSLEhomolog 5vspseudogeneHV10910413YAP1Yes-associated3xprotein 1, 65 kDa110653121ZBTB8Azinc finger and BTB3xxSLEdomain containingvsBAHV11155311ZNF444zinc finger protein3x44411229ABRactive BCR-related4xSLEgenevsAID113118ADD1adducin 1 (alpha)4xSLEvsAID11455256ADI1acireductone4xSLEdioxygenase 1vsHV1159255AIMP1aminoacyl tRNA4xsynthetase complex-interactingmultifunctionalprotein 111654522ANKRD16ankyrin repeat4xSLEdomain 16vsHV117348APOEapolipoprotein E4xSLEvsHV11864333ARHGAP9Rho GTPase4xSLEactivating proteinvs9HV11922994AZI15-azacytidine4SLEinduced 1vsHV12055971BAIAP2L1BAI1-associated4xprotein 2-like 11217919BAT1HLA-B associated4xSLEtranscript 1vsRA1226046BRD2bromodomain4xcontaining 212356912C11orf60chromosome 11 open4xreading frame 6012479415C17orf62chromosome 17 open4xreading frame 6212551300C3orf1chromosome 3 open4xSLEreading frame 1vsRA126128866CHMP4Bchromatin modifying4xSLEprotein 4BvsAID12723122CICcapicua homolog4xSLE(Drosophila)vsAID12810970CKAP4cytoskeleton-4xSLEassociated proteinvs4HV12923122CLASP2cytoplasmic linker4xassociated protein21301311COMPcartilage4xoligomeric matrixprotein1317812CSDE1cold shock domain4xSLEcontaining E1, RNA-vsbindingHV1328642DCHS1dachsous 14xSLE(Drosophila)vsAID1339909DENND4BDENN/MADD domain4xxcontaining 4B1341743DLSTdihydrolipoamide S-4xsuccinyltransferase(E2 component of 2-oxo-glutaratecomplex)13584444DOT1LDOT1-like, histone4xH3 methyltransferase(S. cerevisiae)13651143DYNC1LI1dynein, cytoplasmic4xSLE1, lightvsintermediate chain 1HV13751011FAHD2Afumarylacetoacetate4xhydrolase domaincontaining 2A13892689FAM114A1family with sequence4xsimilarity 114,member A113954463FAM134Bfamily with sequence4xsimilarity 134,member B140100129583FAM47Efamily with sequence4xSLEsimilarity 47,vsmember EHV14193611FBXO44F-box protein 444x14260681FKBP10FK506 binding4xSLEprotein 10, 65 kDavsAID14323360FNBP4formin binding4xprotein 41442300FOXL1forkhead box L14xSLEvsHV14564689GORASP1golgi reassembly4xSLEstacking protein 1,vs65 kDaAID1462934GSNgelsolin4xSLE(amyloidosis,vsFinnish type)HV1473039HBA1hemoglobin, alpha4x1483040HBA2hemoglobin, alpha 24x14938858HES5hairy and enhancer4xof split 5(Drosophilia)15010525HYOU1hypoxia up-regulated4x11513608ILF2interleukin enhancer4xSLEbinding factor 2,vs45 kDaRA15223135KDM6Blysine (K)-specific4xSLEdemethylasae 6BvsAID15356243KIAA1217KIAA12174xSLEvsHV15457662KIAA1543KIAA15434x15557498KIDINS220kinase D-interacting4xsubstrate, 220 kDA1563855KRT7keratin 74xSLEvsHV157729970LOC729970similar to4xhCG20283521589935MAFBv-maf4xmusculoaponeuroticfibrosarcomaoncogene homolog B(avian)15923764MAFFv-maf4xSLEmusculoaponeuroticvsfibrosarcomaHVoncogene homolog F(avian)16022924MAPRE3microtubule-4xassociated protein,RP/EB family, member31618079MLF2myeloid leukemia4xfactor 21624676NAP1L4nucleosome assembly4xprotein 1-like 41634688NCF2neutrophil cytosolic4xSLEfactor 2vsHV1644780NFE2L2nuclear factor4x(erythroid-derived2)-like 216579840NHEJ1nonhomologous end-4xxjoining factor 116622861NLRP1NLR family, pyrin4xSLEdomain containing 1vsHV16765009NDRG4NDRG family member 44xSLEvsHV1684841NONOnon-POU domain4xSLEcontaining, octamer-vsbindingAID16929982NRBF2nuclear receptor4xSLEbinding factor 2vsAID1708439NSMAFneutral4xSLEsphingomyelinase (N-vsSMase)activationHVassociated factor1714926NUMA1nuclear mitotic4xSLEapparatus protein 1vsRA17284759PCGF1polycomb group ring4xfinger 117384306PDCD2Lprogrammed cell4xSLEdeath 2-likevsHV1745195PEX14peroxisomal4xSLEbiogenesis factor 14vsHV1759091PIGQphosphatidylinositol4xSLEglycan anchorvsbiosynthesis, classRAQ176100137049PLA2G4Bphospholipase A2,4xSLEgroup IVBvs(cytosolic)RA17710226PLIN3perilipin 34x1785373PMM2phosphomannomutase 24x17910450PPIEpeptidylprolyl4xisomerase E(cyclophilin E)1805694PSMB6proteasome (prosome,4xmacropain) subunit,beta type, 618122913RALYRNA binding protein,4xSLEautoantigenicvs(hnRNP-associatedHVwith lethal yellowhomolog (mouse))1828241RBM10RNA binding motif4xprotein 101839904RBM19RNA binding motif4xSLEprotein 19vsHV1849743RICSRho GTPase-4xactivating protein1858780RIOK3RIO kinase 3 (yeast)4x1868578SCARF1scavenger receptor4xSLEclass 4, member 1vsAID18723513SCRIBscribbled homolog4xSLE(Drosophila)vsHV188644096SDHAF1succinate4xSLEdehydrogenasevscomplex assemblyRAfactor 118957794SF4splicing factor 44xSLEvsRA1909814SFI1Sfi1 homolog,4xspindle assemblyassociated (yeast)1916421SFPQsplicing factor4xSLEproline/glutamine-vsrich (polypyrimidineAIDtract bindingprotein associated)19283442SH3BGRL3SH3 domain binding4xglutamic acid-richprotein like 31936461SHBSrc homology 24xSLEdomain containingvsadaptor protein BAID19423381SMG5Smg-5 homolog,4xSLEnonsense mediatedvsmRNA decay factorHV(C. elegans)195112574SNX18sorting nexin 184xSLEvsHV19684501SPIRE2spire homolog 24xSLE(Drosophila)vsHV19754961SSH3slingshot homolog 34xSLE(Drosophila)vsAID1989263STK17Aserine/threonine4xkinase 17a19951111SUV420H1suppressor of4xvariegation 4-20homolog 1(Drosophila)2006902TBCAtubulin folding4xcofactor A2017024TFCP2transcription factor4xSLECP2vsHV2027030TFE3transcription factor4xSLEbinding to IGHMvsenhancer 3HV20390326THAP3THAP domain4xSLEcontaining,vsapoptosis associatedAIDprotein 320410043TOM1target of myb14x(chicken)2057168TPM1tropomyosin 14xSLE(alpha)vsHV20654952TRNAU1APtRNA selenocysteine4x1 associated protein120726140TTLL3tubulin tyrosine4xligase-like family,member 32087371UCK2uridine-cytidine4xSLEkinase 2vsHV2099277WDR46WD repeat domain 464xSLEvsHV21055100WDR70WD repeat domain 704xSLEvsAID21123038WDTC1WD and4xSLEtetratricopeptidevsrepeats 1HV2129831ZNF623zinc finger protein4x62321379364ZXDCZXD family zinc4xxSLEfinger CvsAID2147791ZYXzyxin4xSLEvsAID21555964SEPT3septin 35X2165413SEPT5septin 55x21726574AATFapoptosis5xantagonizingtranscription factor21891703ACY3aspartoacylase5x(aminocyclase) 32199509ADAMTS2ADAM6SLEmetallopeptidasevswith thrombospondinRAtype 1 motif, 222010939AFG3L2AFG3 ATPase family6SLEgene 3-like 2vs(yeast)HV2211646AKR1C2aldo-keto reductase6SLEfamily 1, member C2vs(dihydrodiolRAdehydrogenase 2;bile acid bindingprotein; 3-alphahydroxysteroiddehydrogenase, typeIII)222267AMFRautocrine motility6SLEfactor receptorvsRA22310777ARPP-21cyclic AMP-regulated6SLEphosphoprotein, 21vskDRA224421ARVCFarmadillo repeat6SLEgene deletes invsvelocardiofacialRAsyndrome22580150ASRGL1asparaginase like 16SLEvsRA226539ATP50ATP synthase, H+6SLEtransporting,vsmitochondrial F1RAcomplex, O subunit22779870BAALCbrain and acute6SLEleukemia,vscytoplasmicRA2289531BAG3BCL2-associated5xathanogene 32299275BCL7BB-cell CLL/lymphoma6SLE7BvsHV23055108BSDC1BSD domain6SLEcontaining 1vsAID23154934C12orf41chromosome 12 open6SLEreading frame 41vsRA23255049C19orf60chromosome 19 open6SLEreading frame 60vsRA233388799C20orf107chromosome 20 open5xreading frame 107234149840C20orf196chromosome 20 open6SLEreading frame 196vsRA23551507C20orf43chromosome 20 open6SLEreading frame 43vsRA23655684C9orf86chromosome 9 open6SLEreading frame 86vsHV23723523CABIN1calcineurin binding6SLEprotein 1vsRA238157922CAMSAP1calmodulin regulated6SLEspectrin-associatedvsprotein 1RA23923624CBLCCas-Br-M (murine)6SLEecotropic retroviralvstransformingHVsequence c240124808CCDC43coiled-coil domain6SLEcontaining 43vsRA241100133941CD24CD24 molecule5x24211140CDC37cell division cycle6SLE37 homologvs(S. cerevisiae)RA24310153CEBPZCCAAT/enhancer6SLEbinding proteinvs(C/EBP), zetaRA24451510CHMP5chromatin modifying6SLEprotein 5vsRA24563922CHTF18CTF18, chromosome5xtransmissionfidelity factor 18homolog(S. cerevisiae)24651727CMPK1cytidine6SLEmonophosphate (UMP-vsCMP) kinase 1,AIDcytosolic24764708COPS7BCOP9 constitutive5xphotomorphogenichomolog subunit 7B(Arabidopsis)24851117COQ4coenzyme Q4 homolog6SLE(S. cerevisiae)vsRA24927254CSDC2cold shock domain5xcontaining C2, RNAbinding250162989DEDD2death effector6SLEdomain containing 2vsRA2519704DHX34DEAH (Asp-Glu-Ala-6SLEHis) box polypeptidevs34RA25255837EAPPE2F-associated6SLEphosphoproteinvsRA2531915EEF1A1eukaryotic6SLEtranslationvselongation factor 1RAalpha 12541936EEF1Deukaryotic6SLEtranslationvselongation factor 1RAdelta (guaninenucleotide exchangeprotein)2558669EIF3Jeukaryotic6SLEtranslationvsinitiation factor 3,RAsubunit J25655740ENAHenabled homolog6SLE(Drosophila)vsHV2572023ENO1enolase 1, (alpha)6SLEvsHV25811124FAF1Fas (TNFRSF6)5xassociated factor 125911170FAM107Afamily with sequence6SLEsimilarity 107,vsmember AHV26084908FAM136Afamily with sequence6SLEsimilarity 136,vsmember ARA26110144FAM13Afamily with sequence6SLEsimilarity 13,vsmember ARA26226017FAM32Afamily with sequence6SLEsimilarity 32,vsmember AHV26364762FAM59Afamily with sequence6SLEsimilarity 59,vsmember ARA264150946FAM59Bfamily with sequence6SLEsimilarity 59,vsmember BHV26583706FERMT3fermitin family6SLEhomolog 3vs(Drosophila)RA26623307FKBP15FK506 binding6SLEprotein 15, 133 kDavsHV2672670GFAPglial fibrillary6SLEacidic proteinvsRA26851031GLOD4glyoxalase domain6SLEcontaining 4vsAID26981488GRINL1Aglutamate receptor,6SLEionotropic, N-methylvsD-aspartate-like 1ARA2702922GRPgastrin-releasing6SLEpeptidevsRA2712935GSPT1G1 to S phase6SLEtransition 1vsRA27293323HAUS8HAUS augmin-like6SLEcomplex, subunit 8vsHV2733054HCFC1host cell factor C16SLE(VP16-accessoryvsprotein)AID2743069HDLBPhigh density6SLElipoprotein bindingvsproteinRA2753184HNRNPDheterogenous nuclear6SLEribonucleoprotein Dvs(AU-rich element RNAHVbinding protein 1,37 kDa)2763320HSP90AA1heat shock protein6SLE90 kDa alphavs(cytosolic), class ARAmember 12777184HSP90B1heat shock protein6SLE90 kDa beta (Grp94),vsmember 1RA2783304HSPA1Bheat shock 70 kDa6SLEprotein 1BvsRA2793315HSPB1heat shock 27 kDa4xxSLEprotein 1vsRA2805654HTRA1HtrA serine6SLEpeptidase 1vsRA2813382ICA1islet cell6SLEautoantigen 1, 69 kDavsRA2823550IKIK cytokine, down-6SLEregulator of HLA IIvsHV28380895ILKAPintegrin-linked6SLEkinase-associatedvsserine/threonineRAphosphatase 2C28484162KIAA1109KIAA11096SLEvsAID2853856KRT8keratin 86SLEvsRA28623367LARP1La ribonucleoprotein6SLEdomain family,vsmember 1AID2874001LMNB1lamin B16SLEvsRA28879888LPCAT1lysophosphatidylcholine5xSLEacyltransferasevs1HV28910916MAGED2melanoma antigen5xfamily D, 229055700MAP7D1MAP7 domain6SLEcontaining 1vsRA2915602MAPK10mitogen-activated6SLEprotein kinase 10vsHV29222919MAPRE1microtubule-6SLEassociated protein,vsRP/EB family, memberAID12934137MAPTmicrotubule-6SLEassociated protein,vstauRA29423139MAST2microtubule6SLEassociatedvsserine/threonineRAkinase 229553615MBD3methyl-CpG binding6SLEdomain protein 3vsRA29656922MCCC1methylcrotonoyl-6SLECoenzyme Avscarboxylase 1HV(alpha)2971953MEGF6multiple EGF-like-6SLEdomains 6vsRA2984302MLLT6myeloid/lymphoid or6SLEmixed-lineagevsleukemia (trithoraxRAhomolog,Drosophila);translocated to, 629910200MPHOSPH6M-phase6SLEphosphoprotein 6vsRA30010240MRPS31mitochondrial6SLEribosomal proteinvsS31HV30184939MUM1melanoma associated5xantigen (mutated) 13024599MX1myxovirus (influenza6SLEvirus) resistance 1,vsinterferon-inducibleRAprotein p78 (mouse)3034716NDUFB10NADH dehydrogenase6SLE(ubiquinone) 1 betavssubcomplex, 10,RA22 kDa3044796NFKBIL2nuclear factor of6SLEkappa lightvspolypeptide geneHVenhancer in B-cellsinhibitor-like 230511188NISCHnischarin6SLEvsRA30610381TUBB3tubulin, beta 36SLEclass IIIvsRA3078602NOP14NOP14 nucleolar6SLEprotein homologvs(yeast)RA3089722NOS1APnitric oxice6synthase 1(neuronal) adaptorprotein30929959NRBP1nuclear receptor5xbinding protein 1310142PARP1poly (ADP-ribose)6SLEpolymerase 1vsRA3115091PCpyruvate6SLEcarboxylasevsRA31223024PDZRN3PDZ domain6SLEcontaining ringvsfinger 3RA3138682PEA15phosphoprotein6SLEenriched invsastrocytes 15RA3145187PER1period homolog 16SLE(Drosophila)vsHV31557649PHF12PHD finger protein5x1231626227PHGDHphosphoglycerate5xdehydrogenase3171263PLK3polo-like kinase 36SLE(Drosophila)vsRA31823654PLXNB2plexin B26SLEvsRA31956902PNO1partner of NOB16SLEhomologvs(S. cerevisiae)RA3205479PPIBpeptidylprolyl6SLEisomerase Bvs(cyclophilin B)HV32156978PRDM8PR domain6SLEcontaining 8vsHV32255119PRPF38BPRP38 pre-mRNA6SLEprocessing factorvs38 (yeast) domainRAcontaining B3235764PTNpleiotrophin6SLEvsHV3245819PVRL2poliovirus5xreceptor-related 2(herpesvirus entrymediator B)3255831PYCR1pyrroline-5-6SLEcarboxylatevsreductase 1RA32665997RASL11BRAS-like, family6SLE11, member BvsRA32755658RNF126ring finger protein6SLE126vsAID328115992RNF166ring finger protein6SLE166vsHV3299025RNF8ring finger protein6SLE8vsHV3306092ROBO2roundabout, axon5xguidance receptor,homolog 2(Drosophila)33164221ROBO3roundabout, axonxguidance receptor,homolog 3(Drosophila)3324736RPL10Aribosomal protein6SLEL10avsRA3336152RPL24robosomal protein6SLEL24vsRA334148418SAMD13sterile alpha motif6SLEdomain containingvs13HV33557147SCYL3SCY1-like 36SLE(S. cerevisiae)vsAID3366382SDC1syndecan 16SLEvsRA33791461SGK493protein kinase-like5xprotein SgK4933386449SGTAsmall glutamine-6SLErichvstetratricopeptideHVrepeat (TPR)-containing, alpha3399627SNCAIPsynuclein, alpha5xinteracting protein3409552SPAG7sperm associated6SLEantigen 7vsRA34157522SRGAP1SLIT-ROBO Rho6SLEGTPase activatingvsprotein 1RA3426744SSFA2sperm specific6SLEantigen 2vsRA3436487ST3GAL3ST3 beta-6SLEgalactoside alpa-vs2,3-RAsialyltransferase 334423345SYNE1spectrin repeat6SLEcontaining, nuclearvsenvelope 1AID3456879TAF7TAF7 RNA polymerase6SLEII, TATA boxvsbinding proteinHV(TBF)-associatedfactor, 55 kDa3466895TARBP2TAR (HIV-1) RNA6SLEbinding protein 2vsRA3476949TCOF1Treacher Collins-6SLEFranceschettivssyndrome 1RA3487980TFPI2tissue factor5xpathway inhibitor 234956674TMEM9BTMEM9 domain6SLEfamily, member BvsRA35011189TNRC4trinucleotide5xrepeat containing 435110155TRIM28tripartite motif-6SLEcontaining 28vsHV3527204TRIOtriple functional6SLEdomain (PTPRFvsinteracting)RA353203068TUBBtubulin, beta6SLEvsRA3547280TUBB2Atubulin, beta 2ASLEvsRA35527229TUBGCP4tubulin, gamma6SLEcomplex associatedvsprotein 4RA35610422UBAC1UBA domain6SLEcontaining 1vsRA3577316UBCubiquitin C6SLEvsRA35855585UBE2Q1ubiquitin-6SLEconjugating enzymevsE2Q familiy member 1HV35965109UPF3BUPF3 regulator of5xnonsensetranscripts homologB (yeast)3607378UPP1uridine6SLEphosphorylase 1vsAID36164856VWA1von Willebrand6SLEfactor A domainvscontaining 1RA36255884WSB2WD repeat and SOCS5xbox-containing 23639877ZC3H11Azinc finger CCCH-5xtype containing 11A36455854ZC3H15zinc finger CCCH-6SLEtype containing 15vsHV3657592ZNF41zinc finger protein6SLE41vsRA366170959ZNF431zinc finger protein6SLE431vsRA367146542ZNF688zinc finger protein6SLE688vsRA3684670HNRNPMheterogeneous7SLEnuclearvsribonucleoprotein MHV36910540DCTN2dynactin 2 (p50)7SLEvsHV37010938EHD1EH-domain7SLEcontaining 1vsHV37138ACAT1Acetyl-Coenzyme A7SLEacetyltransferase 1vs(acetoacetylHVCoenzyme Athiolase)372684BST2bone marrow stromal7SLEcell antigen 2vsHV3731058CENPAcentromere protein A7SLEvsHV3741665DHX15DEAH (Asp-Glu-Ala-7SLEHis) box polypeptidevs15HV3753092HIP1Huntingtin7SLEinteracting proteinvs1HV3763336HSPE1heating shock 10 kDa7SLEprotein 1vs(chaperonin 10)HV3775455POU3F3POU class 3 homeobox7SLE3vsHV3785918RARRES1retinoic acid7SLEreceptor respondervs(tazarotene induced)HV13796136RPL12ribosomalprotein L127SLEvsHV3806626SNRPAsmall nuclear7SLEribonucleoproteinvspolypeptide AHV3816631SNRPCsmall nuclear7SLEribonucleoproteinvspolypeptide CHV3826757SSX2synovial sarcoma, X7SLEbreakpoint 2vsHV3839788MTSS1metastasis7SLEsuppressor 1vsHV38410134BCAP31B-cell receptor-7SLEassociated proteinvs31HV38510522DEAFldeformed epidermal7SLEautoregulatoryvsfactor 1HV(Drosophila)38610633RASLl0ARAS-like, family 10,7SLEmember AvsHV38754795TRPM4transient receptor7SLEpotential cationvschannel, subfamilyHVM, member 438854913RPP25ribonuclease P/MRP7SLE25 kDa subunitvsHV38954994C20orf11chromosome 20 open7SLEreading frame 11vsHV39055727BTBD7BTB (POZ) domain7SLEcontaining 7vsHV39179140CCDC28Bcoiled-coil domain7SLEcontaining 28BvsHV39279613TMCO7transmembrane and7SLEcoiled-coil domainsvs7HV3935504PPP1R2protein phosphatase7SLE1, regulatoryvs(inhibitor subunit 2HV3948349HIST2H2BEhistone cluster 2,7SLEH2bevsHV39511168PSIPlPC4 and SFRS17SLEinteracting proteinvs1HV396149986LSM14BLSM14B, SCD6 homolog7SLEB (S. cerevisiae)vsHV397655BMP7Bone morphogenetic7SLEprotein 7vs(osteogenic proteinHV1)3981676DFFADNA fragmentation7SLEfactor, 45 kDa, alphavspolypeptideHV3993071NCKAPlLNCK-associated7SLEprotein 1-likevsHV4003727JUNDjun D proto-oncogene7SLEvsHV4013960LGALS4lectin, galactoside-7SLEbinding, soluble, 4vsHV4024920ROR2Receptor tyrosine7SLEkinase-like orphanvsreceptor 2HV4037424VEGFCvascular endothelial7SLEgrowth factor CvsHV4048906AP1G2adaptor-related7SLEprotein complex 1,vsgamma 2 subunitHV40510297APC2adenomatosis7SLEpolyposis coli 2vsHV40610841FTCDFormiminotransferase7SLEcyclodeaminasevsHV40711066SNRNP35small nuclear7SLEribonucleoproteinvs35 kDa (Ull/U12)HV40811345GABARAPL2GABA(A)receptor-7SLEassociated protein-vslike 2HV40925854FAM149Afamily with sequence7SLEsimilarity 149,vsmember AHV41026065LSM14ALSM14A, SCD6 homolog7SLEA (S. cerevisiae)vsHV41128998MRPL13mitochondrial7SLEribosomal proteinvsL13HV41251520LARSleucyl-tRNA7SLEsysthetasevsHV41355747FAM21Bfamily with sequence7SLEsimilarity 21,vsmember BHV41464841GNPNAT1glucosamine-7SLEphosphate N-vsacetyltransferase 1HV41583483PLVAPPlasmalemma vesicle7SLEassociated proteinvsHV41684968PNMA6Aparaneoplastic7SLEantigen like 6AvsHV417118430MUCLlMucin-like 17SLEvsHV418122830NAT12N-acetyltransferase7SLE12vsHV419221092HNRNPUL2heterogeneous7SLEnuclearvsribonucleoprotein U-HVlike 2420388962BOLA3bolA homolog 37SLE(E. coli)vsHV421729230FLJ78302Similar to c-c7SLEchemokine receptorvstype 2 (C-C CKR-2)HV(CC-CKR-2) (CCR-2)(CCR2) (Monocytechemoattractantprotein 1 receptor)(MCP-1-R) (CD192antigen)422729447GAGE2AG antigen 2A7SLEvsHV4231152CKBNo Gene Name;7SLEcreatine kinase,vsbrainHV424972CD74CD74 molecule, major7SLEhistocompatibilityvscomplex, class IIHVinvariant chain4251397CRIP2cysteine-rich7SLEprotein 2vsHV4262040STOMstomatin7SLEvsHV4272316FLNAfilamin A, alpha7SLEvsHV4284000LMNAlamin A/C7SLEvsHV4294582MUClmucin 1, cell7SLEsurface associatedvsHV4305230PGKlPhosphoglycerate7SLEkinase 1vsHV4315340PLGplasminogen7SLEvsHV4326525SMTNsmoothelin7SLEvsHV4338936WASFlWAS protein family,7SLEmember 1vsHV43423647ARFIP2ADP-ribosylation7SLEfactor interactingvsprotein 2HV4356712SPTBN2spectrin, beta, non-7SLEerythrocytic 2vsHV4366729SRP54signal recognition7SLEparticle 54 kDavsHV4379987HNRPDLheterogeneous7SLEnuclearvsribonucleoprotein D-HVlike438337APOA4Apolipoprotein A-IV7SLEvsHV439950SCARB2scavenger receptorclass B, member 24403183HNRNPCheterogeneous7SLEnuclearvsribonucleoprotein CHV(Cl/C2)4413185HNRPFHeterogeneous7SLEnuclearvsribonucleoprotein FHV4423313HSPA9heat shock 70 kDa7SLEprotein 9 (mortalin)vsHV4433467IFNWlInterferon, omega 17SLEvsHV4443799KIF5Bkinesin family7SLEmember 5BvsHV4457918BAT4HLA-B associated7SLEtranscript 4vsHV4468337HIST2H2AA3histone cluster 2,7SLEH2aa3vsHV44710195ALG3asparagine-linked7SLEglycosylation 3,vsalpha-1,3-HVmannosyltransferasehomolog(S. cerevisiae)44823299BICD2bicaudal D homolog 27SLE(Drosophila)vsHV44980184CEP290centrosomal protein7SLE290 kDavsHV45090861HNlLhematological and7SLEneurologicalvsexpressed 1-likeHV451349136WDR86WD repeat domain 867SLEvsHV452no Gene IDdsDNAdsDNA7SLEvsHV45360ACTBactin, beta8SLEvsHV454498ATP4A1ATP synthase, H+8SLEtransporting,vsmitochondrial FlHVcomplex, alphasubunit 1, cardiacmuscle455506ATP5BATP synthase, H+8SLEtransporting,vsmitochondrial FlHVcomplex, betapolypeptide456563AZGPlalpha-2-8SLEglycoprotein 1,vszinc-bindingHV457602BCL3B-cell CLL/lymphoma8SLE3vsHV4581729DIAPHldiaphanous-related8SLEformin 1vsHV4591937EEFlGeukaryotic8SLEtranslationvselongation factor 1HVgamma4601973EIF4Aleukaryotic8SLEtranslationvsinitiation factorHV4A14612280FKBPlAFK506 binding8SLEprotein lA, 12 kDavsHV4622495FTHlferritin, heavy8SLEpolypeptide 1vsHV4632597GAPDHglyceraldehyde-3-8SLEphosphatevsdehydrogenaseHV4642819GPDlglycerol-3-8SLEphosphatevsdehydrogenase 1HV(soluble)4653295HSD17B4hydroxysteroid 17-8SLEbeta) dehydrogenasevs4HV4663305HSPAlLheat shock 70 kDa8SLEprotein 1-likevsHV4673312HSPA8heat shock 70 kDa8SLEprotein 8vsHV4684174MCM5minichromosome8SLEmaintenance complexvscomponent 5HV4694215MAP3K3mitogen-activated8SLEprotein kinasevskinase kinase 3HV4704591TRIM37tripartite motif8SLEcontaining 37vsHV4714691NCLnucleolin8SLEvsHV4724898NRDlnardilysin (N-8SLEarginine dibasicvsconvertase)HV4734904YBXlY box binding8SLEprotein 1vsHV4745037PEBPlphosphatidylethanol8SLEamine bindingvsprotein 1HV4755315PKM2pyruvate kinase,8SLEmusclevsHV4765481PPIDpeptidylprolyl8SLEisomerase DvsHV4775684PSMA3proteasome8SLE(prosome,vsmacropain)subunit,HValpha type, 34786128RPL6ribosomal protein L68SLEvsHV4796129RPL7ribosomal protein L78SLEvsHV4806130RPL7Aribosomal protein8SLEL7avsHV4816132RPL8ribosomal protein LB8SLEvsHV4826187RPS2ribosomal protein S28SLEvsHV4836189RPS3Aribosomal protein8SLES3AvsHV4846249CLIPlCAP-GLY domain8SLEcontaining linkervsprotein 1HV4856793STKl0serine/threonine8SLEkinase 10vsHV4866880TAF9TAF9 RNA polymerase8SLEII, TATA box bindingvsprotein (TBP)-HVassociated factor,32 kDa4877001PRDX2peroxiredoxin 28SLEvsHV4887552ZNF711zinc finger protein8SLE711vsHV4898260ARDlAN(alpha)-8SLEacetyltransferasevs10, NatA catalyticHVsubunit4908317CDC7cell division cycle8SLE7vsHV4918667EIF3Heukaryotic8SLEtranslationvsinitiation factorHVsubunit H4929223MAGilmembrane associated8SLEguanylate kinase, WWvsand PDZ domainHVcontaining 14939230RABllBRABllB, member RAS8SLEoncogene familyvsHV4949425CDYLchromodomain8SLEprotein, Y-likevsHV4959694EMC2ER membrane protein8SLEcomplex subunit 2vsHV49610075HUWElHECT, UBA and WWE8SLEdomain containing 1,vsE3 ubiquitin proteinHVligase49710109ARPC2actin related8SLEprotein 2/3 complex,vssubunit 2, 34 kDaHV49810180RBM6RNA binding motif8SLEprotein 6vsHV49910273STUBlSTIPl homology and8SLEU-box containingvsprotein 1, E3HVubiquitin proteinligase50010432RBM14RNA binding motif8SLEprotein 14vsHV50110539GLRX3glutaredoxin 38SLEvsHV50210806SDCCAG8serologically8SLEdefined colon cancervsantigen 8HV50311108PRDM4PR domain containing8SLE4vsHV50423002DAAMldishevelled8SLEassociated activatorvsof morphogenesis 1HV50523351KHNYNKH and NYN domain8SLEcontainingvsHV50623589CARHSP1calcium regulated8SLEheat stable proteinvs1, 24 kDaHV50726986PABPClpoly (A) binding8SLEprotein, cytoplasmicvs1HV50827072VPS41vacuolar protein8SLEsorting 41 homologvs(S. cerevisiae)HV50930836DNTTIP2deoxynucleotidyltransferase,8SLEterminal,vsinteracting proteinHV251051028VPS36vacuolar protein8SLEsorting 36 homologvs(S. cerevisiae)HV51151082POLRlDpolymerase (RNA) I8SLEpolypeptide D, 16 kDavsHV51251138COPS4COP9 signalosome8SLEsubunit 4vsHV51351466EVLEnah/Vasp-like8SLEvsHV51454869EPS8LlEPS8-like 18SLEvsHV51554903MKSlMeckel syndrome,8SLEtype 1vsHV51657017COQ9coenzyme Q98SLEvsHV51757026PDXPpyridoxal8SLE(pyridoxine, vitaminvsB6) phosphataseHV51857221ARFGEF3ARFGEF family8SLEmember 3vsHV51964753CCDC136coiled-coil domain8SLEcontaining 136vsHV52080208SPGllspastic paraplegia8SLE11 (autosomalvsrecessive)HV52183858ATAD3BATPase family, AAA8SLEdomain containing 3BvsHV52284893FBXO18F-box protein,8SLEhelicase, 18vsHV523129563DIS3L2DIS3 like 3′-5′8SLEexoribonuclease 2vsHV524144097Cllorf84chromosome 11 open8SLEreading frame 84vsHV525256364EML3echinorm microtubule8SLEassociated proteinvslike 3HV526347733TUBB2Btubulin, beta 2B8SLEclass IIbvsHV5273303HSPAlAheat shock 70 kDa8SLEprotein 1AvsHV5285163PDKlpyruvate8SLEdehydrogenasevskinase, isozyme 1HV5291001CDH3cadherin 3, type 1,8SLEP-cadherinvs(placental)HV Example 9: Identification of Autoantibody Reactivities in ENA-4-Negative SLE Patients In order to identify new SLE-specific autoantigens, the autoantibody profiles of new SLE-specific autoantigens were the autoantibody profiles of the group of SLE patients seropositive for the autoantigens Sm-protein, U1-RNP, Rho52/SS-A and Ro60/SS-B, with which the seronegative was compared. The result of the statistical test is summarised in Table 2. Group 4 comprises additional antigens suitable for the identification of ENA-4-negative SLE patients. FIG.5shows the volcano plot of the autoantibody reactivities of ENA-4-positives compared to ENA-4-negative SLE patients. Example 10: Calculation of Antigen Panels for Improved Diagnosis of SLE Due to the high clinical and serological heterogeneity of the SLE disease, it is not possible to diagnose this disease using just one biomarker. It is therefore necessary to combine (where possible) uncorrelated biomarker panels to form what are known as biomarker panels. Group 1 of the antigens in Table 2 comprises the most important 24 antigens used for the calculation of biomarker panels for the diagnosis of SLE. Table 4 shows different combinations of antigens which were used for the calculation of the biomarker panels (ENA-4, ENA-4+anti-rib, PI, PII, PIII, PVI, PV). FIG.6shows the sensitivity and specificity and also the area under the curve (AUC) for the known 4 antigens compared with antigen panels that were calculated using a combination of the antigens from Table 2. Due to the inclusion of the 3 ribosomal antigens anti-rib) RPLP0, RPLP1 and RPLP2, the sensitivity could be increased already by 10% compared with the known 4 ENA antigens from 0.63 to 0.72. However, only a freely selected combination of known and new antigens could increase the sensitivity by 20% compared with the ENA-4 test to 0.8. Antigens which have an adjusted p-value for the non-parametric mean value comparison between groups of <0.05, alongside a fold change of >1.5 and additionally an AUC resulting from the ROC analysis of >0.75 were selected on the basis of the univariate results for the generation of panels. In addition, the ENA-4 antigens were selected. For this pool of selected candidates, an L1-penalised logistic regression model was established within the scope of a nested cross validation. Antigens which were not taken into consideration within the scope of the model formation were removed from the further consideration. Within the remaining pools, panel contents were defined, for example in accordance with established markers and new markers. Group 4 in Table 2 contains further statistically significant antigens which can be used for the identification of ENA-4-negative patients and for the definition of biomarker panels. Group 6 in Table 2 contains further statistically significant antigens which can be used for the diagnosis and differential diagnosis of SLE compared with healthy controls and other autoimmune diseases. TABLE 4Composition of the diagnostic SLE PanelPanelsENA-4 +Gene SymbolGene NameAntigenENA-4anti-ribPIPIIPIIIPVIPVSNRPNsmall nuclearSmXXXXSEQ IDribonucleoproteinprotein DNO. 14polypeptide NTRIM 21tripartite motif-SSA/ROXXXXXSEQ IDcontaining 21NO. 19TROVE2TROVE domainSSA/Ro60XXXXXSEQ IDfamily, member 2NO. 20SSBSjogren sindromeSSB/LaXXXXXSEQ IDantigen BNO. 17(autoantigen La)SNRNP70small nuclearUl-RNPXXXXXSEQ IDribonucleoproteinNO. 1270 kDa (Ul)SNRPBsmall nuclearSmXXXXXSEQ IDribonucleoproteinproteinNO. 13polypeptides BB/B′and BlRPLP0ribosomalanti-ribXXXXSEQ IDprotein, large,NO. 8PORPLP2ribosomalanti-ribXXXSEQ IDprotein, large,NO. 10P2RPLPlribosomalanti-ribXXXSEQ IDprotein, large,NO. 9PlXRCCSX-ray repairKu80XSEQ IDcomplementingNO. 22defective repairin Chinesehamster cells 5(double-strand-break rejoining)VIMvimentinXXSEQ IDNO. 21SPTBspectrin, beta,XXSEQ IDerythrocyticNO. 16DBTdihydrolipoamideXXXXXSEQ IDbranched chainNO. 1transacylase E2EZRezrinXXXXXSEQ IDNO. 3HNRNPA2B1heterogeneousXXSEQ IDnuclearNO. 6ribonucleoproteinA2/B1TMPOthymopoietinXXXXSEQ IDNO. 18MVPmajor vaultXXXXSEQ IDproteinNO. 7ZNF574zinc fingerXXXSEQ IDprotein 574NO. 24HIST1H2BDhistone clusteranti-XXSEQ ID1, H2bdhistoneNO. 4SH3KBP1SH3-domain kinaseXSEQ IDbinding protein 1NO. 11ZNF217zinc fingerXXSEQ IDprotein 217NO. 23SPl00SPl00 nuclearXXXXXSEQ IDantigenNO. 15HNRNPAlheterogeneousXXXXXSEQ IDnuclearNO. 5ribonucleoproteinAlDLATdihydrolipoamidePDC-E2, M2XXXXSEQ IDS-acetyltransferaseantigenNO. 2 TABLE 5AUC, sensitivity and specificity of the SLE panelsSLE vs PSSAUCCl (AUC)Sans.Cl (Sens.)Spec.Cl (Spec)a) SLE versus healthy controlsPanel PI0.99[0.94, 0.98]0.83[0.77, 0.9]0.98[0.96, 1.0]Panel PII0.90[0.84, 0.95]0.63[0.53, 0.73]0.94[0.91, 0.98]Panel PIII0.91[0.87, 0.95]0.61[0.5, 0.72]0.95[0.92, 0.98]Panel PIV0.90[0.86, 0.94]0.57[0.44, 0.71]0.95[0.91, 0.99]Panel PV0.91[0.87, 0.96]0.64[0.52, 0.76]0.95[0.91, 0.99]ENA-40.89[0.84, 0.94]0.63[0.49, 0.77]0.96[0.9, 0.98]ENA-4 +0.93[0.88, 0.98]0.72[0.6, 0.84]0.97[0.95, 0.99]anti-ribb) SLE versus SSc (PSS)Panel PI0.9[0.85, 0.95]0.78[0.73, 0.83]0.83[0.71, 0.94]Panel PII0.83[0.78, 0.88]0.72[0.61, 0.83]0.76[0.98, 0.85]Panel PIII0.81[0.74, 0.88]0.68[0.56, 0.79]0.75[0.6, 0.89]Panel PIV0.83[0.78, 0.88]0.69[0.58, 0.81]0.77[0.67, 0.88]Panel PV0.84[0.79, 0.9]0.71[0.62, 0.8]0.76[0.65, 0.87]ENA-40.75[0.66, 0.85]0.6[0.5, 0.7]0.8[0.71, 0.88]ENA-4 +0.82[0.74, 0.9]0.63[0.51, 0.75]0.83[0.74, 0.93]anti-ribc) SLE versus all AID (early RA, SSc, SPA)SLE vs Pool(EA, PSS, SPA)AUCCl (AUC)Sans.Cl (Sens.)Spec.Cl (Spec)Panel PI0.94[0.91, 0.96]0.6[0.51, 0.69]0.98[0.98, 0.99]Panel PII0.83[0.78, 0.89]0.26[0.16, 0.35]0.98[0.97, 0.99]Panel PIII0.83[0.74, 0.92]0.27[0.16, 0.37]0.99[0.98, 1]Panel PIV0.83[0.79, 0.87]0.19[0.1, 0.28]0.98[0.97, 0.99]Panel PV0.85[0.78, 0.91]0.34[0.22, 0.46]0.99[0.98, 0.99]ENA-40.84[0.79, 0.9]0.35[0.22, 0.47]0.99[0.98, 0.99]ENA-4 +0.91[0.88, 0.93]0.49[0.41, 0.58]0.98[0.97, 0.99]anti-rib Example 11: Identification of Lupus Nephritis Patients The autoantibody profiles of SLE patients with lupus nephritis were compared with those of SLE patients without lupus nephritis. Following univariate statistical evaluation, a threshold value of p<0.05 and a 1.5 times modified reactivity compared with the control group were applied. 85 antigens met these criteria and are detailed in Table 2. FIG.7shows the volcano plot of the sera compared with selected lupus nephritis antigens. Group 2 in Table 2 contains 30 additional and important antigens which can be used for the generation of lupus nephritis biomarker panels. An L1-penalised logistic regression model with five-fold cross validation and twenty times repetition was computed for the selection of the best candidates. The antigens selected most frequently in this model computation with a frequency of more than 50% constituted the best candidates for the diagnosis of lupus nephritis. FIG.8shows the frequency distribution of the lupus nephritis antigens. Group 5 comprises further statistically significant antigens suitable for the diagnosis of lupus nephritis. Example 12: Identification of SLE Subforms and Subgroups The large clinical heterogeneity of SLE constitutes a big problem both for diagnosis and active substance development. The identification of specific antibody signatures in SLE patient subgroups thus constitutes a key step for the improved definition of patient groups in clinical studies. By way of example, as presented under Example 9, specific antibodies for lupus nephritis could be used to recruit this subgroup for drug studies. A large number of new active substances and therapeutic antibodies are currently undergoing clinical development: inter alia, therapeutic antibodies against cell-surface receptors of immune cells, such as anti-CD20, anti-CD22, or against pro-inflammatory cytokines, such as anti-IL6, are being developed. It is therefore now possible, due to the identification of serologically-defined subgroups of SLE, to link this to a target-specific response to a drug. It was first examined whether, on the basis of the typical ENA antigens and ribosomal antigens, different autoantibody signatures can already be identified in SLE patients and thus patient subgroups. FIGS.9aandbshow a dendogram of the SLE antigens after calculation of Spearman's rank correlation coefficient FIG.9ashows a dendogram for the antigens Sm, SS-B, Ro-52/SS-A, Ro60-SS-B and three ribosomal proteins. 3 antigen clusters can be already be defined on the basis of these 7 antigens. For an improved definition of SLE subgroups, however, a larger number of antigens are necessary. 50 antigens from Table 2 were therefore selected, and the correlation thereof in SLE patients was examined by calculation of Spearman's rank correlation coefficient. Group 3 contains 37 of the most important antigens necessary for the characterisation of SLE subgroups. Further antigens have already been defined in group 1 and group 2. The presentation of the antigens as a dendogram shows groups of antigens of which the reactivities in SLE patients are correlated with one another. As illustrated inFIG.9b, at least 6 groups of correlated antigens can be identified as a result. Interestingly, one of the clusters includes the antigens MVP, MIER2, CCS, DCAF6, which were identified in the table as biomarkers for lupus nephritis. Due to the calculation of a PPLS-DA-based regression model, it is possible to visualise how well the selected antigens contribute to the discrimination of the SLE patients from healthy controls. FIGS.10aandbshow the PPLS-DA biplot of the SLE patients and healthy controls with use of the SLE antigens. FIG.10ashows a PPLS-DA biplot for the selected ENA antigens and ribosomal proteins and measured values thereof in the SLE patients.FIG.10ashows that the separation of healthy and SLE is not complete and that some SLE patients coincide with the group of healthy samples. However, there is already a division of the SLE patients into 2 clusters with just few antigens. FIG.10bshows a PPLS-DA biplot for 50 antigens which are contained in Table 2. The selection of further antigens results in a practically perfect separation of the SLE patients and healthy samples. A further subdivision of the SLE patients into possible subgroups is provided by 50 antigens. These subgroups can be defined by specific antigens, some of which have been highlighted by way of example. Example 13: Validation of SLE Antigens in an Independent Test Cohort II For validation of the SLE-associated autoantigens specified in Table 2, the autoantibody reactivity in serum samples of a further independent cohort of 101 SLE patients, 105 healthy controls and 89 samples of the SLE cohort from Example 6 was measured. For this purpose the 529 human proteins specified in Table 2 (SEQ ID No. 1057 to 1584), and double-stranded DNA (dsDNA) thereof, were coupled to Luminex beads, and the antigen-coupled beads were measured in a multiplex assay with the patient samples. The binding of autoantibodies was measured by means of a PE-conjugated autoantibody in a Luminex instrument. Following univariate statistical evaluation, a threshold value of p<0.05 (Wilcoxon rank-sum test) compared with the control group was applied. A list of the significance values (p-values) for autoantibodies against 50 antigens in the SLE cohort II is shown in Table 6. Of the 50 antigens, 43 antigens in cohort I and cohort II achieved a p-value<0.05. The frequency (in %) of autoantibodies against 50 antigens in the three SLE cohorts is shown in Table 7. FIG.11: shows the calculated p-value of the antigens from Table 2 and also the frequency of SLE patients who were classified as autoantibody-positive for this antigen. Example 14: Validation of SLE Autoantigens in a Third Independent Test Cohort III For validation of the SLE-associated autoantigens specified in Table 2, the autoantibody reactivity in serum samples of an independent cohort of 183 SLE patients and 109 healthy controls was measured. For this purpose, 6,912 human proteins were coupled to Luminex beads and the protein-coupled beads were measured in a multiplex assay with the patient samples. The binding of autoantibodies was measured by means of a PE-conjugated autoantibody in a Luminex instrument. After univariate statistical evaluation, a threshold value of p<0.05 and a Cohen's d effect size of greater than 0.3 compared to the control group was provided. A list of the significance values is shown in Table 6. The table contains selected markers which are part of the panels ENA+anti-rib, panel I, panel VI, panel VII and panel VIII. The table contains further markers which achieved a p-value of <0.05 in all three cohorts. TABLE 6significance values (p-values) of 50 antigens in 3 SLE cohortsGeneSLESLESLESeq. NrGenIDSymbolcohort Icohort IIcohort IIIPanel11629DBT1.28E−042.16E−033.82E−03Panel I; II; III;IV; V; VII21737DLAT6.33E−084.12E−111.31E−04Panel I; III;IV; VI; VII37430EZR1.44E−032.68E−019.79E−02panel I; II; III;IV43017HIST1H2BD9.43E−034.08E−014.00E−02Panel II, V53178HNRNPA11.99E−093.60E−062.81E−04Panel I; II, III;IV; V; VI; VII63181HNRNPA2B17.31E−082.81E−051.70E−05Panel III; V; VI;VII79961MVP1.40E−033.90E−061.56E−04Panel I; II; III;V; VI; VII86175RPLP04.59E−134.75E−123.51E−08ENA-4 + anti-rib,Panel I; III; VI;VII96176RPLP14.61E−113.05E−137.05E−07ENA-4 + anti-rib;Pane lIII; IV;VII106181RPLP22.38E−105.18E−102.94E−09ENA-4 + anti-rib;Penal I; VI; VII1130011SH3KBP11.68E−042.22E−082.64E−02Panel V126625SNRNP704.87E−029.02E−021.22E−08ENA-4 + anti-rib;Panel I; II; IV136628SNRPB2.95E−095.57E−072.49E−09ENA-4 + anti-rib;Panel I; II; IV;VI; VII146638SNRPN7.44E−052.61E−023.64E−02EN-4 + anti-rib,Panel II; PanelIV156672SP1001.98E−042.47E−043.04E−07Panel I; II;III; IV; V; VII166710SPTB7.76E−065.13E−018.37E−01Panel III; V176741SSB1.75E−033.20E−027.82E−02ENA-4 + anti-rib;Panel I; II; IV187112TMPO2.20E−033.15E−021.15E−05Panel I; II; III;IV; VI196737TRIM215.07E−121.96E−106.35E−10Panel I; II; iV;VI; VII206738TROVE26.11E−042.59E−019.27E−05ENA-4 + anti-rib;Panel I; II; IV217431VIM2.73E−012.25E−041.06E−03Panel III; V227520XRCC52.06E−062.01E−057.83E−04Panel V; VI; VII237764ZNF2173.28E−011.55E−015.04E−05Panel III; V2464763ZNF5741.02E−028.36E−041.33E−03Panel I; II; V;VII319973CCS6.65E−065.28E−066.62E−09Panel VII956629SNRPB21.06E−038.68E−033.66E−06Panel VIII12810970CKAP41.52E−057.26E−084.60E−05Panel VIII1341743DLST1.63E−051.78E−064.95E−05Panel VII1684841NONO1.27E−043.76E−075.17E−04Panel VI; VII16929982NRBF22.22E−042.44E−027.44−03Panel VIII1714926NUMA11.62E−034.28E−054.31−03Panel VIII2147791ZYX4.54E−041.77E−052.57E−03Panel VII3684670HNRNPM7.61E−038.98E−032.34E−05Panel VII36910540DCTN21.11E−061.69E−082.49E−05Panel VII37010938EHD15.60E−051.73E−051.01E−03Panel VII372684BST2251E−021.44E−021.13E−08Panel VIII3731058CENPA4.45E−048.70E−062.51E−05Panel VIII3753092HIP14.42E−028.73E−072.03E−04Panel VIII3763336HSPE12.15E−021.42E−021.73E−03Panel VIII3775455POU3F31.31E−031.02E−046.18E−03Panel VIII3806626SNRPA7.16E−121.85E−112.60E−16Panel VIII3816631SNRPC1.42E−061.06E−073.73E−15Panel VIII3826757SSX22.40E−031.78E−022.09E−04Panel VIII38410134BCAP314.10E−021.04E−054.34E−08Panel VIII39055727BTBD73.33E−023.68E−041.79E−04Panel VIII4284000LMNA1.59E−037.49E−043.41E−04Panel VIII4294582MUC11.35E−041.66E−056.29E−04Panel VIII4315340PLG1.22E−031.64E−025.61E−03Panel VIII4326525SMTN2.05E−031.53E−029.78E−03Panel VIII452dsDNAdsDNA1.65E−065.35E−17NAdsDNA TABLE 7List of the frequency of SLE patients positively tested for autoantibodiesin 3 independent cohorts. The frequency in % of individualspositively tested for autoantibodies from Table 6 was calculatedby means of the 95% quantile of healthy controls.Proportion of SLEpatients positivelytested forautoantibodies (%)based on the 95%quantile of thecontrol groupSeq.GeneSLESLESLENr.GeneIDSymbolcohort Icohort IIcohort IIIPanel11629DBT1.28E−042.16E−033.82E−03Panel I: II; III;IV; V; VII21737DLAT6.33E−084.12E−111.31E−04Panel I; III; IV;VI; VII37430EZR1.44E−032.68E−019.79E−02panel I; II; III;IV43017HIST1H2BD9.43E−014.08E−014.00E−01Panel II, V53178HNRNPA11.99E−093.60E−062.81E−04Panel I; II, III;IV; V; VI; VII63181HNRNPA2B17.31E−082.81E−051.70E−05Panel III; V; VI;VII79961MVP1.40E−033.90E−061.56E−04Panel I; II; III;V; VI; VII86175RPLP04.59E−134.75E−123.51E−08ENA-4 + anti-rib,Panel I; III; VI;VII96176RPLP14.61E−113.05E−137.50E−07ENA-4 + anti-rib;Pane 1III; IV; VII106181RPLP22.38E−105.18E−102.94E−09ENA-4 + anti-rib;Panel I; VI; VII1130011SH3KBP11.68E−042.22E−082.64E−02Panel V126625SNRNP704.87E−029.02E−021.22E−08ENA-4 + anti-rib;Panel I; II; IV136628SNRPB2.95E−095.57E−072.49E−09ENA-4 + anti-rib;Panel I; II; IV;VI; VII146638SNRPN7.44E−052.61E−023.64E−02ENA-4 + anti-rib,Panel II; Panel IV156672SP1001.98E−042.47E−043.04E−07Panel I; II; III;IV; V; VII166710SPTB7.76E−065.13E−018.37E−01Panel III; V176741SSB1.75E−033.20E−027.82E−02ENA-4 + anti-rib;Panel I; II; IV187112TMPO2.20E−033.15E−021.15E−05Panel I; II; III;IV; VI196737TRIM215.07E−121.96E−106.35E−10Panel I; II; iV;VI; VII206738TROVE26.11E−042.59E−019.27E−05ENA-4 + anti-rib;Panel I; II; IV217431VIM2.37E−012.25E−041.06E−03Panel III; V227520XRCC52.06E−062.01E−057.83E−04Panel V; VI; VII237764ZNF2173.28E−011.55E−015.04E−05Panel III; V2464763ZNF5741.02E−028.36E−041.33E−03Panel I; II; V; VII319973COS6.65E−065.28E−066.62E−09Panel VII956629SNRPB21.06E−038.68E−033.66E−06Panel VIII12810970CKAP41.52E−057.26E−084.60E−05Panel VIII1341743DLST1.63E−051.78E−064.95E−05Panel VII1684841NONO1.27E−043.76E−075.17E−04Panel VI; VII16929982NRBF22.22E−042.44E−027.44E−03Panel VIII1714926NUMA11.62E−034.28E−054.31E−03Panel VIII2147791ZYX4.54E−041.77E−052.57E−03Panel VII3684670HNRNPM7.61E−038.98E−032.34E−05Panel VII36910540DCTN21.11E−061.69E−082.49E−05Panel VII37010938EHD15.60E−051.73E−051.01E−03Panel VII372684BST22.51E−021.44E−021.13E−08Panel VIII3731058CENPA4.45E−048.70E−062.51E−05Panel VIII3753092HIP14.42E−028.73E−072.03E−04Panel VIII3763336HSPE12.15e−021.42E−021.73E−03Panel VIII3775455POU3F31.31E−031.02E−046.18E−03Panel VIII3806626SNRPA7.16E−121.85E−112.60E−16Panel VIII3816631SNRPC1.42E−061.06E−073.73E−15Panel VIII3826757SSX22.40E−031.78E−022.09E−04Panel VIII38410134BCAP314.10E−021.04E−054.34E−08Panel VIII39055727BTBD73.33E−023.68E−041.79E−04Panel VIII4284000LMNA1.59E−037.49E−043.41E−04Panel VIII4294582MUC11.35E−041.66E−056.29E−04Panel VIII4315340PLG1.22E−031.64E−025.61E−03Panel VIII4326525SMTN2.05E−031.53E−029.78E−03Panel VIII452dsDNAdsDNA1.65E−065.35E−17NAdsDNA Example: Calculation of Biomarker Panels As shown in Table 7, only at most approximately 60% of the SLE patients had antibodies for a specific autoantigen. In order to therefore increase the sensitivity of the diagnostic autoantibodies, such as anti-dsDNA, SSA-Ro (TRIM21/TROVE2) and U1-RNP (SNRNP70, SNRPNA, SNRNPC), new methods with which autoantibodies can be combined to form what are known as biomarker panels were tested. For this pool of selected candidates, a logistic regression was carried out for panels PI to PVII. An L1-penalised logistic regression model was established within the scope of a nested cross validation for panels PVIII to PXI. Antigens which were not considered within the scope of the model formation were removed from the further consideration. The content of panels was defined within the remaining pool, for example in accordance with established markers and new markers. The antigens specified in Table 2 were used for the calculation of biomarker panels for the diagnosis of SLE. Table 4 shows different combinations of antigens which were used for the calculation of the biomarker panels (ENA-4, ENA-4+anti-rib, PI, PII, PIII, PVI, PV). Table 8 shows further different combinations of antigens which were used for the calculation of panels and which were selected on account of their significance and reactivity in three SLE cohorts. TABLE 8Combinations of antigens from Table 2:Seq. IDGenePanel+antiPanelPanelPanelPanelPanelPanelPanelPanelPanelPanelPanelNrGeneIDSymbolENA-4ribPIPIIIIIPIVVVIVIIVIIIIXX *XI *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* The panels X and XI can be supplemented by 20 or more markers from the other available 1587 markers, in particular proteins.Panel VI comprises 11 antigens which were measured in all three SLE cohorts with a p-value < 0.05.Panel VII comprises 19 antigens which were measured in the three SLE cohorts with a p-value < 0.05.Panel VIII comprises panel VII and a further 52 antigens which were found in cohort 3 and at least one of the other SLE cohorts with a p-value < 0.05 for the comparison of SLE against healthy controls.Panel IX comprises panel VII, panel VIII and a further 110 antigens which, in one or two SLE cohorts for the comparison of SLE against healthy controls, achieved a p-value of 0.05.Panel X comprises panel VII, panel VIII, panel IX and a further 227 antigens which, as specified in Table 2, originate from different comparisons and achieved a p-value < 0.05 in at least one SLE cohort. Tables 9a, 9c and 9e show the area under the curve (AUC) confidence interval, sensitivity and specificity of different biomarker combinations in the three different SLE cohorts. Tables 9b and 9d show the area under the curve (AUC), confidence interval, sensitivity and specificity of the different panels in the three SLE cohorts in combination with anti-dsDNA autoantibodies. TABLE 9aArea under the curve (AUC), sensitivity and specificityof the different panels in the SLE cohort I.AUCSensitivitySpecificityCohort IlowerupperlowerupperlowerupperPanelmeanCICImeanCICImeanCICIPI0.860.840.870.790.760.810.840.820.86PII0.880.870.900.810.790.840.820.800.85PIII0.840.830.860.740.710.760.800.770.83PIV0.850.840.870.800.770.820.830.810.85PV0.810.790.830.730.700.750.760.730.79PVI0.870.860.890.790.770.820.830.810.85Panel.0.870.850.880.730.710.760.860.840.89ENAPanel.0.870.850.880.780.750.800.830.810.85ENA +antiRibPVII0.790.770.810.750.720.780.770.740.79PVIII0.850.830.860.750.720.780.820.790.84PIX0.830.810.840.730.710.760.810.780.83PX0.830.810.840.730.700.760.780.760.81PXI0.830.810.840.740.710.760.790.760.81 TABLE 9bArea under the curve (AUC), upper and lower confidence interval(CI), sensitivity and specificity of the biomarker panels inSLE cohort I in combination with anti-dsDNA autoantibodies.Cohort IPanelAUCSensitivitySpecificitypluslowerupperlowerupperlowerupperdsDNAmeanCICImeanCICImeanCICIPI0.860.840.870.790.770.810.830.810.85PII0.870.850.880.800.770.820.810.790.83PIII0.830.810.850.740.710.770.780.760.81PIV0.860.840.870.790.760.820.830.810.85PV0.800.780.820.720.700.750.760.730.78PVI0.860.850.880.790.770.820.820.800.85Panel.0.860.850.880.730.710.760.860.840.89ENAPanel.0.860.850.880.760.740.780.830.800.85ENA +antiRibPVII0.790.770.810.740.710.770.760.730.78PVIII0.890.880.900.800.780.830.850.830.87PIX0.900.890.920.810.790.830.850.830.87PX0.840.820.860.730.700.750.810.790.84PXI0.890.880.900.810.780.830.840.820.86 TABLE 9cArea under the curve (AUC), shows the sensitivity and specificityof the different panels in the SLE cohort II.CohortAUCSensitivitySpecificityIIlowerupperlowerupperlowerupperPanelmeanCICImeanCICImeanCICIPI0.840.830.860.740.720.760.790.770.81PII0.780.770.800.680.650.700.720.700.75PIII0.830.810.840.730.700.750.780.760.81PIV0.860.840.870.760.740.780.810.790.84PV0.770.750.780.670.650.690.740.720.76PVI0.870.860.880.770.740.790.820.800.84Panel.0.760.740.770.590.560.61780.750.80ENAPanel.0.840.830.860.730.710.760.830.820.85ENA +antiRibPVII0.840.820.860.760.740.780.800.770.82PVIII0.850.830.860.760.740.780.800.780.82PIX0.840.830.860.760.740.780.790.770.81PX0.830.810.850.760.730.780.780.760.80PXI0.820.810.840.740.710.760.790.760.81 TABLE 9dArea under the curve (AUC), sensitivity and specificityof the different panels in SLE cohort II inCohortIIPanelAUCSensitivitySpecificitypluslowerupperlowerupperlowerupperdsDNAmeanCICImeanCICImeanCICIPI0.840.820.850.730.710.760.780.760.81PII0.780.760.800.670.650.700.730.700.75PIII0.820.810.840.730.710.750.760.740.79PIV0.850.840.870.760.730.780.800.780.83PV0.770.750.780.670.650.690.710.690.74PVI0.870.850.880.770.750.790.820.800.84Panel.0.770.760.790.600.580.630.770.750.80ENAPanel.0.850.840.860.730.710.760.830.810.85ENA +antiRibPVII0.840.820.850.750.730.780.790.770.82PVIII0.850.830.860.720.700.750.830.800.85PIX0.780.770.800.640.620.670.780.750.80PX0.840.830.850.720.700.750.830.810.85PXI0.870.850.880.750.730.770.840.820.86 TABLE 9eArea under curve (AUC), sensitivity and specificityof the different panels in the SLE cohort III.CohortIIPanelAUCSensitivitySpecificitypluslowerupperlowerupperlowerupperdsDNAmeanCICImeanCICImeanCICIPI0.830.820.840.710.690.730.800.780.81PII0.820.810.840.710.690.720.800.790.81PIII0.790.780.800.650.630.670.760.740.78PIV0.820.810.830.710.690.730.790.780.81PV0.770.760.780.660.640.670.760.740.78PVI0.840.830.850.710.700.730.810.790.82Panel.0.780.770.800.650.630.670.820.810.84ENAPanel.0.790.780.800.670.660.690.830.820.85ENA +antiRibPVII0.830.820.840.720.710.740.790.770.81PVIII0.830.820.840.730.720.750.770.760.79PIX0.810.790.820.720.700.740.760.740.77PX0.790.780.810.730.710.750.760.740.78PXI0.780.770.800.700.690.720.750.730.77 FIG.11: The figure shows the comparison of the calculated p-values and autoantibody frequencies (% positive classified observations) for the antigens from Table 2 in the three SLE cohorts. The antigens are illustrated as circles with the consecutive number. The horizontal line marks the threshold value of p<0.05 for the comparison of SLE compared with healthy controls. LITERATURE Li P H, Wong W H, Lee T L, Lau C S, Chan T M, Leung A M, Tong K L, Tse N K, Mok C C, Wong S N, Lee K W, Ho M H, Lee P P, Chong C Y, Wong R W, Mok M Y, Ying S K, Fung S K, Lai W M, Yang W, Lau Y L. Relationship between autoantibody clustering and clinical subsets in SLE: cluster and association analyses in Hong Kong Chinese. Rheumatology (Oxford). 2013 February; 52(2):337-45. doi: 10.1093/rheumatology/kes261.Epub 2012 Oct. 4. PubMed PMID: 23038697.Liu C C, Kao A H, Manzi S, Ahearn J M. Biomarkers in systemic lupus erythematosus: challenges and prospects for the future. Ther Adv Musculoskelet Dis. 2013 August; 5(4):210-33.Ching K H, Burbelo P D, Tipton C, Wei C, Petri M, Sanz I, Iadarola M J. Two major autoantibody clusters in systemic lupus erythematosus. PLoS One. 2012; 7(2):e32001. doi: 10.1371/journal.pone.0032001. Epub 2012 Feb. 21. PubMed PMID: 22363785; PubMed Central PMCID: PMC3283706.Stohl W. Future prospects in biologic therapy for systemic lupus erythematosus. Nat Rev Rheumatol. 2013 Sep. 10. doi: 10.1038/nrrheum.2013.136. [Epub ahead of print] PubMed PMID: 24018550.Thanou A, Merrill J T. Treatment of systemic lupus erythematosus: new therapeutic avenues and blind alleys. Nat Rev Rheumatol. 2013 Oct. 8. doi:10.1038/nrrheum.2013.145. [Epub ahead of print] PubMed PMID: 24100460.Sherer Y, Gorstein A, Fritzler M J, Shoenfeld Y. Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients. Semin Arthritis Rheum. 2004 October; 34(2):501-37. Review. PubMed PMID: 15505768. | 70,665 |
11860163 | 5. DETAILED DESCRIPTION The present invention relates to compositions and methods for identifying patients considered for or undergoing a chimeric antigen receptor (CAR) T cell therapy who are at higher risk for poor response to the CAR T cell therapy or who are likely to achieve a partial or complete response by analyzing the intestinal microbiome of those patients before or during the CAR T cell therapy, and related therapeutic compositions and methods to reduce the risk of poor response and improve the likelihood of partial or complete response to the CAR T cell therapy. In a CAR T cell therapy, a patient's own T cells are harvested, then genetically modified outside of the patient's body so that the T cells begin to express a chimeric antigen receptor on their surface. The CAR targets cancer cells in the patient's body. After modification, the T cells are then injected back into the patient, where they proceed to recognize the cancer cells and cause an immune response to the cancer cells. The cancer cells are destroyed by the immune response, treating the cancer. The presently disclosed invention is based in part on the discovery that certain intestinal microbial modules are predictive of a poor response to a CAR T cell therapy, and that certain intestinal microbial modules are predictive of a strong or a complete response to the CAR T cell therapy. The discovery is based on experiments, including those in the Examples disclosed herein, in which the intestinal microbiota of patients were characterized and compared prior to and after a CAR T cell therapy, and their relationship with the patients' response to the CAR T cell therapy. In certain embodiments, microbiota of the Peptostreptococcaceae family, such as theRomboutsiagenus, particularlyRomboutsia ileitis, the Bacteroidaceae family, particularlyBacteroides uniformis, or the Clostridiaceae family, particularlyClostridium butyricumare predictive of a poor response to a CAR T cell therapy. In certain embodiments, microbiota of the Lachnospiraceae family, such as members of theRoseburiagenus, members of thePseudobutyrivibriogenus, particularlyPseudobutyrivibrioruminis, or members of theLachnospiragenus, particularlyLachnospira pectinoschiza, or particularly orClostridium amygdalinum, Clostridium saccharolyticum, orCoprococcus comes, the Rikenellaceae family, particularlyAlistipes indistinctus, the Lactobacillaceae family, such as theLactobacillusgenus, particularlyLactobacillus fermentumorLactobacillus rogosae, the Oscillospiraceae family, particularlyOscillibacter valericigenes, and the Ruminococcaceae family, such as members of Ruminococcaceae UCG-004 genus, members of theAnaerotruncusgenus, particularlyAnaerotruncus colihominis, or particularlyClostridium methylpentosum, or the Acidaminococcaceae family, such as members of thePhascolarctobacteriumgenus, particularlyPhascolarctobacterium faecium, bacteria of the Peptococcaceae family are predictive of a strong or complete response to a CAR T cell therapy. In particular, microbiota of the Lachnospiraceae family were found in abundance in patients who achieved a strong or complete response to a CAR T cell therapy, while microbiota of the Peptostreptococcaceae family were found in abundance in patients who exhibited a poor response to a CAR T cell therapy. In addition, a higher than normal abundance of genes associated with B vitamin biosynthesis and genes associated with secondary bile acid biosynthesis and degradation was observed in the intestinal microbiome of patients with a poor response to a CAR T cell therapy, or who were unable to achieve a complete response. In particular a higher than normal abundance of genes for thiamine biosynthesis (e.g., thiH), pantothenic acid biosynthesis (e.g., panC), and pyroxidine biosynthesis (e.g., pdxJ, gapA, dxs) was observed in the intestinal microbiome of patients with poor response to the CAR T cell therapy, or who were unable to achieve a complete response. For clarity of description, and not by way of limitation, this section is divided into the following subsections:5.1 Methods of Predicting Responsiveness to a CAR T cell therapy;5.2 Therapeutic bacteria;5.3 Pharmaceutical compositions;5.4 Methods of Treatment; and5.5 Kits. The following are terms relevant to the present invention: As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Still further, the terms “having,” “including,” “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms. The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. An “individual” or “subject” or “patient” herein is a vertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include birds, such a poultry, including chickens, turkeys, ducks, and geese; rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys. In certain embodiments, the level or the abundance of a bacterium can be determined by quantification of bacterial DNA or RNA in the sample. In certain embodiments, the bacterial DNA or RNA comprises 16s rDNA or RNA encoded by a bacterial gene unique to the bacterial species. In certain embodiments, the bacterial DNA (e.g., 16s rDNA) or RNA level is determined by a sequencing method, e.g., metagenomic sequencing or shotgun metagenomic sequencing. In certain embodiments, the sequencing is performed using a Illumina MiSeq platform or Illumina HiSeq 2000 platform. In certain embodiments, the bacterial DNA or RNA level (e.g., copy number) is determined by an amplification-based method, e.g., by polymerase chain reaction (PCR), including reverse transcription-polymerase chain reaction (RT-PCR) for RNA quantitative analysis. In certain embodiments, amplification of the bacterial DNA or RNA in a sample may be accomplished by any known method, including but not limited to ligase chain reaction (LCR), transcription-mediated amplification, and self-sustained sequence replication or nucleic acid sequence-based amplification (NASBA). In certain embodiments, the level of a bacterial DNA or RNA level can be determined by size fractionation (e.g., gel electrophoresis), whether or not proceeded by an amplification step. In certain embodiments, the level of a bacterial DNA or RNA level can be determined by sequence-specific probe hybridization. In certain embodiments, the level of a bacterial DNA or RNA level can be determined by mass spectroscopy, PCR, microarray hybridization, thermal sequencing, capillary array sequencing, or solid phase sequencing. In certain embodiments, the level or the abundance of the bacterium is determined by quantification of one or more proteins unique to the bacteria. In certain embodiments, the protein that is indicative of a bacterium's identity, can be detected but not limited using Western Blot, microarray, gel electrophoresis (such as 2-dimensional gelelectrophoresis), and immunohistochemical assays. In certain embodiments, the level or the abundance of the bacterium refers to a relative abundance of the bacterium in a sample. The relative abundance of a bacterium refers to the proportion occupied by the particular bacterium in the whole bacterial flora in the sample. The relative abundance of a bacterium can be determined from, for example, the total number of bacterial cells constituting the bacterial flora and the number of the particular bacterial cells included in the bacterial flora. More specifically, for example, genes having a nucleotide sequence that is common in the bacteria included in the bacterial flora and nucleotide sequences characteristic to each bacterial species (for example, 16S rRNA gene) are comprehensively decoded, and the relative abundance of a particular bacterium can be determined by designating the total number of decoded genes and the total number of genes belonging to particular bacterial species as the total number of bacterial cells constituting the bacterial flora and the number of particular bacterial cells, respectively. In certain embodiments, the level of a bacterial gene is determined by measuring a level of a bacterial nucleic acids include DNA and RNA including at least a portion of the bacterial gene, a bacterial mRNA or cDNA that is transcribed from the bacterial gene, or a sequence complementary or homologous thereto (including but not limited to antisense or small interfering RNA). Said nucleic acid may be included of natural nucleotides and may optionally include nucleotide bases which are not naturally occurring. In certain embodiments, the level of a bacterial gene is determined by measuring a level of a bacterial protein that is encoded by the bacterial gene. Any suitable methods known in the art for measuring nucleic acid and protein levels can be used with the presently disclosed methods. In certain embodiments, methods for measuring nucleic acid levels include, but not limited to, real-time PCR (RT-PCR), quantitative PCR, quantitative real-time polymerase chain reaction (qRT-PCR), fluorescent PCR, RT-MSP (RT methylation specific polymerase chain reaction), PicoGreen™ (Molecular Probes, Eugene, OR) detection of DNA, radioimmunoassay or direct radio-labeling of DNA, in situ hybridization visualization, fluorescent in situ hybridization (FISH), microarray, sequencing. In certain embodiments, methods for measuring protein levels include, but are not limited to, mass spectrometry techniques, 1-D or 2-D gel-based analysis systems, chromatography, enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), enzyme immunoassays (EIA), Western Blotting, immunoprecipitation and immunohistochemistry. In certain non-limiting embodiments, the subject suffers from a cancer. In certain non-limiting embodiments, the subject is receiving or can receive CAR T cell therapy. “CART cell therapy” is the killing of cancer cells using a T cell genetically modified to express a chimeric antigen receptor (CAR) that binds to the cancer cells, resulting in activation of the patient's immune system to kill the cancer cells. CAR T cell therapy can be particularly useful in treating acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, CD19 malignancies, myeloma other B cell-related or hematologic malignancies, or in treating solid tumors, such as ovarian cancer. As used herein, a “response to a CAR T cell therapy” refers to a complete response or a partial response to the CAR T cell therapy. A “complete response” or “complete remission” is defined for any given cancer type as the absence of cancer cells detectable by imaging or molecular methods conventionally used for detection of that type of cancer. A “complete response” does not necessarily mean that all cancer cells are absent from the patient. For cancers in which multiple conventional imaging or molecular methods are conventionally used for detection, the absence of detectable cancer cells using any one of such multiple methods is sufficient to indicate a “complete response” for purposes of the present specification. A “partial response” or “partial remission” is defined for any given cancer type as at least a 50% reduction in estimated number of cancer cells or tumor burden detectable by imaging or molecular methods conventionally used for detection of that type of cancer. For cancers in which multiple conventional imaging or molecular methods are conventionally used for detection, a 50% reduction of detectable cancer cells using any one of such multiple methods is sufficient to indicate a “partial response” for purposes of the present specification. A “poor response” is any response to a CAR T treatment that is not a “complete response” or a “partial response.” A poor response can include an increase in cancer cells or tumor burden as detectable using conventional imaging or molecular methods for detection of that type of cancer. A poor response can also include a minimal decrease in cancer cells that is still not sufficient to be considered “partial remission.” “CAR T toxicity” is an early response to CAR T cell therapy and includes cytokine release syndrome and neurotoxicity. Although CAR T toxicity is often considered an adverse reaction, it results from T cell activity and, thus, is also an indicator of likely efficacy of the CAR T cell therapy. “Cytokine release syndrome” or “CRS” is characterized by high fever, myalgias, malaise, respiratory insufficiency, hemodynamic instability and capillary leak with hypotension, tachycardia, hypoxia, tachypnea, hemophagocytic lymphohistiocytosis/macrophage activation syndrome, or other organ toxicity associated with elevated serum cytokine concentrations. Elevated cytokines and associated molecules include interferon (IFN)-γ, IL-2, soluble IL-2Rα, IL-6, soluble IL-6R, granulocyte-macrophage colony-stimulating factor (GM-CSF), and other cytokines primarily secreted by the monocytes and/or macrophages such as IL-1, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF)-α, IFN-α, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP) la. CRS usually occurs within a few days of administration of the genetically modified T cells to the patient. “Neurotoxicity” associated with CAR T cell therapy is characterized by encephalopathy, headache, delirium, anxiety, tremor, aphasia, decreased level of consciousness, confusion, seizures, or cerebral edema. Neurotoxicity can be associated with elevated serum concentrations of IL-6, IFN-γ, and TNF-α. An “effective amount” of a substance as that term is used herein is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In the context of administering a composition to reduce the risk of a poor response to CAR T cell therapy or to increase the chance of a complete response or partial response to CAR T cell therapy, an effective amount of a composition described herein is an amount sufficient to decrease the likelihood of a poor response or to increase the likelihood of a complete response or partial response by at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%. As used herein, and as well-understood in the art, “treatment” or administration of a “therapeutic agent” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this subject matter, beneficial or desired clinical results include, but are not limited to decreased risk of a poor response to CAR T cell therapy, increased likelihood of a complete response or partial response to CAR T cell therapy, or a combination thereof. A “probiotic” is a microorganism or group of microorganisms that provides health benefits, or that is non-pathogenic, to a subject when consumed, ingested, or otherwise administered to a subject, for example, a reduction in the likelihood of relapse following cancer treatment. As used herein, the term probiotic can be used to describe, for example, probiotic bacteria and can include the bacteria described herein as well as other bacteria. A “prebiotic” is a substance that promotes the growth, proliferation and/or survival of one or more bacteria or yeast. As used herein, the term prebiotic can be used to describe, for example, a nutritional supplement including plant fiber, or one or more of poorly-absorbed complex carbohydrates, oligosaccharides, inulin-type fructans or arabinoxylans. A “postbiotic” is a substance derived from a probiotic organism. As used herein, the term postbiotic can be used to describe, for example, a protein expressed by one or more bacteria, a metabolic product of one or more bacteria, or media from a culture of one or more strains of bacteria. 5.1 Methods of Predicting Responsiveness to a CAR T Cell Therapy The present disclosure provides methods of identifying a subject as likely to have a response to a CAR T cell therapy. In certain embodiments, the response is a complete response or a partial response. The present disclosure also provides methods of identifying a subject as likely to have no response or a poor response to a CAR T cell therapy. In certain embodiments, the methods disclosed herein comprise determining the level of a bacterium or spores thereof in a sample from the subject, comparing the level of the bacterium or spores thereof to a reference level, identifying the subject as likely to have a response to the CAR T cell therapy based on the comparison, or identifying the subject as likely to have no response or a poor response to the CAR T cell therapy based on the comparison. In certain embodiments, the methods comprise determining the level of a bacterial gene in a sample from the subject, comparing the level of the bacterial gene to a reference level, identifying the subject as likely to have a response to the CAR T cell therapy based on the comparison, or identifying the subject as likely to have no response or a poor response to the CAR T cell therapy based on the comparison. In certain embodiments, the methods further comprise treating the subject that is identify as likely to have a response to the CAR T cell therapy with the CAR T cell therapy. In certain embodiments, the methods further comprise treating the subject that is identify as likely to have no response or have a poor response to the CAR T cell therapy with the presently disclosed therapeutic bacteria or the pharmaceutical compositions (e.g., as disclosed in the Sections 5.2 and 5.3). The present disclosure further provides methods of identifying a subject as likely to have a CAR-T cell associated toxicity. In certain embodiments, the CAR-T cell associated toxicity is a cytokine release syndrome or a neurotoxicity. In certain embodiments, the methods comprise determining the level of a bacterium or spores thereof in a sample from the subject, comparing the level of the bacterium or spores thereof to a reference level, identifying the subject as likely to have a CAR-T cell associated toxicity based on the comparison. In certain embodiments, the methods comprise determining the level of a bacterial gene in a sample from the subject, comparing the level of the bacterial gene to a reference level, identifying the subject as likely to have a CAR-T cell associated toxicity based on the comparison. In certain embodiments, the methods further comprise treating the CAR-T cell associated toxicity in the subject. An increased or decreased level of the bacterium or spores thereof or of the bacterial gene is determined with respect to a reference bacterium or spores thereof level or a reference bacterial gene level. In certain embodiments, the level (e.g., the measured level and the reference level) can be based on a relative abundance in the intestinal microbiome. For instance, the level can represent a percentage of the bacterium or spores thereof of all the bacteria or spores thereof in the intestinal microbiome. The level can also be an absolute number. In certain embodiments, the reference level is a predetermined level of a bacterium or spores thereof or of a bacterial genetic module that a level higher or lower than the reference level indicates the subject is likely to have a response to the CAR T cell therapy, or is likely to have no response or a poor response to the CAR T cell therapy. In certain embodiments, the reference level is the level of a bacterium or spores thereof or of a bacterial gene from a subject or a population of subjects that have a response to the CAR T cell therapy. In certain embodiments, the reference level is the level of a bacterium or spores thereof or of a bacterial gene from a population of subjects that are candidates for a CAR T cell therapy or subjects with cancer that have not received a CAR T cell therapy. In certain embodiments, the reference level is the level of a bacterium or spores thereof or of a bacterial gene from a sample of the same subject collected at an earlier time point. In certain embodiments, the reference level can be based on a prior test in the same patient, or on levels found in a patient population, such as patients who are candidates for CAR T cell therapy or patients with cancer who have not undergone CAR T cell therapy. In certain embodiments, the bacterium determined in the sample of the subject is selected from the group consisting of bacteria of the Peptostreptococcaceae family (e.g., theRomboutsiagenus, e.g.,Romboutsia ileitis), bacteria of the Bacteroidaceae family (e.g.,Bacteroides uniformis), bacteria of the Clostridiaceae family (e.g.,Clostridium butyricum, Clostridium saccharolyticum, Clostridium amygdalinum), bacteria of the Lachnospiraceae family (e.g., theRoseburiagenus, thePseudobutyrivibriogenus, e.g.,Pseudobutyrivibrioruminis, e.g., theLachnospiragenus, e.g.,Lachnospira pectinoschiza, Coprococcus comes), bacteria of the Rikenellaceae family (e.g.,Alistipes indistinctus), bacteria of the Lactobacillaceae family (e.g.,Lactobacillusgenus, particularlyLactobacillus fermentumorLactobacillus rogosae), bacteria of the Oscillospiraceae family (e.g.,Oscillibacter valericigenes), bacteria of the Ruminococcaceae family (e.g., the Ruminococcaceae UCG-004 genus, theAnaerotruncusgenus, e.g.,Anaerotruncus colihominis, Clostridium methylpentosum), bacteria of the Acidaminococcaceae family (e.g., thePhascolarctobacteriumgenus, e.g.,Phascolarctobacterium faecium), bacteria of the Peptococcaceae family and any combinations thereof. In certain embodiments, the bacterial gene determined in the sample of the subject is selected from the group consisting of the genes involved in B vitamin biosynthesis (e.g., riboflavin (B2), pantothenate (B5) and thiamine (B1), genes involved in secondary bile acid biosynthesis and degradation, and any combinations thereof. In certain embodiments, the genes involved in B vitamin biosynthesis include thiH, panC, pdxJ, gapA, dxs, and a combination thereof. In certain embodiments, the genes involved in secondary bile acid biosynthesis and degradation include baiA1, baiF, baiE, baiCD, or a combination thereof. In certain embodiments, the methods disclosed herein further comprise identifying the subject as likely to have a response to the CAR T cell therapy, or as likely to have a CAR T cell associated toxicity, if the level of the bacterium or spores thereof is lower than the reference bacterium or spores thereof level, wherein the bacterium is selected from the group consisting of bacteria of the Peptostreptococcaceae family (e.g., theRomboutsiagenus, e.g.,Romboutsia ileitis), bacteria of the Bacteroidaceae family (e.g.,Bacteroides uniformis), bacteria of the Clostridiaceae family (e.g.,Clostridium butyricum), and any combinations thereof. In certain embodiments, the methods disclosed herein further comprise identifying the subject as likely to have a response to the CAR T cell therapy, or as likely to have a CAR T cell associated toxicity, if the level of the bacterium or spores thereof is higher than the reference bacterium or spores thereof level, wherein the bacterium is selected from the group consisting of bacteria of the Lachnospiraceae family (e.g., theRoseburiagenus, thePseudobutyrivibriogenus, e.g.,Pseudobutyrivibrioruminis, e.g., theLachnospiragenus, e.g.,Lachnospira pectinoschiza, e.g.,Coprococcus comes), bacteria of the Rikenellaceae family (e.g.,Alistipes indistinctus), bacteria of the Lactobacillaceae family (e.g., theLactobacillusgenus, e.g.,Lactobacillus fermentumorLactobacillus rogosae), bacteria of the Oscillospiraceae family (e.g.,Oscillibacter valericigenes), bacteria of the Ruminococcaceae family (e.g., the Ruminococcaceae UCG-004 genus, theAnaerotruncusgenus, e.g.,Anaerotruncus colihominis, Clostridium methylpentosum), bacteria of the Acidaminococcaceae family (e.g., thePhascolarctobacteriumgenus, e.g.,Phascolarctobacterium faecium), bacteria of the Clostridiaceae family (e.g.,Clostridium amygdalinum, Clostridium saccharolyticum), bacteria of the Peptococcaceae family and any combinations thereof. In certain embodiments, the methods disclosed herein further comprise identifying the subject as likely to have a response to the CAR T cell therapy, or as likely to have a CAR T cell associated toxicity, if the level of the bacterial gene is lower than the reference bacterial gene level, wherein the gene is selected from the group consisting of the genes involved in B vitamin biosynthesis (e.g., riboflavin (B2), pantothenate (B5) and thiamine (B1), genes involved in secondary bile acid biosynthesis and degradation, and any combinations thereof. In certain embodiments, the genes involved in B vitamin biosynthesis include thiH, panC, pdxJ, gapA, dxs, or a combination thereof. In certain embodiments, the genes involved in secondary bile acid biosynthesis and degradation include baiA1, baiF, baiE, baiCD, or a combination thereof. In certain embodiments, the methods disclosed herein further comprise identifying the subject as likely to have no response or a poor response the CAR T cell therapy, if the level of the bacterium or spores thereof is higher than the reference bacterium or spores thereof level, wherein the bacterium is selected from the group consisting of bacteria of the Peptostreptococcaceae family (e.g., theRomboutsiagenus, e.g.,Romboutsia ileitis), bacteria of the Bacteroidaceae family (e.g.,Bacteroides uniformis), bacteria of the Clostridiaceae family (e.g.,Clostridium butyricum), and combinations thereof. In certain embodiments, the methods disclosed herein further comprise identifying the subject as likely to have no response or a poor response the CAR T cell therapy, if the level of the bacterium or spores thereof is lower than the reference bacterium or spores thereof level, wherein the bacterium is selected from the group consisting of bacteria of the Lachnospiraceae family (e.g., theRoseburiagenus, thePseudobutyrivibriogenus, e.g.,Pseudobutyrivibrioruminis, e.g., theLachnospiragenus, e.g.,Lachnospira pectinoschiza, e.g.,Coprococcus comes), bacteria of the Rikenellaceae family (e.g.,Alistipes indistinctus), bacteria of the Lactobacillaceae family (e.g., theLactobacillusgenus, e.g.,Lactobacillus fermentumorLactobacillus rogosae), bacteria of the Oscillospiraceae family (e.g.,Oscillibacter valericigenes), bacteria of the Ruminococcaceae family (e.g., the Ruminococcaceae UCG-004 genus, theAnaerotruncusgenus, e.g.,Anaerotruncus colihominis, Clostridium methylpentosum), bacteria of the Acidaminococcaceae family (e.g., thePhascolarctobacteriumgenus, e.g.,Phascolarctobacterium faecium), bacteria of the Clostridiaceae family (e.g.,Clostridium amygdalinum, Clostridium saccharolyticum), bacteria of the Peptococcaceae family and any combinations thereof. In certain embodiments, the methods disclosed herein further comprise identifying the subject as likely to have no response or a poor response the CAR T cell therapy, if the level of the bacterial gene is higher than the reference bacterial gene level, wherein the gene is selected from the group consisting from the genes involved in B vitamin biosynthesis (e.g., riboflavin (B2), pantothenate (B5) and thiamine (B1), genes involved in secondary bile acid biosynthesis and degradation, and any combinations thereof. In certain embodiments, the genes involved in B vitamin biosynthesis is selected from the group consisting of thiH, panC, pdxJ, gapA, dxs, and combinations thereof. In certain embodiments, the genes involved in secondary bile acid biosynthesis and degradation include baiA1, baiF, baiE, baiCD, or a combination thereof. The sample from the subject can be a fecal sample or an intestinal content sample, for example, a rectal swab. In certain embodiments, the subject has a cancer. In certain embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer, CD19 malignancies, and other B cell-related or hematologic malignancies. In certain embodiments, the cancer is an ovarian cancer, a multiple myeloma, or a B-cell malignancy (e.g., a B-cell ALL, CLL, or non-Hodgkin lymphoma), and any combinations thereof. In certain embodiments, the subject or the patient is a human. Any CAR T cell therapy known in the art can be used with the presently disclosed subject matter. In certain embodiments, the CAR T cell therapy comprises a CAR T cell comprising an extracellular binding domain that binds to mucin 16 (MUC16), B-cell maturation antigen (BCMA), CD19, or a combination thereof. The amount and/or type of bacteria present in a sample can be determined by measuring the amount or presence of bacterial nucleic acid specific for the type of bacteria, such as 16S rRNA. The amount and/or type of bacteria present in a sample can be determined by shotgun sequencing of bacterial DNA, PCR amplification of specific genes carried by the bacteria, quantitative PCR of transcripts expressed specifically by the bacteria, antibody based methods of bacterial detection, metabolomic detection of bacterial metabolites, proteomic detection of bacterial proteins, and/or by methods of culturing the microbiota sample. The amount and/or type of bacterial genes present in a sample can be determined by PCR amplification of the specific genes or quantitative PCR of transcripts expressed specifically by the bacteria, or by tests for the effects of the expression of such genes, such as degradation of secondary bile acids by the microbiota sample. In certain embodiments, the subject is a candidate for a CAR T cell therapy and has not received the CAR T cell therapy. In certain embodiments, the subject has previously received a CAR T cell therapy. In certain embodiments, the subject is receiving a CAR T cell therapy. The microbiota sample can be collected from the patient up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more days before cells will be harvested from the patient for modification in CAR T cell therapy, or before modified T cells will be administered to the patient in CAR T cell therapy. The microbiota sample can be collected from the subject after cells are harvested from the patient for CAR T cell therapy, but prior to administration of the modified T cell. The microbiota sample can also be collected from the patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after administration of the modified T cells to the patient, or after the patient exhibits a symptom of toxicity. A patient identified as likely to have a poor response to CAR T cell therapy can receive prophylactically therapeutic bacteria or a pharmaceutical composition as described herein (e.g., Sections 5.2 and 5.3) prior to harvesting of cells for modification, between harvesting cells and administration of genetically modified T cells, or after administration of genetically modified T cells in CAR T cell therapy. A patient identified as likely to have a CAR T cell associated toxicity can be subject to increased monitoring for signs of severe toxicity, can received prophylactic treatments to decrease the chances or effects of severe toxicity, without unduly hampering the effectiveness of CAR T cell therapy, or a combination thereof. 5.2 Therapeutic Bacteria The present disclosure provides therapeutic bacteria or spores thereof for treating cancer in combination with a CAR T cell therapy, or improving a subject's responsiveness to a CAR T cell therapy. In certain embodiments, the therapeutic bacteria comprise bacteria of the Lachnospiraceae family (e.g., theRoseburiagenus, thePseudobutyrivibriogenus, e.g.,Pseudobutyrivibrioruminis, e.g., theLachnospiragenus, e.g.,Lachnospira pectinoschiza, e.g.,Coprococcus comes), bacteria of the Rikenellaceae family (e.g.,Alistipes indistinctus), bacteria of the Lactobacillaceae family (e.g., theLactobacillusgenus, e.g.,Lactobacillus fermentumorLactobacillus rogosae), bacteria of the Oscillospiraceae family (e.g.,Oscillibacter valericigenes), bacteria of the Ruminococcaceae family (e.g., the Ruminococcaceae UCG-004 genus, theAnaerotruncusgenus, e.g.,Anaerotruncus colihominis, Clostridium methylpentosum), bacteria of the Acidaminococcaceae family (e.g., thePhascolarctobacteriumgenus, e.g.,Phascolarctobacterium faecium), bacteria of the Clostridiaceae family (e.g.,Clostridium amygdalinum, Clostridium saccharolyticum), bacteria of the Peptococcaceae family or a combination thereof. In certain embodiments, the present disclosure provides a composition comprising at least one of the presently disclosed bacteria or spores thereof, or a cluster including at least one of the presently disclosed bacteria. The presently disclosed therapeutic bacteria can be administered in the vegetative or dormant state, or as spores, or a mixture thereof. Therapeutic bacteria as described herein, any combinations thereof, or a cluster including any one or more of the therapeutic bacteria, can be administered in the form of purified bacteria or spores or other progenitors thereof, or alternatively can be administered as a constituent in a mixture of types of bacteria, optionally including one or more species or cluster of additional bacteria, for example, probiotic bacteria, a probiotic yeast, prebiotic, postbiotic and/or antibiotic. The present disclosure provides pharmaceutical compositions, and therapeutic uses thereof, as described herein, including such forms of therapeutic bacteria, a combination thereof, or a cluster including any one or more of the therapeutic bacteria, and optionally including one or more species or cluster of additional bacteria, for example, probiotic bacteria, a probiotic yeast, prebiotic, postbiotic and/or antibiotic. The presently disclosed bacteria can be administered in the form of a liquid, a suspension, a dried (e.g., lyophilized) powder, a tablet, a capsule, or a suppository, and can be administered orally, nasogastrically, or rectally. The bacteria can be administered in a food product, for example, a yogurt food product. A “food product” can mean a product or composition that is intended for consumption by a human or a non-human animal. Such food products include any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and/or consumable toys), meal substitute or meal replacement. The present disclosure provides a composition including an isolated presently disclosed therapeutic bacteria, a combination of any isolate therapeutic bacteria with one another, or a cluster including any one or more of the isolated therapeutic bacteria. The bacteria can be in a formulation for administration to a patient. The composition can include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, or between twenty and one hundred distinct species of the presently disclosed therapeutic bacteria. The present disclosure provides a composition including an isolated therapeutic bacteria, which can be one or more of the therapeutic bacteria described herein, but alternate or additional bacteria can be included in other compositions described herein, for example, bacteria which can be naturally occurring bacteria that are in a cluster with any one or more of therapeutic bacteria. 5.3 Pharmaceutical Compositions The present disclosure provides for pharmaceutical compositions, and therapeutic uses thereof as described herein, which include a therapeutic composition, as described herein, such as, for example, a therapeutic bacteria, as described herein. Such pharmaceutical compositions can further include at least one other agent, such as a stabilizing compound or additional therapeutic agent, for example, a probiotic, prebiotic, postbiotic, and/or antibiotic, and can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, glycerol, polyethylene glycol, and water. The pharmaceutical composition can be in a liquid or lyophilized or freeze-dried form. In some non-limiting embodiments, a formulation includes a diluent (for example, a buffer such as Tris, citrate, acetate or phosphate buffers) having suitable pH values and ionic strengths, solubilizer such as polysorbate (e.g., Tween®), carriers such as human serum albumin or gelatin. In some cases, a preservative can be included that does not affect viability of the organisms in the pharmaceutical composition. Examples of preservatives include thimerosal, parabens, benzylalconium chloride or benzyl alcohol, antioxidants such as ascorbic acid or sodium metabisulfite, and other components such as lysine or glycine. Selection of a particular composition will depend upon a number of factors, including the condition being treated, the route of administration and the pharmacokinetic parameters desired. A more extensive survey of components suitable for pharmaceutical compositions is found in Remington's Pharmaceutical Sciences, 18th ed. A. R. Gennaro, ed. Mack, Easton, PA (1980). The therapeutic methods and pharmaceutical compositions of the present disclosure can be used for treating a subject having a cancer, decreasing the risk of a poor response to a CAR T cell therapy, increasing the chance of a partial response or complete response to a CAR T cell therapy, improving a subject's responsiveness to a CAR T cell therapy, or a combination thereof. Such therapeutic bacteria are administered to the patient in a pharmaceutically acceptable carrier. The route of administration eventually chosen will depend upon a number of factors and can be ascertained by one skilled in the art. The pharmaceutical compositions of the present disclosure can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral, nasogastric, or rectal administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral, rectal or nasal ingestion by a patient to be treated. In some non-limiting embodiments, the formulation includes a capsule or tablet formulated for gastrointestinal delivery, e.g., an enteric coated capsule or pill. Pharmaceutical compositions suitable for use in the present disclosure can include compositions where the active ingredients are contained in an effective amount to achieve the intended purpose. The amount will vary from one individual to another and will depend upon a number of factors, including the intestinal microbiota of the subject, whether cells for modification have been collected from the patient, whether modified T cells have been administered to the patient, the type and dose of cancer treated by the CART cell therapy, the results of any methods described herein to assess the risk of the patient exhibiting a poor response to the CAR T cell therapy or achieving a partial response to complete response to the CAR T cell therapy, the chances of the patient developing toxicity, including severe toxicity, and the overall physical condition of the patient. The compositions of the present disclosure can be administered for therapeutic treatments, which can include prophylactic treatments. For example, pharmaceutical compositions of the present disclosure can be administered in an amount sufficient to reduce the risk of a poor response to a CAR T cell therapy, or to increase the chance or a partial response or a complete response to a CAR T cell therapy. As is well known in the medical arts, dosages for any one patient depends upon many factors, including stage of the disease or condition, the severity of the disease or condition, the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and interaction with other drugs being concurrently administered. A therapeutic bacteria can be administered to a patient alone, or in combination with one or more other drugs, nucleotide sequences, lifestyle changes, etc. used in combination with a CAR T cell therapy, including those designed to treat or reduce the risk of toxicity, including severe toxicity, and/or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers. Single or multiple administrations of formulations can be given depending on the dosage and frequency as required and tolerated by the patient. The formulations can provide a sufficient quantity of active agent to effectively reduce the risk of a poor response to a CAR T cell therapy or to increase the chance of a partial response or a complete response to a CAR T cell therapy. 5.4 Methods of Treatment and Use of Therapeutic Bacteria The present disclosure provides methods of treating subjects having cancer. In certain embodiments, the present disclosure provides a method of reducing the risk of a poor response to a CAR T cell therapy. In certain embodiments, the present disclosure provides a method of increasing the chance of a partial response to a CAR T cell therapy. In certain embodiments, the present disclosure provides a method of increasing the chance of a complete response to a CAR T cell therapy. In certain embodiments, the present disclosure provides methods of improving a subject's responsiveness to a CAR T cell therapy. In certain non-limiting embodiments, the methods disclosed herein, include administering to the subject, at least one presently disclosed therapeutic bacteria or spores thereof, or a composition comprising thereof (e.g., therapeutic bacteria and pharmaceutical compositions disclosed in Sections 5.2 and 5.3). In certain embodiments, the methods further comprise administering to the subject a CAR-T cell therapy. In certain embodiments, the therapeutic bacteria or spores thereof, or the composition comprising thereof is administered to the subject prior to or during the CAR-T cell therapy. A single method can achieve any two or all three of the previous outcomes. Patients in need of such treatment or compositions include patients who are receiving or are being considered for or can receive CAR T cell therapy. Such patients typically include patients with certain cancers. In certain embodiments, the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute myelogenous leukemia, biliary cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectal cancer, endometrial cancer, esophageal, gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, liver cancer, prostate cancer, and urinary bladder cancer, CD19 malignancies, and other B cell-related or hematologic malignancies. In certain embodiments, the cancer is an ovarian cancer, a multiple myeloma, or a B-cell malignancy (e.g., a B-cell ALL, CLL, or non-Hodgkin lymphoma), and any combinations thereof. In certain embodiments, the subject or the patient is a human. Such patients can, in particular, include those identified using methods disclosed herein to have an increased risk of poor response to CAR T cell therapy, a decreased risk of partial response or complete response to CAR T cell therapy, or a combination thereof. The present disclosure provides for a method of decreasing the risk of a poor response to CAR T cell therapy, increasing the chances of a partial response or complete response to CAR T cell therapy, or a combination thereof, by administering, to a patient in need of such treatment, an effective amount of a probiotic including at least one therapeutic bacteria. The present disclosure provides for a method of treating checkpoint blockade therapy associated colitis by administering, to a patient in need of such treatment, an effective amount of a prebiotic. The prebiotic can be administered separately from the therapeutic bacteria and can promote the growth, proliferation and/or survival of at least one therapeutic bacteria. The prebiotic can include one or more agents, for example, a nutritional supplement, that increases growth and survival of at least one therapeutic bacteria. The prebiotic can include one or more of poorly-absorbed complex carbohydrates, oligosaccharides, inulin-type fructans or arabinoxylans. The present disclosure provides for a method of decreasing the risk of a poor response to CAR T cell therapy, increasing the chances of a partial response or complete response to CAR T cell therapy, or a combination thereof, by administering, to a patient in need of such treatment, an effective amount of a postbiotic. The postbiotic can be administered separately from the therapeutic bacteria. The present disclosure provides for a method of decreasing the risk of a poor response to CAR T cell therapy, increasing the chances of a partial response or complete response to CAR T cell therapy, or a combination thereof, by including determining the risk of a poor response to CAR T cell therapy, the chances of a partial response or complete response to CAR T cell therapy, or both. The present disclosure provides the use of any composition described herein, including the use of any therapeutic bacteria described herein for decreasing the risk of a poor response to CAR T cell therapy, increasing the chances of a partial response or complete response to CAR T cell therapy, or a combination thereof in a patient. The use can be further characterized by aspects of the methods described above and elsewhere herein. 5.5 Kits The presently disclosed subject matter provides for kits for diagnosing a subject receiving or considered for CAR T cell therapy, including determining the risk of a poor response to CAR T cell therapy, chances of a partial response or complete response to CAR T cell therapy, or a combination thereof. The kit can include an agent for determining whether a sample (e.g., a feces sample or an intestinal content sample) of a subject contains an increased or decreased level of a bacterium or spores thereof, or a bacterial gene as compared to a reference level. An increased or decreased level of the bacterium or spores thereof or of the bacterial gene is determined with respect to a reference bacterium or spores thereof level or a reference bacterial gene level. In certain embodiments, the level (e.g., the measured level and the reference level) can be based on a relative abundance in the intestinal microbiome. For instance, the level can represent a percentage of the bacterium or spores thereof of all the bacteria or spores thereof in the intestinal microbiome. The level can also be an absolute number. In certain embodiments, the reference level is a predetermined level of a bacterium or spores thereof or of a bacterial genetic module that a level higher or lower than the reference level indicates the subject is likely to have a response to the CAR T cell therapy, or is likely to have no response or a poor response to the CAR T cell therapy. In certain embodiments, the reference level is the level of a bacterium or spores thereof or of a bacterial gene from a subject or a population of subjects that have a response to the CAR T cell therapy. In certain embodiments, the reference level is the level of a bacterium or spores thereof or of a bacterial gene from a population of subjects that are candidates for a CAR T cell therapy or subjects with cancer that have not received a CAR T cell therapy. In certain embodiments, the reference level is the level of a bacterium or spores thereof or of a bacterial gene from a sample of the same subject collected at an earlier time point. In certain embodiments, the reference level can be based on a prior test in the same patient, or on levels found in a patient population, such as patients who are candidates for CAR T cell therapy or patients with cancer who have not undergone CAR T cell therapy. In certain embodiments, the bacterium determined in the sample of the subject is selected from the group consisting of bacteria of the Peptostreptococcaceae family (e.g., theRomboutsiagenus, e.g.,Romboutsia ileitis), bacteria of the Bacteroidaceae family (e.g.,Bacteroides uniformis), bacteria of the Clostridiaceae family (e.g.,Clostridium butyricum, Clostridium saccharolyticum, Clostridium amygdalinum), bacteria of the Lachnospiraceae family (e.g., theRoseburiagenus, thePseudobutyrivibriogenus, e.g.,Pseudobutyrivibrio ruminis, e.g., theLachnospiragenus, e.g.,Lachnospira pectinoschiza, Coprococcus comes), bacteria of the Rikenellaceae family (e.g.,Alistipes indistinctus), bacteria of the Lactobacillaceae family, such as theLactobacillusgenus, particularlyLactobacillus fermentumorLactobacillus rogosae), bacteria of the Oscillospiraceae family (e.g.,Oscillibacter valericigenes), bacteria of the Ruminococcaceae family (e.g., the Ruminococcaceae UCG-004 genus, theAnaerotruncusgenus, e.g.,Anaerotruncus colihominis, Clostridium methylpentosum), bacteria of the Acidaminococcaceae family (e.g., thePhascolarctobacteriumgenus, e.g.,Phascolarctobacterium faecium), bacteria of the Peptococcaceae family and any combinations thereof. In certain embodiments, the bacterial gene determined in the sample of the subject is selected from the group consisting of the genes involved in B vitamin biosynthesis (e.g., riboflavin (B2), pantothenate (B5) and thiamine (B1), genes involved in secondary bile acid biosynthesis and degradation, and any combinations thereof. In certain embodiments, the genes involved in B vitamin biosynthesis include thiH, panC, pdxJ, gapA, dxs, and a combination thereof. In certain embodiments, the genes involved in secondary bile acid biosynthesis and degradation include baiA1, baiF, baiE, baiCD, or a combination thereof. The agent can include nucleic acid primers specific for said bacteria or genes, such as nucleic acid primers are specific for 16S rRNA sequencing. The presently disclosed subject matter also provides for kits for treating a patient who has received or can receive CAR T cell therapy. Such a kit can include one or more therapeutic bacteria or compositions as described herein (e.g., disclosed in Sections 5.2 and 5.3). The kit can include instructions for administering the therapeutic bacteria or compositions. The instructions can include information about the use of the therapeutic bacterial or compositions in conjunction with CAR T cell therapy. The instructions can include at least one of the following: description of the therapeutic bacteria or composition; dosage schedule and administration; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions can be printed directly on a container (when present) containing the therapeutic bacteria or composition, or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. The kit can include both components for diagnosing whether a subject receiving or considered for CAR T cell therapy is at an increased or decreased risk of a poor response, partial response, or complete response, or a combination thereof, and components for treating a patient who has or can receive or can CAR T cell therapy. The kit can include instructions for administering components for treating the patient based upon results obtained using the components for diagnosing the patient. 6 EXAMPLES The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of aspects of the invention, and not by way of limitation. Example 6.1: Microbiota and Response to CAR T Cell Therapy Stool samples were collected from intended recipients of CAR T cell therapy after T cell collection and prior to administration of modified T cells. Patients varied in conditioning regimen, CAR construct, and underlying cancer diagnosis. CAR constructs include those targeting mucin 16 (MUC16), B cell maturation antigen (BCMA), and cluster of differentiation 19 (CD 19). Cancers included CD19 malignancies, myeloma, and ovarian cancer. Representative patients included those with solid tumors and hematologic malignancies. 16S RNA was sequenced from the samples and operational taxonomic units (OTUs) were classified using the sequence data with reference to the National Center for Biotechnology Information (NCBI) Reference Sequence Database. After administration of T cells in connection with the CAR T cell therapy, patients were assessed for signs of toxicity, complete response, or both. For purposes of these Examples, partial responses were not separately identified; patients were assessed solely for a complete response or lack of a complete response based on computed tomography (CT) scan results in patients with solid tumors and lymphoma, or on bone marrow biopsy results in patients with leukemia. Toxicity was determined using clinical grading to be Grade 1 to 4 CRS or Grade 1 to 4 neurotoxicity. A graph showing relative 16S RNA abundance for various bacterial families in a representative patient is presented inFIG.1. For all patients, the composition of the microbiota prior to administration of CAR T cells was diverse, as defined by an inverse Simpson diversity index of greater than 4 (FIG.2). LEfSe was used to identify microbiota associated with complete response, lack of complete response, toxicity, or combinations thereof using relative abundances of corresponding 16S RNA with a linear discriminant analysis score threshold of greater than 2.5. LEfSe analysis of patients who exhibited a complete response or lack of a complete response found increased abundance of certain microbiota correlated with complete response or lack of complete response. A graph presenting these results, with relevant linear discriminant analysis (LDA) scores, is presented inFIG.3. Bacteria in the Oscillospiraceae, Ruminococcaceae, Lachnospiraceae, Acidaminococcaceae, Rikenellaceae, and Lactobacilaceae families were associated with a complete response, while bacteria in the Peptostreptococceceae family were associated with no complete response. Toxicity exhibited a correlation with a complete response in the patient population (FIG.4) and, therefore, was used as an indicator of the tendency to have a complete response to CAR T cell therapy. Toxicity typically occurs within days to one to two weeks of administering genetically modified CAR T cells, and therefore can be a suitable early indicator the response to CAR T cell therapy. Accordingly LEfSe analysis was also conducted based on whether patients exhibited toxicity. Results are presented inFIG.5. Bacteria in the Lachnospiraceae and Lactobacillaceae families were associated with toxicity. Bacteria in the Bacteroidaceae, Clostridiaceae, and Peptostreptococcaceae families were associated with no toxicity. In particular, the data ofFIG.3andFIG.5show that Lachnospiraceae are associated with both a complete response and toxicity, while Peptostreptococcaceae are associated with lack of a complete response and lack of toxicity. Example 6.2: Gene Expression and Response to CAR T Cell Therapy Fecal samples from eighteen patients of the patient population in Example 1 were subjected to metagenomic sequencing. thiH is associated with thiamin biosynthesis. Additional fecal samples were collected after administration of genetically modified T cells. pdxJ, gapA, and dxs are associated with pyridoxine biosynthesis. panC is associated with pantothenate biosynthesis. baiA1 is associated with secondary bile acid biosynthesis. baiF, baiE, and baiCD are associated with secondary bile acid degradation. hdgA, gctA, and gtcB are associated with glutarate biosynthesis. atoA and atoD are associated with acetate biosynthesis. Results are presented as heatmap data in reads per kilobase per million reads (RPKMs) for patients who exhibited a complete response (CR) or no complete response (noCR) (FIG.6). Patients who did not exhibit a complete response had an increased abundance of bacterial genes associated with B vitamin biosynthesis, secondary bile acid biosynthesis, and secondary bile acid degradation. Results are also presented as heatmap data in reads per kilobase per million reads (RPKMs) for patients who exhibited a toxicity or no toxicity (FIG.7). Patients who did not exhibit toxicity response had an increased abundance of bacterial genes associated with B vitamin biosynthesis, secondary bile acid biosynthesis, and secondary bile acid degradation. Analysis of the relative abundance of bacterial genes associated with biosynthesis of various B vitamins in patients with CD19 malignancies (CD19+ group), myeloma, or ovarian cancer who exhibited a complete response or did not exhibit a complete response to CAR T cell therapy (FIG.8) showed an increased abundance in vitamin B synthesis genes in patients who did not exhibit a complete response. This is particularly true of myeloma and ovarian cancer patients. Analysis of the relative abundance of bacterial genes associated with biosynthesis of various B vitamins in patients with CD19 malignancies (CD19+ group), myeloma, or ovarian cancer who exhibited toxicity or did not exhibit toxicity in response to CAR T cell therapy (FIG.9) showed a somewhat increased abundance in vitamin B synthesis genes in patients who did not exhibit toxicity. This is particularly true of ovarian cancer patients. Example 6.3: Intestinal Microbiome Analyses Identify Biomarkers for Patient Response to CAR T Cell Therapy Forty-four (44) patients receiving chimeric antigen receptor T cell therapy (median 63 years) were selected for the presently disclosed cohort. The primary inclusion criteria were adult patients who received cellular therapy with CAR T cells, and for whom a microbiota stool specimen was obtained prior to cell infusion. Patients varied in terms of conditioning regimen, CAR construct and underlying diagnosis, with diffuse large B cell lymphoma (DLBCL) being the most prevalent in this cohort. Patient characteristics and clinical outcomes were shown inFIG.11andFIG.12. As shown inFIG.10, microbiota stool specimens were collected from each patient at baseline prior to CAR T cell infusion and weekly for four weeks following CAR T cells infusions. The four weeks following CAR T cell infusion was relevant as this was the time period during which toxicity, due to cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS)/neurotoxicity, may occur. BCMA CAR T cells were used for the treatment of multiple myeloma, CD19 CAR T cells were used for the treatment of B cell malignancies, such as B-cell ALL, CLL, and non-Hodgkin lymphoma. A total of 112 samples were collected from 44 patients. Stool samples were aliquoted and frozen for subsequent processing and batch sequencing. Stool specimens were sequenced using multiple high-throughput techniques. For 16S rRNA sequencing, silica bead-beating were used to disrupt the bacterial cell walls, then the nucleic acids were isolated using phenol-chloroform extraction. Polymerase chain reaction (PCR) was used to amplify the V4-V5 region of the 16S rRNA gene, which was then sequenced using the Illumina MiSeq platform (Jenq R R et al BBMT 2015, Turnbaugh P J et al Nature 2009). 16S data were analyzed using the DADA2 pipeline (Callahan B J et al Nat Methods 2016). Shotgun metagenomic sequences from 38 of the 44 samples was performed and the sequences were functionally annotated using the shortBRED pipeline. For shotgun metagenomic sequencing, DNA was extracted as described above and then sheared to a target size of 650 bp using a Covaris ultrasonicator. DNA was then prepared for sequencing using the Illumina TruSeq DNA library preparation kit and sequenced using the Illumina HiSeq system targeting ˜10-20×106reads per sample with 100 bp, paired-end reads. The abundances of genes assigned to three hypothesized immunological relevant pathways (B vitamin synthesis, bile acid biosynthesis, and short-chain fatty acid production) were inspected. LeFSe (Segata N et al, Genome Biol. 2011) was performed to identify differential bacterial taxa that were associated with CR and no CR. LeFSe assessed all of levels of the bacterial taxa down to the genus level to identify the bacteria that are most associated with the clinical outcome. Taxa were identified with the Silva Database. LEfSe analysis of patients who exhibited a complete response or lack of a complete response found increased abundance of certain microbiota correlated with complete response or lack of complete response. These results, with relevant linear discriminant analysis (LDA) scores, were presented inFIG.13AandFIG.13B. Based on these data, the increased abundance of the genera—Roseuriaand Ruminococcaceae UCG-004—were strongly associated with complete response to CAR T cell therapy. Members of these genera can be good consortia as supplement to improve the response to the CAR T cell therapy. Peptococcaceae was also associated with complete response. Peptostreptococcaceae was strongly associated with a lack of a complete response. The above disclosed subject matter is to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other embodiments which fall within the true spirit and scope of the present disclosure. Thus, to the maximum extent allowed by law, the scope of the present disclosure is to be determined by the broadest permissible interpretation of the following claims and their equivalents, and shall not be restricted or limited by the foregoing detailed description. | 63,491 |
11860164 | DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS This technology disclosed herein is described in one or more exemplary embodiments in the following description with reference to the Figures. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present technology disclosed herein. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment. The described features, structures, or characteristics of the technology disclosed herein may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are recited to provide a thorough understanding of embodiments of the technology disclosed herein. One skilled in the relevant art will recognize, however, that the technology disclosed herein may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the technology disclosed herein. Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding embodiments; however, the order of description should not be construed to imply that these operations are order dependent. The following explanations of terms and methods are provided to better describe the present compounds, compositions and methods, and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terminology used in the disclosure is for the purpose of describing particular embodiments and examples only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). As used herein, “one or more” or at least one can mean one, two, three, four, five, six, seven, eight, nine, ten or more, up to any number. As used herein, the term “comprises” means “includes.” Hence “comprising A or B” means including A, B, or A and B. It is further to be understood that all base sizes and all molecular weight or molecular mass values given for peptides and nucleic acids are approximate and are provided for description. With respect to the use of any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); and other similar references. Suitable methods and materials for the practice or testing of this disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which this disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Also, it is noted that embodiments may be described as a process depicted as a flowchart, a flow diagram, a dataflow diagram, a structure diagram, or a block diagram. Although a flowchart may describe the operations as a sequential process, many of the operations may be performed in parallel, concurrently, or simultaneously. In addition, the order of the operations may be re-arranged. A process may be terminated when its operations are completed, but may also have additional steps not included in the figure(s). A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, and the like. When a process corresponds to a function, its termination may correspond to a return of the function to the calling function and/or the main function. Furthermore, a process may be implemented by hardware, software, firmware, middleware, microcode, hardware description languages, or any combination thereof. When implemented in software, firmware, middleware or microcode, the program code or code segments to perform the necessary tasks may be stored in a machine or computer readable medium. A code segment may represent a procedure, a function, a subprogram, a program, a routine, a subroutine, a module, program code, a software package, a class, or any combination of instructions, data structures, program statements, and the like. As used hereinafter, including the claims, the term “circuitry” may refer to, be part of, or include an Application Specific Integrated Circuit (ASIC), an electronic circuit, a processor (shared, dedicated, or group), and/or memory (shared, dedicated, or group) that execute one or more software or firmware programs, a combinational logic circuit, and/or other suitable hardware components that provide the described functionality. In some embodiments, the circuitry may implement, or functions associated with the circuitry may be implemented by, one or more software or firmware modules. As used hereinafter, including the claims, the term “memory” may represent one or more hardware devices for storing data, including random access memory (RAM), magnetic RAM, core memory, read only memory (ROM), magnetic disk storage mediums, optical storage mediums, flash memory devices and/or other machine readable mediums for storing data. The term “computer-readable medium” may include, but is not limited to, memory, portable or fixed storage devices, optical storage devices, wireless channels, and various other mediums capable of storing, containing or carrying instruction(s) and/or data. As used hereinafter, including the claims, the term “computing platform” may be considered synonymous to, and may hereafter be occasionally referred to, as a computer device, computing device, client device or client, mobile, mobile unit, mobile terminal, mobile station, mobile user, mobile equipment, user equipment (UE), user terminal, machine-type communication (MTC) device, machine-to-machine (M2M) device, M2M equipment (M2ME), Internet of Things (IoT) device, subscriber, user, receiver, etc., and may describe any physical hardware device capable of sequentially and automatically carrying out a sequence of arithmetic or logical operations, equipped to record/store data on a machine readable medium, and transmit and receive data from one or more other devices in a communications network. Furthermore, the term “computing platform” may include any type of electronic device, such as a cellular phone or smartphone, a tablet personal computer, a wearable computing device, an autonomous sensor, personal digital assistants (PDAs), a laptop computer, a desktop personal computer, a video game console, a digital media player, an in-vehicle infotainment (IVI) and/or an in-car entertainment (ICE) device, an in-vehicle computing system, a navigation system, an autonomous driving system, a vehicle-to-vehicle (V2V) communication system, a vehicle-to-everything (V2X) communication system, a handheld messaging device, a personal data assistant, an electronic book reader, an augmented reality device, and/or any other like electronic device. As used hereinafter, including the claims, the term “link” or “communications link” may refer to any transmission medium, either tangible or intangible, which is used to communicate data or a data stream. Additionally, the term “link” may be synonymous with and/or equivalent to “communications channel,” “data communications channel,” “transmission channel,” “data transmission channel,” “access channel,” “data access channel,” “channel,” “data link,” “radio link,” “carrier,” “radiofrequency carrier,” and/or any other like term denoting a pathway or medium through which data is communicated. As used hereinafter, including the claims, the terms “module”, “input interface”, “converter”, “analyzer”, “artificial neural network”, “trained neural network”, “partially retrained artificial neural network”, or “retrained artificial neural network” may refer to, be part of, or include one or more Application Specific Integrated Circuits (ASIC), electronic circuits, programmable combinational logic circuits (such as field programmable gate arrays (FPGA)) programmed with logic to perform operations described herein, a processor (shared, dedicated, or group) and/or memory (shared, dedicated, or group) that execute one or more software or firmware programs generated from a plurality of programming instructions with logic to perform operations described herein, and/or other suitable components that provide the described functionality In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided: Administration: To provide or give a subject an agent by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Alteration or difference: An increase or decrease in the amount of something, such as a cell surface molecule expression. In some examples, the difference is relative to a control or reference value or range of values, such as an amount of a protein that is expected in a subject who does not have a particular condition or disease being evaluated. Detecting an alteration or differential expression/activity can include measuring a change in expression, concentration or activity, such as by ELISA, Western blot and/or mass spectrometry. “Analysis” or “analyzing,” as used herein, are used interchangeably and refer to any of the various methods of separating, detecting, isolating, purifying, solubilizing, detecting and/or characterizing molecules of interest. Examples include, but are not limited to, solid phase extraction, solid phase micro extraction, electrophoresis, mass spectrometry, e.g., Multiplexed targeted selected ion monitoring (SIM)-MS followed by iterative MS2 DDA, ESI-MS, SPE HILIC, or MALDI-MS, liquid chromatography, e.g., high performance, e.g., reverse phase, normal phase, or size exclusion, ion-pair liquid chromatography, liquid-liquid extraction, e.g., accelerated fluid extraction, supercritical fluid extraction, microwave-assisted extraction, membrane extraction, soxhlet extraction, precipitation, clarification, electrochemical detection, staining, elemental analysis, Edmund degradation, nuclear magnetic resonance, infrared analysis, flow injection analysis, capillary electrochromatography, ultraviolet detection, and combinations thereof. Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen). The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2ndEd., Springer Press, 2010). A single-chain antibody (scFv) is a genetically engineered molecule containing the VH and VL domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al, Science, 242:423-426, 1988; Huston et al, Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al, Clin. Dev. Immunol, 2012, doi: 10.1 155/2012/980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH-domain and the VL-domain in a scFv, is typically not decisive for scFvs. Thus, scFvs with both possible arrangements (VH-domain-linker domain-VL-domain; VL-domain-linker domain-VH-domain) may be used. In a dsFv the VH and VL have been mutated to introduce a disulfide bond to stabilize the association of the chains. Diabodies also are included, which are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, for example, Holliger et ai, Proc. Natl. Acad. ScL, 90:6444-6448, 1993; Poljak of ai, Structure, 2: 1121-1123, 1994). Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rdEd., W.H. Freeman & Co., New York, 1997. An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. Antibody competition assays are known, and an exemplary competition assay is provided herein. An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites. Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007).) In several embodiments, the VH and VL combine to specifically bind the antigen. In additional embodiments, only the VH is required. For example, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Any of the disclosed antibodies can include a heterologous constant domain. For example the antibody can include constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutations) to increase half-life. References to “VH” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab. The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al, (JMB 273,927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VHCDR3 is the CDR3 from the VHof the antibody in which it is found, whereas a VLCDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3. A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples, monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nded. Cold Spring Harbor Publications, New York (2013).) A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences. A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody. A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas of aZ. Phage display: A Laboratory Manuel. 1stEd. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008). A variety of immunoassay formats are appropriate for selecting antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term “antigen” includes all related antigenic epitopes. An “antigenic polypeptide” is a polypeptide to which an immune response, such as a T cell response or an antibody response, can be stimulated. “Epitope” or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and multi-dimensional nuclear magnetic resonance spectroscopy. The term “antigen” denotes both subunit antigens, (for example, antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes. An “antigen,” when referring to a protein, includes a protein with modifications, such as deletions, additions and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. B-cell: One of the two major types of lymphocytes. B-cells arise from bone marrow progenitor cells, which progress through multiple stages such as the pro-, pre- and transitional stages into the naive B-cell. The antigen receptor on B lymphocytes is a cell-surface immunoglobulin molecule. Upon activation by an antigen, B-cells differentiate into cells producing antibody of the same specificity as their initial receptor. An “immature B cell” is a cell that can develop into a mature B cell. Generally, pro-B cells (that express, for example, CD10) undergo immunoglobulin heavy chain rearrangement to become pro B pre B cells, and further undergo immunoglobulin light chain rearrangement to become an immature B cells. Immature B cells include T1 and T2 B cells. Thus, one example of an immature B cell is a T1 B that is an AA41hiCD23locell. Another example of an immature B cell is a T2 B that is an AA41hiCD23hicell. Thus, immature B cells include B220 expressing cells wherein the light and the heavy chain immunoglobulin genes are rearranged, and that express AA41. Immature B cells can develop into mature B cells, which can produce immunoglobulins (e.g., IgA, IgG or IgM). Mature B cells express characteristic markers such as CD21 and CD23 (CD23hiCD21hicells), but do not express AA41. In some examples, a B cell is one that expresses CD179hi, CD24, CD38 or a combination thereof. B cells can be activated by agents such as lippopolysaccharide (ITS) or IL-4 and antibodies to IgM. B-cells have many functions. For example, a B-cell can serve as an antigen presenting cell (APC) (which activates T-cytotoxic cells toward effector function), activate naive or memory Th1 cells, or evolve into long-lived memory cell and transform into an antibody secreting plasma cell with T-cell help, and perpetuate antibody responses to autoantigens. Antigen is sensed by the B-cell via the B-cell receptor, or the immunoglobulin molecule. Chromatography: A process of separating a mixture, for example a mixture containing peptides, proteins, polypeptides and/or antibodies. It involves passing a mixture through a stationary phase, which separates molecules of interest from other molecules in the mixture and allows one or more molecules of interest to be isolated. Contacting: “Contacting” includes in solution and solid phase. “Contacting” can occur in vitro with, e.g., samples, such as biological samples containing a target biomolecule. “Contacting” can also occur in vivo. Control: A reference standard. A control can be a known value or range of values indicative of basal levels or amounts or present in a tissue or a cell or populations thereof. A control can also be a cellular or tissue control, for example a tissue from a non-diseased state. A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. Detecting: Identifying the presence, absence or relative or absolute amount of the object to be detected. Diagnosis: The process of identifying a condition or disease by its signs, symptoms, results of various tests and presence of diagnostic indicators. The conclusion reached through that process is also called “a diagnosis.” Immunoassay: A biochemical test that measures the presence or concentration of a substance in a sample, such as a biological sample, using the reaction of an antibody to its cognate antigen, for example the specific binding of an antibody to a protein. Both the presence of antigen and the amount of antigen present can be measured. For measuring proteins, for each the antigen and the presence and amount (abundance) of the protein can be determined or measured. Measuring the quantity of antigen can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody with a detectable label. An “enzyme linked immunosorbent assay (ELISA)” is type of immunoassay used to test for antigens (for example, proteins present in a sample, such as a biological sample). A “competitive radioimmunoassay (RIA)” is another type of immunoassay used to test for antigens. A “lateral flow immunochromatographic (LFI)” assay is another type of immunoassay used to test for antigens. Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages (such as horseradish peroxidase), radioactive isotopes (for example14C,32P,125I,3H isotopes and the like) and particles such as colloidal gold. In some examples a protein, such as a protein associated with a particular infection, is labeled with a radioactive isotope, such as14C,32P,125I,3H isotope. In some examples an antibody that specifically binds the protein is labeled. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N. Y., 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998), Harlow & Lane (Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, 1988). Liquid chromatography: A process in which a chemical mixture carried by a liquid can be separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reverse phase liquid chromatography, ion-exchange chromatography, size exclusion chromatography, affinity chromatography, and hydrophobic chromatography. Mass spectrometer: A device capable of detecting specific molecular species and accurately measuring their masses. The term can be meant to include any molecular detector into which a polypeptide or peptide may be eluted for detection and/or characterization. A mass spectrometer consists of three major parts: the ion source, the mass analyzer, and the detector. The role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters can be transferred into gas phase and ionized either concurrently (such as in electrospray ionization). The choice of ion source depends on the application. Measure: To detect, quantify or qualify the amount (including molar amount), concentration or mass of a physical entity or chemical composition either in absolute terms in the case of quantifying, or in terms relative to a comparable physical entity or chemical composition. Merkel Cell Carcinoma: A rare form of skin cancer that originates in Merkel cells. Merkel cells are found at the base of the epidermis. Some forms of Merkel cell carcinoma are caused by the virus, Merkel cell polyomavirus, which lives on the surface of the skin. Merkel cell carcinoma usually appears as a flesh-colored or bluish-red nodule, often on the face, head or neck. Merkel cell carcinoma is also known as neuroendocrine carcinoma of the skin. Prognosis: A prediction of the course of a condition or disease. The prediction can include determining the likelihood of a subject to develop aggressive, recurrent disease, to survive a particular amount of time (e.g., determine the likelihood that a subject will survive 1, 2, 3 or 5 years), to respond to a particular therapy or combinations thereof. Protein: The terms “protein,” “peptide,” “polypeptide” refer, interchangeably, to a polymer of amino acids and/or amino acid analogs that are joined by peptide bonds or peptide bond mimetics. The twenty naturally-occurring amino acids and their single-letter and three-letter designations are as follows: Alanine A Ala; Cysteine C Cys; Aspartic Acid D Asp; Glutamic acid E Glu; Phenylalanine F Phe; Glycine G Gly; Histidine H His; Isoleucine I He; Lysine K Lys; Leucine L Leu; Methionine M Met; Asparagine N Asn; Proline P Pro; Glutamine Q Gln; Arginine R Arg; Serine S Ser; Threonine T Thr; Valine V Val; Tryptophan w Trp; and Tyrosine Y Tyr. Sample: A sample, such as a biological sample, includes biological materials (such as nucleic acids) obtained from an organism or a part thereof, such as a plant, or animal, and the like. In particular embodiments, the biological sample is obtained from an animal subject, such as a human subject. A biological sample is any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated). For example, a biological sample can be bone marrow, tissue biopsies, whole blood, serum, plasma, blood cells, endothelial cells, circulating tumor cells, lymphatic fluid, ascites fluid, interstitial fluid (also known as “extracellular fluid” and encompasses the fluid found in spaces between cells, including, inter alia, gingival cervicular fluid), cerebrospinal fluid (CSF), saliva, mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids. Sensitivity: The percent of diseased individuals (individuals with prostate cancer) in which the biomarker of interest is detected (true positive number/total number of diseased×100). Non-diseased individuals diagnosed by the test as diseased are “false positives”. Sequence identity: As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.). Signs or symptoms: Any subjective evidence of disease or of a subject's condition, e.g., such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state. A “sign” is any abnormality indicative of disease, discoverable on examination or assessment of a subject. A sign is generally an objective indication of disease. Specificity: The percent of non-diseased individuals for which the biomarker of interest is not detected (true negative/total number without disease×100). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay, are termed “true negatives.” Standard: A substance or solution of a substance of known amount, purity or concentration. A standard can be compared (such as by spectrometric, chromatographic, or spectrophotometric analysis) to an unknown sample (of the same or similar substance) to determine the presence of the substance in the sample and/or determine the amount, purity or concentration of the unknown sample. In one embodiment, a standard is a peptide standard. An internal standard is a compound that is added in a known amount to a sample prior to sample preparation and/or analysis and serves as a reference for calculating the concentrations of the components of the sample. In one example, nucleic acid standards serve as reference values for tumor or non-tumor expression levels of specific nucleic acids. In some examples, peptide standards serve as reference values for tumor or non-tumor expression levels of specific peptides. Isotopically-labeled peptides are particularly useful as internal standards for peptide analysis since the chemical properties of the labeled peptide standards are almost identical to their non-labeled counterparts. Thus, during chemical sample preparation steps (such as chromatography, for example, HPLC) any loss of the non-labeled peptides is reflected in a similar loss of the labeled peptides. T-Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4+ T cells and CD8+ T cells. A CD4+T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+ T cells carry the “cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 T cells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell is a suppressor T cell. Variants: sequences derived by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end, and/or addition of one or more bases to the 5′ or 3′ end of the nucleic acid sequence; deletion or addition of one or more amino acids/nucleic acids at one or more sites in the sequence; or substitution of one or more amino acids/nucleic acids at one or more sites in the sequence. The antibodies and antibody fragments described herein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the enzyme can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. The substitution may be a conserved substitution. A “conserved substitution” is a substitution of an amino acid with another amino acid having a similar side chain. A conserved substitution would be a substitution with an amino acid that makes the smallest change possible in the charge of the amino acid or size of the side chain of the amino acid (alternatively, in the size, charge or kind of chemical group within the side chain) such that the overall enzyme retains its spatial conformation but has altered biological activity. For example, common conserved changes might be Asp to Glu, Asn or Gln; His to Lys, Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanine is commonly used to substitute for other amino acids. The 20 essential amino acids can be grouped as follows: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine having nonpolar side chains; glycine, serine, threonine, cystine, tyrosine, asparagine and glutamine having uncharged polar side chains; aspartate and glutamate having acidic side chains; and lysine, arginine, and histidine having basic side chains. General Description The inventors developed a non-parametric gating method for cytometry experiments named full annotation using shaped-constrained trees (FAUST,FIGS.1A-1E). FAUST defines cell sub-populations as modes of the joint-distribution of protein expression within each sample. Due to its dimensionality and throughput, direct non-parametric estimation of the joint distribution is infeasible for cytometry data. Instead, FAUST selects a subset of consistently well-separated protein markers using a novel depth score, bounds a standardized set of phenotypic regions containing modes of interest for the selected markers alone, and annotates those regions relative to data-derived annotation boundaries. Standardization means that the number of regions is fixed across samples but the location of the boundaries of those regions can vary from sample to sample. Consequently, FAUST clusters are annotated with biologically interpretable labels and each represents a cell sub-population with a homogeneous phenotype. FAUST's standardization of phenotypic regions provides a common solution to three major challenges posed by sample- and batch-heterogeneity in cytometry experiments: cluster discovery, cluster matching, and cluster labeling. Since each discovered cluster is merely a collection of cells falling within a phenotypic region, clusters can have distributions whose shape varies broadly across samples. Consequently, FAUST can accommodate significant sample-to-sample heterogeneity. Similarly, since each region (and therefore each cluster) is assigned exactly one phenotypic label, the labels can be used to match clusters across samples and interpret the cell type of each cluster. An additional benefit of matching regions by phenotypic labels is robustness to sparsity since cell counts within a region can vary by orders of magnitude across samples. Here, an unbiased FAUST procedure was applied to analyze data generated from four cancer immunotherapy clinical trials and demonstrate how the disclosed approach can be used to discover candidate biomarkers correlated with outcome and perform cross-study analyses in the presence of heterogeneous marker panels. Referring toFIGS.1A-1E, FAUST estimates annotation boundaries for an experimental unit. An experimental unit is user defined and can be a sample, stimulation condition, subject, batch, or site. This schematic overview of FAUST provided inFIG.1Aassumes the experimental unit is an individual sample. Panel A) To estimate annotation boundaries, FAUST grows an exhaustive forest of 1-dimensional, depth-3 gating strategies, constrained by shape: if, prior to depth-3, the cells in a node of the gating strategy have unimodal expression along all markers, the gating strategy along that path terminates. Panel B) Annotation boundaries are estimated for markers within an experimental unit by averaging over gates drawn for that marker over the entire annotation forest. A “depth score” is derived for each marker and it quantifies how well-gated the marker is in each experimental unit. The distribution of scores across experimental units is used to determine whether a marker should be included in the discovery process and to determine the number of annotation boundaries a marker should receive. Panel C) This procedure ensures that FAUST selects a standard set of markers for discovery and annotation as well as a standard number of annotation boundaries per selected marker. Panel D) For each experimental unit, FAUST then relaxes the depth-3 constraint and conducts a search of 1-dimensional gating strategies in order to discover and select phenotypes present in the experimental unit. Each discovered phenotype is given a score that quantifies the homogeneity of cells in an experimental unit with that phenotype; high-scoring phenotypes are then selected for annotation. Each selected phenotype is annotated using all selected markers from Panel C), regardless of the specific gating strategy that led to the phenotype's discovery. Panel E) FAUST returns an annotated count matrix with counts of cells in each phenotypic region discovered and selected in Panel D) that also survives down-selection by frequency of occurrence across experimental units. In some embodiments, the disclosed method utilizes a computer device.FIG.7provides a block diagram of a computer device suitable for practicing the present disclosure, in accordance with various embodiments. As shown, computer device800can include one or more processors802, memory controller803, and system memory804. Each processor802can include one or more processor cores, and hardware accelerator805. An example of hardware accelerator805can include, but is not limited to, programmed field programmable gate arrays (FPGA). In embodiments, processor802can also include a memory controller (not shown). In embodiments, system memory804can include any known volatile or non-volatile memory. Additionally, computer device800can include mass storage device(s)806(such as solid state drives), input/output device interface808(to interface with various input/output devices, such as, mouse, cursor control, display device (including touch sensitive screen), and so forth) and communication interfaces810(such as network interface cards, modems and so forth). In embodiments, communication interfaces810can support wired or wireless communication, including near field communication. The elements can be coupled to each other via system bus812, which can represent one or more buses. In the case of multiple buses, they can be bridged by one or more bus bridges. Each of these elements may perform its conventional functions known in the art. In particular, system memory804and mass storage device(s)806can be employed to store a working copy and a permanent copy of the executable code of the programming instructions of an operating system, one or more applications, and/or various software implemented components of a converter, binary file input interface, binary to 8-bit vector conversion, 2D array conversion, array resize, analyzer, partially retrained neural network, training, retrain and validation and classification, and computing logic822. The programming instructions implementing computing logic822can comprise assembler instructions supported by processor(s)802or high-level languages, such as, for example, C, that can be compiled into such instructions. In embodiments, some of computing logic can be implemented in hardware accelerator805. In embodiments, part of computational logic822, e.g., a portion of the computational logic822associated with the runtime environment of the compiler may be implemented in hardware accelerator805. The permanent copy of the executable code of the programming instructions or the bit streams for configuring hardware accelerator805can be placed into permanent mass storage device(s)806and/or hardware accelerator805in the factory, or in the field, through, for example, a distribution medium (not shown), such as a compact disc (CD), or through communication interface810(from a distribution server (not shown)). While for ease of understanding, the compiler and the hardware accelerator that executes the generated code that incorporate the predicate computation teaching of the present disclosure to increase the pipelining and/or parallel execution of nested loops are shown as being located on the same computing device, in alternate embodiments, the compiler and the hardware accelerator can be located on different computing devices. The number, capability and/or capacity of these elements810-812can vary, depending on the intended use of example computer device800. The constitutions of these elements810-812are otherwise known, and accordingly will not be further described. FIG.8illustrates an example computer-readable storage medium having instructions configured to implement all (or portion of) software implementations. As illustrated, computer-readable storage medium can include the executable code of a number of programming instructions or bit streams. Executable code of programming instructions (or bit streams) can be configured to enable a device, e.g., computer device800, in response to execution of the executable code/programming instructions (or operation of an encoded hardware accelerator875), to perform (aspects of) various processes. In alternate embodiments, executable code/programming instructions/bit streams904can be disposed on multiple non-transitory computer-readable storage medium902instead. In embodiments, computer-readable storage medium902can be non-transitory. In still other embodiments, executable code/programming instructions904can be encoded in transitory computer readable medium, such as signals. Referring back toFIG.7, for one embodiment, at least one of processors802can be packaged together with a computer-readable storage medium having some or all of computing logic822(in lieu of storing in system memory804and/or mass storage device806) configured to practice all or selected ones of the operations. For one embodiment, at least one of processors802is packaged together with a computer-readable storage medium having some or all of computing logic822to form a System in Package (SiP). For one embodiment, at least one of processors802can be integrated on the same die with a computer-readable storage medium having some or all of computing logic822. For one embodiment, at least one of processors802can be packaged together with a computer-readable storage medium having some or all of computing logic822to form a System on Chip (SoC). For at least one embodiment, the SoC can be utilized in, e.g., but not limited to, a hybrid computing tablet/laptop. The disclosed methods can be used to identify cell populations by identifying candidate biomarkers associated with specific cell populations. In some examples, the markers, such as sets of markers, are used for monitoring disease states, such as cancer, in an organism, such as a mammalian subject, for example a human subject. In some examples, the method can be used to monitor the response to a therapy, disease progression and/or make treatment decisions for subjects. In some particular examples, the disclosed markers are used to monitor therapy response to immunotherapy treatment in Merkel cell carcinoma. In some examples, the disclosed markers are used to predict responsiveness to immunotherapy treatment in Merkel cell carcinoma which is virus mediated (Merkel cell polyomavirus). In particular embodiments, disclosed are candidate biomarkers discovered and annotated by the FAUST method, and associated with patient response to immunotherapy, such as anti-PD-1 therapy, for Merkel cell carcinoma, and in particular, Merkel cell carcinoma of viral origin. In one example, candidate biomarkers were found in baseline fresh blood samples from patients with Merkel cell carcinoma. In some examples, the phenotypes of T cell biomarkers include one or more of the follow combinations:1. CD4− CD3+ CD8+ CD45RA− HLADR+ CD28+ PD1 Dim CD25− CD127− CCR7−2. CD4+ CD3+ CD8− CD45RA− HLADR− CD28+ PD1 Dim CD25− CD127− CCR7−3. CD4+ CD3+ CD8− CD45RA+ HLADR− CD28− PD1 Dim CD25− CD127+ CCR7+4. CD4− CD3+ CD8+ CD45RA− HLADR+ CD28+ PD1 Bright CD25− CD127− CCR7−5. CD4+ CD3+ CD8− CD45RA− HLADR+ CD28+ PD1 Dim CD25− CD127− CCR7−6. CD4+ CD3+ CD8− CD45RA+ HLADR− CD28+ PD1− CD25− CD127+ CCR7−7. CD4− CD3+ CD8+ CD45RA− HLADR− CD28+ PD1 Dim CD25− CD127− CCR7−8. CD4− CD3+ CD8+ CD45RA+ HLADR− CD28− PD1 Bright CD25− CD127− CCR7−9. CD4+ CD3+ CD8− CD45RA+ HLADR− CD28+ PD1 Dim CD25− CD127+ CCR7−10. CD4− CD3+ CD8+ CD45RA+ HLADR− CD28− PD1 Dim CD25− CD127− CCR7−11. CD4− CD3+ CD8+ CD45RA+ HLADR− CD28− PD1 Dim CD25+ CD127− CCR7−12. CD4+ CD3+ CD8− CD45RA− HLADR− CD28+ PD1 Dim CD25− CD127− CCR7+13. CD4+ CD3+ CD8− CD45RA− HLADR+ CD28+ PD1 Dim CD25− CD127− CCR7+14. CD4− CD3+ CD8+ CD45RA− HLADR− CD28+ PD1 Bright CD25− CD127− CCR7−15. CD4− CD3+ CD8− CD45RA+ HLADR+ CD28+ PD1 Dim CD25− CD127− CCR7−16. CD4+ CD3+ CD8− CD45RA+ HLADR− CD28− PD1 Dim CD25− CD127− CCR7−17. CD4+ CD3+ CD8− CD45RA+ HLADR− CD28+ PD1 Dim CD25− CD127− CCR7+. In the aforementioned listing, the phenotypes of T cell candidate biomarkers are listed in ranked order, from strongest association with responsiveness to treatment to weakest (but still statistically significant at FDR-adjusted 0.20 level). In some examples, phenotypes of myeloid candidate biomarkers were discovered and annotated by the FAUST method, and were associated with patient response to immunotherapy. These candidate biomarkers were also found in baseline fresh blood samples from patients with Merkel cell carcinoma. For example, in some embodiments, one of more of the following combinations of candidate biomarkers were discovered to be associated with response to immunotherapy in Merkel cell carcinoma:1. CD33 Bright CD16− CD15− HLADR Bright CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+2. CD33 Bright CD16− CD15− HLADR Bright CD14− CD3− CD11B+ CD20− CD19− CD56− CD11C+3. CD33 Bright CD16− CD15+ HLADR Bright CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+4. CD33 Bright CD16+ CD15+ HLADR Bright CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+5. CD33 Bright CD16− CD15− HLADR Bright CD14− CD3− CD11B− CD20− CD19− CD56− CD11C+6. CD33 Dim CD16+ CD15+ HLADR Bright CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+7. CD33− CD16− CD15− HLADR Dim CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+8. CD33 Dim CD16− CD15+ HLADR Dim CD14− CD3− CD11B+ CD20− CD19− CD56− CD11C−9. CD33 Dim CD16+ CD15+ HLADR Bright CD14− CD3− CD11B+ CD20+CD19+ CD56− CD11C−10. CD33 Bright CD16− CD15− HLADR Dim CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+11. CD33 Dim CD16− CD15+ HLADR Bright CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C+12. CD33 Dim CD16+ CD15+ HLADR Dim CD14− CD3+ CD11B+ CD20− CD19− CD56− CD11C−13. CD33 Dim CD16− CD15− HLADR Bright CD14− CD3− CD11B+ CD20− CD19− CD56− CD11C+14. CD33 Dim CD16− CD15+ HLADR− CD14− CD3− CD11B+ CD20− CD19− CD56− CD11C−15. CD33 Bright CD16− CD15− HLADR Dim CD14+ CD3− CD11B+ CD20− CD19− CD56− CD11C−16. CD33 Dim CD16− CD15+ HLADR− CD14− CD3− CD11B− CD20− CD19− CD56− CD11C−17. CD33 Dim CD16− CD15− HLADR Dim CD14− CD3− CD11B− CD20− CD19− CD56− CD11C+18. CD33− CD16− CD15− HLADR Bright CD14− CD3− CD11B+ CD20− CD19− CD56+ CD11C+19. CD33 Dim CD16− CD15− HLADR Dim CD14− CD3− CD11B− CD20− CD19− CD56− CD11C−. In the aforementioned listing, the phenotypes are listed in ranked order, from strongest association with responsiveness to treatment to weakest (but still statistically significant at FDR-adjusted 0.20 level). Appropriate samples for use in the methods disclosed herein include any conventional biological sample obtained from an organism (including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). In some particular examples, the biological sample is blood sample. The biological sample may be examined using any assay format that is capable of detecting desired molecules including immunohistology techniques, ELISA, Western Blotting, and the like. The disclosed markers may be used to identify as well predict a subject's responsiveness to immunotherapy treatment of Merkel cell carcinoma and in particular, Merkel cell carcinoma of viral origin. Changes in the profile can also represent the progression (or regression) of the disease process. Methods for monitoring the efficacy of therapeutic agents are described herein. The diagnostic methods of the present disclosure are valuable tools for practicing physicians including for monitoring a subject for onset and/or advancement of a particular condition and/or disease, including Merkel cell carcinoma. The methods disclosed herein can also be used to monitor the effectiveness of a therapy. Following the measurement of the expression levels of one or more of the molecules identified herein, the assay results, findings, diagnoses, predictions and/or treatment recommendations can be recorded and communicated to technicians, physicians and/or patients, for example. In certain embodiments, computers will be used to communicate such information to interested parties, such as, patients and/or the attending physicians. Based on the measurement, the therapy administered to a subject can be modified. In one embodiment, a diagnosis, prediction and/or treatment recommendation based on the expression level in a test subject of one or more of the Merkel cell carcinoma biomarkers disclosed herein is communicated to the subject as soon as possible after the assay is completed and the diagnosis and/or prediction is generated. The results and/or related information may be communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to a test subject by any means of communication, including writing, such as by providing a written report, electronic forms of communication, such as email, or telephone. Communication may be facilitated by use of a computer, such as in case of email communications. In certain embodiments, the communication containing results of a diagnostic test and/or conclusions drawn from and/or treatment recommendations based on the test, may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in U.S. Pat. No. 6,283,761; however, the present disclosure is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the disclosure, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions. In several embodiments, identification of a subject as having Merkel cell carcinoma results in the physician treating the subject, such as prescribing one or more therapeutic agents for inhibiting or delaying one or more signs and symptoms associated with Merkel cell carcinoma, including use of anti-PD-1 therapy. In additional embodiments, the dose or dosing regimen is modified based on the information obtained using the methods disclosed herein. The subject can be monitored while undergoing treatment using the methods described herein in order to assess the efficacy of the treatment protocol. In this manner, the length of time or the amount give to the subject can be modified based on the results obtained using the methods disclosed herein. The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. EXAMPLE A Method for Unbiased Cell Population Discovery and Annotation Reveals Correlates of Clinical Response to Cancer Immunotherapy FAUST identifies baseline CD8+ T cells in blood that correlate with outcome in CITN-09, a Merkel cell carcinoma anti-PD-1 trial. FAUST was used to perform cell sub-population discovery in cytometry data generated by the Cancer Immunotherapy Trials Network (CITN) from study CITN-09, a phase 2 clinical trial of the Pembrolizumab anti-PD-1 therapy in Merkel cell carcinoma (MCC), with the goal of identifying baseline correlates of response to treatment (Nghiem et al., Durable tumor regression and overall survival in patients with advanced merkel cell carcinoma receiving pembrolizumab as first-line therapy. Journal of Clinical Oncology, 37(9):693-702, 2019. PMID: 30726175). Seventy-eight stained samples were analyzed to identify T cells. FAUST selected 10 markers for discovery and annotation, including the marker CD3. Since the panel was designed to investigate T cells, only these CD3+ sub-populations were used for downstream correlates analysis. Binomial generalized linear mixed models (GLMMs) (Nowicka et al., Cytof workflow: differential discovery in high-throughput high-dimensional cytometry datasets. F1000Research, 6, 2017, which is hereby incorporated by reference in its entirety) were used to test each sub-population for differential abundance between responders and non-responders in the 27 subjects at the baseline time point, prior to receiving anti-PD-1 therapy (equation (4.5) specifies the model). Responders were defined as subjects that exhibited either a complete (CR) or partial (PR) response (per RECIST1.1 (Eisenhauer et al., New response evaluation criteria in solid tumours: Revised (RECIST) guideline (version 1.1). European Journal of Cancer, 45(2):228-247, 2009. Response assessment in solid tumours (RECIST): Version 1.1 a), and non-responders as subjects exhibiting progressive (PD) or stable (SD) disease. At a false discovery rate (FDR)-adjusted 5% level (Yoav Benjamini and Yosef Hochberg. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the royal statistical society. Series B (Methodological), pages 289-300, 1995), four FAUST phenotypes were associated with response to therapy. Two had a CD28+ HLA-DR+ CD8+ annotation, with PD-1 dim or PD-1 bright, respectively. The third had an HLA-DR− CD28+ CD4+ PD-1 dim annotation, while the fourth had an HLA-DR− CD28− CD4+ PD-1 dim annotation. Effect sizes with 95% confidence intervals for the correlates are reported in Table 6. Three of the four correlates were annotated CD45RA− and CCR7−, indicating they represented effector-memory T cells. The complete phenotypes are described inFIGS.2A-2C. The primary flow cytometry data was inspected to confirm that the discovered population phenotypes matched the underlying protein expression. By plotting cluster densities against samples (FIG.2A), the FAUST annotations accurately described the observed cellular phenotypes in these sub-populations. These data were also visualized using UMAP embeddings (McInnes et al., UMAP: Uniform Manifold Approximation and Projection for Dimension Reduction. ArXiv e-prints, February 2018) with “qualitative” parameter settings (Becht et al., Dimensionality reduction for visualizing single-cell data using umap. Nature biotechnology, 37(1):38, 2019) (FIGS.2B,2C). FAUST clusters were not typically separated into disjoint “islands” in the UMAP embedding (FIG.2C), and that single UMAP “islands” contained significant variation in expression of some of the measured protein markers (FIG.2B). Taken together, these observations demonstrate that dimensionality reduction does not necessarily reflect all variation measured in the underlying proteins, and that any method that relies on UMAP for population discovery would likely miss these candidate biomarkers. The association between the abundance of the discovered correlates and tumor viral status of each subject was next investigated. The differential abundance GLMM were adapted to test for an interaction between response to therapy and tumor viral status. This interaction was statistically significant for both CD8+ correlates (FIG.3A), indicating that these T cells are particularly relevant in subjects with virus-positive tumors. In order to further investigate the relevance of these T cells measured in blood, data on PD-1 immunohistochemistry (IHC) staining in tumor biopsies from the same patients was acquired (Miller et al., Merkel cell polyomavirus-specific immune responses in patients with merkel cell carcinoma receiving anti-pd-1 therapy. Journal for immunotherapy of cancer, 6(1):131, 2018). Importantly, the in-tumor PD-1 measurement is a known outcome correlate in MCC (Giraldo et al., Multidimensional, quantitative assessment of pd-1/pd-11 expression in patients with merkel cell carcinoma and association with response to pembrolizumab. Journal for immunotherapy of cancer, 6(1):99, 2018). Limited overlap between the assays resulted in only five subjects where both flow cytometry and tumor biopsy anti-PD-1 IHC staining were available, and only four of these were virus-positive. Nonetheless, the frequencies of the CD8+ PD-1 dim T cells were strongly correlated (ρ=0.945) with the PD-1 total IHC measurements within the four virus-positive subjects (FIG.3B). TCR clonality data generated from patient tumor samples was obtained (Miller et al., Merkel cell polyomavirus-specific immune responses in patients with merkel cell carcinoma receiving anti-pd-1 therapy. Journal for immunotherapy of cancer, 6(1):131, 2018). Ten subjects passing clonality QC were common to the two data sets, six of which were virus positive. Frequencies of the FAUST populations within these six subjects were strongly correlated (ρ=0.952) with the measurement of productive clonality (FIG.3C). Normalizing the correlate cell counts by the total number of CD3+ annotated FAUST sub-populations (i.e., total T cells, the recommended normalization constant for T cell clonality) instead of total lymphocyte count improved the observed correlation to ρ=0.972 (FIGS.10A-C). FIGS.11A-11Cprovide the FAUST sub-population annotated CD4− CD3+CD8+ CD45RA− HLA-DR+ CD28+ PD-1 bright CD25− CD127− CCR7− that is associated with clinical outcome at the FDR-adjusted 5% level, with tumor measurements inFIGS.11B and11C.FIGS.12A-12Cprovide the FAUST sub-population annotated CD4 bright CD3+ CD8− CD45RA− HLA-DR− CD28+ PD-1 dim CD25− CD127− CCR7− that is associated with clinical outcome at the FDR-adjusted 5% level, with tumor measurements inFIGS.12B and12C.FIGS.13A-13Cprovide the FAUST sub-population annotated CD4 bright CD3+ CD8− CD45RA+ HLA-DR− CD28− PD-1 dim CD25− CD127+ CCR7+ that is associated with clinical outcome at the FDR-adjusted 5% level, with tumor measurements inFIGS.13B and13C. Together, these results suggest that the CD8+ T cell correlate discovered by FAUST in blood may represent a circulating population of tumor-associated virus-specific T cells that are also detectable in the tumor. Subsequent to this initial analysis, FAUST was used to test the hypothesis that the increased pre-treatment abundance of the top CD8+ correlate and top CD4+ correlate discovered by FAUST in the MCC trial are associated with positive response to pembrolizumab treatment by applying FAUST to the public dataset described in Subramanyam et al. (Distinct predictive biomarker candidates for response to anti-ctla-4 andanti-pd-1 immunotherapy in melanoma patients. Journal for immunotherapy of cancer, 6(1):18, 2018). FAUST was applied only to unstimulated baseline PBMC samples. Before applying FAUST to the public dataset, the pre-gating strategy reported by Subramanyam et al. was replicated using the computational package openCyto (Finak et al., Opencyto: an open source infrastructure for scalable, robust, reproducible, and automated, end-to-end flow cytometrydata analysis. PLoS computational biology, 10(8):e1003806, 2014). FAUST was used to estimate data-driven annotation threshold for the 10 annotating markers selected in the unbiased MCC analysis. These thresholds were used to extract per-sample counts of the top CD8+ and CD4+ phenotypes matching those produced by FAUST in the MCC analysis. In the CyTOF dataset of Subramanyam et al., FAUST defined one standardized annotation threshold for all ten markers (including CD4 and PD-1). On the other hand, in the MCC study FAUST defined two thresholds for CD4 and PD-1, and one threshold for the other markers. Therefore, to extract corresponding counts in the CyTOF dataset, PD-1+ cells were treated as comparable to the FAUST phenotypes PD-1 dim and PD-1 bright in the MCC study; the same map was used for CD4+ cells in the CyTOF dataset. The extracted counts for the top CD8+ phenotype and top CD4+ phenotype were then tested for differential abundance between responders and non-responders in pre-treatment samples from subjects that went on to receive the anti-PD-1 therapy pembrolizumab. Subjects in the CyTOF dataset were defined as subjects that exhibited progression-free survival for at least 180 days. Increased abundance was observed in responding subjects for the top CD8+ phenotype and top CD4+ phenotype defined by FAUST (FIGS.30A-30B), validating the associations detected by FAUST in the MCC analysis, and demonstrating that FAUST can be used to perform targeted validation across studies and technologies. FAUST Sub-Populations Capture Underlying Biological and Technical Signals in Longitudinal Studies FAUST's sample-specific processing and phenotypic standardization enables the identification of cell sub-populations whose abundances differ or may be absent across different subsets of experimental samples. In the MCC anti-PD-1 trial, all CD8+ T cells with the PD-1-bright phenotype were examined. The temporal abundance of these cells is shown in (FIG.4A) and reveals that these cells are not detectable in most samples after subjects have received pembrolizumab therapy. The absence of these phenotypes post-therapy is hypothesized to occur due to pembrolizumab blocking the detecting antibody. FAUST was also applied to data generated from CITN-07, a randomized phase II trial studying how a DEC-205/NY-ESO-1 fusion protein (CDX-1401) and a neoantigen-based melanoma poly-ICLC vaccine (poly-ICLC) therapy work when given with or without recombinant FLT3 ligand (CDX-301) in treating patient with stage IIB to stage IV melanoma. The cytometry data consisted of fresh PBMCs stained for myeloid cell phenotyping. Examination of the longitudinal profile of clusters with phenotypic annotations consistent with dendritic cells (FIG.4B) revealed dynamic expansion and contraction of the total DC compartment in the FLT3-L stimulated cohort but not in the unstimulated-by-FLT3-L-pre-treatment cohort. The expansion peaked at day 8 after FLT3-L simulation in cycles 1 and 2. This dynamic is consistent with observations from manual gating of the DC population (Bhardwaj et al., A phase ii randomized study of cdx-1401, a dendritic cell targeting ny-eso-1 vaccine, in patients with malignant melanoma pre-treated with recombinant cdx-301, a recombinant human flt3 ligand. Journal of Clinical Oncology, 34(15):9589, 2016) and consistent with the expected biological effect of FLT3-L (Fong et al., Altered peptide ligand vaccination with flt3 ligand expanded dendritic cells for tumor immunotherapy. Proc. Natl. Acad. Sci. U.S.A., 98(15):8809-8814, July 2001) as well as the timing of FLT3 administration. These results demonstrate that FAUST is able to detect, annotate, and correctly assign abundance to cell sub-populations, including those that are missing in some samples. The longitudinal behavior of PD-1 bright T cell populations in the MCC anti-PD-1 trial and the dendritic cells in the FLT3 ligand+CDX-1401 trial serve as an internal validation of the methodology. FAUST Identifies Phenotypically Similar Myeloid Sub-Populations Associated with Clinical Response Across Multiple Cancer Immunotherapy Trials. Both the MCC anti-PD-1 and FLT3-L+therapeutic Vx trials had data sets stained with a myeloid phenotyping panel. FAUST was applied to two additional myeloid phenotyping data sets (one CyTOF discovery and one FACS validation assay) from an anti-PD-1 trial in metastatic melanoma (Krieg et al., High-dimensional single-cell analysis predicts response to anti-pd-1 immunotherapy. Nature medicine, 24(2):144, 2018). In the following, these are referred to as the melanoma anti-PD-1 FACS and melanoma anti-PD-1 CyTOF data sets. In each study, a different staining panel was used to interrogate the myeloid compartment. A principal finding of the melanoma anti-PD-1 trial was that the frequency of CD14+ CD16− HLA-DRhi cells associated with response to therapy. In all four data sets FAUST identified cell sub-populations associated with clinical outcome (FDR-adjusted 5% level, using binomial GLMMs to test for differential abundance) whose phenotype was consistent with the previously-published CD14+ CD16− HLA-DRhi phenotype (FIGS.5A-5D). Complete phenotypes, effect sizes and confidence intervals for the myeloid baseline predictors discovered in the MCC anti-PD-1 myeloid phenotyping data are in Table 1; those discovered in the FLT3-L+therapeutic Vx trial are in Table 2. These results demonstrate the power of the disclosed method to detect candidate biomarkers in a robust manner across different platforms, staining panels, and experimental designs. TABLE 1All statistically significant (Bonferroni adjusted significancethreshold of 5%) from the MCC anti-PD-1 trial.PopulationEffect SizeLower 2.5%Upper 97.5%BonferroniPCD33 Bright CD16− CD15− HLADR2.6111.0734.1620.041Bright CD14− CD3− CD11B−CD20− CD19− CD56− CD11C+CD33 Bright CD16− CD15− HLADR2.7761.3164.2740.009Bright CD14− CD3− CD11B+CD20− CD19− CD56− CD11C+CD33 Bright CD16− CD15− HLADR3.5581.8375.2840.002Bright CD14+ CD3− CD11B+CD20− CD19− CD56− CD11C+CD33 Bright CD16− CD15+ HLADR3.6891.8595.8230.007Bright CD14+ CD3− CD11B+CD20− CD19− CD56 − CD11C+CD33 Bright CD16+ CD15+ HLADR4.7542.3537.5780.011Bright CD14+ CD3− CD11B+CD20− CD19− CD56− CD11C+ The complete set of baseline predictors from the FLT3-L+therapeutic Vx trial are listed in Table 2. The top populations, by magnitude, were CD14+CD16-monocyte populations. TABLE 2All statistically significant (Bonferroni adjusted significancethreshold of 5%) from the FLT3-L + therapeutic Vx trial.adjusted.Populationestimatelowerupperstd. errorstatisticp. valuep. valueCD8−CD3−HLA DRbright CD4−CD19−2.441.163.730.663.739.70e−050.01CD14+CD11C+CD123−CD16−CD56−CD8−CD3−HLA DRdimCD4−CD19−2.261.223.310.544.231.17e−050.00CD14+CD11C+CD123−CD16−CD56−CD8dim CD3−HLA DRbright CD4−CD19−2.140.983.290.593.621.46e−040.02CD14+CD11C+CD123−CD16−CD56−CD8dim CD3−HLA DRdim CD4−CD19−1.910.932.890.503.826.67e−050.01CD14−CD11C−CD123−CD16+CD56+CD8dim CD3−HLA DR−CD4−CD19−1.600.732.470.443.591.64e−040.02CD14−CD11C−CD123−CD16+CD56+CD8−CD3−HLA DRdim CD4−CD19−1.600.692.500.463.472.65e−040.03CD14−CD11C−CD123−CD16+CD56+CD8bright CD3+HLA DRbright CD4−CD19−1.500.712.290.403.721.01e−040.01CD14−CD11C−CD123−CD16−CD56−CD8dim CD3−HLA DRdim CD4−CD19−1.230.751.700.245.072.01e−070.00CD14−CD11C−CD123−CD16+CD56−CD8−CD3−HLA DRbright CD4−CD19−1.220.601.830.323.855.90e−050.01CD14−CD11C−CD123−CD16−CD56−CD8−CD3−HLA DRdimCD4−CD19−1.120.681.560.225.022.64e−070.00CD14−CD11C−CD123−CD16+CD56−CD8dim CD3−HLA DR−CD4−CD19−0.970.581.350.204.924.34e−070.00CD14−CD11C−CD123−CD16+CD56−CD8−CD3−HLA DR−CD4−CD19−CD14−0.920.531.300.204.671.49e−060.00CD11C−CD123−CD16+CD56−CD8−CD3−HLA DRdim CD4−CD19−0.750.311.190.223.383.64e−040.05CD14−CD11C−CD123−CD16−CD56− FAUST Enables Cross-Study Comparisons Between Different Marker Panels FAUST annotations enable the use of prior biological knowledge of hierarchical relationships among cell types to test hypotheses that cannot immediately be tested in the absence of cluster labels. By jointly modeling the annotated populations related through biological hierarchy, FAUST can be used to account for this dependence structure when conducting secondary tests of interests. This is analogous to the techniques used to perform gene set enrichment analysis in gene expression data (Weimar et al., Peripheral blood manipulation significantly affects the result of dendritic cell monitoring. Transplant immunology, 17(3):169-177, 2007). This approach is contrasted against aggregating (i.e., summing) cell sub-population counts on the basis of their common annotations to derive ancestral populations that resemble those obtained by manual gating. To demonstrate this FAUST was used to test each of four different myeloid sub-compartments for association with outcome at baseline in each of the three trials with heterogeneous marker panels. FAUST annotations were used to define membership in the myeloid compartment (described below). All FAUST sub-populations that were annotated as lineage negative (CD3−, CD56−, CD19−) and expressing HLA-DR (either dim or bright) were selected as part of the myeloid compartment. Myeloid sub-compartments were defined in terms of a sub-population's CD14 and CD16 expression, with CD14− CD16− cells defined as dendritic cells, and other combinations as double-positive, CD14+, or CD16+ monocytes, respectively. Two models were fit to each data set. First, a multivariate model of all candidate cell sub-populations was fit, and the cell sub-populations' model coefficients were aggregated over each sub-compartment to test for increased abundance in responders vs. non-responders at baseline. This model represents the cell population analog of gene set enrichment analysis. Second, a univariate model was fit to cell counts derived by summing over each myeloid sub-compartment, producing a single coefficient to test for increased abundance in responders vs. non-responders at baseline. This represents the modeling approach one would undertake if the myeloid sub-compartments were defined using a manual gating strategy. 95% confidence intervals were adjusted to 99% to account for multiple comparisons (Bonferroni correction for five tests). Details of both models are provided in the Methods disclosed herein. In the aggregate model, significantly increased abundance of the CD14+ CD16− sub-compartment among responders (FIG.6A) was observed only in the melanoma anti-PD-1 trial FACS data set, consistent with the validation analysis where they manually gated monocytes. Statistically significant differential abundance was not detected in either CITN trial data set using the aggregate model. In contrast, when differential abundance tests were performed by aggregating model coefficients over homogeneous sub-populations (thereby accounting for dependence structure), significantly increased abundance was observed in the CD14+ CD16− monocyte sub-compartment across all data sets. These results highlight that sub-populations defined by manual gating may not exhibit differential abundance when they don't capture all the heterogeneity in a cell population. The multivariate modeling strategy also detected a significant association between outcome and differential abundance in the CD14− CD16− dendritic cell sub-compartment (FIG.6B) in the two CITN trials, consistent with the analysis of baseline predictors in those trials. Such an association was not detected in the DC sub-compartment in the Melanoma anti-PD1 trial. The latter used frozen PBMC samples while both the CITN trials used fresh blood samples for analysis. Reports have shown that the functional characteristics of monocytes are not adversely affected by cryopreservation, while the relative abundance of pDCs and mDCs can be affected. Difference in cryopreservation status could provide an explanation for the observed differences in modeling outcomes for the DC sub-compartment (Pardali et al., Cryopreservation of primary human monocytes does not negatively affect their functionality or their ability to be labelled with radionuclides: basis for molecular imaging and cell therapy. EJNMMI research, 6(1):77, 2016); Gerrits et al., Peripheral blood manipulation significantly affects the result of dendritic cell monitoring. Transplant immunology, 17(3):169-177, 2007). This multivariate modeling approach demonstrates how FAUST can enable cross-study data integration and analysis even in the presence of heterogeneous staining panels.FIG.20provides results for the remaining compartments from the multivariate and aggregate myeloid compartment analysis described herein showing the multivariate modeling also reveals evidence of increased abundance in responders across the entire myeloid compartment. FAUST is Robust to Different Data Generating Processes The clustering methods densityCut (Ding et al., densitycut: An efficient and versatile topological approach for automatic clustering of biological data. Bioinformatics, 32(17):2567-2576, 2016, which is hereby incorporated by reference in its entirety), FlowSOM (Van Gassen et al., Flowsom: Using self-organizing maps for visualization and interpretation of cytometry data. Cytometry Part A, 87(7):636-645, 2015, which is hereby incorporated by reference), Phenograph (Levine et al., Data-driven phenotypic dissection of aml reveals progenitor-like cells that correlate with prognosis. Cell, 162(1):184-197, 2015) and FAUST were applied to live lymphocytes from all 78 experimental samples as well as from the 27 baseline samples alone in the MCC anti-PD-1 T cell dataset, transforming samples using both the biexponential as well as the hyperbolic arcsine. For all non-FAUST methods, samples were combined before clustering in all scenarios. Tuning parameters were set to the settings reported in (Lukas M Weber and Mark D Robinson. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data. Cytometry Part A, 89(12):1084-1096, 2016, which is hereby incorporated by reference] when possible. After testing for differential abundance between responders and non-responders using counts derived from each method's clusterings, three clusters defined by densityCut were significantly associated with response to therapy at the FDR-adjusted 0.20 level, but none of these represented T cells (FIG.14). No other clusters produced by densityCut, FlowSOM, or Phenograph were associated with response to therapy at this level of significance. On the other hand, FAUST repeatedly found that CD28+ HLA-DR+ PD-1 expressing effector-memory CD8 T cells as well as CD28+ PD-1 expressing CD4 T cells were associated with response to therapy at baseline across all tested conditions at the FDR-adjusted 5% level. Simulate studies that generated data according to FAUST's methodological assumptions were conducted. The study generated data sets from a variety mixture models incorporating different combinations of assumptions detailed in the methods disclosed herein. These assumptions are quite general, so the study begins by simulating data from multivariate gaussian distributions (data sets which are favorable to many existing methods) and progressively simulates data that more closely represents flow cytometry and CyTOF data sets. In the study, FAUST is compared to FlowSOM since FlowSOM is computationally efficient. Each simulated mixture component (representing a cell sub-population) is partially parameterized by a mean vector and is given a phenotypic label that describes the phenotype of the component. By treating these phenotypic labels as ground-truth, it is possible to measure how well the count matrix produced by FAUST agrees with the simulated count matrix, matching discovered and simulated cell populations based on their phenotypes. The simulation studies demonstrate that FAUST's discovery and annotation strategy does not severely over partition the data under a variety of generative regimes (FIG.26). Results also show that the cell counts derived from FAUST's discovered clusters strongly correlate with the underlying true counts across all simulation settings. A median correlation of 0.91 was observed between FAUST and the simulated truth, when cluster counts are correlated between FAUST clusters and the ground truth using only cluster annotations to perform the comparison (FIG.25). The simulated data sets always include a sub-population that is differentially abundant between 50% of the subjects. These results show that when a causal relationship of varying strength is simulated between this differential sub-population and a simulated response to therapy, FAUST discovers the differential sub-population, annotates it correctly, and often identifies that the differential population is associated with response to therapy (FIGS.27-29). In the present simulation, FlowSOM clusters were tested for differential abundance under the same causal regimes as FAUST. The results show that FlowSOM's ability to detect the causal association is adversely affected when the simulation departs from multivariate normality or when the simulated data contains 50 true clusters and batch effects and/or nuisance variables, even when FlowSOM is provided with the true number of clusters as a tuning parameter. This study confirms the empirical finding that FAUST is able to robustly detect signals in data that are not found by other discovery methods. FAUST Methodology FAUST assumes the following criteria are met in a cytometry experiment consisting of n experimental units Ei, 1≤i≤n. Assumption 1. Each Sample in the Cytometry Experiment has been Pre-Gated to Remove Debris and Dead Cells. If pre-gating has not been performed by an investigator, computational methods can be used before applying FAUST to cytometry data in order to guarantee this assumption is met. Assumption 2. In Each Sample, Measurements on the Live Cells are Made Using a Common Set of p Transformed Protein Markers. Let nidenote the number of events in the ithexperimental unit. FAUST supposes each event Ei,jin an experimental unit Ei, of dimension p (the number of markers), arises as a sample from a finite mixture model Ei,j∼∑Mm=1ωmfm,i(x),(4.1) for 1≤j≤ni, with M∈N, 0≤ωm≤1 and ΣMm=1ωm=1 for all 1≤i≤n. FAUST assumes the mixture components fm,iof an experimental unit in (4.1) belong to the class of densities on the space of protein measurements i≡{fm,i|∃λm,i∈ℝ,σm,i∈ℝsuchthatfm,i+λm,iσm,i∈∀1≤m≤M}(4.2) for each experimental unit i, with the common class F is defined as ≡{fm|fis unimodal along all margins}. (4.3) (4.2) expresses the fundamental modeling assumption: each mixture (4.1) that generates an experimental unit consists of a common set of densities (4.3), with unit-specific changes to location (the translations λm,i) and scale (the scalar multiples σm,i) of the component densities. These unit-specific modifications represent technical and biological effects. FAUST only assumes marginal unimodality for the f in (4.3), but makes no assumptions about the joint-distribution of these densities. FAUST Method: Schematic Overview FAUST is designed to perform independent approximate modal clustering of each mixture in each experimental unit. Its approximation strategy is to use 1-dimensional densities to grow an exhaustive forest of gating strategies, from which it estimates a standardized set of annotation boundaries for all markers in a mixture, which exhibit 1-dimensional multimodality either marginally or across a large number of conditional 1-dimensional density estimates. Annotation boundaries are estimated by taking a weighted average of marginal and conditional 1-dimensional antimodes for a marker that FAUST selects, using a score that quantifies if the marker has persistent multimodality in the experimental unit. FAUST also uses the distribution of the depth score across units to select a subset of markers to use for cluster discovery and annotation. FAUST defines a cluster as a subset of events in an experimental unit that fall inside either a conical or hyper-rectangular region bounded by the Cartesian product of the standardized set of annotation boundaries. FAUST discovers cluster phenotypes by growing a random forest of partition trees for each experimental unit (following a strategy related to growing the annotation forest), and locating a sub-collection of homogeneous leaf nodes in the forest relative to the standardized phenotypic boundaries. FAUST collects a list of phenotypes discovered in each experimental unit and counts how often each phenotype appears across the set of lists. If a phenotype exceeds a user-specified filtering threshold, FAUST will annotate that cluster in each experimental unit relative to the standardized annotation boundaries. Intuitively, an annotation is a pointer to a modal regions of each experimental unit's mixture distribution. FAUST concludes by deriving a count matrix, with each row corresponding to a sample in the experiment, each column an annotated cluster, and each entry the cell count corresponding the annotated cluster in the sample. 1.1 FAUST Method: Growing the Annotation Forest For all markers in a sample, all cells for each marker are tested for unimodality using the dip test (John A Hartigan and P M Hartigan. The Dip Test of Unimodality. The Annals of Statistics, pages 70-84, 1985). The hypothesis of unimodality is rejected for any marker that has dip test p-values below 0.25. All markers which are deemed multimodal according to this dip criterion are then used to start gating strategies. Gate locations for each strategy are determined using the taut string density estimator (P Laurie Davies and Arne Kovac. Densities, spectral densities and modality. The Annals of Statistics, 32(3):1093-1136, 2004). The location of each gate is the mid-point of any anti-modal component of the taut string. Since the taut string makes no assumptions about the number of modes present in a density, in principle this approach can lead to estimating an arbitrary number of gates in a given strategy. In practice, FAUST only pursues strategies containing 4 or fewer gates under the assumption that marker expression of 5 expression categories does not reflect biological signal. Once the initial set of gates are computed for a given marker, events are divided into sub-collections relative to the gates for that marker and the procedure recurses and repeats along each sub-collection. Algorithm 1 gives an overview of the procedure. A gating strategy terminates when it meets any of the following stopping conditions. First, once a strategy involves any three combinations of markers, it terminates. This is because the space of gating strategies grows factorially with the number of markers. Due to this growth rate, nodes in the forest are penalized factorially relative to their depth in the gating strategy when FAUST subsequently computes the depth score. Second, if at any point in a strategy FAUST fails to reject the null hypothesis of unimodality for all tested markers, the strategy terminates regardless of depth. Finally, a gating strategy terminates along a branch if all nodes along the branch contain 25 or fewer cells. Algorithm 1 Grow Annotation Forest1:function growAnnotationForest(currentCells, currentDepth, activeMarkers)2:if (length(currentCells) < 25) or (currentDepth > 3) then3:return strategy1> Gating strategy stops due to depth, event constraints.4:else5:currentDepth ← currentDepth + 16:multimodalList ← empty list7:for markerIndex ∈ (columns(expressionMatrix) activeMarkers) do8:if pValue(dipTest(expressionMatrix[currentCells,markerIndex])) < 0.25 then9:append(multimodalList, markerIndex)10:if length(multimodalList) = = 0 then11:return strategy1> Gating strategy stops due to shape constraint.12:else13:for markerIndex in multimodalList do14:boundaryList ← empty list15:Compute taut string density estimate of expressionMatrix[currentCells,markerIndex]16:boundaryList ← mid-points of antimodal components of taut string17:remainingMarkers ← activeMarkers markerIndex18:for i in [1,length(boundaryList)] do19:lg ← boundaryList[(i−1)]20:ug ← boundaryList[i]21:newCells ← rows of expressionMatrix[currentCells,markerIndex] between lg and ug22:growAnnotationForest(newCells, currentDepth,remainingMarkers) FAUST Method: Depth Score Computation Suppose there are p>1 active markers in a sample. To compute the depth score for any of the p markers, the annotation forest is first examined to determine the following quantities: d1, the number of times different markers are gated in the root population; d2, the number of times children of the root are gated; and d3the number of times grandchildren of the root are gated. For i∈{1,2,3} define δi=¯1di.For1≤m≤p,let≡{Nm,1,Nm,2,…,Nm,n}. be the set of all n parent nodes in the annotation forest for which the null hypothesis of unimodality is rejected for marker m. For a parent node 1≤j≤n, let 1Rdenote the indicator function that is 1 when Nm,jis the root population. Similarly, let 1Cdenotes an indicator of a child of the root, and 1Ga grandchild of the root. Define the scoring function Q(Nm,j)≡(1−αR)1R(Nm,j)+(1−αR)(1−αC)1C(Nm,j)+(1−αR)(1−αC)(1−αG)1G(Nm,j), definingαR≡αR(Nm,j)≡the dip test p-value in the root population of the gating strategy that led to Nm,j. Define αCand αGsimilarly. The function Q can be interpreted as a measure of the quality of the gating strategy that led to node Nm,j. In the case of a grandchild node that had clear modal separation along all markers in the strategy, Q(Nm,j)≈1, while a grandchild node that had p-values of 0.25 at each ancestral node, Q(Nm,j)≈27/64=0.753. Let Pmbe the population size for marker m in the root population. Next define P(Nm,j)≡#ofcellsinnodeNm,j. Finally, define D(Nm,j)≡δ1·1R(Nm,j)+δ2·1C(Nm,j)+δ3·1G(Nm,j). The depth score is defined to be the normalized sum DS(𝒩m)≡Σi=1nQ(Nm,i)·P(Nm,i)·D(Nm,i)max1≤q≤pDS(𝒩q)≡Σi=1nω(Nm,i)max1≤q≤pDS(𝒩q).(4.4.) The depth score maps into [0,1] with at least one marker in a gated sample achieving the maximal score of 1. This is taken as a measure of separation quality: the best scoring marker according to the depth score is taken to be the best separated marker in that sample at the root population, and conditionally along all other gating strategies. Normalizing to the unit interval allows depth scores to be compared across experimental units for given markers. By using the factorial weights δi, the depth score also explains why FAUST only explores gating strategies involving, at most, combinations of three markers in its scoring and marker selection phase. Adding more combinations of markers induces a factorial increase in computational cost. But any marker that enters a gating strategy at depth 4 (or beyond) will be dominated in depth score by those markers initially gated in the annotation forest at or near the root population. Consequently, after normalization in experiments with a large number of markers, such markers have depth score an above zero, and are effectively never selected by FAUST for discovery and annotation. Hence the restriction to 3-marker gating strategies. FAUST Method: Phenotypic Boundary Estimation The depth score is also used to estimate annotation boundaries. Recalling FAUST only explores gating strategies with 4 or fewer annotation boundaries, FAUST partitions the set ≡∪∪∪. Define 1≡{Nm,i∈|Nm,ihas a single gate determined by the taut string}. Define2,3, and4similarly. In other words, each1is the subset of nodes in the annotation forest for marker m with i gates. Recalling (4.4), the score can be partitioned as ∑i=1nω(Nm,i)=∑j=14∑N∈𝒢jω(N). FAUST selects the number of annotation boundaries for the marker m by choosing the setjwith the maximal sum ∑N∈𝒢jω(N) Letting g1(Nm,j) denote the smallest gate location estimated by the taut string in node Nm,j(which is the only gate location if FAUST selects1), FAUST estimates the phenotypic boundary locations for the marker by taking the weighted average ∑N∈𝒢jω(N)g1(N)∑N∈𝒢jω(N) In the event FAUST selectsj, j>1 (i.e., multiple annotation boundaries), similar weighted averages are taken for g2(Nm,j), etc. FAUST Method: Marker Selection Markers are selected by comparing the user-selected, empirical depth score quantile across experimental units to a user-selected threshold value. All markers whose empirical quantile exceeds the threshold are used for discovery and annotation. FAUST Method: Boundary Standardization FAUST standardizes the number of annotation boundaries for each marker by majority vote. The most frequently occurring number of annotation boundaries across experimental units is chosen as the standard number. This behavior can be over-ridden via the preference list tuning parameter in order to incorporate prior biological information into FAUST. Next, for a given marker, FAUST selects the set of samples where the number of annotation boundaries for that marker matches the standard. Then, by rank, FAUST computes the median absolute deviation of the location of each phenotypic boundary across experimental units. In the following, the median boundary locations are referred to as the standard boundaries. FAUST enforces standardization of annotation boundaries for non-conforming experimental units by imputation or deletion. Imputation in an experimental unit occurs when FAUST estimates fewer boundaries than the standard. In this case, each boundary in the non-conforming unit is matched to one of the standards by distance. Unmatched standards are used to impute the missing boundaries. Similar distance computations are done in the case of deletion, but FAUST deletes boundaries that are farthest from the standards. For both imputation and deletion, if multiple boundaries match the same standard, then the boundary minimizing the distance is kept, and the other boundaries are deleted. Should this result in standards that don't map to any boundaries, then those unmatched standards are used to impute the missing boundaries. FAUST Method: Phenotype Discovery and Cluster Annotation For each experimental unit, FAUST constructs a forest of partition trees (randomly sampled) and annotates selected leaves from this forest relative to the standardized annotation boundaries. Partition tree construction is similar to tree construction for the annotation forest, but they are not depth-constrained: a tree continues to grow following the previously described strategy until each leaf is unimodal according to the dip test (John A Hartigan and P M Hartigan. The Dip Test of Unimodality. The Annals of Statistics, pages 70-84, 1985) or contains fewer than 25 cells. Consequently, a single partition tree defines a clustering of an experimental unit. Clusterings from the forest of partition trees are combined into a single clustering in the following manner. To ensure cells are not assigned to multiple clusters, a subset of leaves of the partition forest are selected by scoring leaves according to shape criteria, and then selecting a subset of leaves across partition trees that share no cells to maximize their total shape score. Only the selected leaves are given phenotypic annotations. FAUST keeps a list of discovered phenotypes for each experimental unit, and concludes by returning exact counts of cells in each sample whose phenotypes exceed a user-specified occurrence frequency threshold. Details of the scoring and selection procedure are described in Greene et al. (Selective clustering annotated using modes of projections. arXiv preprint arXiv:1807.10328, 2018, which is hereby incorporated by reference in its entirety). FAUST Method: Tuning Parameters 1.1.1 Starting Cell Population The name of the population in the manual gating strategy where FAUST conducts discovery and annotation. 1.1.2 Active Markers A list of all markers in the experiment that can possibly be used for discovery and annotation in the starting cell population. FAUST will only compute the depth score for markers in this initial set. 1.1.3 Marker Boundary Matrix A 2×n matrix of lower and upper protein expression bounds. By default, it is set for inf and inf for all markers in a flow experiment. When the manual gating strategy does not remove all debris or doublets from the starting cell population, samples can appear to have clusters of events along at very low or very high expression values for some markers. By setting boundaries for those markers to exclude these doublet or debris clusters, FAUST treats all events below the lower and above the upper bounds as default low or high, respectively. These events are not dropped from the experiment. However, they are ignored when testing for multimodality and subsequent density estimation. In the case of mass cytometry experiments, the default lower boundary is set to 0 for all markers in an experiment in order to accommodate the zero-inflation common to mass cytometry data. The number of events in a marker that fall between the lower and upper marker boundaries in the starting cell population define the effective sample size for that marker. 1.1.4 Depth-Score Selection Quantile The empirical quantile of a marker's depth-score across all experimental units that is used to compare against a user-selected depth-score threshold. By default, this parameter is set to the median. 1.1.5 Depth-Score Selection Threshold A value in [0, 1] used to select a subset of markers to be used in discovery and annotation based on their empirical depth score selection quantile. By default, this parameter is set to 0.01. 1.1.6 Supervised Boundary Estimation List Allows the user to modify FAUST's default gate standardization methodology for each marker. This parameter is one way to incorporate prior (biological) knowledge in the FAUST procedure: if a marker is known to have a certain range of expression, such as low-dim-bright, this can be used to encourage or force FAUST to estimate the corresponding number of annotation boundaries from the data. Similarly, if FMO controls have been collected for a marker, this parameter can be used to set the phenotypic boundary according to the controls. 1.1.7 Phenotype Occurrence Threshold An integer value used to include or exclude discovered phenotypes in the final count matrix returned by FAUST. If a phenotype appears at least Phenotype Occurrence Threshold times across experimental units, it is included in the final counts matrix. By default, all discovered phenotypes are included. Phenotypes exceeding the threshold are assumed to be biological signal while those that fall below it are assumed to be sample- or batch-specific effects. A consequence of this assumption is that all cells in a sample associated with any phenotype falling below the threshold are re-annotated with a common non-informative label indicating those phenotypes ought not be analyzed due to their rarity. 1.2 CITN-09 T Cell Panel Analysis FAUST was applied to samples generated in this study. Between one and four samples were collected from 27 patients with stage IV and unresectable stage IIIB Merkel Cell Carcinoma (Nghiem et al., Durable tumor regression and overall survival in patients with advanced merkel cell carcinoma receiving pembrolizumab as first-line therapy. Journal of Clinical Oncology, 37(9):693-702, 2019. PMID: 30726175); Nghiem et al., New England Journal of Medicine, 374(26):2542-2552, 2016. PMID: 27093365) and spanning the course of treatment. All 27 patients had samples collected at baseline (cycle C01, before initiation of anti-PD-1 therapy); 16 at cycle C02 (3 weeks post-treatment of the second cycle of therapy); 22 at cycle C05 (12 weeks post-treatment of the fifth cycle of therapy); and 13 at end of trial (EOT, patient specific). 18 of 27 subjects responded to therapy for an observed response rate of 67%. Each sample was pre-gated to remove debris and identify live lymphocytes. The manual gating strategy is displayed herein: Let ci,kdenote the number of events in FAUST cluster k for sample i. Let nidenote the number of events in the ithsubject's baseline sample. Similar to Nowicka et al., (Cytof workflow: differential discovery in high-throughput high-dimensional cytometry datasets. F1000Research, 6, 2017), it is assumed that ci,k˜Binomial(ni, μi,k). The model is logit−1(μi,k)=β0+β1·Responder+ξi,k, (4.5) where Responder is an indicator variable equal to 1 when the subject exhibits complete or partial response to therapy, and 0 otherwise, and each ξi,k˜N(0, σi,k2) is a subject-level random effect. The R package lme4 was used to fit all GLMMs (Bates et al., Fitting linear mixed-effects models using lme4. arXiv preprint arXiv:1406.5823, 2014). 1.3 CITN-09 Myeloid Panel This data set consisted of 69 samples stained to investigate myeloid cells. An initial screen comparing the ratio of the number of events in the singlet gate to the number of events in the root population led us to remove 14 samples from analysis due to low quality. FAUST was applied to remaining 55 samples which consisted of 16 samples collected at cycle C01, before initiation of anti-PD-1 therapy; 15 at cycle C02; 15 at cycle C05; and 9 at EOT. Discovery and annotation was run at the individual sample level using cells in the “45+” node of the manual gating strategy.FIG.16provides an exemplary gating strategy for CD4− CD3+ CD8+ CD45RA− HLA-DR+ CD28+ PD-1 dim CD25− CD127− CCR7− in two baseline samples from CITN-09. FAUST selected 11 markers: CD33, CD16, CD15, HLA-DR, CD14, CD3, CD11B, CD20, CD19, CD56, CD11C. CITN-07 Phenotyping Panel Analysis FAUST was applied to this data set comprising of a total of 358 longitudinal samples from 35 subjects in two cohorts (Cohort 1: with FLT-3 pre-treatment and Cohort 2: without pre-treatment), with between 4 and 12 samples per subject over four cycles of therapy and at end of trial. Subjects were given FLT-3 ligand seven days prior to the start of the first two of four treatment cycles. FLT-3 ligand was given to promote the expansion of myeloid and dendritic cell compartments in order to investigate whether expansion improved response to therapy. FAUST was configured to perform cell population discovery and annotation per sample in order to account for biological and technical heterogeneity. Debris, dead cells and non-lymphocytes were excluded by pre-gating (FIG.19). Each cell populations discovered by FAUST was tested at the cohort-specific baseline for association with recurrence of disease (14 subjects had disease recur, 18 did not have disease recur). Similar to model (4.5), here the model adjusted for subject-to-subject variability using a random effect. Cohort status, recurrence, and NYESO-1 staining of the tumor by immunohistochemistry (measured as positive, negative, or undetermined) were modeled as population effects. 1.4 Analysis of CyTOF Data Published by Krieg et al. FAUST was used to discover and annotate cell populations in the mass cytometry data sets stained to investigate myeloid cells as reported by Krieg et al. (High-dimensional single-cell analysis predicts response to anti-pd-1 immunotherapy. Nature medicine, 24(2):144, 2018). Following Krieg et al., samples with fewer than 50 cells were removed from the analysis. To account for batch effects and small sample sizes, all samples within a batch were concatenated and processed by FAUST. FAUST selected 11 markers for discovery and annotation: CD16, CD14, CD11b, CD11c, CD33, ICAM1, CD62L, PD-L1, CD7, CD56, and HLA-DR. The baseline model was similar to (4.5), but was modified by logit−1(μi,k)β0+β1·Responder+ξi,k+ηi,j, where j∈{1, 2}, and ηi,j˜N(0, σj2) is a random effect included to model the batch effects. 1.5 Analysis of FACS Data Published by Krieg et al. FAUST processed 31 flow cytometry samples from responders and non-responders to therapy. FAUST was run at the individual sample level on live cells from the manual gating strategy used by Krieg et al. QC and review of the manual gating strategy let one make manual adjustments to the “Lymphocytes” gate of 7 samples in this data set. An example of this gate adjustment is shown in which FAUST selected 9 markers for discovery and annotation: CD3, CD4, HLA-DR, CD19, CD14, CD11b, CD56, CD16, and CD45RO. The statistical model used here is identical to (4.5), with ci,know denoting the 40 clusters in the FACS data, and nirefers to the baseline FACS sample counts.FIGS.15A and15Bprovide an example of modification to the manual gating strategy of the Krieg et al. FACS data. 1.6 Compartment Multivariate Analysis All FAUST clusters annotated as CD3−, CD56−, and CD19− and included in the univariate analysis were included in the multivariate analysis. Within this set, sub-populations annotated as HLA-DR− were further excluded. This defined the Myeloid compartment for CITN-07, CITN-09, and the Krieg et al. FACS data. Let k* denote the number of FAUST clusters within a given study. Let n denote the number of subjects at baseline, and N=n·k*. For 1≤i≤N, 1≤j≤k*the statistical model is logit−1(μi,j)=β0+βR·Responderi+Σj=1k*1(βc,j·Clusteri,j+βi,j·Clusteri,j·Responderi)+ηi, (4.6) where Clusteri,jis an indicator variable that is 1 when observation i is from cluster j and 0 otherwise, Responderiis an indicator variable when observation i is taken from a responding subject, and ηi˜N(0, σi2)iis an observation-level random effect. To test for differential abundance across a compartment, linear combination of the coefficients βi,jin (4.6) are tested for positivity. For example, the test for differential abundance across an entire compartment is specified H0:βR+1k*·∑j=1k*βi,j≤0,H1:βR+1k*·∑j=1k*βi,j>0. 1.7 Compartment Aggregate Analysis For the aggregate analysis, compartment definitions are the same as presented in section 4.15. Counts are derived by summing across FAUST clusters within each compartment. The model (4.5) is then used to test each derived compartment for differential abundance. A1 Effect Sizes and Confidence Intervals in CITN-09PopulationEffect SizeLower 2.5%Upper 97.5%CD4− CD3+ CD8+ CD45RA− HLA-DR+1.8440.7842.955CD28+ PD-1+ CD25− CD127− CCR7−CD4− CD3+ CD8+ CD45RA− HLA-DR+1.8960.8982.981CD28+ PD-1 dim CD25− CD127− CCR7−CD4 bright CD3+ CD8− CD45RA− HLA-DR−1.9070.9292.941CD28+ PD-1 dim CD25− CD127− CCR7−CD4 bright CD3+ CD8− CD45RA+ HLA-DR−2.9991.3874.837CD28− PD-1 dim CD25− CD127+ CCR7+A2 Alternative analysis of CITN-09 T cell panelMethodNum ClustersTransformationInput DataBest FDRSecond FDRDensityCut2599BiexpBaseline0.091.00DensityCut2570AsinhBaseline0.830.83DensityCut6389BiexpAll0.000.22DensityCut6166AsinhAll0.001.00FlowSOM100AsinhBaseline0.620.62FlowSOM400AsinhBaseline0.530.53FlowSOM100BiexpBaseline0.490.71FlowSOM400BiexpBaseline0.580.58FlowSOM100AsinhAll0.640.64FlowSOM400AsinhAll0.430.43FlowSOM100BiexpAll0.650.65FlowSOM400BiexpAll0.290.29Phenograph46AsinhBaseline0.350.35Phenograph45BiexpBaseline0.360.36Phenograph47AsinhAll0.310.31Phenograph49BiexpAll0.340.34FAUST267BiexpBaseline0.030.03FAUST290AsinhBaseline0.000.02FAUST238BiexpAll0.020.02FAUST239AsinhAll0.000.00Table 3: Results of applying the clustering methods DensityCut, FAUST, FlowSOM, and Phenograph to flow cytometry data stained to investigate T cell activity from the MCC anti-PD-1 trial.Tuning parameters for FlowSOM and Phenograph (including the number of clusters for FlowSOM) were set.The supporting table “cytoa23030-sup-0001-suppinfo.xlsx” reports that Phenograph is run with k = 30 neighbors and using the Euclidean metric.“FlowSOM pre” is reported as running with 100 and 400 clusters with “transform = FALSE” and “scale = FALSE”.Flow cytometry data are reported as being transformed by the hyperbolic arcsine transformation with cofactor 120.Data is transformed using both the biexponential transformation (used by CITN on the “Biexp” rows) as well as the hyperbolic arcsine with cofactor 120 (The “Asinh” rows).DensityCut was run totally unsupervised: the K parameter is set to its default value 1og2(N).Samples were concatenated before analysis by each method except FAUST, which was run at the sample level for all analyses.Rows with “Input data” listing “Baseline” only combine patient samples from the baseline time point, while that list All″ have samples from all time points combined prior to analysis.The reported FAUST number of clusters is the number of clusters with “CD3+” annotations.The tuning parameters for FAUST in the “Asinh” runs and the baseline “Biexp” run were taken from the FAUST “Biexp” all run, which is reported in the paper.The channel bounds matrix was transformed to the “Asinh” runs by computing the empirical quantiles of the concatenated biexponentially transformed data corresponding to the bounds reported herein, and then computing those quantiles on the transformed concatenated data.Similarly, the baseline phenotypic filtering threshold was scaled from the setting of 5 for the 78 all sample runs to the setting of 2 for the 27 baseline sample runs. A3 Staining Panels Used in FAUST Analyses Staining panels from the experiments used in FAUST analyses are provided herein, including described below. Additionally,FIGS.17-19provide visualization of the manual gating used in initial analysis of CITN-09 T cell staining panel, CITN-09 Myeloid staining panel and CITN-07 phenotyping staining panel. A.3.1 CITN-09 T Cell Staining Panel namedesc$P1FSC-A$P2FSC-H$P3SSC-A$P4SSC-H$P5<PE-A>CD278 ICOS$P6<FITC-A>CD3$P7<BV 421-A>CD127$P8<Alexa Fluor 700-A>CD197 CCR7$P9<PE-Cy7-A>CD279 PD-1$P10<PerCP-Cy5-5-A>CD8$P11<APC-Cy7-A>CD4$P12<ECD-A>CD28$P13<APC-A>CD25$P14PE-Cy5-A$P15<AmCyan-A>CD45$P16<BV 605-A>HLA DR$P17<BV 650-A>CD45RA$P18Time A.3.2 CITN-09 Myeloid Staining Panel namedesc$P1FSC-A$P2FSC-H$P3SSC-A$P4SSC-H$P5<PE-A>CD11B$P6<FITC-A>CD20$P7<BV 421-A>CD14$P8<Alexa Fluor 700-A>CD11C$P9<PE-Cy7-A>CD56$P10<PerCP-Cy5-5-A>CD33$P11<APC-Cy7-A>CD16$P12<ECD-A>CD3$P13<APC-A>CD15$P14<PE-Cy5-A>CD19$P15<AmCyan-A>CD45$P16<BV605-A>HLA DR$P17BV 650-A$P18Time CITN-07 Phenotyping Staining Panel namedesc$P1FSC-A$P2FSC-H$P3SSC-A$P4SSC-H$P5<PE-A>CD123$P6<FITC-A>CD4$P7<BV 421-A>CD14$P8<Alexa Fluor 700-A>CD11C$P9<PE-Cy7-A>CD56$P10<PerCP-Cy5-5-A>CD8$P11<APC-Cy7-A>CD16$P12<ECD-A>CD3$P13<APC-A>CD122$P14<PE-Cy5-A>CD19$P15<AmCyan-A>CD45$P16<BV 605-A>HLA DR$P17BV 650-A$P18Time Myeloid CyTOF Panel namedesc$P1Bi209Di209Bi_CD11b$P2Dy162Di162Dy_CD11c$P3Dy163Di163Dy_CD7$P4Er166Di166Er_CD209$P5Er167Di167Er_CD38$P6Eu151Di151Eu_CD123$P7Eu153Di153Eu_CD62L$P8Gd152Di152Gd_CD66b$P9Gd154Di154Gd_ICAM-1$P10Gd155Di155Gd_CD1c$P11Gd156Di156Gd_CD86$P12Gd160Di160Gd_CD14$P13Ho165Di165Ho_CD16$P16Lu175Di175Lu_PD-L1$P18Nd146Di146Nd_CD64$P22Sm147Di147Sm_CD303$P23Sm148Di148Sm_CD34$P24Sm149Di149Sm_CD141$P25Sm150Di150Sm_CD61$P26Tm169Di169Tm_CD33$P27Y89Di89Y_CD45$P29Yb173Di173Yb_CD56$P30Yb174Di174Yb_HLA-DR FACS Panel namedesc$P1FSC-A$P2FSC-H$P3FSC-W$P4SSC-A$P5SSC-H$P6SSC-W$P7Comp-Brilliant Violet 785-ACD3$P8Comp-Brilliant Violet 711-ACD4$P9Comp-Brilliant Violet 421-ACD11b$P10Comp-PerCP-Cy5-5-ACD33$P11Comp-FITC-AHLA-DR$P12Comp-PE-Cy7-ACD56$P13Comp-PE-Texas Red-ACD45RO$P14Comp-APC-Cy7-ANIR$P15Comp-Alexa Fluor 700-ACD11c$P16Comp-APC-ACD16$P17Comp-PE-ACD14$P18Comp-Brilliant Violet 605-ACD19$P19Time Simulation Study A.1.1 Simulation Goals The purpose of this simulation study is to assess the performance of the FAUST algorithm, both as a clustering tool and as a discovery tool, when experimental data are simulated from mixture models following the assumptions disclosed herein. The simulation measures how well FAUST recovers the underlying mixture under a variety of parametric scenarios. The simulation also measures how well FAUST is able to detect a sub-population, elevated in half the samples, that is simulated to have causal relationship with a subject's response to therapy. The performance of FAUST is compared to the performance of the flowSOM clustering algorithm (Gassen et al., Flowsom: Using self-organizing maps for visualization and interpretation of cytometry data. Cytometry Part A, 87(7):636-645, 2015). A.1.2 Baseline Simulation Description The basic simulation generates data from an experiment containing 100 independent samples of 10-dimensional data from a Gaussian mixture model with 10 components. A probability vector p˜Dirichlet(α≡(1,1, . . . ,1)) (A.7) of dimension equal to the number of mixture components is generated. In a given simulation iteration, sampling from the Dirichlet continues until all elements are greater or equal to 0.001. There are four tuning parameters that modify this baseline setting. A complete description of how the simulation study works under default parameters is given, after which a description of how the tuning parameters modify the baseline study is given. In the basic setting, the size of each of the 100 samples is nj=max(5000, s), 1≤j≤100, where s˜T(μ=10000,v=3) is a sample from a non-central T distribution with 3 degrees of freedom and non-centrality parameter 10000. Each sample is meant to represent a sample taken from a subject in an immunology study and then interrogated via flow cytometry. Before generating the samples, a fixed collection of mean vectors ˜c, 1≤c≤10 is determined for the ten Gaussian mixture components that is used across all simulated samples. Each of the ten entries of μcare randomly selected from the columns of Table 4, and represent whether or not the measured variable exhibits a signal. When an entry of μcis from the “No Signal” row of Table 4, the corresponding variable is labeled “−”. Similarly, when an entry of μcis from the “Signal” row of Table 4, the corresponding variable is labeled “+”. In an example, the annotation “V1− V2− V3+ V4− V5+ V6− V7− V8− V9+ V10−” indicates the mean vector μcof the mixture component contains 0 for V1, V2, V4, V6, V7, V8, and V10, while it is 7 for V3, 6 for V5, and 4 for V9. Each mean vector is associated with an element of the probability vector (A.7). Covariance matrices Σcare always constrained to have variances between 1 and 2, but otherwise are randomly generated sample by sample and component by component. TABLE 4Possible mean vector entries for the ten simulation variables.V1V2V3V4V5V6V7V8V9V10No Signal0000000000Signal8877665544 Each simulation iteration, 50 of the 100 samples are randomly selected to have a mixture component elevated. Without loss of generality, suppose (A.7) is in sorted order, so that the first entry p1is the largest value, the tenth entry p10is the smallest value, and intermediate entries correspond to their order statistics. In the non-elevated samples, the mean-vector μcassociated with the smallest element of the probability vector (A.7), p10, is identified as the cluster component to elevate. In the samples randomly selected for elevation, the probability vector (A.7) is modified as follows. The numerical value ptarget≡p7 is fixed. Next, the intermediate probability vector pint≡(p1+p109,p2+p109,…p9+p109,0)≡(q1,q2,…,q9,0)(A.8) is generated. Then (A.8) is modified so that pelevated≡(q1−q1·ptarget,q2−q2·ptarget, . . . ,q9−q9·ptarget,ptarget)≡(r1,r2, . . . ,r9,r10) (A.9). The transformation from (A.7) to (A.9) causes the identified population to be, on average, the 7th largest mixture component in half the samples, and the smallest mixture component in the other half. A sample of size njwith 1≤j≤100 is generated by first determining the relative size of each mixture component within the sample. When the sample is selected as having the elevated population, the size of mixture components is determined by taking a sample from a multinomial distribution with njtrials and cell probabilities determined by (A.9). Otherwise, the size of mixture components is determined by taking a sample from a multinomial distribution with njtrials and cell probabilities determined by (A.7). In both cases, the resulting multinomial vector is then used to sample multivariate Gaussian samples of the corresponding size, with mean vectors μc+ec,jand covariance matrices Σc, for 1≤c≤10. The vector ec,j=(ec,j,1, . . . , ec,j,10) is determined by taking a 10 independent samples,j,k˜N(0, ½), 1≤k≤10, and then rounding ec,j,k=round(,j,k) to the nearest integer. The vector ec,jmodels sample-specific perturbations (corresponding to subject-level effects) without modifying (with high probability) the semantic interpretation of the annotations corresponding to pc. A visualization of the baseline experiment is provided inFIGS.21A-21C. Once the experimental data is generated, it is processed by FAUST in a completely unsupervised setting. FAUST is set to use individual samples as the experimental unit. All simulated variables are taken as admissible and the channel boundaries are set to the entire real line for all markers. The depth score selection threshold is set to 0.01, the depth score selection quantile is set to the median, and the phenotype occurrence number is set to 25. The 100 samples are also concatenated and clustered by the flowSOM algorithm in two different ways. First, the flowSOM grid is set to 1 by the number of mixture components to simulate one best case scenario: an oracle provides flowSOM with the true number of clusters. Second, flowSOM over partitions the data by setting the grid to 5 by 5 (assuming 25 clusters when in truth there are 10). To test how well each of the three methods discover sub-populations associated with differential abundance, a binary response is generated for each sample in the experiment. For samples where the identified population is elevated, a probability of response presponseis varied from 0.50 to 0.80 in increments of 0.05. Each elevated sample is then associated with a response status by sampling from a Bernoulli (presponse). Similarly, samples where the identified population is not elevated are given a probability of response qresponse≡1−presponse. Each non-elevated sample is then associated with a response status by sampling from a Bernoulli(qresponse). Once samples are associated with a binary outcome, the clusters produced by each of the three approaches are tested for differential abundance following the strategy described herein, including in section A equation (4.5). P-values are adjusted for FDR (q-values). In the event FAUST discovers the elevated population by exact annotation, the associate q-value is recorded. For flowSOM, the “best” q-value is defined as follows. Both the cluster containing the largest number of observations from the elevated population in terms of absolute counts, and the cluster containing proportionally the most observation from the elevated population are identified. The minimum q-value from the two clusters (when different) is recorded for both the oracle flowSOM and over partitioned flowSOM clusterings. This modeling procedure is repeated 50 times for each setting of presponse. The median q-values across each of the 50 iterations is recorded in a single simulation iteration. After this, the entire experimental simulation is repeated 50 times, and the median of median q-values across those 50 simulation runs is reported. In addition, F-measures of the clusterings are computed, along with several other measures of the quality of the FAUST clusterings. A.1.3 Simulation Tuning Parameters The first simulation parameter that is variated is the underlying number of mixture components: this parameter is set to 25 components and 50 components, in addition to the baseline of 10. While the sample sizes are random, the underlying sampling scheme is fixed, which introduces rarer and rarer populations appear across simulations as the number of mixture components increase. In both the 25 and 50 component setting, the probability vector (A.7) is expanded accordingly; a continues to be set to 1 for each component. In all cases, sampling from the Dirichlet continues until all elements are greater or equal to 0.001. In the 25-component setting, the elevated population has ptargetset to p18; in the 50 component setting, ptargetis set to p35. The second simulation parameter varied is used to add a batch effect to the simulation. The batch effect is modeled as a translation of the underlying mean vector. Batches are modeled as groups of 10 samples. After the initial 10 samples are generated, the mean vectors of the Gaussian mixture components (sampled from (S4)) are translated by a constant vector λ1=(⅓, ⅓, . . . , ⅓). After the next 10 samples are generated, the translate increases to the constant vector λ2=(⅔, ⅔, . . . , ⅔). This continues in groups of 10 until the final 10 samples are translated by λ9=(9/3, 9/3, . . . , 9/3).FIGS.22A-22Cillustrate an example of a simulated experiment with 50 mixture components and the batch effect parameter turned on. The third simulation parameter controls whether or not nuisance variables are added to the simulation. This parameter is meant to generate data under the scenario that several markers in the panel are uninformative because of staining issues. When this parameter is turned on the following occurs. Each time a sample of size njis generate, an independent sample of size njit taken from a Multivariate Gaussian distribution centered at μnuisance=(5, 5, 5, 5, 5), and Σnuisanceconstrained to have variances between 1 and 2 but otherwise random. The independent Gaussian sample is then adjoined to the mixture of size nj, producing a simulated data set in 15 dimensions. Since nuisance variables are independently generated, they do not affect the mixture structure of a given simulation. Consequently, the underlying annotations of observations by their cluster component mean vector are not changed when the nuisance variables are added to the simulation. The final simulation parameter is used to investigate departures from normality. Two settings are available: after generating each sample, the data are transformed coordinate-by-coordinate through the square map f(x)=x2or the gamma map g(x)=Γ(1+(x/4)). The square map was used to investigate a mild departure from Normality; the gamma map is used to transform the mixture into data that looked similar to CyTOF. Under the gamma map, the space possible Gaussian mean vectors are modified to those determined by Table 5.FIGS.23A-23Cillustrate an example of a simulated experiment with 25 mixture components, both the batch effect parameter and nuisance variable parameters turned on, and data are transformed by the Gamma map. TABLE 5Possible mean vector entries for the ten simulationvariables when data subsequently transformed bythe map g(x) = Γ(1 + |(|x/4)).V1V2V3V4V5V6V7V8V9V10No Signal0000000000Signal8887777666 A.1.1 Simulation Results By adjusting the tuning parameters described in section A.14.3, 36 distinct scenarios are explored in silico. Each simulation setting is run 50 times, with three exceptions which are now reported. The scenario of 25 Clusters with no batch effect but with nuisance variables transformed by the gamma map completed 34 iterations. The scenario of 25 Clusters with batch effect but with no nuisance variables transformed by the gamma map completed 37 iterations. The scenario of 50 Clusters with batch effect and with nuisance untransformed (the identity map) completed 35 iterations. Based on their log files, these three scenarios did not complete 50 iterations in 7 days of compute time due to generating experiments in which the regression modeling took unusually long to fit to each cluster. This simulation study shows that departures from multivariate-normality as well as batch-effects combined with large numbers of clusters impair FlowSOM's ability to define clusters that correlate with outcome. FAUST, on the other hand, performed robustly across simulation settings since its key methodological assumption is that some subset of the measured markers in a cytometry data set are marginally separated into modal groups. Plots of the observed expression data show the MCC anti-PD1 data set has non-Gaussian characteristics, and also has sample-to-sample variation which is common in most cytometry experiments (FIGS.9A-9D). Hence, the non-Gaussian nature of the MCC anti-PD1 trial data combined with sample-to-sample variation both contribute to the discovery differences observed between FlowSOM and FAUST. A.1.2 Additional Simulation Results Additional simulation studies were conducted to compare the FAUST methodology to existing computational approaches. Disclosed herein are the results of two studies. In both, multivariate gaussian data was again simulated in a fashion analogous to that described in section “A.1.2 Baseline simulation description”. Each simulation iteration, 50 samples were simulated independently, with each sample containing 75 clusters specified by the mean vector listed in Table 6, after transformation by the map g(x)=Γ(1+|(|x/4)). In the first study, a fixed probability vector was sampled from the Dirichlet distribution with 75 components. Across simulation iterations, the mass of the 70th component was then incremented prior to generating 25 of the simulated samples. Samples that had the 70th mixture component elevated were called responders; samples with the 70th component unmodified were called non-responders. Different computational discovery methods were then applied to the samples. If a method produced a clustering of the simulated dataset, the resulting clusters were tested for association with responder status using a binomial GLMM. The frequency of the best-associated cluster was then used in logistic regression model to predict responder status and 5-fold cross-validated AUC were computed. For methods that did not produce a ranked clustering of the dataset, a derived best cluster was computed by combining all simulated observations in subsets deemed relevant by the method into a single derived cluster, which was then used in a logistic regression model to predict responder status. FIG.31contains the result of this study and demonstrates that the FAUST methodology is able to detect and use the simulated biomarker to correctly predict responder status when the simulated expected fold change of the biomarker in responders exceeds 1.5. All methods were run with default parameter settings where possible. The methods CITRUS, FlowSOM, k-means, and rclusterpp were provided information about the number of clusters in the experiment. The reported cross-validated AUC for the cytoDx method is based on fitting a predictive model to all 50 simulated samples, and then predicting responder status for those same samples based on the simulated datasets alone. The CITRUS method was also run without providing information about the number of clusters in the experiment—results are reported as dsCitrus (stating for default settings). In the second study, datasets were again generated according to the scheme just described. However, the prevalence of responders in the population was set (in expectation) to 50%, but the strength of the association between samples with the elevated biomarker and responder status was varied. The goal of this study was to investigate the performance of methods when a relevant biomarker did not perfectly predict responder status, a situation which might arise in observed datasets should different pathways exist that produce a positive response to a therapy. Consequently, for each simulated dataset, responder status was sampled 10 times for each tested strength of association. Clusters produced by each method were then tested for association with the simulated response status, and the best associated cluster was used to predict responder status in a logistic regression model. Model coefficients were recorded for 5 simulated datasets, and ultimately used to compute boot-strapped estimates of the log-odds of the association between the observed frequencies in the best cluster and responder status. FIG.32contains the result of this study and demonstrates that, for log-odds larger than 1.7, FAUST is able to detect a statistically significant effect. In all examined simulation settings, no other method tested in this study was able to detect statistically significant associations between the simulated responder status and simulated biomarker, as their 95% boot-strapped confidence interval all include 0. TABLE 6Possible mean vector entries for the ten simulation variables.V1V2V3V4V5V6V7V8V9V10No Signal0000000000Signal8888888888 In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. | 132,951 |
11860165 | DETAILED DESCRIPTION OF THE INVENTION Definitions Disorder: The term ‘disorder’ used herein refers to a disease or medical problem, and is an abnormal condition of an organism that impairs bodily functions, associated with specific symptoms and signs. It may be caused by external factors, such as invading organisms, or it may be caused by internal dysfunctions, such as impaired catecholamine production or transport. In particular, a disorder as used herein is cancer. Glycan: The term glycan as used herein refers to a polysaccharide or oligosaccharide. Glycan may also be used to refer to the carbohydrate portion of a glycoconjugate, such as a glycoprotein, glycolipid, or a proteoglycan. Glycans usually consist solely of O-glycosidic linkages of monosaccharides. For example, cellulose is a glycan (or more specifically a glucan) composed of beta-1,4-linked D-glucose, and chitin is a glycan composed of beta-1,4-linked N-acetyl-D-glucosamine. Glycans can be homo or heteropolymers of monosaccharide residues, and can be linear or branched. Glycans of the invention are listed below. N-Acetylgalactosamine (GalNAc), is an amino sugar derivative of galactose (2-Acetamido-2-deoxy-D-galactose) with the molecular formula C8H15NO6. N-Acetylglucosamine (GlcNAc), is an amino sugar derivative of glucose (2-Acetamido-2-deoxy-D-glucose) with the molecular formula C8H15NO6. Galactosamine is a hexosamine derived from galactose with the molecular formula C6H13NO5. Sialic acid is a generic term for N-acetylneuraminic acid (Neu5Ac or NANA). The amino group can be varied with either an acetyl or glycolyl group but other modifications have been described. The hydroxyl substituents may vary considerably: acetyl, lactyl, methyl, sulfate, and phosphate groups have been found.STn or sialyl-Tn antigen: (Neu5Acα2-6GalNAc-Ser/Thr)Tn or Tn antigen: (GalNAc-Ser/Thr)Core-2: Galβ3(GlcNAcβ6)GalNAc-Ser/Thr)Core-3: GlcNAcβ3GalNAc-Ser/Thr)Core-4: GlcNAcβ3(GlcNAcβ6)GalNAc-Ser/Thr)ST or sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr) Pharmaceutical composition: or drug, medicament or agent refers to any chemical or biological material, compound, or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Some drugs are sold in an inactive form that is converted in vivo into a metabolite with pharmaceutical activity. For purposes of the present invention, the terms “pharmaceutical composition” and “medicament” encompass both the inactive drug and the active metabolite. Polypeptide: The term “polypeptide” as used herein refers to a molecule comprising at least two amino acids. The amino acids may be natural or synthetic. “Oligopeptides” are defined herein as being polypeptides of length not more than 100 amino acids. The term “polypeptide” is also intended to include proteins, i.e. functional biomolecules comprising at least one polypeptide; when comprising at least two polypeptides, these may form complexes, be covalently linked or may be non-covalently linked. The polypeptides in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups. Protein: The term ‘protein’ used herein refers to an organic compound, also known as a polypeptide, which is a peptide having at least, and preferably more than two amino acids. The generic term amino acid comprises both natural and non-natural amino acids any of which may be in the ‘D’ or ‘L’ isomeric form. Methods for Detecting Disease The present inventors have developed a chemo-enzymatic approach to produce libraries of disease-associated O-glycopeptides such as cancer-associated O-glycopeptides printed on a microarray platform allowing high through-put detection of auto-antibodies to the O-glycopeptidome. Based on this the inventors have created an expanded mucin glycopeptide array to identify disease-associated glycopeptide targets. The inventors have for example created an expanded mucin glycopeptide array useful in identifying cancer associated glycopeptide targets being specific for detection of colon cancer specific autologous antibodies. While the invention is useful in the detection of autoantibodies and corresponding glycopeptide epitopes recognised by said autoantibodies, the invention is also applicable to any glycopeptide associated with disease or disorder. The data provides clear support for the utility of this approach and provides guidance for the person skilled in the art to further improve specificity and sensitivity of the targets provided. As a solution to the need for biomarkers for the early detection of disease such as, but not limited to cancer the present inventors have found that specific changes in post-translational modifications, such as glycosylation pattern of the translated proteins are useful in detecting disease. In relation to cancer, these changes also provide recognizable patterns that may be used to discriminate between cancer such as colorectal cancer and inflammatory disease of the gastrointestinal tract. In principle the present invention is applicable to any glycosylated peptide epitope and a corresponding antibody, acting in a lock and key-manner, for the detection of various disorders and diseases. The present inventors have found that the efficiency of detection is particularly good when two or more different glycosylated peptides are used for the detection of disease. In one aspect the present invention concerns a method for detecting a disease in a host organism wherein said disease is characterised in that autoantibodies are produced by the individual suffering from the disease, said method comprising(i) contacting a sample from said host organism with at least two different O-glycosylated peptides, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to the peptides of step (i) are indicative of disease or disorder in the host organism. In one embodiment, the at least two different O-glycosylated peptides are mucin peptides. In one embodiment of the above method, the disease is cancer. In a further embodiment the cancer is selected from the group consisting of colorectal cancer, breast cancer, oral cancer, gastric cancer, esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer, basal cell cancer, squamous cell cancer, malignant melanoma, bladder cancer, endometrial cancer and cervix cancer. The glycopeptide targets in MUC1(Tn-MUC1, STn-MUC1, Core-3-MUC1) found by the inventors, are recognised by auto-antibodies in colorectal cancer patients. In addition the inventors have found epitopes in Tn-MUC4 (GalNAc-al MUC4), which allowed the inventors to distinguish between colorectal cancer patients and healthy individuals and patients with inflammatory bowel disease, respectively. Thus in one embodiment the cancer detected or treated by the present invention is colorectal cancer. The novel concept for the simple and non-invasive identification of patients with e.g. colorectal cancer is a significant advantage over current screening techniques by its use of immunological amplified signals present at early stages of the disease. The strategy can be performed alone or in combination with current screening efforts such a colonoscopy. Conventional endoscopic screening is limited due to lack of molecular specificity and because only anatomical changes are revealed through a macroscopic view of the surface mucosa. Therefore, flat or depressed neoplasms are difficult to detect using endoscopic methods, and especially patients with chronic inflammatory bowel disease are at increased risk of developing malignancy due to undetected dysplastic lesions. There is thus a need for improved methods for detecting early changes in high-risk individuals. New approaches includes the use of novel imaging techniques as well as combining endoscopic efforts with fluorescent imaging with either non-specific or specific dyes (Hsiung, Hardy et al. 2008). Another perspective is to combine these approaches with serum markers, which could serve as indicators for the presence of disease and hence warrant further examination by existing techniques. In this way serum marker strategies could be complementary to anatomical data provided by imaging techniques. In summary, the inventors have provided enabling support for a novel O-glycopeptide array for the sensitive detection of disease-associated auto-antibodies, such as cancer associated auto-antibodies. The data furthermore provides clear support for the utility of this approach and provides guidance for the person skilled in the art to further improve specificity and sensitivity of the targets provided. The invention thus provides a method for screening patients routinely for possible presence of a disease, imaging agents for in situ disclosure and definition of the volume of diseased tissue, and highly specific therapeutic agents to treat the disease. In relation to colorectal cancer, the inventors have found that patients afflicted with colorectal cancer selectively generate antibodies recognizing Tn-MUC1, STn-MUC1 and Tn-MUC4, while auto-antibodies against Core-3-MUC1 were generated in both colorectal cancer and inflammatory bowel patients. Combining the cancer associated glycoforms of MUC1 and MUC4 as target antigens resulted in detection of 82% of the cancer patients with a specificity of 95% (See table II). The inventors have found that the predominant epitope identified by auto-antibodies against STn and Core-3-MUC1 is located in the -GSTAP- motif of the MUC1 tandem repeat sequence carrying two glycans, while a few patients have additional immunoreactivity with glyco-peptides with one glycan in either T in -VTS- or T in -PDTR (SeeFIG.2and Table II). The rather large difference in the auto-antibody levels between patients is noteworthy and indicates important biological variations in auto-antibody production and responses in cancer patients. First, interpersonal variations in the amount of expressed antigen (glycan-MUC1) could vary in colorectal cancer. However, using Tn/STn-MUC1 specific antibodies the inventors found that 92% (23/25) of the examined patients express Tn/STn-MUC1. In contrast only 50% of the patients have STn-MUC1 antibodies. Additional explanations therefore exist for the variations in auto-antibody levels. Among these, the subject's ability to recognise and present STn- and Core3-MUC1 could be important. In order to test if patients with high levels of auto-antibodies corresponded to patients in which peripheral T cell response could be identified, the inventors isolated T-cells from 15 selected colon cancer patients. Furthermore, variance in local stromal factors such as the secretion of TGF beta among other factors are known to down-regulate the immune-response to cancer targets causing immunological escape (Tinder, Subramani et al. 2008). This is a possible explanation to the lack of selected immunological reaction such as anti-STn-MUC1 in some individuals. Finally, many of these patients could be immuno-compromised due to the progression of their disease. In this respect the inventors detected a deterioration of both the Core-3-MUC1 and STn-MUC1 response in later stage cancers with liver metastasis (FIG.3C). This could reflect the immuno-compromised state of the patients or changes in expression of the antigen. Alternatively, the decrease in serum antibodies in late stage cancers is due to chelation of circulating antibodies by large tumour and metastasis mass along with the possibility of immune-complex formation, which would render the antibodies undetectable. In summary, the results show that the method of the present invention is useful in detecting cancer at an early stage. The immunogenic nature of MUC1 provides an explanation for MUC1 glycopeptide responses in cancers other than gastrointestinal cancers, such as breast, ovarian, and prostate cancer. In these patients, however, only few have circulating MUC1 auto-antibodies. In one embodiment the cancer detected or treated by the present invention is colorectal cancer. In one embodiment the at least two different O-glycosylated peptides of the method of the present invention as defined herein above comprises at least 5 consecutive amino acid residues of a mucin selected from the group consisting of MUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variant thereof, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin. In one embodiment the O-glycosylated peptide of the method of the present invention is not a MUC2 peptide. In one embodiment the at least 5 consecutive amino acid residues of a mucin is/are selected from the group consisting of MUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variant thereof, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin are selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ ID NO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), and PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25), or a fragment of said peptides, or variants of said peptides in which variants any amino acid has been changed to a different amino acid, provided that no more than 5 of the amino acid residues in the sequence are so changed. In one embodiment of the method of the present invention as defined herein, the at least two O-glycosylated peptides are a first and a second O-glycosylated mucin peptide wherein the first O-glycosylated mucin peptide is selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn), and the second O-glycosylated mucin peptide is selected from the group consisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In principle the peptides of the invention may be glycosylated by any physiological O-glycan at a potential O-glycosylation site. Thus, in one embodiment the optional glycan is Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr). In another embodiment the optional glycan is Tn (GalNAc-α-Ser/Thr). In yet another embodiment the optional glycan is STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr). In yet another embodiment the optional glycan is Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)). In another embodiment the optional glycan is a Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr). In yet another embodiment the optional glycan is ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In one embodiment of the present invention the at least two different O-glycosylated peptides are at least three different O-glycosylated peptides selected from the group consisting of: VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24), PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr) In one embodiment of the present invention the at least two different O-glycosylated peptides are at least four different O-glycosylated peptides selected from the group consisting of: VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24), PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)), Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In one embodiment of the present invention the at least two different O-glycosylated peptides are five or more different O-glycosylated peptides selected from the group consisting of: VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24), PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)), Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr) In one embodiment the five or more different O-glycosylated peptides are six or more O-glycosylated peptides, such as seven or more O-glycosylated peptides, for example eight or more O-glycosylated peptides, such as nine or more O-glycosylated peptides, for example ten or more O-glycosylated peptides, such as eleven or more O-glycosylated peptides, for example twelve or more O-glycosylated peptides, such as 13 or more O-glycosylated peptides. An object of the present invention is to use the general method defined above for detecting specific disorders characterised in that autoantibodies are produced by the individual suffering from said disease. One such disease is cancer. An individual afflicted with cancer significantly benefits from an early stage diagnosis. Accordingly, in one aspect the present invention relates to a method for detecting cancer, said method comprising(i) contacting a sample with one or more O-glycosylated mucin peptides, wherein said peptide is selected from the group consisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24), PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), or a fragment of said peptides, or variants of said peptides in which variants any amino acid has been changed to a different amino acid, provided that no more than 5 of the amino acid residues in the sequence are so changed, and wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)), Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr) and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. The glycosylated peptides of the present invention preferably comprises at least 5 amino residues. In one aspect the present invention relates to a method for detecting cancer, said method comprising(i) contacting a sample with one or more O-glycosylated mucin peptides, wherein said peptide comprises at least 5, such as at least 8, e.g. at least 10, such as at least 15 consecutive amino acid residues of a mucin selected from the group consisting of MUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), or a fragment or variant thereof, wherein said variant is at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, such as at least 98%, for example at least 99% identical to said at least 5 consecutive amino acid residues of said mucin selected from the group consisting of MUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. The peptides of the invention may be produced either by digesting full length or truncated mucin polypeptides or by automated peptide synthesis. The peptides are then optionally glycosylated. These methods are known to those skilled in the art. In one aspect the present invention relates to a method for detecting cancer, said method comprising(i) contacting a sample with one or more mucin peptides, wherein said peptide is selected from the group consisting of:a) MUC4 Tn selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orb) a MUC4 non glycosylated mucin peptide selected from the group consisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ ID NO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tn glycosylated mucin peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orc) a Tn glycosylated MUC4 peptide selected from the group consisting of PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), ord) an all-Tn MUC4 peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylation site, wherein the glycan is Tn (GalNAc-α-Ser/Thr), ore) a recombinant MUC4 Tn having the sequence PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orf) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptide selected from the group consisting of VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG, VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30),wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), org) a MUC1 STn and a MUC4 selected from the group consisting of VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), and wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. In one embodiment the cancer detected by the method of the present invention is selected from the group consisting of colorectal cancer, breast cancer, oral cancer, gastric cancer, esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer, basal cell cancer, squamous cell cancer, malignant melanoma, bladder cancer, endometrial cancer and cervix cancer. In one embodiment the detected cancer is colorectal cancer. In one aspect the present invention relates to a method for detecting colorectal cancer in a host organism, said method comprising(i) contacting a sample from said host organism with one or more O-glycosylated mucin peptides, wherein said peptide is selected from the group consisting of: PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*H DAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), VT*S*APDT*RPAPGS*T*APPAHG (SEQ ID NO: 24), PT*T*T*PIT*T*T*T*T*VT*PT*PT*PT*GT*QT*PT*T*T*PIS*T*T*C (SEQ ID NO: 25) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), or a fragment of said peptides, or variants of said peptides in which variants any amino acid has been changed to a different amino acid, provided that no more than 5 of the amino acid residues in the sequence are so changed, and wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. As demonstrated in table II of the present application, and as discussed herein above, it may be useful to utilise one or more, such as two different O-glycosylated peptides for detecting colorectal cancer, and for distinguishing between colorectal cancer and inflammatory bowel disease. Thus, in one aspect the present invention relates to a method for detecting colorectal cancer in a sample host, said method comprising(i) contacting a sample from said sample host with one or more mucin peptides, wherein said peptide is selected from the group consisting of:a) MUC4 Tn selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orb) a MUC4 non glycosylated mucin peptide selected from the group consisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ ID NO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tn glycosylated mucin peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orc) a Tn glycosylated MUC4 peptide selected from the group consisting of PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), ord) an all-Tn MUC4 peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*TTS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylation site, wherein the glycan is Tn (GalNAc-α-Ser/Thr), ore) a recombinant MUC4 Tn having the sequence PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orf) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptide selected from the group consisting of VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG, VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30),wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), org) a MUC1 STn and a MUC4 selected from the group consisting of VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), and wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. In a further aspect the invention relates to a method for detecting colorectal cancer in a host organism, said method comprising(i) contacting a sample from said host organism with at least two different O-glycosylated peptides, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to the peptides of step (i) are indicative of disease or disorder in the host organism. In principle, any suitable O-glycosylated peptide can be used for detecting auto-antibodies binding to this glycosylated peptide epitope. It is preferred that the peptide epitope comprises at least 5 amino acid residues. In one aspect the present invention thus relates to a method for detecting colorectal cancer in a host organism, said method comprising(i) contacting a sample from said host organism with one or more O-glycosylated peptides, wherein said peptide comprises at least 5 consecutive amino acid residues of a mucin selected from the group consisting of MUC4 (SEQ ID NO: 1), MUC1 (SEQ ID NO: 2), MUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD, MUC2 (SEQ ID NO: 26), MUC3A, MUC3B, MUC4, MUC5AC (SEQ ID NO: 27), MUC5B, MUC6 (SEQ ID NO: 28), and MUC7 (SEQ ID NO: 29), MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variant thereof, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin, or a naturally occurring fragment or variant of said mucin, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. In one embodiment said O-glycosylated mucin peptide is selected from the group consisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ ID NO: 22) and PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23). In another embodiment said O-glycosylated mucin peptide is a MUC4 selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In another embodiment said O-glycosylated mucin peptide is a MUC4 Tn selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr). In another embodiment said O-glycosylated mucin peptide is a MUC4 non glycosylated or Tn glycosylated mucin selected from the group consisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ ID NO: 22) and PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the glycan is Tn (GalNAc-α-Ser/Thr). In another embodiment said O-glycosylated mucin peptide is a MUC4 fragment Tn selected from the group consisting of PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16), GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is a Tn (GalNAc-α-Ser/Thr). In another embodiment said O-glycosylated mucin peptide is an all-Tn MUC4 peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr). In another embodiment the at least two different O-glycosylated mucin peptides are a first O-glycosylated mucin peptide selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ ID NO: 23) and a second O-glycosylated mucin peptide selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) respectively, wherein said first O-glycosylated mucin peptide is glycosylated on one or more Thr or Ser residues and wherein said glycosylation is(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. In another embodiment, wherein the one or more O-glycosylated mucin peptides and/or the two or more O-glycosylated mucin peptides as defined anywhere in the present application, are a first O-glycosylated mucin peptide and a second O-glycosylated mucin peptide wherein said first O-glycosylated mucin peptide is a MUC1 O-glycosylated mucin peptide and wherein said second O-glycosylated mucin peptide is a MUC4 O-glycosylated mucin peptide. In another embodiment said MUC1 O-glycosylated mucin peptide is a MUC1STn and/or MUC1Tn and/or MUC1Core3 O-glycosylated mucin peptide selected from the group consisting of VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG, VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, and wherein said MUC4 O-glycosylated mucin peptide is selected from the group consisting of PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the glycan is independently and optionally selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In one aspect the present invention relates to a method for detecting colorectal cancer, said method comprising:(i) contacting a sample with one or more O-glycosylated MUC1 peptides and optionally at least one second mucin peptide wherein the O-glycosylated MUC1 peptides are selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and wherein said at least one second mucin peptide optionally is/are selected from:PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. The peptide can be any peptide sharing a significant sequence identity with the peptides specified herein above, or a peptide sharing secondary or tertiary structure with the present peptides. Thus, in a further aspect the present invention relates to a method for detecting colorectal cancer in a host organism, said method comprising(i) contacting a sample from said host organism with one or more O-glycosylated mucin peptides, wherein said peptide comprises, or said peptides comprise, at least 5 consecutive amino acid residues of a mucin, or a fragment or variant thereof, wherein said variant is at least 70% identical, such as at least 75% identical to, e.g. at least 80% identical to, such as at least 85% identical to, e.g. at least 90% identical to, such as at least 95% identical to, e.g. at least 98% identical to, such as at least 99% identical to said at least 5 consecutive amino acid residues of said mucin, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the host organism. All mucin peptides defined herein above may occur as tandem repeats. Thus in one embodiment, the glycopeptide of the present invention is a tandem repeat peptide. In one embodiment the number of repeats is two. In another embodiment the number of repeats is three. In yet another embodiment the number of repeats is four. In yet another embodiment the number of repeats is five or more, such as six, for example seven, such as eight, for example nine, such as ten, for example eleven, such as 12, for example 13, such as 14, for example 15, such as 16, for example 17, such as 18, for example 19, such as 20, for example 21, such as 22, for example 23, such as 24, for example 25, such as 26, for example 27, such as 28, for example 29, such as 30, for example 31, such as 32, for example 33, such as 34, for example 35, such as 36, for example 37, such as 38, for example 39, such as 40, for example 41, such as 42, for example 43, such as 44, for example 45, such as 46, for example 47, such as 48, for example 49, such as 50 or more. The term variant as used herein should be understood as functional equivalent and can be used interchangeably. In one preferred embodiment of the invention there is also provided variants of the mucin glycopeptides and variants or fragments thereof. When being polypeptides, variants are determined on the basis of their degree of identity or their homology with a predetermined amino acid sequence, said predetermined amino acid sequence being one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29 and SEQ ID NO: 30, or, when the variant is a fragment, a fragment of any of the aforementioned amino acid sequences, respectively. Accordingly, variants preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 91% sequence identity, for example at least 91% sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example 99% sequence identity with the predetermined sequence. Sequence identity is determined in one embodiment by utilising fragments of the glycosylated peptides comprising at least 5 contiguous amino acids and having an amino acid sequence which is at least 80%, such as 85%, for example 90%, such as 95%, for example 99% identical to the amino acid sequence of any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29 or SEQ ID NO: 30, respectively, wherein the percent identity is determined with the algorithm GAP, BESTFIT, or FASTA in the Wisconsin Genetics Software Package Release 7.0, using default gap weights. Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected. The term “sequence identity” means that two peptide or polypeptide sequences are identical (i.e., on an amino acid to amino acid basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid (e.g., A, G, T, S etc.) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denotes a characteristic of a peptide or polypeptide sequence, wherein the peptide or polypeptide sequence comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a predetermined sequence over a comparison window of at least 20 amino acid positions, wherein the percentage of sequence identity is calculated by comparing the predetermined sequence to the peptide or polypeptide sequence which may include deletions or additions which total 20 percent or less of the predetermined sequence over the window of comparison. The predetermined sequence may be a subset of a larger sequence, for example, as a segment of the full-length mucin polypeptide sequences illustrated herein. Furthermore, a degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of homology or similarity of amino acid sequences is a function of the number of amino acids, i.e. structurally related, at positions shared by the amino acid sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, though preferably less than 25% identity, with one of the peptide or polypeptide sequences of the present invention. The term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine, a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Additionally, variants are also determined based on a predetermined number of conservative amino acid substitutions as defined herein below. Conservative amino acid substitution as used herein relates to the substitution of one amino acid (within a predetermined group of amino acids) for another amino acid (within the same group), wherein the amino acids exhibit similar or substantially similar characteristics. Within the meaning of the term “conservative amino acid substitution” as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below:i) Amino acids having polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gln, Ser, Thr, Tyr, and Cys,)ii) Amino acids having non-polar side chains (Gly, Ala, Val, Leu, Ile, Phe, Trp, Pro, and Met)iii) Amino acids having aliphatic side chains (Gly, Ala Val, Leu, Ile)iv) Amino acids having cyclic side chains (Phe, Tyr, Trp, His, Pro)v) Amino acids having aromatic side chains (Phe, Tyr, Trp)vi) Amino acids having acidic side chains (Asp, Glu)vii) Amino acids having basic side chains (Lys, Arg, His)viii) Amino acids having amide side chains (Asn, Gln)ix) Amino acids having hydroxy side chains (Ser, Thr)x) Amino acids having sulphur-containing side chains (Cys, Met),xi) Neutral, weakly hydrophobic amino acids (Pro, Ala, Gly, Ser, Thr)xii) Hydrophilic, acidic amino acids (Gln, Asn, Glu, Asp), andxiii) Hydrophobic amino acids (Leu, Ile, Val) Accordingly, a variant or a fragment thereof according to the invention may comprise, within the same variant of the sequence or fragments thereof, or among different variants of the sequence or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another. It is clear from the above outline that the same variant or fragment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above. The addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as additions from 50 to 100 amino acids, addition of 100 to 150 amino acids, addition of 150-250 amino acids, are also comprised within the present invention. The deletion and/or the addition may—independently of one another—be a deletion and/or an addition within a sequence and/or at the end of a sequence. The polypeptide fragments according to the present invention, including any functional equivalents thereof, may in one embodiment comprise less than 250 amino acid residues, such as less than 240 amino acid residues, for example less than 225 amino acid residues, such as less than 200 amino acid residues, for example less than 180 amino acid residues, such as less than 160 amino acid residues, for example less than 150 amino acid residues, such as less than 140 amino acid residues, for example less than 130 amino acid residues, such as less than 120 amino acid residues, for example less than 110 amino acid residues, such as less than 100 amino acid residues, for example less than 90 amino acid residues, such as less than 85 amino acid residues, for example less than 80 amino acid residues, such as less than 75 amino acid residues, for example less than 70 amino acid residues, such as less than 65 amino acid residues, for example less than 60 amino acid residues, such as less than 55 amino acid residues, for example less than 50 amino acid residues, such as less than amino acid residues, for example less than 40 amino acid residues, such as less than 35 amino acid residues, for example less than 30 amino acid residues, such as less than 25 amino acid residues, for example less than 20 amino acid residues, such as less than 15 amino acid residues, for example less than 12 amino acid residues, such as less than 10 amino acid residues, for example less than 8 amino acid residues, such as less than 7 amino acid residues, for example less than 6 amino acid residues, such as less than 5 amino acid residues, for example less than 4 amino acid residues, such as less than 3 amino acid residues. “Functional equivalency” as used in the present invention is according to one preferred embodiment established by means of reference to the corresponding functionality of a predetermined fragment of the sequence. Functional equivalents or variants of a glycosylated mucin peptide will be understood to exhibit amino acid sequences gradually differing from the preferred predetermined sequence, as the number and scope of insertions, deletions and substitutions including conservative substitutions increases. This difference is measured as a reduction in homology between the preferred predetermined sequence and the fragment or functional equivalent. All fragments or functional equivalents of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29 and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69 are included within the scope of this invention, regardless of the degree of homology that they show to the respective, predetermined peptide sequences disclosed herein. The reason for this is that some regions of the peptide are most likely readily mutatable, or capable of being completely deleted, without any significant effect on the binding activity of the resulting fragment. The homology between amino acid sequences may be calculated using well known scoring matrices such as any one of BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, and BLOSUM 90. Fragments sharing homology with fragments of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29 and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68 and SEQ ID NO:69, respectively, are to be considered as falling within the scope of the present invention when they are preferably at least about 90 percent homologous, for example at least 92 percent homologous, such as at least 94 percent homologous, for example at least 95 percent homologous, such as at least 96 percent homologous, for example at least 97 percent homologous, such as at least 98 percent homologous, for example at least 99 percent homologous with said predetermined fragment sequences, respectively. According to one embodiment of the invention the homology percentages refer to identity percentages. Additional factors that may be taken into consideration when determining functional equivalence according to the meaning used herein are i) the ability of antisera to detect a peptide fragment according to the present invention, or ii) the ability of the functionally equivalent peptide fragment to compete with the corresponding peptide in an assay, such as an inhibition assay. One method of determining a sequence of immunogenically active amino acids within a known amino acid sequence has been described by Geysen in U.S. Pat. No. 5,595,915 and is incorporated herein by reference. A further suitably adaptable method for determining structure and function relationships of peptide fragments is described by U.S. Pat. No. 6,013,478, which is herein incorporated by reference. Also, methods of assaying the binding of an amino acid sequence to a receptor moiety are known to the skilled artisan. In addition to conservative substitutions introduced into any position of a preferred predetermined sequence, or a fragment thereof, it may also be desirable to introduce non-conservative substitutions in any one or more positions of the glycopeptides of the invention. A non-conservative substitution leading to the formation of a functionally equivalent peptide fragment would for example i) differ substantially in polarity, for example a residue with a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met) substituted for a residue with a polar side chain such as Gly, Ser, Thr, Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, or Lys, or substituting a charged or a polar residue for a non-polar one; and/or ii) differ substantially in its effect on polypeptide backbone orientation such as substitution of or for Pro or Gly by another residue; and/or iii) differ substantially in electric charge, for example substitution of a negatively charged residue such as Glu or Asp for a positively charged residue such as Lys, His or Arg (and vice versa); and/or iv) differ substantially in steric bulk, for example substitution of a bulky residue such as His, Trp, Phe or Tyr for one having a minor side chain, e.g. Ala, Gly or Ser (and vice versa). Variants obtained by substitution of amino acids may in one preferred embodiment be made based upon the hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. In addition to the variants described herein, sterically similar variants may be formulated to mimic the key portions of the variant structure and that such compounds may also be used in the same manner as the variants of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention. In a further embodiment the present invention relates to functional variants comprising substituted amino acids having hydrophilic values or hydropathic indices that are within +/−4.9, for example within +/−4.7, such as within +/−4.5, for example within +/−4.3, such as within +/−4.1, for example within +/−3.9, such as within +/−3.7, for example within +/−3.5, such as within +/−3.3, for example within +/−3.1, such as within +/−2.9, for example within +/−2.7, such as within +/−2.5, for example within +/−2.3, such as within +/−2.1, for example within +/−2.0, such as within +/−1.8, for example within +/−1.6, such as within +/−1.5, for example within +/−1.4, such as within +/−1.3 for example within +/−1.2, such as within +/−1.1, for example within +/−1.0, such as within +/−0.9, for example within +/−0.8, such as within +/−0.7, for example within +/−0.6, such as within +/−0.5, for example within +/−0.4, such as within +/−0.3, for example within +/−0.25, such as within +/−0.2 of the value of the amino acid it has substituted. The importance of the hydrophilic and hydropathic amino acid indices in conferring interactive biologic function on a protein is well understood in the art (Kyte & Doolittle, 1982 and Hopp, U.S. Pat. No. 4,554,101, each incorporated herein by reference). The amino acid hydropathic index values as used herein are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5) (Kyte & Doolittle, 1982). The amino acid hydrophilicity values are: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1); glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4) (U.S. Pat. No. 4,554,101). In addition to the peptidyl compounds described herein, sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention. Peptides with N-terminal alkylations and C-terminal esterifications are also encompassed within the present invention. Functional equivalents also comprise glycosylated and covalent or aggregative conjugates formed with the same or other peptide fragments, including dimers or unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art. Functional equivalents may thus comprise fragments conjugated to aliphatic or acyl esters or amides of the carboxyl terminus, alkylamines or residues containing carboxyl side chains, e.g., conjugates to alkylamines at aspartic acid residues; O-acyl derivatives of hydroxyl group-containing residues and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g. conjugates with fMet-Leu-Phe or immunogenic proteins. Derivatives of the acyl groups are selected from the group of alkyl-moieties (including C3 to C10 normal alkyl), thereby forming alkanoyl species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species. The reactive groups preferably are difunctional compounds known per se for use in cross-linking proteins to insoluble matrices through reactive side groups. Covalent or aggregative functional equivalents and derivatives thereof are useful as reagents in immunoassays or for affinity purification procedures. For example, a fragment of a mucin of the invention may be made insoluble by covalent bonding to cyanogen bromide-activated Sepharose by methods known per se or adsorbed to polyolefin surfaces, either with or without glutaraldehyde cross-linking, for use in an assay or purification of anti-mucin antibodies or cell surface receptors. Fragments may also be labelled with a detectable group, e.g., radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in e.g. diagnostic assays. Mutagenesis of a preferred predetermined fragment of a mucin epitope of the invention can be conducted by making amino acid insertions, usually on the order of about from 1 to 10 amino acid residues, preferably from about 1 to 5 amino acid residues, or deletions of from about from 1 to 10 residues, such as from about 2 to 5 residues. In one embodiment the fragment of the mucin epitope is synthesised by automated synthesis. Any of the commercially available solid-phase techniques may be employed, such as the Merrifield solid phase synthesis method, in which amino acids are sequentially added to a growing amino acid chain. (See Merrifield, J. Am. Chem. Soc. 85:2149-2146, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied Biosystems, Inc. of Foster City, Calif., and may generally be operated according to the manufacturer's instructions. Solid phase synthesis will enable the incorporation of desirable amino acid substitutions into any fragment according to the present invention. It will be understood that substitutions, deletions, insertions or any subcombination thereof may be combined to arrive at a final sequence of a functional equivalent. Insertions shall be understood to include amino-terminal and/or carboxyl-terminal fusions, e.g. with a hydrophobic or immunogenic protein or a carrier such as any polypeptide or scaffold structure capable as serving as a carrier. Oligomers including dimers including homodimers and heterodimers of fragments of mucin glycopeptides according to the invention are also provided and fall under the scope of the invention. Mucin peptide equivalents and variants can be produced as homodimers or heterodimers with other amino acid sequences or with native mucin sequences. Heterodimers include dimers containing immunoreactive mucin fragments as well as mucin fragments that need not have or exert any biological activity. Mucin glycopeptide fragments according to the invention may be synthesised both in vitro and in vivo. Method for in vitro synthesis are well known, and methods being suitable or suitably adaptable to the synthesis in vivo are also described in the prior art. The GalNAc moiety of the glycans of the invention can be included in the peptide synthesis by the inclusion of GalNAc-S/T building blocks. When synthesized in vivo, a host cell is transformed with vectors containing DNA encoding mucin fragments or a fragment thereof. A vector is defined as a replicable nucleic acid construct. Vectors are used to mediate expression of mucin fragments. An expression vector is a replicable DNA construct in which a nucleic acid sequence encoding the predetermined mucin fragment, or any functional equivalent thereof that can be expressed in vivo, is operably linked to suitable control sequences capable of effecting the expression of the fragment or equivalent in a suitable host. Such control sequences are well known in the art. Cultures of cells derived from multicellular organisms represent preferred host cells. In principle, any higher eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture. Examples of useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI38, BHK, COS-7, 293 and MDCK cell lines. Preferred host cells are eukaryotic cells known to synthesize endogenous mucins. Cultures of such host cells may be isolated and used as a source of the fragment, or used in therapeutic methods of treatment, including therapeutic methods aimed at promoting or inhibiting a growth state, or diagnostic methods carried out on the human or animal body. Glycosylation Glycosylation is the enzymatic process that links saccharides to produce glycans, attached to proteins, lipids, or other organic molecules. This enzymatic process produces one of the fundamental biopolymers found in cells (along with DNA, RNA, and proteins). Glycosylation is a form of co-translational and post-translational modification. Glycans serve a variety of structural and functional roles in membrane and secreted proteins. The majority of proteins synthesised in the rough ER undergo glycosylation. It is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of glycation. Glycosylation also occurs in the cytoplasm and nucleus, for example the O-GlcNAc modification. Five classes of glycan modification of peptides can be produced: N-linked glycans attached to a nitrogen of asparagine or arginine side chains, O-linked glycans attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains, or to oxygens on lipids such as ceramide; phospho-glycans linked through the phosphate of a phospho-serine; C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side chain, and glypiation which is the addition of a GPI anchor which links proteins to lipids through glycan linkages. The glycosylation of main interest in the present invention is O-linked glycosylation. This type of glycosylation occurs at a late stage during protein processing, in the Golgi apparatus. This is the addition of N-acetyl-galactosamine to serine or threonine residues by the enzyme UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41), followed by other carbohydrates (such as galactose and sialic acid). This process is important for certain types of proteins such as proteoglycans, which involves the addition of glycosaminoglycan chains to an initially unglycosylated “proteoglycan core protein.” These additions are usually serine O-linked glycoproteins, which seem to have one of two main functions. One function involves secretion to form components of the extracellular matrix, adhering one cell to another by interactions between the large sugar complexes of proteoglycans. The other main function is to act as a component of mucosal secretions, and it is the high concentration of carbohydrates that tends to give mucus its consistency. In addition, O-linked glycans are involved in directing cleavage of membrane proteins, intracellular sorting, secretion, protease resistance and in intracellular signalling. In one embodiment of the present invention, at least one amino acid residue, such as at least two amino acid residues, for example at least three amino acid residues, such as at least at least four amino acid residues, for example at least five amino acid residues of the mucin peptides defined herein above is/are glycosylated by an O-linked glycan selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). Each Serine (S) or Threonine (T) residue of the polypeptides selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ ID NO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25) and PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNGHTTQAPTTALQ AAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMT HTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETT RFSSNPSRDSHTT (SEQ ID NO: 30) may be individually and optionally glycosylated by one of the glycans selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). Thus, in one embodiment of the present invention, one or more amino acid residues of the mucin peptide is/are Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr) glycosylated. In another embodiment of the present invention, one or more amino acid residues of the mucin peptide is/are Tn (GalNAc-α-Ser/Thr) glycosylated. In another embodiment of the present invention, one or more amino acid residues of the mucin peptide is/are STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr) glycosylated. In another embodiment of the present invention, one or more amino acid residues of the mucin peptide is/are Core-2 ((Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) glycosylated. In yet another embodiment of the present invention, one or more amino acid residues of the mucin peptide is/are Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr) glycosylated. In yet another embodiment of the present invention, one or more amino acid residues of the mucin peptide is/are ST/sialyl-T (Neu5Acα2-3Galβ1GalNAc-Ser/Thr) glycosylated. In one embodiment, the peptide used in the methods for detecting cancer defined herein above, is selected from the group consisting of: VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and PVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn) T(Tn)GHAT(Tn). In one embodiment a glycopeptide selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and PVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn) T(Tn)GHAT(Tn) is used in a method of detecting IgG autoantibodies in a sample from a subject, wherein the presence of said autoantibodies to any of said glycopeptides indicates colorectal cancer. In one embodiment, the peptide of the present invention is selected from the group consisting of: VTSAPDT(Core3)RPAPGSTAPPAHG, VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG, VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG, VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG, VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG, VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and PVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn) T(Tn)GHAT(Tn), wherein Core-3 is GlcNAcβ1-3GalNAc-α-Ser/Thr, and wherein Tn is GalNAc-α-Ser/Thr, and wherein STn/sialyl-Tn is Neu5Acα2-6GalNAc-α-Ser/Thr, and wherein Core-2 is Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr and wherein Core-4 is GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr, and wherein the glycosylation is on the serine (S) or Threonine (T) amino acid residue preceding the glycan denotation. In one embodiment a glycopeptide selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG, VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG, VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG, VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG, VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG, VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) and PVT(Tn)YAS(Tn)S(Tn)AS(Tn)T(Tn)GDT(Tn)T(Tn)PLPVT(Tn)DT(Tn)S(Tn)S(Tn)VS(Tn) T(Tn)GHAT(Tn), is used in a method of detecting IgA autoantibodies in a sample from a subject, wherein the presence of said autoantibodies to any of said glycopeptides indicates colorectal cancer. Core-3 is an abbreviation for GlcNAβ1-3GalNAc-α-Ser/Thr, and Tn is an abbreviation for GalNAc-α-Ser/Thr. STn or sialyl-Tn is an abbreviation for Neu5Acα2-6GalNAc-α-Ser/Thr. Core-2 is an abbreviation for Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr. Core-4 is an abbreviation for GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr. Glycosylation occurs on the serine (S) or Threonine (T) amino acid residue preceding the glycan denotation as defined herein above. In one embodiment, the peptide of the invention is selected from the group consisting of PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (MUC4-TR Tn) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (MUC4 Tn), wherein the asterisk (*) indicates a potential O-glycosylation site. Accordingly, each asterisk signifies that the amino acid residue preceding said asterisk can be glycosylated by an O-linked glycan selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). Antibodies Other aspects of the present invention are antibodies prepared using the glycopeptides defined herein above, methods for preparation of said antibodies and use of said antibodies in therapy and diagnosis. Also part of the invention are antibodies capable of recognising the IO-glycosylated peptides of the present invention, wherein said antibodies are produced by state-of-the art recombinant methods. An antibody binds tightly to a particular target molecule, thereby either inactivating it directly or marking it for destruction. The antibody recognizes its target (antigen) with remarkable specificity and strength dictated by the sum of many chemical forces, including hydrogen bonds, hydrophobic and van der Waal's forces, as well as ionic interactions. In general, the more complex the target is chemically, the more immunogenic it will be. The antigenic determinant may encompass short linear amino acid stretches or a more complicated, three-dimensional protein module. Conceptually, antibodies directed against a target receptor may inhibit ligand binding in two ways: competitive or allosteric. Competitive inhibition involves the direct binding of the antibody to or near the ligand binding site on the receptor, thereby displacing the ligand from its receptor or sterically inhibiting the approach of the ligand to the ligand binding site. Allosteric inhibition involves the binding of the antibody to a site on the receptor polypeptide that is distinct from the ligand binding epitope. However, binding to this site will induce a conformational change in the overall structure of the receptor that makes it more difficult or even impossible for the ligand to bind to its cognate recognition site. The antibody or functional equivalent thereof may be any antibody known in the art, for example a polyclonal or a monoclonal antibody derived from a mammal or a synthetic antibody, such as a single chain antibody or hybrids comprising antibody fragments. Furthermore, the antibody may be mixtures of monoclonal antibodies or artificial polyclonal antibodies. In addition functional equivalents of antibodies may be antibody fragments, in particular epitope binding fragments. Furthermore, antibodies or functional equivalent thereof may be a small molecule mimicking an antibody. Naturally occurring antibodies are immunoglobulin molecules consisting of heavy and light chains. In preferred embodiments of the invention, the antibody is a monoclonal antibody. Monoclonal antibodies (Mab's) are antibodies, wherein every antibody molecule are similar and thus recognises the same epitope. Monoclonal antibodies are in general produced by a hybridoma cell line. Methods of making monoclonal antibodies and antibody-synthesizing hybridoma cells are well known to those skilled in the art. Antibody producing hybridomas may for example be prepared by fusion of an antibody producing B lymphocyte with an immortalized B-lymphocyte cell line. Monoclonal antibodies according to the present invention may for example be prepared as described in Antibodies: A Laboratory Manual, By Ed Harlow and David Lane,Cold Spring Harbor Laboratory Press,1988. Said monoclonal antibodies may be derived from any suitable mammalian species, however frequently the monoclonal antibodies will be rodent antibodies for example murine or rat monoclonal antibodies. It is preferred that the antibodies according to the present invention are monoclonal antibodies or derived from monoclonal antibodies. Polyclonal antibodies is a mixture of antibody molecules recognising a specific given antigen, hence polyclonal antibodies may recognise different epitopes within said antigen. In general polyclonal antibodies are purified from serum of a mammal, which previously has been immunized with the antigen. Polyclonal antibodies may for example be prepared by any of the methods described in Antibodies: A Laboratory Manual, By Ed Harlow and David Lane,Cold Spring Harbor Laboratory Press,1988. Polyclonal antibodies may be derived from any suitable mammalian species, for example from mice, rats, rabbits, donkeys, goats, sheeps, cows or camels. The antibody is preferably not derived from a non-mammalian species, i.e. the antibody is for example preferably not a chicken antibody. The antibody may also for example be an artificial polyclonal antibody as for example described in U.S. Pat. No. 5,789,208 or U.S. Pat. No. 6,335,163, both patent specifications are hereby incorporated by reference into the application in their entirety. The antibodies according to the present invention may also be recombinant antibodies. Recombinant antibodies are antibodies or fragments thereof or functional equivalents thereof produced using recombinant technology. For example recombinant antibodies may be produced using a synthetic library or by phage display. Recombinant antibodies may be produced according to any conventional method for example the methods outlined in “Recombinant Antibodies”, Frank Breitling, Stefan Dübel, Jossey-Bass, September 1999. The antibodies according to the present invention may also be bispecific antibodies, i.e. antibodies specifically recognising two different epitopes. Bispecific antibodies may in general be prepared starting from monoclonal antibodies, or from recombinant antibodies, for example by fusing two hybridoma's in order to combine their specificity, by Chemical crosslinking or using recombinant technologies. Antibodies according to the present invention may also be tri-specific antibodies. Functional equivalents of antibodies may in one preferred embodiment be a fragment of an antibody, preferably an antigen binding fragment or a variable region. Examples of antibody fragments useful with the present invention include Fab, Fab′, F(ab′)2and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual “Fc” fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab′)2fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc′). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. As used herein, “functional fragment” with respect to antibodies, refers to Fv, F(ab) and F(ab′)2fragments. Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor. Some preferred fragments are defined as follows:(1) Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.(2) Fab′ is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab′ fragments are obtained per antibody molecule. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.(3) (Fab′)2is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. F(ab′)2is a dimer of two Fab′ fragments held together by two disulfide bonds.(4) Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VLdimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VLdimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. In one embodiment of the present invention the antibody is a single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as “single-chain Fv” or “scFv” antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. In another embodiment of the present invention the functional equivalent of an antibody is a small molecule mimicking an antibody. Such molecules may be a non-immunoglobulin binding members. Thus the epitope polypeptide of the present invention binding may be derived from a naturally occurring protein or polypeptide; said protein or polypeptide may for example be designed de novo, or may be selected from a library. The binding member may be a single moiety, e.g., a polypeptide or protein domain, or it may include two or more moieties, e.g., a pair of polypeptides such as a pair polypeptides. The binding polypeptide may for example, but not exclusively, be a lipocalin, a single chain MHC molecule, an Anticalin™ (Pieris), an Affibody™, or a Trinectin™ (Phylos), Nanobodies (Ablynx). The binding member may be selected or designed by recombinant methods known by people well known in the art. Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein,Nature256:495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes. Immunizations To generate fully human monoclonal antibodies to the epitopes of interest to the present invention, transgenic or transchromosomal mice containing human immunoglobulin genes can be immunized with an enriched preparation of the antigen and/or cells expressing the epitopes of the receptor targets of the present invention, as described, for example, by Lonberg et al. (1994), supra; Fishwild et al. (1996), supra, and WO 98/24884. Alternatively, mice can be immunized with DNA encoding the CaOU-1 epitope. Preferably, the mice will be 6-16 weeks of age upon the first infusion. Cumulative experience with various antigens has shown that the HuMAb transgenic mice respond best when initially immunized intraperitoneally (i.p.) or subcutaneously (s.c.) with antigen expressing cells in complete Freund's adjuvant, followed by every other week i.p. immunizations (up to a total of 10) with the antigen expressing cells in PBS. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by FACS analysis, and mice with sufficient titers of anti-antigen human immunoglobulin can be used for fusions. Mice can be boosted intravenously with antigen expressing cells for example 4 and 3 days before sacrifice and removal of the spleen. Use of Partial Antibody Sequences to Express Intact Antibodies Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998)Nature332:323-327; Jones, P. et al. (1986)Nature321:522-525; and Queen, C. et al. (1989)Proc. Natl. Acad. Sci. USA86:10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody which contains mutations throughout the variable gene but typically clustered in the CDRs. For example, somatic mutations are relatively infrequent in the amino terminal portion of framework region 1 and in the carboxy-terminal portion of framework region 4. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to determine which germline variable and joining gene segments contributed to the recombined antibody variable genes. The germline sequence is then used to fill in missing portions of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons. The nucleotide sequences of heavy and light chain transcripts from hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and HindIII sites are engineered upstream of the translation initiation sites. For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding, strand sequences are broken down into 30-50 nucleotides approximately at the midpoint of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150-400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150-400 nucleotides. Typically, a single variable region oligonucleotide set will be broken down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the BbsI site of the kappa light chain, or the AgeI site of the gamma heavy chain) in the PCR amplification to generate fragments that can easily be cloned into the expression vector constructs. The reconstructed heavy and light chain variable regions are then combined with cloned promoter, leader, translation initiation, constant region, 3′ untranslated, polyadenylation, and transcription termination, sequences to form expression vector constructs. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains. Monovalent Antibodies The monospecific binding polypeptide may be monovalent, i.e. having only one binding domain. For a monovalent antibody, the immunoglobulin constant domain amino acid residue sequences comprise the structural portions of an antibody molecule known in the art as CH1, CH2, CH3 and CH4. Preferred are those binding polypeptides which are known in the art as CL. Preferred CLpolypeptides are selected from the group consisting of Ckappaand Clambda. Furthermore, insofar as the constant domain can be either a heavy or light chain constant domain (CHor CL, respectively), a variety of monovalent binding polypeptide compositions are contemplated by the present invention. For example, light chain constant domains are capable of disulfide bridging to either another light chain constant domain, or to a heavy chain constant domain. In contrast, a heavy chain constant domain can form two independent disulfide bridges, allowing for the possibility of bridging to both another heavy chain and to a light chain, or to form polymers of heavy chains. Thus, in another embodiment, the invention contemplates an isolated monovalent binding polypeptide wherein the constant chain domain C has a cysteine residue capable of forming at least one disulfide bridge, and where at least two monovalent polypeptides are covalently linked by said disulfide bridge. In preferred embodiments, the constant chain domain C can be either CLor CH. Where C is CL, the CLpolypeptide is preferably selected from the group consisting of Ckappaand Clambda. In another embodiment, the invention contemplates a binding polypeptide composition comprising a monovalent polypeptide as above except where C is CLhaving a cysteine residue capable of forming a disulfide bridge, such that the composition contains two monovalent polypeptides covalently linked by said disulfide bridge. Multispecificity, Including Bispecificity In a preferred embodiment the present invention relates to multispecific binding polypeptides, which have affinity for and are capable of binding at least two different entities. Multispecific binding polypeptides can include bispecific binding polypeptides. In one embodiment the multispecific molecule is a bispecific antibody (BsAb), which carries at least two different binding domains, where preferably at least one of which is of antibody origin. A bispecific molecule of the invention can also be a single chain bispecific molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding domain, or a single chain bispecific molecule comprising two binding domains. Multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. The multispecific, including bispecific, antibodies may be produced by any suitable manner known to the person skilled in the art. The traditional approach to generate bispecific whole antibodies was to fuse two hybridoma cell lines each producing an antibody having the desired specificity. Because of the random association of immunoglobulin heavy and light chains, these hybrid hybridomas produce a mixture of up to 10 different heavy and light chain combinations, only one of which is the bispecific antibody. Therefore, these bispecific antibodies have to be purified with cumbersome procedures, which considerably decrease the yield of the desired product. Alternative approaches include in vitro linking of two antigen specificities by chemical cross-linking of cysteine residues either in the hinge or via a genetically introduced C-terminal Cys as described above. An improvement of such in vitro assembly was achieved by using recombinant fusions of Fab's with peptides that promote formation of heterodimers. However, the yield of bispecific product in these methods is far less than 100%. A more efficient approach to produce bivalent or bispecific antibody fragments, not involving in vitro chemical assembly steps, was described by Holliger et al. (1993). This approach takes advantage of the observation that scFv's secreted from bacteria are often present as both monomers and dimers. This observation suggested that the VHand VLof different chains could pair, thus forming dimers and larger complexes. The dimeric antibody fragments, also named “diabodies” by Hollinger et al., are in fact small bivalent antibody fragments that assembled in vivo. By linking the VHand VLof two different antibodies 1 and 2, to form “cross-over” chains VH1VL2 and VH2-VL1, the dimerisation process was shown to reassemble both antigen-binding sites. The affinity of the two binding sites was shown to be equal to the starting scFv's, or even to be 10-fold increased when the polypeptide linker covalently linking VHand VLwas removed, thus generating two proteins each consisting of a VHdirectly and covalently linked to a VLnot pairing with the VH. This strategy of producing bispecific antibody fragments was also described in several patent applications. Patent application WO 94/09131 (SCOTGEN LTD; priority date Oct. 15, 1992) relates to a bispecific binding protein in which the binding domains are derived from both a VHand a VLregion either present at two chains or linked in an scFv, whereas other fused antibody domains, e.g. C-terminal constant domains, are used to stabilise the dimeric constructs. Patent application WO 94/13804 (CAMBRIDGE ANTIBODY TECHNOLOGY/MEDICAL RESEARCH COUNCIL; first priority date Dec. 4, 1992) relates to a polypeptide containing a VHand a VLwhich are incapable of associating with each other, whereby the V-domains can be connected with or without a linker. Mallender and Voss, 1994 (also described in patent application WO 94/13806; DOW CHEMICAL CO; priority date Dec. 11, 1992) reported the in vivo production of a single-chain bispecific antibody fragment inE. coli. The bispecificity of the bivalent protein was based on two previously produced monovalent scFv molecules possessing distinct specificities, being linked together at the genetic level by a flexible polypeptide linker. Traditionally, whenever single-chain antibody fragments are referred to, a single molecule consisting of one heavy chain linked to one (corresponding) light chain in the presence or absence of a polypeptide linker is implicated. When making bivalent or bispecific antibody fragments through the “diabody” approach (Holliger et al., (1993) and patent application WO 94/09131) or by the “double scFv” approach (Mallender and Voss, 1994 and patent application WO 94/13806), again the VHis linked to a (the corresponding) VL. The multispecific molecules described above can be made by a number of methods. For example, all specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the multispecific molecule is a mAb X mAb, mAb X Fab, Fab X F(ab′)2or ligand X Fab fusion protein. Various other methods for preparing bi- or multivalent antibodies are described for example described in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858. By using a bispecific or multispecific binding polypeptide according to the invention the invention offers several advantages as compared to monospecific/monovalent binding polypeptides. It may be preferred that the at least one other binding domain is capable of binding an immunoactive cell, such as a leucocyte, a macrophage, a lymphocyte, a basophilic cell, and/or an eosinophilic cell, in order to increase the effect of the binding polypeptide in a therapeutic method. This may be accomplished by establishing that the at least one other binding domain is capable of specifically binding a mammalian protein, such as a human protein, such as a protein selected from any of the cluster differentiation proteins (CD), in particular CD64 and/or CD89. A method for producing bispecific antibodies having CD64 specificity is described in U.S. Pat. No. 6,071,517 to Medarex, Inc. The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in WO 88/00052 and in U.S. Pat. No. 4,954,617. While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies. Such murine, chimeric and humanized monoclonal antibodies can be prepared by methods known in the art. Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kranz et al. (1981)Proc. Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (see U.S. Pat. No. 4,474,893), or recombinant DNA techniques. When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation. Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a mAb×mAb, mAb×Fab, Fab×F(ab′)2or ligand×Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule can be a single chain molecule, such as a single chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for preparing bi- and multispecific molecules are described for example in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858. Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. Humanised Antibody Framework It is not always desirable to use non-human antibodies for human therapy, since the non-human “foreign” epitopes may elicit immune response in the individual to be treated. To eliminate or minimize the problems associated with non-human antibodies, it is desirable to engineer chimeric antibody derivatives, i.e., “humanized” antibody molecules that combine the non-human Fab variable region binding determinants with a human constant region (Fc). Such antibodies are characterized by equivalent antigen specificity and affinity of the monoclonal and polyclonal antibodies described above, and are less immunogenic when administered to humans, and therefore more likely to be tolerated by the individual to be treated. Humanised antibodies are in general chimeric antibodies comprising regions derived from a human antibody and regions derived from a non-human antibody, such as a rodent antibody. Humanisation (also called Reshaping or CDR-grafting) is a well-established technique for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent), increasing the homology to a human immunoglobulin, and for improving their activation of the human immune system. Thus, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. It is further important that humanized antibodies retain high affinity for the antigen and other favourable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of certain residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is maximized, although it is the CDR residues that directly and most substantially influence antigen binding. One method for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody, preferably a human antibody. Methods for carrying out such chimerisation procedures are for example described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023 (Genentech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University) and EP-A-0 194 276 (Celltech Limited). A more complex form of humanisation of an antibody involves the re-design of the variable region domain so that the amino acids constituting the non-human antibody binding site are integrated into the framework of a human antibody variable region (Jones et al., 1986). The humanized antibody of the present invention may be made by any method capable of replacing at least a portion of a CDR of a human antibody with a CDR derived from a non-human antibody. Winter describes a method which may be used to prepare the humanized antibodies of the present invention (UK Patent Application GB 2188638A, filed on Mar. 26, 1987), the contents of which is expressly incorporated by reference. The human CDRs may be replaced with non-human CDRs using oligonucleotide site-directed mutagenesis as described in the examples below. As an example the humanized antibody of the present invention may be made as described in the brief explanation below. The humanized antibodies of the present invention may be produced by the following process:(a) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding an antibody heavy chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention;(b) constructing, by conventional techniques, an expression vector containing an operon with a DNA sequence encoding a complementary antibody light chain in which the CDRs and such minimal portions of the variable domain framework region that are required to retain donor antibody binding specificity are derived from a non-human immunoglobulin, and the remaining parts of the antibody chain are derived from a human immunoglobulin, thereby producing the vector of the invention;(c) transfecting the expression vectors into a host cell by conventional techniques to produce the transfected host cell of the invention; and(d) culturing the transfected cell by conventional techniques to produce the humanised antibody of the invention. The host cell may be cotransfected with the two vectors of the invention, the first vector containing an operon encoding a light chain derived polypeptide and the second vector containing an operon encoding a heavy chain derived polypeptide. The two vectors contain different selectable markers, but otherwise, apart from the antibody heavy and light chain coding sequences, are preferably identical, to ensure, as far as possible, equal expression of the heavy and light chain polypeptides. Alternatively, a single vector may be used, the vector including the sequences encoding both the light and the heavy chain polypeptides. The coding sequences for the light and heavy chains may comprise cDNA or genomic DNA or both. The host cell used to express the altered antibody of the invention may be either a bacterial cell such asE. coli, or a eukaryotic cell. In particular a mammalian cell of a well defined type for this purpose, such as a myeloma cell or a Chinese hamster ovary cell may be used. The general methods by which the vectors of the invention may be constructed, transfection methods required to produce the host cell of the invention and culture methods required to produce the antibody of the invention from such host cells are all conventional techniques. Likewise, once produced, the humanized antibodies of the invention may be purified according to standard procedures as described below. Antigenic epitope(s) such as the O-glycosylated peptides of the invention may be administered to a mammal in an amount sufficient to stimulate an immunological response against the antigenic epitope(s). The antigenic epitope(s) may be combined in a therapeutic composition and administered in several doses over a period of time that optimizes the immunological response of the mammal. Such an immunological response can be detected and monitored by observing whether antibodies directed against the epitopes of the invention are present in the bloodstream of the mammal. Such antibodies can be used alone or conjugated to, or combined with, therapeutically useful agents. Antibodies can be administered to mammals suffering from any cancer that displays the cancer-associated epitope(s). Such administration can provide both therapeutic treatment, and prophylactic or preventative measures. For example, therapeutic methods can be used to determine the spread of a cancer and lead to its remission. Antibodies of the invention can be used for passive immunization of patient, i.e. administering the antibodies or as antibody fragments such as Fab fragments in isolated form to the patient. Furthermore, medicaments such as toxins or chemotherapeutic agents can be conjugated to the antibodies of the invention by methods known to those skilled in the art. All of the above antibodies can subsequent to administration target cancer cells specifically, based on the knowledge that the pattern of multiple and aberrantly O-glycosylated mucins on cancer cells distinguishes them from healthy cells. Therapeutically useful agents which may be conjugated to the antibodies of the invention include but is not limited to the group comprising adrimycin, aminoglutethimide, aminopterin, azathioprine, bleomycin sulfate, bulsulfan, carboplatin, carminomycin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, cytosine arabinoside, cytoxin dacarbazine, dactinomycin, daunomycin, daunorubicin, doxorubicin, esperamicins, etoposide, fluorouracil, ifosfamide, interferon-α, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin C, mitotane, mitoxantrone, procarbazine HCl, taxol, taxotere (docetaxel), teniposide, thioguanine, thiotepa, vinblastine sulfate, vincristine sulfate and vinorelbine. Additional agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, pp. 1202-1263, of Goodman and Gilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division). Toxins can be proteins such as, for example, pokeweed anti-viral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, orPseudomonasexotoxin. Toxin moieties can also be high energy-emitting radionuclides such as cobalt-60, I-131, I-125, Y-90 and Re-186, and enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. Chemotherapeutic agents can be used to reduce the growth or spread of cancer cells and tumors that express the tumor associated epitope of the invention. Animals that can be treated by the chemotherapeutic agents of the invention include humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, rodents and the like. In all embodiments human tumor antigens and human subjects are preferred. Species-dependent antibodies can be used in therapeutic methods. Such a species-dependent antibody has constant regions that are substantially non-immunologically reactive with the chosen species. Such species-dependent antibody is particularly useful for therapy because it gives rise to substantially no immunological reactions. The species-dependent antibody can be of any of the various types of antibodies as defined above, but preferably is mammalian, and more preferably is a humanized or human antibody. The present inventors have found glycopeptide epitopes associated with colorectal cancer and antibodies useful in detecting said glycopeptides. In another aspect, the present invention relates to a method for detecting colorectal cancer, said method comprising(i) contacting a sample from said host organism with one or more O-glycosylated mucin peptides, wherein said peptide comprises, or said peptides comprise, at least 5 consecutive amino acid residues of a mucin, or a fragment or variant thereof, wherein said variant is at least 70% identical, such as at least 75% identical to, e.g. at least 80% identical to, such as at least 85% identical to, e.g. at least 90% identical to, such as at least 95% identical to, e.g. at least 98% identical to, such as at least 99% identical to said at least 5 consecutive amino acid residues of said mucin, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the host organism. The present inventors have raised several antibodies specific for different glycosylated parts of the mucin proteins. Accordingly, in one aspect the present invention relates to a method for detecting cancer, said method comprising(i) contacting a sample with one or more antibodies capable of recognising one or more O-glycosylated mucin peptides, wherein said peptide comprises at least 5 consecutive amino acid residues of a mucin, or a fragment or variant thereof, wherein said variant is at least 70% identical, such as at least 75% identical to, e.g. at least 80% identical to, such as at least 85% identical to, e.g. at least 90% identical to, such as at least 95% identical to, e.g. at least 98% identical to, such as at least 99% identical to said at least consecutive amino acid residues of said mucin, and(ii) removing unbound sample and/or unbound antibody, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to said antibodies indicate cancer in the sample host. In one embodiment said O-glycosylated mucin peptide is selected from the group consisting ofa) MUC4 Tn selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orb) a MUC4 non glycosylated mucin peptide selected from the group consisting of PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21) STGDTLPLPVTDTSSV (SEQ ID NO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), or a Tn glycosylated mucin peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 22), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orc) a Tn glycosylated MUC4 peptide selected from the group consisting of PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO: 7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), ord) an all-Tn MUC4 peptide selected from the group consisting of PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30),PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), wherein the asterisk (*) indicates an O-glycosylation site, wherein the glycan is Tn (GalNAc-α-Ser/Thr), ore) a recombinant MUC4 Tn having the sequence PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is Tn (GalNAc-α-Ser/Thr), orf) a MUC1Tn/STn/Core3 glycosylated or MUC4 glycosylated mucin peptide selected from the group consisting of VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG, VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG, PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), and PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30),wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), org) a MUC1 STn and a MUC4 selected from the group consisting of VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG, PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22) and PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23), and wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In one aspect the present invention relates to a method for detecting a gastrointestinal disease in a host organism wherein said disease is characterised in that O-glycosylated mucin peptides are shed from the diseased host and secreted in the gastrointestinal tract of the sample organism suffering from the disease, said method comprising(i) contacting a sample from said host organism with one or more antibodies capable of recognising said O-glycosylated mucin peptides, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to the antibodies of step (i) are indicative of disease or disorder in the host organism. In another aspect the present invention relates to a method for detecting a gastrointestinal disease in a host organism wherein said disease is characterised in that O-glycosylated mucin peptides are shed from the diseased cells of the host into an extracellular volume, such as secreted into, the lumen of the bladder, milk ducts of the breast, lumen of the uterus, the vagina, into pancreatic fluid, into ascites fluid, onto bronchiolar surface of the lung, ductal surfaces of the prostate, lumen of the seminiferous tubules, the oesophagus or the gastrointestinal tract of the sample organism suffering from the disease, said method comprising(i) contacting a sample from said host organism with one or more antibodies capable of recognising said O-glycosylated mucin peptides, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to the antibodies of step (i) are indicative of disease or disorder in the host organism. In one embodiment the disease detected by the above method is cancer wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, oral cancer, gastric cancer, esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer, basal cell cancer, squamous cell cancer, malignant melanoma, bladder cancer, endometrial cancer and cervix cancer. In one embodiment the disease detected by the above method is colorectal cancer. In one embodiment of the above method, the antibody the present invention is capable of recognising at least one O-glycosylated peptide, said peptide comprising at least 5 consecutive amino acid residues of a mucin selected from the group consisting of MUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variant thereof, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin. In one embodiment of the present invention the at least 5 consecutive amino acid residues of a mucin as defined herein above are from a mucin selected from the group consisting of MUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG. In one embodiment the antibody is capable of recognising at least one O-glycosylated peptide variant wherein said variant is at least 70% identical a peptide selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), STGDTLPLPVTDTSSV (SEQ ID NO: 22), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), and PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25). In another embodiment the antibody is capable of recognising an O-glycosylated mucin peptide selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn), PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*H DAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30),wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In one aspect the invention relates to a method for detecting colorectal cancer, said method comprising(i) contacting a sample with a polyclonal antibody serum wherein said polyclonal antibody is capable of recognising at least two different O-glycosylated mucin peptides, wherein said peptides comprises at least 5 consecutive amino acid residues selected from the group consisting of MUC1 Variant CT58, MUC1 Variant CT80, MUC1 Variant SEC, MUC1 Variant X, MUC1 Variant Y, MUC1 Variant ZD, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, MUC20, MUC21 and MUC-HEG, or a fragment or variant thereof, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin, and(ii) removing unbound sample and/or unbound antibody, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to said antibodies indicate cancer in the sample host. In one embodiment the at least two different O-glycosylated mucin peptides as defined herein above are a first O-glycosylated mucin peptide selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ ID NO: 23) and PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNGHTTQAPTTALQ AAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMT HTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETT RFSSNPS (SEQ ID NO: 30) wherein at least one serine and/or threonine residue is optionally O-glycosylated and wherein the optional glycan is selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and a second O-glycosylated mucin peptide selected from the group consisting of VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn) respectively, and(ii) removing unbound sample, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein antibodies bound to said peptides indicate cancer in the sample host. In one aspect the invention relates to a method for detecting cancer, said method comprising(i) contacting a sample with at least one first and at least one second antibody, wherein said first antibody is capable of recognising a first mucin peptide selected from the group consisting of the O-glycosylated MUC1 peptides VTSAPDT(Core3)RPAPGSTAPPAHG (MUC1 Core3), VT(Core3)SAPDTRPAPGS(Core3)T(Core3)APPAHG (MUC1 9Core3), VT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHG (MUC1 15Core3), VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHG (MUC 1 (15STn), VT(STn)SAPDTRPAPGS(STn)T(STn)APPAHG (MUC1 9STn), VT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHG (MUC1 15Tn), VT(Tn)SAPDTRPAPGS(Tn)T(Tn)APPAHG (MUC1 9Tn), VT(Tn)SAPDTRPAPGST(Tn)APPAHG (MUC1 6Tn),and wherein said second antibody is capable of recognising another mucin peptide selected from:PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO:3), PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4), S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5), T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6), T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7), APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8), S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9), NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10), T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11), AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12), EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13), PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14), T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID NO: 15), S*GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16) and GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17), PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P (SEQ ID NO: 18), LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH (SEQ ID NO: 19), LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 20), PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ (SEQ ID NO: 21), S*T*GDT*LPLPVT*DT*S*S*V (SEQ ID NO: 22), PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*TTS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30), wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr), and(ii) removing unbound sample and/or unbound antibody), and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to said antibodies indicate cancer in the sample host. In another aspect the invention relates to a method for detecting cancer, said method comprising(i) contacting a sample with at least one antibody, wherein said antibody is capable of recognising a glycosylated mucin peptide, wherein said peptide comprises at least 5 consecutive amino acid residues of a mucin selected from the group consisting of MUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), or a fragment or variant thereof, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin selected from the group consisting of MUC4 (SEQ ID NO: 1) and/or MUC1 (SEQ ID NO: 2), and(ii) removing unbound sample and/or unbound antibody, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to said antibodies indicate cancer in the sample host. In one aspect the invention relates to a method for detecting colorectal cancer, said method comprising(i) contacting a sample with at least one antibody, wherein said antibody is capable of recognising a glycosylated mucin peptide selected from the group consisting of PMTDTKTVTTPGSSFTA (SEQ ID NO: 3), PGSSFTASGHSPSEIVPQD (SEQ ID NO: 4), SEIVPQDAPTISAATTFAPA (SEQ ID NO: 5), TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6), TTQAPTTALQAAPSSHD (SEQ ID NO: 7), APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8), SLSKTGALTLANSVVSTP (SEQ ID NO: 9), NSVVSTPGGPEGQWTSASAS (SEQ ID NO: 10), TSASASTSPRTAAAMTHT (SEQ ID NO: 11), AAAMTHTHQAESTEASGQT (SEQ ID NO: 12), EASGQTQTSEPASSGSRTT (SEQ ID NO: 13), PASSGSRTTSAGTATPSSS (SEQ ID NO: 14), TATPSSSGASGTTPSGSEGI (SEQ ID NO: 15), SGSEGISTSGETTRFSSN (SEQ ID NO: 16), GETTRFSSNPSRDSHTT (SEQ ID NO: 17), PVTSPSSASTGHTTPLPVTDTSSASTGDTTP (SEQ ID NO: 18), LPVTSLSSVSTGDTTPLPVTSPSSASTGH (SEQ ID NO: 19), LPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 20), PLPVTSPSSASTGHASPLLVTDASSASTGQ (SEQ ID NO: 21), PVTYASSASTGDTTPLPVTDTSSVSTGHAT (SEQ ID NO: 22), STGDTLPLPVTDTSSV (SEQ ID NO: 23), VTSAPDTRPAPGSTAPPAHG (SEQ ID NO: 24), and PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC (SEQ ID NO:25) or a fragment of said peptides, or variants of said peptides in which variants any amino acid has been changed to a different amino acid, provided that no more than 5 of the amino acid residues in the sequence are so changed, and(ii) removing unbound sample and/or unbound antibody, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to said antibodies indicate cancer in the sample host. In another aspect the invention relates to a method for detecting colorectal cancer, said method comprising(i) contacting a sample with at least two different antibodies, wherein said antibodies are capable of recognising two different glycosylated mucin peptides, wherein said peptide comprises at least 5 consecutive amino acid residues of a mucin selected from the group consisting of MUC4 (SEQ ID NO:1), MUC1 (SEQ ID NO: 2), MUC2 (SEQ ID NO: 26), MUC5AC (SEQ ID NO: 27), MUC6 (SEQ ID NO: 28), and MUC7 (SEQ ID NO: 29) or a naturally occurring fragment or variant of said mucin, wherein said variant is at least 70% identical to said at least 5 consecutive amino acid residues of said mucin, and(ii) removing unbound sample and/or unbound antibody, and(iii) qualitatively and/or quantitatively characterise the bound material, wherein peptides bound to said antibodies indicate cancer in the sample host. The antibodies of the present invention have been raised against different parts of glycosylated mucin polypeptides, Thus in one main aspect, the present invention relates to a an antibody, in particular a monoclonal antibody as defined in the claims1to5. The invention also relates to antigen binding fragment of said antibody, wherein said antibody or the antigen binding fragment of said antibody is capable of specifically recognising a mucin glycopeptide as defined in any of the claims. In one embodiment the antibody is selected from the group consisting of IgA, IgG, IgD, IgE and IgM antibodies. In a further embodiment the IgA antibody is an IgA1 or an IgA2 antibody. In a further embodiment the IgG antibody is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 antibodies. In a further embodiment the IgG antibody is selected from the group consisting of mouse IgG1, mouse IgG2A, mouse IgG2B and mouse IgG3 antibodies. In a further embodiment the IgG antibody is selected from the group consisting of human IgG1, human IgG2, human IgG3 and human IgG4 antibodies. In a further embodiment the IgG antibody is selected from the group consisting of rabbit IgG1, rabbit IgG2A, rabbit IgG2B and rabbit IgG3 antibodies. In a further embodiment the IgG antibody is selected from the group consisting of goat IgG1, goat IgG2A, goat IgG2B and goat IgG3 antibodies. In one embodiment the antibodies of the invention is raised using any other mammal suitable, as known to the person skilled in the art, for the purpose of raising antibodies. In one embodiment the antibody of the present invention is selected from the group consisting of MAb 5C10, MAb 3C9, MAb 4D9, MAb 6C11 and MAb 6E3. In one embodiment the antibody is MAb 4D9 produced by the cell line 4D9 deposited under the Budapest Treaty with the European Collection of Authenticated Cell Cultures (ECACC) on Dec. 1, 2009, and which was given accession number 09120102. The address of the depository is Public Health England Culture Collections, Porton Down, Salisbury SP4 OJG, United Kingdom. In one embodiment the antibody is MAb 5C10 produced by the cell line 5C10 deposited under the Budapest Treaty with the ECACC on Dec. 1, 2009, and which was given accession number 09120101. In one embodiment the antibody is MAb 6E3 produced by the cell line 6E3 deposited under the Budapest Treaty with the ECACC on Dec. 1, 2009, and which was given accession number 09120103. In one embodiment the antibody is MAb 3C9. In one embodiment the antibody is MAb 6C11. In embodiment the MAb 5C10 antibody as defined herein above binds to one or more amino acid residues of the glycosylated MUC1 epitope having the sequence VTSAPDT(Core3)RPAPGSTAPPAHG (SEQ ID NO: 24) In embodiment the MAb 4D9 antibody as defined herein above binds to one or more amino acid residues of the glycosylated MUC4 epitope having the sequence PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In embodiment the MAb 3C9 antibody as defined herein above binds to one or more amino acid residues of the glycosylated MUC4 epitope having the sequence PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2(Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In embodiment the MAb 6C11 antibody as defined herein above binds to one or more amino acid residues of the glycosylated MUC4 epitope having the sequence PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT* (SEQ ID NO: 23) wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). In embodiment the MAb 6E3 antibody as defined herein above binds to one or more amino acid residues of the glycosylated MUC4 epitope having the sequence PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT* T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEG QWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T* S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 30) wherein the asterisk (*) indicates a potential O-glycosylation site, wherein the optional glycan is independently selected from the group consisting of Core-3 (GlcNAcβ1-3GalNAc-α-Ser/Thr), Tn (GalNAc-α-Ser/Thr), STn/sialyl-Tn (Neu5Acα2-6GalNAc-α-Ser/Thr), Core-2 (Galβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr)) Core-4 (GlcNAcβ1-3(GlcNAcβ1-6)GalNAc-α-Ser/Thr), and ST/sialyl-T (Neu5Acα2-3Galβ3GalNAc-Ser/Thr). Antibodies can be used as carriers for functional groups conjugated to the antibodies. The functional groups may be for example agents suitable for detection by imaging methods or toxins useful in killing or destroying cells, for example cancer cells. In one aspect the present invention relates to a method of detecting a peptide and/or an antibody as defined herein above, said method comprising conjugating an imaging agent to said peptide or antibody. In one embodiment said imaging agent is detectable by at least one of the technologies selected from the group consisting of: computer tomography, ultrasound, magnetic resonance, nuclear imaging, optical and/or electron optical imaging. In a further embodiment said optical and/or electron optical imaging is selected from the group consisting of diffuse optical tomography, optical coherence tomography, confocal laser scanning, microscopy, electron microscopy, fluorescence correlation microscopy, fluorescence resonance energy transfer, and fluorescence lifetime imaging. In a further embodiment said nuclear imaging is selected from the group consisting of PET, SPECT and MRI. In one embodiment said imaging agent is selected from the group consisting of antibodies, small molecules, peptides and metal ions, wherein said metal ion is selected from ions of transition metals or lanthanides and actinides. In one embodiment the metal ion is an ion of Hf, Ho or Gd. In one embodiment the antibody of the present invention, further comprises a toxin conjugated to said antibody. The antibodies of the present invention have been raised against different parts of glycosylated mucin polypeptides, Thus in one aspect, the present invention relates to a monoclonal antibody or an antigen binding fragment of said antibody, wherein said antibody or the antigen binding fragment of said antibody is capable of specifically recognising a mucin glycopeptide as defined herein above. The method used by the present inventors for raising antibodies is also part of the invention. Thus, in one aspect, the present invention relates to a method for producing the antibody defined herein above, said method comprising the steps of:i) providing a host organism,ii) immunizing the host organism with an O-glycosylated mucin as defined herein above, andiii) obtaining said antibody. The present invention is applicable to a subject undergoing therapy, such as surgery. In surgery the glycopeptides epitopes identified by the present inventors can be targeted with antibodies of the invention wherein the antibodies are conjugated to a visualization label, such as a fluorescent label. The epitopes can thus be visualised during surgery with the effect of more efficiently removing e.g. cancer tumours. Accordingly, in one aspect, the present invention relates to a method for detecting a cancer tumour in a patient undergoing therapy and/or examination, said method comprising the steps of:(i) administering to an area of the patient, the antibody as defined herein above, or an antigen binding fragment of said antibody, suitably conjugated to a visualisation label, and(ii) removing unbound antibodies, and(iii) detecting antibodies bound to glycopeptide epitopes as defined herein above, wherein labelling indicates presence of a tumour. In one embodiment of the present invention the therapy is surgery. In one embodiment of the present invention the examination is examination for colorectal cancer. In one embodiment of the present invention the examination for colorectal cancer is by visualisation means such as endoscopy. Another aspect of the present invention is a method for the preparation of hybridoma cells, which secrete monoclonal antibodies specific for the immunogenic glycopeptide characterized in that a suitable mammal is immunized with the immunogenic glycopeptide, antibody-producing cells of said mammal are fused with cells of a continuous cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. Still another aspect is a monoclonal antibody selected from the group consisting of a monoclonal antibody produced by the hybridoma cells prepared by the method described above, a monoclonal antibody prepared by molecular display techniques, such as mRNA display, ribosome display, phage display and covalent display against the immunogenic glycopeptide. Traditionally, monoclonal antibodies have been prepared using hybridoma technology. However, alternative techniques such as mRNA display, ribosome display, phage display and covalent display are now available. These are all display techniques where a peptide library is selected against the immunogenic glycopeptide. Such techniques can e.g. be used to identify humanized or fully human antibodies. In one embodiment, the monoclonal antibody binds the MUC4 or MUC1 glycopeptides specified herein, on cancer cells but not on a non-malignant counterpart. Preferably said antibody binds glycopeptide epitopes associated with colorectal cancer. In one aspect, the present invention relates to a monoclonal antibody or an antigen binding fragment of said antibody, wherein said antibody or the antigen binding fragment of said antibody is capable of specifically recognising a mucin glycopeptide as defined herein above. Quantitative and Qualitative Analysis of Bound Material The step of the method of the present invention comprising qualitatively and/or quantitatively characterising the bound peptide or antibody is based on an ELISA-type, or ELISA-analogous method. ELISA-analogous methods may comprise using e.g. microbeads to which peptides or antibodies can be used. One example of a microbead method is the Luminex method (www.luminexcorp.com). Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence. Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. In one embodiment the step of qualitatively and/or quantitatively characterising bound material in the method of the present invention is by Enzyme-linked immunosorbent assay (ELISA). In one embodiment the step of qualitatively and/or quantitatively characterising bound material in the method of the present invention is by a bead assay such as a Luminex assay. Pharmaceutical Compositions and Administration Forms The main routes of drug delivery, in the treatment method are intravenous, oral, and topical. Other drug-administration methods, such as intraveneous, subcutaneous and intramuscular injection or via inhalation, which are effective to deliver the drug to a target site or to introduce the drug into the bloodstream, are also contemplated. The mucosal membrane to which the pharmaceutical preparation of the invention may be administered can be any mucosal membrane of the mammal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, preferably the mucosa of the nose, mouth or vagina. Compounds of the invention may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration. The subcutaneous and intramuscular forms of parenteral administration are generally preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques. The compounds may also be administered by inhalation, which is by intranasal and oral inhalation administration. Appropriate dosage forms for such administration, such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques. The compounds according to the invention may be administered with at least one other compound. The compounds may be administered simultaneously, either as separate formulations or combined in a unit dosage form, or administered sequentially. In one embodiment of the present invention, the dosage of the active ingredient of the pharmaceutical composition as defined herein above, is between 10 μg to 500 mg per kg body mass. Formulations Whilst it is possible for the compounds or salts of the present invention to be administered as the raw glycopeptide or antibody preparation, it is preferred to present them in the form of a pharmaceutical formulation. Accordingly, the present invention further provides a pharmaceutical formulation, for medicinal application or for use during in situ detection of autoantibodies or glycopeptides relating to disease. The pharmaceutical composition comprises a compound of the present invention or a pharmaceutically acceptable salt thereof, as herein defined, and a pharmaceutically acceptable carrier therefore. The compounds of the present invention may be formulated in a wide variety of oral administration dosage forms. The pharmaceutical compositions and dosage forms may comprise the compounds of the invention or its pharmaceutically acceptable salt or a crystal form thereof as the active component. The pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material. Preferably, the composition will be about 0.5% to 75% by weight of a compound or compounds of the invention, with the remainder consisting of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. In powders, the carrier is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Powders and tablets preferably contain from one to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be as solid forms suitable for oral administration. Drops according to the present invention may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 98-100° C. for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container aseptically. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. Other forms suitable for oral administration include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations. Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. Solid form preparations include solutions, suspensions, and emulsions, and may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. The compounds of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or nonaqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. Oils useful in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides; (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-.beta.-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof. The parenteral formulations typically will contain from about 0.5 to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. The compounds of the invention can also be delivered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat. Compositions for topical administration via the skin and mucous membranes should not give rise to signs of irritation, such as swelling or redness. The topical composition may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry. The compounds of the present invention may be formulated for topical administration to the epidermis as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also containing one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Formulations suitable for topical administration in the mouth include lozenges comprising active agents in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives or a fatty acid such as steric or oleic acid together with an alcohol such as propylene glycol or a macrogel. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included. Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil orarachisoil. Transdermal Delivery The pharmaceutical agent-chemical modifier complexes described herein can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like. Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body. See Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), Marcel Dekker, Inc., (1989); Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekker Inc., (1987); and Transdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC Press, (1987). Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel. Passive Transdermal Drug Delivery A variety of types of transdermal patches will find use in the methods described herein. For example, a simple adhesive patch can be prepared from a backing material and an acrylate adhesive. The pharmaceutical agent-chemical modifier complex and any enhancer are formulated into the adhesive casting solution and allowed to mix thoroughly. The solution is cast directly onto the backing material and the casting solvent is evaporated in an oven, leaving an adhesive film. The release liner can be attached to complete the system. Alternatively, a polyurethane matrix patch can be employed to deliver the pharmaceutical agent-chemical modifier complex. The layers of this patch comprise a backing, a polyurethane drug/enhancer matrix, a membrane, an adhesive, and a release liner. The polyurethane matrix is prepared using a room temperature curing polyurethane prepolymer. Addition of water, alcohol, and complex to the prepolymer results in the formation of a tacky firm elastomer that can be directly cast only the backing material. A further embodiment of this invention will utilize a hydrogel matrix patch. Typically, the hydrogel matrix will comprise alcohol, water, drug, and several hydrophilic polymers. This hydrogel matrix can be incorporated into a transdermal patch between the backing and the adhesive layer. The liquid reservoir patch will also find use in the methods described herein. This patch comprises an impermeable or semipermeable, heat sealable backing material, a heat sealable membrane, an acrylate based pressure sensitive skin adhesive, and a siliconized release liner. The backing is heat sealed to the membrane to form a reservoir which can then be filled with a solution of the complex, enhancers, gelling agent, and other excipients. Foam matrix patches are similar in design and components to the liquid reservoir system, except that the gelled pharmaceutical agent-chemical modifier solution is constrained in a thin foam layer, typically a polyurethane. This foam layer is situated between the backing and the membrane which have been heat sealed at the periphery of the patch. For passive delivery systems, the rate of release is typically controlled by a membrane placed between the reservoir and the skin, by diffusion from a monolithic device, or by the skin itself serving as a rate-controlling barrier in the delivery system. See U.S. Pat. Nos. 4,816,258; 4,927,408; 4,904,475; 4,588,580, 4,788,062; and the like. The rate of drug delivery will be dependent, in part, upon the nature of the membrane. For example, the rate of drug delivery across membranes within the body is generally higher than across dermal barriers. The rate at which the complex is delivered from the device to the membrane is most advantageously controlled by the use of rate-limiting membranes which are placed between the reservoir and the skin. Assuming that the skin is sufficiently permeable to the complex (i.e., absorption through the skin is greater than the rate of passage through the membrane), the membrane will serve to control the dosage rate experienced by the patient. Suitable permeable membrane materials may be selected based on the desired degree of permeability, the nature of the complex, and the mechanical considerations related to constructing the device. Exemplary permeable membrane materials include a wide variety of natural and synthetic polymers, such as polydimethylsiloxanes (silicone rubbers), ethylenevinylacetate copolymer (EVA), polyurethanes, polyurethane-polyether copolymers, polyethylenes, polyamides, polyvinylchlorides (PVC), polypropylenes, polycarbonates, polytetrafluoroethylenes (PTFE), cellulosic materials, e.g., cellulose triacetate and cellulose nitrate/acetate, and hydrogels, e.g., 2-hydroxyethylmethacrylate (HEMA). Other items may be contained in the device, such as other conventional components of therapeutic products, depending upon the desired device characteristics. For example, the compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like. These pharmaceutical compositions also can contain other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics, and antipruritic agents. The compounds of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the active component is dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and to solidify. The active compound may be formulated into a suppository comprising, for example, about 0.5% to about 50% of a compound of the invention, disposed in a polyethylene glycol (PEG) carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]. The compounds of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate. The compounds of the present invention may be formulated for nasal administration. The solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in a single or multidose form. In the latter case of a dropper or pipette this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomizing spray pump. The compounds of the present invention may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compound will generally have a small particle size for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization. The active ingredient is provided in a pressurized pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by a metered valve. Alternatively the active ingredients may be provided in a form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form for example in capsules or cartridges of e.g., gelatin or blister packs from which the powder may be administered by means of an inhaler. When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. Pharmaceutically Acceptable Salts Pharmaceutically acceptable salts of the instant compounds, where they can be prepared, are also intended to be covered by this invention. These salts will be ones which are acceptable in their application to a pharmaceutical use. By that it is meant that the salt will retain the biological activity of the parent compound and the salt will not have untoward or deleterious effects in its application and use in treating diseases. Pharmaceutically acceptable salts are prepared in a standard manner. If the parent compound is a base it is treated with an excess of an organic or inorganic acid in a suitable solvent. If the parent compound is an acid, it is treated with an inorganic or organic base in a suitable solvent. The compounds of the invention may be administered in the form of an alkali metal or earth alkali metal salt thereof, concurrently, simultaneously, or together with a pharmaceutically acceptable carrier or diluent, especially and preferably in the form of a pharmaceutical composition thereof, whether by oral, rectal, or parenteral (including subcutaneous) route, in an effective amount. Examples of pharmaceutically acceptable acid addition salts for use in the present inventive pharmaceutical composition include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, for example. Therapeutically useful agents can be formulated into a composition with the antibodies of the invention and need not be directly attached to the antibodies of the invention. However, in some embodiments, therapeutically useful agents are attached to the antibodies of the invention using methods available to one of skill in the art, for example, standard coupling procedures. Compositions may contain antibodies, antigenic epitopes or trypsin-like protease inhibitors. Such compositions are useful for detecting the antigenic peptide epitopes (glycopeptides) and for therapeutic methods involving prevention and treatment of cancers associated with the presence of said antigenic epitopes. The antibodies, (and for example antigenic epitopes and protease inhibitors) can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration. Routes for administration include, for example, intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal and other routes selected by one of skill in the art. Solutions of the antibodies, (and for example antigenic epitopes and protease inhibitors) can be prepared in water or saline, and optionally mixed with a nontoxic surfactant. Formulations for intravenous or intra-arterial administration may include sterile aqueous solutions that may also contain buffers, liposomes, diluents and other suitable additives. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions comprising the active ingredient that are adapted for administration by encapsulation in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. Sterile injectable solutions are prepared by incorporating the antibodies, antigenic epitopes and protease inhibitors in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In one aspect the present invention relates to a pharmaceutical composition comprising an active ingredient, wherein said active ingredient is selected from (i) one or more of the O-glycosylated peptides defined herein above, or (ii) the antibody capable of recognising the peptide of (i), or any other antibody defined herein above, and wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or diluent. In one embodiment the pharmaceutical composition is formulated for administration by injection, suppository, oral administration, sublingual tablet or spray, cutaneous administration or inhalation. In one embodiment said injection is intramuscular, intravenous, intranasal, intraperitoneal, subcutaneous, a bolus or a continuous administration. In one embodiment the pH of the pharmaceutical composition is between pH 4 and pH 10. In one embodiment administration of the pharmaceutical composition occurs at intervals of 30 minutes to 24 hours. In one embodiment administration of the pharmaceutical composition occurs at intervals of 1 to 6 hours. In one embodiment the duration of the treatment with the pharmaceutical composition is from 6 to 72 hours. In one embodiment the duration of the treatment with the pharmaceutical composition is life long. In one embodiment the dosage of the active ingredient is between 10 μg to 500 mg per kg body mass. Diagnostic Kits Kits for detection of the antigenic peptide epitopes of the present invention can be provided. A kit for detection of the antigenic epitope of the invention may contain a container containing an antibody capable of binding to an antigenic epitope of the invention. Such an antibody may be labeled for easy detection. Individual kits may be adapted for performing one or more of the methods of the invention. Optionally, the subject kit may further comprise at least one other reagent required for performing the method that the kit is adapted to perform. Examples of such additional reagents include: a label, a standard, a control, a buffer, a solution for diluting the test sample, or a reagent that facilitates detection of the label. The reagents included in the kits of the invention may be supplied in premeasured units so as to provide for greater precision and accuracy. Typically, kits reagents and other components are placed and contained in separate vessels. A reaction vessel, test tube, microwell tray, microtiter dish or other container can also be included in the kit. Different labels can be used on different reagents so that each reagent can be distinguished from another. The kit can also be for the treatment of cancer comprising a pharmaceutical composition and an instructional material. Such a kit may contain a container having an antigenic epitope, an antibody or an inhibitor of the invention. The antigenic epitope may act as a vaccine for preventing formation of metastatic adenocarcinoma. The antibody is directed against an antigenic epitope of the invention and can be administered to treat or prevent the spread of adenocarcinomas. Any one of these antigenic epitopes, antibodies or inhibitors may be contained within an appropriate container in the kit. Alternatively, a combination of antigenic epitopes, antibodies or inhibitors may be contained within an appropriate container in the kit. Further, a kit comprising a pharmaceutical composition and a delivery device for delivering the composition to a mammal, for example, a human patient who may have an adenocarcinoma can also be provided. By way of example, the delivery device may be a squeezable spray bottle, a metered-dose spray bottle, an aerosol spray device, an atomizer, a dry powder delivery device, a self-propelling solvent/powder-dispensing device, a syringe, a needle, a tampon, or a dosage measuring container. In one aspect, the present invention relates to a method for identifying a disease (e.g. cancer such as colorectal cancer) associated with shedding of O-glycosylated peptides or peptide fragments, said method comprising the steps of:(i) selecting potential target polypeptides containing potential O-glycosylation sites, and(ii) producing recombinant fragments covering specific areas of interest from each potential target, and/or(iii) producing synthetic peptides covering specific areas of interest from each potential target, and(iv) in vitro glycosylate the fragments of (ii) and/or (iii) using recombinant glycosyltransferases(v) purifying the fragments of (iv), and(vi) characterizing the purified products of (v), and (vii) printing of non-glycosylated and glycosylated targets,(viii) screening the printed targets of (vii) with sera from a potentially diseased sample host and(ix) screening the printed targets of (vii) with sera from a healthy sample host as control,wherein the presence of auto-antibodies bound to the printed targets of (viii) indicates disease in the potentially diseased sample host. In one embodiment of the method for identifying a disease defined herein above, the recombinant fragments covering specific areas of interest from each potential target are between 10 to 30 kDa. In one embodiment of the method for identifying a disease defined herein above, the synthetic fragments covering specific areas of interest from each potential target are between 10 and 30 amino acid residues. In one embodiment of the method for identifying a disease defined herein above, the purification is by HPLC. In one embodiment of the method for identifying a disease defined herein above, the characterization of the glycosylation products is by MALDI-TOF. In one embodiment the kit of the invention comprises items useful in the method defined herein above. Device The glycopeptides of the invention have been used to construct a device for detecting disease, in particular cancer and especially colorectal cancer. One way of constructing the device is the Mucin O-glycopeptide array print method demonstrated in example 2. Thus, in one aspect the method defined herein, wherein the at least two, or the one or more O-glycosylated peptides are conjugated to a surface form a glycopeptide array device discussed above. In one aspect the invention is directed to a device comprising at least two different O-glycosylated peptides conjugated to a surface, wherein the at least two different peptides are selected from the peptides defined herein above. In an analogous embodiment the method is reversed such that the antibodies defined herein above are conjugated to a surface thus forming an antibody array device of the present invention. In one such embodiment the invention relates to a device comprising a plurality of different antibodies conjugated to a surface, wherein one or more of the antibodies is/are selected from the antibodies defined herein above In a further embodiment the invention relates to the use of the device defined herein above. In another aspect, the present invention relates to a device comprising a plurality of glycosylated peptides attached to a surface, wherein at least a part of the peptides are selected from the peptides defined herein above. As discussed above, the glycopeptides and the antibodies of the invention act in a lock-and-key manner. Thus, in one aspect, the present invention relates to a device comprising a plurality of antibodies covalently attached to a surface, wherein at least a part of the antibodies are selected from the antibodies as defined herein, i.e. including but not limited to the antibodies selected from the group consisting of MAb 5C10, MAb 3C9, MAb 4D9, MAb 6C11 and MAb 6E3. In one aspect, the present invention relates to a mucin peptide as defined herein above for use as a medicament, in particular for use in a method of treatment of cancer. Said mucin peptide used in said method of treatment of cancer can e.g. include immunisation of an individual by administering to said individual the glycosylated mucin peptide. The medical use also applies to the other part of the lock-and-key invention. Thus, in this aspect the present invention relates to a method of immunising an individual, by administering to said individual an antibody of the invention In one aspect, the present invention relates to a device comprising a plurality of glycosylated peptides attached to a surface, wherein at least a part of the peptides are selected from the peptides defined herein above. In a further aspect, the present invention relates to a device comprising a plurality of antibodies covalently attached to a surface, wherein at least a part of the antibodies are selected from the antibodies defined herein above. In a further embodiment, the device comprises a mixture of glycosylated peptides and antibodies according to the present invention, and may thus be used as a multi-detection tool. In one aspect the invention relates to a device comprising a plurality of glycosylated peptides conjugated to a surface, wherein at least a part of the peptides are selected from the peptides defined herein above. In one aspect the invention relates to a device comprising a plurality of antibodies conjugated to a surface, wherein at least a part of the antibodies are selected from the antibodies defined herein above. In one aspect the present invention relates to the use of the device defined herein above, in a method of identifying auto-antibodies associated with disease, said method comprising contacting said device with a sample from a host organism. In one aspect the present invention relates to the use of the device defined herein above in a method of identifying O-glycosylated peptides associated with disease, said method comprising contacting said device with a sample from a host organism. In one embodiment the disease identifiable using said device is cancer, wherein the cancer is selected from the group consisting of colorectal cancer, breast cancer, oral cancer, gastric cancer, esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer, basal cell cancer, squamous cell cancer, malignant melanoma, bladder cancer, endometrial cancer and cervix cancer. In one embodiment the disease identifiable using said device is colorectal cancer. Methods of Treating Cancer Passive Immunisation Passive immunity is the transfer of active humoral immunity in the form of readymade antibodies, such as the antibodies of the present invention. Passive immunity can occur naturally, when maternal antibodies are transferred to the fetus through the placenta, and can also be induced artificially, when high levels of human antibodies specific for e.g. an O-glycosylated mucin peptide of the invention is transferred to non-immune individuals. One method of treating cancer, such as colorectal cancer is passive immunisation. In one aspect the antibody defined herein can be used in a method of treatment of cancer comprising passive immunisation. The cancer is selected from the group consisting of colorectal cancer, breast cancer, oral cancer, gastric cancer, esophageal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, lung cancer, renal cancer, prostate cancer, hepatocellular carcinoma, testis cancer, basal cell cancer, squamous cell cancer, malignant melanoma, bladder cancer, endometrial cancer and cervix cancer. In one embodiment, one or more antibodies of the invention have been conjugated to one or more toxins capable of destroying the cells such as cancer cells. A toxin is understood as an agent having cytotoxic properties. A toxin may be e.g. a biotoxin such as a toxin produced by microorganisms or a protein isolated from the venom of the cone snail, spider, snake, scorpion, jellyfish, wasp, bee, ant, termite, honeybee, wasp, poison dart frog. In one embodiment the toxin is selected from the group consisting of cyanotoxins, hemotoxins, necrotoxins, cytotoxins such as but not limited to ricin and apitoxin. In one embodiment the toxin conjugated to the antibody of the invention is selected from the group consisting of small molecules, metals, metal ions, small inorganic molecules, proteins, peptides, glycopeptides, RNA, DNA and siRNA. In one embodiment the toxin conjugated to the antibody of the invention is a venom from spider, snake, scorpion, jellyfish, wasp, bee, ant, termite, honeybee, wasp or poison dart frog. In one embodiment the toxin conjugated to the antibody of the invention is selected from the group consisting of cyanotoxin, hemotoxin, necrotoxin and cytotoxin. In one embodiment a toxin has been conjugated to the antibody of the invention as defined herein above. In one embodiment the toxin-conjugated antibodies of the invention are administered to a patient afflicted with disease, such as cancer. In one embodiment, the combination of toxins is such that individually they are not potent cell killers, but when presented simultaneously to an individual cell their combined effects are lethal to that cell. In one embodiment the toxin conjugated antibody of the invention targets a cancer cell that presents a matching O-glycosylated mucin peptide epitope on the cell surface, resulting in that the conjugated toxin can act to destroy the cancer cell presenting the O-glycosylated mucin peptide epitope on the cell surface. In a further embodiment of the invention, one or more antibodies capable of recognising different O-glycosylated mucin targets presented by cells of a particular cancer are administered to the patient; these antibodies can similarly be conjugated to one or more toxins capably of destroying the cancer cells. In a particular embodiment, the combination of toxins is such that individually they are not potent cell killers, but when presented simultaneously to an individual cell their combined effects are lethal to that cell. 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EXAMPLES Example 1: Sequences of the Invention Overview of Sequences SEQ ID NO: 1 MUC4SEQ ID NO: 2 MUC1SEQ ID NO: 3 MUC4 peptideSEQ ID NO: 4 MUC4 peptideSEQ ID NO: 5 MUC4 peptideSEQ ID NO: 6 MUC4 peptideSEQ ID NO: 7 MUC4 peptideSEQ ID NO: 8 MUC4 peptideSEQ ID NO: 9 MUC4 peptideSEQ ID NO: 10 MUC4 peptideSEQ ID NO: 11 MUC4 peptideSEQ ID NO: 12 MUC4 peptideSEQ ID NO: 13 MUC4 peptideSEQ ID NO: 14 MUC4 peptideSEQ ID NO: 15 MUC4 peptideSEQ ID NO: 16 MUC4 peptideSEQ ID NO: 17 MUC4 peptideSEQ ID NO: 18 MUC4 peptideSEQ ID NO: 19 MUC4 peptideSEQ ID NO: 20 MUC4 peptideSEQ ID NO: 21 MUC4 peptideSEQ ID NO: 22 MUC4 peptideSEQ ID NO: 23 MUC4 peptideSEQ ID NO: 24 MUC1 peptideSEQ ID NO: 25 MUC2 peptideSEQ ID NO: 26 MUC2SEQ ID NO: 27 MUC5ACSEQ ID NO: 28 MUC6SEQ ID NO: 29 MUC7SEQ ID NO: 30 recMUC4 peptideSEQ ID NO: 31 recMUC1 peptideSEQ ID NO: 32 recMUC2 peptideSEQ ID NO: 33 recMUC5AC peptideSEQ ID NO: 34 recMUC6 peptideSEQ ID NO: 35 recMUC7 peptideSEQ ID NO: 36 MUC3SEQ ID NO: 37 MUC3BSEQ ID NO: 38 MUC5BSEQ ID NO: 39 MUC8SEQ ID NO: 40 MUC12SEQ ID NO: 41 MUC13SEQ ID NO: 42 MUC14SEQ ID NO: 43 MUC15SEQ ID NO: 44 MUC16SEQ ID NO: 45 MUC17SEQ ID NO: 46 MUC19SEQ ID NO: 47 MUC20SEQ ID NO: 48 MUC 21SEQ ID NO: 49 MUC HEGSEQ ID NO: 50 MUC9SEQ ID NO: 51 MUC18SEQ ID NO: 52 p53 peptideSEQ ID NO: 53 p53 peptideSEQ ID NO: 54 p53 peptideSEQ ID NO: 55 p53 peptideSEQ ID NO: 56 p53 peptideSEQ ID NO: 57 p53 peptideSEQ ID NO: 58 p53 peptideSEQ ID NO: 59 p53 peptideSEQ ID NO: 60 p53 peptideSEQ ID NO: 61 p53 peptideSEQ ID NO: 62 p53 peptideSEQ ID NO: 63 p53 peptideSEQ ID NO: 64 p53 peptideSEQ ID NO: 65 p53 peptideSEQ ID NO: 66 p53 peptideSEQ ID NO: 67 p53 peptideSEQ ID NO: 68 p53 peptideSEQ ID NO: 69 p53 peptide Sequence List (MUC4; human)>sp|Q99102|MUC4_HUMAN Mucin-4 OS =Homo sapiensGN = MUC4 PE = 1 SV = 2SEQ ID NO: 1MKGARWRRVPWVSLSCLCLCLLPHVVPGTTEDTLITGSKTPAPVTSTGSTTATLEGQSTAASSRTSNQDISASSQNHQTKSTETTSKAQTDTLTQMMTSTLFSSPSVHNVMETVTQETAPPDEMTTSFPSSVTNTLMMTSKTITMTTSTDSTLGNTEETSTAGTESSTPVTSAVSITAGQEGQSRTTSWRTSIQDTSASSQNHWTRSTQTTRESQTSTLTHRTTSTPSFSPSVHNVTGTVSQKTSPSGETATSSLCSVTNTSMMTSEKITVTTSTGSTLGNPGETSSVPVTGSLMPVTSAALVTVDPEGQSPATFSRTSTQDTTAFSKNHQTQSVETTRVSQINTLNTLTPVTTSTVLSSPSGFNPSGTVSQETFPSGETTISSPSSVSNTFLVTSKVFRMPISRDSTLGNTEETSLSVSGTISAITSKVSTIWWSDTLSTALSPSSLPPKISTAFHTQQSEGAETTGRPHERSSFSPGVSQEIFTLHETTTWPSSFSSKGHTTWSQTELPSTSTGAATRLVTGNPSTRAAGTIPRVPSKVSAIGEPGEPTTYSSHSTTLPKTTGAGAQTQWTQETGTTGEALLSSPSYSVIQMIKTATSPSSSPMLDRHTSQQITTAPSTNHSTIHSTSTSPQESPAVSQRGHTRAPQTTQESQTTRSVSPMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATTFAPAPTGNGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDSHTTQSTTELLSASASHGAIPVSTGMASSIVPGTFHPTLSEASTAGRPTGQSSPTSPSASPQETAAISRMAQTQRTGTSRGSDTISLASQATDTFSTVPPTPPSITSSGLTSPQTQTHTLSPSGSGKTFTTALISNATPLPVTSTSSASTGHATPLAVSSATSASTVSSDSPLKMETSGMTTPSLKTDGGRRTATSPPPTTSQTIISTIPSTAMHTRSTAAPIPILPERGVSLFPYGADAGDLEFVRRTVDFTSPLFKPATGFPLGSSLRDSLYFTDNGQIIFPESDYQIFSYPNPLPTGFTGRDPVALVAPFWDDADFSTGRGTTFYQEYETFYGEHSLLVQQAESWIRKITNNGGYKARWALKVTWVNAHAYPAQWTLGSNTYQAILSTDGSRSYALFLYQSGGMQWDVAQRSGNPVLMGFSSGDGYFENSPLMSQPVWERYRPDRFLNSNSGLQGLQFYRLHREERPNYRLECLQWLKSQPRWPSWGWNQVSCPCSWQQGRRDLRFQPVSIGRWGLGSRQLCSFTSWRGGVCCSYGPWGEFREGWHVQRPWQLAQELEPQSWCCRWNDKPYLCALYQQRRPHVGCATYRPPQPAWMFGDPHITTLDGVSYTFNGLGDFLLVGAQDGNSSFLLQGRTAQTGSAQATNFIAFAAQYRSSSLGPVTVQWLLEPHDAIRVLLDNQTVTFQPDHEDGGGQETFNATGVLLSRNGSEVSASFDGWATVSVIALSNILHASASLPPEYQNRTEGLLGVWNNNPEDDFRMPNGSTIPPGSPEEMLFHFGMTWQINGTGLLGKRNDQLPSNFTPVFYSQLQKNSSWAEHLISNCDGDSSCIYDTLALRNASIGLHTREVSKNYEQANATLNQYPPSINGGRVIEAYKGQTTLIQYTSNAEDANFTLRDSCTDLELFENGTLLWTPKSLEPFTLEILARSAKIGLASALQPRTVVCHCNAESQCLYNQTSRVGNSSLEVAGCKCDGGTFGRYCEGSEDACEEPCFPSVHCVPGKGCEACPPNLTGDGRHCAALGSSFLCQNQSCPVNYCYNQGHCYISQTLGCQPMCTCPPAFTDSRCFLAGNNFSPTVNLELPLRVIQLLLSEEENASMAEVNASVAYRLGTLDMRAFLRNSQVERIDSAAPASGSPIQHWMVISEFQYRPRGPVIDFLNNQLLAAVVEAFLYHVPRRSEEPRNDVVFQPISGEDVRDVTALNVSTLKAYFRCDGYKGYDLVYSPQSGFTCVSPCSRGYCDHGGQCQHLPSGPRCSCVSFSIYTAWGEHCEHLSMKLDAFFGIFFGALGGLLLLGVGTFVVLRFWGCSGARFSYFLNSAEALP(MUC1; human)>sp|P15941|MUC1_HUMAN Mucin-1 OS =Homo sapiensGN = MUC1 PE = 1 SV = 2SEQ ID NO: 2MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAAASANL(human MUC4 peptide)SEQ ID NO: 3PMTDTKTVTTPGSSFTA(human MUC4 peptide)SEQ ID NO: 4PGSSFTASGHSPSEIVPQD(human MUC4 peptide)SEQ ID NO: 5SEIVPQDAPTISAATTFAPA(human MUC4 peptide)SEQ ID NO: 6TTFAPAPTGNGHTTQAPTTA(human MUC4 peptide)SEQ ID NO: 7TTQAPTTALQAAPSSHD(human MUC4 peptide)SEQ ID NO: 8APSSHDATLGPSGGTSLSKT(human MUC4 peptide)SEQ ID NO: 9SLSKTGALTLANSVVSTP(human MUC4 peptide)SEQ ID NO: 10NSVVSTPGGPEGQWTSASAS(human MUC4 peptide)SEQ ID NO: 11TSASASTSPRTAAAMTHT(human MUC4 peptide)SEQ ID NO: 12AAAMTHTHQAESTEASGQT (human MUC4 peptide)SEQ ID NO: 13EASGQTQTSEPASSGSRTT(human MUC4 peptide)SEQ ID NO: 14PASSGSRTTSAGTATPSSS(human MUC4 peptide)SEQ ID NO: 15TATPSSSGASGTTPSGSEGI(human MUC4 peptide)SEQ ID NO: 16SGSEGISTSGETTRFSSN(human MUC4 peptide)SEQ ID NO: 17GETTRFSSNPSRDSHTT(human MUC4 peptide)SEQ ID NO: 18PVTSPSSASTGHTTPLPVTDTSSASTGDTTP(human MUC4 peptide)SEQ ID NO: 19LPVTSLSSVSTGDTTPLPVTSPSSASTGH(human MUC4 peptide)SEQ ID NO: 20LPVTSPSSASTGHASPLLVTDASSASTGQ(human MUC4 peptide)SEQ ID NO: 21PLPVTSPSSASTGHASPLLVTDASSASTGQ(human MUC4 peptide)SEQ ID NO: 22STGDTLPLPVTDTSSV(human MUC4 peptide)SEQ ID NO: 23PVTYASSASTGDTTPLPVTDTSSVSTGHAT(human MUC4 peptide)SEQ ID NO: 24VTSAPDTRPAPGSTAPPAHG(human MUC4 peptide)SEQ ID NO: 25PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC(MUC2 human)>sp|Q02817|MUC2_HUMAN Mucin-2 OS =Homo sapiensGN = MUC2 PE = 1 SV = 2SEQ ID NO: 26MGLPLARLAAVCLALSLAGGSELQTEGRTRYHGRNVCSTWGNFHYKTFDGDVFRFPGLCDYNFASDCRGSYKEFAVHLKRGPGQAEAPAGVESILLTIKDDTIYLTRHLAVLNGAVVSTPHYSPGLLIEKSDAYTKVYSRAGLTLMWNREDALMLELDTKFRNHTCGLCGDYNGLQSYSEFLSDGVLFSPLEFGNMQKINQPDVVCEDPEEEVAPASCSEHRAECERLLTAEAFADCQDLVPLEPYLRACQQDRCRCPGGDTCVCSTVAEFSRQCSHAGGRPGNWRTATLCPKTCPGNLVYLESGSPCMDTCSHLEVSSLCEEHRMDGCFCPEGTVYDDIGDSGCVPVSQCHCRLHGHLYTPGQEITNDCEQCVCNAGRWVCKDLPCPGTCALEGGSHITTFDGKTYTFHGDCYYVLAKGDHNDSYALLGELAPCGSTDKQTCLKTVVLLADKKKNAVVFKSDGSVLLNQLQVNLPHVTASFSVFRPSSYHIMVSMAIGVRLQVQLAPVMQLFVTLDQASQGQVQGLCGNFNGLEGDDFKTASGLVEATGAGFANTWKAQSTCHDKLDWLDDPCSLNIESANYAEHWCSLLKKTETPFGRCHSAVDPAEYYKRCKYDTCNCQNNEDCLCAALSSYARACTAKGVMLWGWREHVCNKDVGSCPNSQVFLYNLTTCQQTCRSLSEADSHCLEGFAPVDGCGCPDHTFLDEKGRCVPLAKCSCYHRGLYLEAGDVVVRQEERCVCRDGRLHCRQIRLIGQSCTAPKIHMDCSNLTALATSKPRALSCQTLAAGYYHTECVSGCVCPDGLMDDGRGGCVVEKECPCVHNNDLYSSGAKIKVDCNTCTCKRGRWVCTQAVCHGTCSIYGSGHYITFDGKYYDFDGHCSYVAVQDYCGQNSSLGSFSIITENVPCGTTGVTCSKAIKIFMGRTELKLEDKHRVVIQRDEGHHVAYTTREVGQYLVVESSTGIIVIWDKRTTVFIKLAPSYKGTVCGLCGNFDHRSNNDFTTRDHMVVSSELDFGNSWKEAPTCPDVSTNPEPCSLNPHRRSWAEKQCSILKSSVFSICHSKVDPKPFYEACVHDSCSCDTGGDCECFCSAVASYAQECTKEGACVFWRTPDLCPIFCDYYNPPHECEWHYEPCGNRSFETCRTINGIHSNISVSYLEGCYPRCPKDRPIYEEDLKKCVTADKCGCYVEDTHYPPGASVPTEETCKSCVCTNSSQVVCRPEEGKILNQTQDGAFCYWEICGPNGTVEKHFNICSITTRPSTLTTFTTITLPTTPTSFTTTTTTTTPTSSTVLSTTPKLCCLWSDWINEDHPSSGSDDGDREPFDGVCGAPEDIECRSVKDPHLSLEQHGQKVQCDVSVGFICKNEDQFGNGPFGLCYDYKIRVNCCWPMDKCITTPSPPTTTPSPPPTTTTTLPPTTTPSPPTTTTTTPPPTTTPSPPITTTTTPLPTTTPSPPISTTTTPPPTTTPSPPTTTPSPPTTTPSPPTTTTTTPPPTTTPSPPMTTPITPPASTTTLPPTTTPSPPTTTTTTPPPTTTPSPPTTTPITPPTSTTTLPPTTTPSPPPTTTTTPPPTTTPSPPTTTTPSPPTITTTTPPPTTTPSPPTTTTTTPPPTTTPSPPTTTPITPPTSTTTLPPTTTPSPPPTTTTTPPPTTTPSPPTTTTPSPPITTTTTPPPTTTPSSPITTTPSPPTTTMTTPSPTTTPSSPITTTTTPSSTTTPSPPPTTMTTPSPTTTPSPPTTTMTTLPPTTTSSPLTTTPLPPSITPPTFSPFSTTTPTTPCVPLCNWTGWLDSGKPNFHKPGGDTELIGDVCGPGWAANISCRATMYPDVPIGQLGQTVVCDVSVGLICKNEDQKPGGVIPMAFCLNYEINVQCCECVTQPTTMTTTTTENPTPPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTPTTTPITTTTTVTPTPTPTGTQTGPPTHTSTAPIAELTTSNPPPESSTPQTSRSTSSPLTESTTLLSTLPPAIEMTSTAPPSTPTAPTTTSGGHTLSPPPSTTTSPPGTPTRGTTTGSSSAPTPSTVQTTTTSAWTPTPTPLSTPSIIRTTGLRPYPSSVLICCVLNDTYYAPGEEVYNGTYGDTCYFVNCSLSCTLEFYNWSCPSTPSPTPTPSKSTPTPSKPSSTPSKPTPGTKPPECPDFDPPRQENETWWLCDCFMATCKYNNTVEIVKVECEPPPMPTCSNGLQPVRVEDPDGCCWHWECDCYCTGWGDPHYVTFDGLYYSYQGNCTYVLVEEISPSVDNFGVYIDNYHCDPNDKVSCPRTLIVRHETQEVLIKTVHMMPMQVQVQVNRQAVALPYKKYGLEVYQSGINYVVDIPELGVLVSYNGLSFSVRLPYHRFGNNTKGQCGTCTNTTSDDCILPSGEIVSNCEAAADQWLVNDPSKPHCPHSSSTTKRPAVTVPGGGKTTPHKDCTPSPLCQLIKDSLFAQCHALVPPQHYYDACVFDSCFMPGSSLECASLQAYAALCAQQNICLDWRNHTHGACLVECPSHREYQACGPAEEPTCKSSSSQQNNTVLVEGCFCPEGTMNYAPGFDVCVKTCGCVGPDNVPREFGEHFEFDCKNCVCLEGGSGIICQPKRCSQKPVTHCVEDGTYLATEVNPADTCCNITVCKCNTSLCKEKPSVCPLGFEVKSKMVPGRCCPFYWCESKGVCVHGNAEYQPGSPVYSSKCQDCVCTDKVDNNTLLNVIACTHVPCNTSCSPGFELMEAPGECCKKCEQTHCIIKRPDNQHVILKPGDFKSDPKNNCTFFSCVKIHNQLISSVSNITCPNFDASICIPGSITFMPNGCCKTCTPRNETRVPCSTVPVTTEVSYAGCTKTVLMNHCSGSCGTFVMYSAKAQALDHSCSCCKEEKTSQREVVLSCPNGGSLTHTYTHIESCQCQDTVCGLPTGTSRRARRSPRHLGSG(MUC5AC, human)>tr|O75372|O75372_HUMAN Gastric mucin (Fragment) OS =Homo sapiensGN = MUC5AC PE = 1 SV = 1SEQ ID NO: 27MSVGRRKLALLWALALALACTRHTGHAQDGSSESSYKHHPALSPIARGPIGVPLRGATVFPSLRTIPVVRASNPAHNGRVCSTWGSFHYKTFDGDVFRFPGLCNYVFSEHCGAAYEDFNIPATPQPGVSGPHAEQGPHEGGWRGHPADQGLRPGQRPPGPAALQPVWGPHSARAAATPRWKPGWALSSCGTTMTACCWKLDTKYANKNLWALWGLQRDARGORAPLPQHQADTHGIREPAERWTNPRSSVRTLSLNPRRTAPLALASCEELLHGQLFSGCVALVDVGSYLEACRQDLCFCEDTDLLSCVCHTLAEYSROCTHAGGLPQDWRGPDFCPQKCPNNMQYHECRSPCADTCSNQEHSRACEDHCVAGCFCPEGTVLDDIGQTGCVPVSKCACVYNGAAYAPGATYSTDCTNCTCSGGRWSCQEVPCPGTCSVLGGAHFSTFDGKQYTVHGDCSYVLTKPCDSSAFTVLAELRRCGLTDSETCLKSVTLSLDGAQTVVVIKASGEVFLNQIYTQLPISAANVTIFRPSTFFIIAQTSLGLQLNLQLVPTMQLFMQLAPKLRGQTCGLCGNFNSIQADDFRTLSGVVEATAAAFFNTFKTQAACPNIRNSFEDPCSLSVENEKYAQHWCSQLTDADGPFGRCHAAVKPGTYYSNCMFDTCNCERSEDCLCAALSSYVHACAAKGVQLGGWRDGVCTKPMTTCPKSMTYHYHVSTCQPTCRSLSEGDITCSVGFIPVDGCICPKGTFLDDTGKCVQASNCPCYHRGSMIPNGESVHDSGAICTCTHGKLSCIGGQAPAPVCAAPMVFFDCRNATPGDTGAGCQKSCHTLDMTCYSPQCVPGCVCPDGLVADGEGGCITAEDCPCVHNEASYRAGQTIRVGCNTCTCDSRMWRCTDDPCLATCAVYGDGHYLTFDGQSYSFNEETASTRWCRTAVAGKTAPRTPFVLSPRTSPAAPQGPPAPRPSRFSWGNFELKLSHGKVEVIGTDESQEVPYTIRQMGIYLVVDTDIGLVLLWDKKTSIFINLSPEFKGRVCGLCGNFDDIAVNDFATRSRSVVGDVLEFGNSWKLSPSCPDALAPKDPCTANPFRKSWAQKQCSILHGPTFAACHAHVEPARYYEACVNDACACDSGGDCECFCTAVARYAQACHEVGTCVCLRTPSICPLFCDYYNPEGQCEWHYQPCGVPCLRTCRNPRGDCLRDVRGLEGCYPKCPPEAPIFDEDKMQCVATCPTPPLPPRCHVHGKSYRPGAVVPSDKNCQSCLCTERGVECTYKAEACVCTYNGQRFHPGDVIYHTTDGTGGCISARCGANGTIERRVYPCSPTTPVPPTTFSFSTPPLVVSSTHTPSNGPSSAHTGPPSSAWPTTAGTSPRT(MUC6, human)>sp|Q6W4X9|MUC6_HUMAN Mucin-6 OS =Homo sapiensGN = MUC6 PE = 1 SV = 2SEQ ID NO: 28MVQRWLLLSCCGALLSAGLANTSYTSPGLQRLKDSPQTAPDKGQCSTWGAGHFSTFDHHVYDFSGTCNYIFAATCKDAFPSFSVQLRRGPDGSISRIIVELGASVVTVSEAIISVKDIGVISLPYTSNGLQITPFGQSVRLVAKQLELELEVVWGPDSHLMVLVERKYMGQMCGLCGNFDGKVTNEFVSEEGKFLEPHKFAALQKLDDPGEICTFQDIPSTHVRQAQHARGCTQLLTLVAPECSVSKEPFVLSCQADVAAAPQPGPQNSSYATLSEYSRQCSMVGQPVALRSPGLCSVGQCPANQVYQECGSACVKTCSNSEHSCSSSCTFGCFCPEGTDLNDLSNNHTCVPVTQCPCVLHGAMYAPGEVTIAACQTCRCTLGRWVCTERPCPGHCSLEGGSFVTTFDARPYRFHGTCTYILLQSPQLPEDGALMAVYDKSGVSHSETSLVAVVYLSRQDKIVISQDEVVTNNGEAKWLPYKTRNITVFRQTSTHLQMATSFGLELVVQLRPIFQAYVTVGPQFRGQTRGLCGNFNGDTTDDFTTSMGIAEGTASLFVDSWRAGNCPDALERETDPCSMSQLNKVCAETHCSMLLRTGTVFERCHATVNPAPIYKRCMYQACNYEETFPHICAALGDYVHACSLRGVLLWGWRSSVDNCTIPCTGNTTFSYNSQACERTCLSLSDRATECHHSAVPVDGCNCPDGTYLNOKGECVRKAQCPCILEGYKFILAEQSTVINGITCHCINGRLSCPQRLQMFLASCQAPKTFKSCSQSSENKFLHTDCRTCSCSRGRWACQQGTHCPSTCTLYGEGHVITFDGQRFVFDGNCEYILATDVCGVNYSQPTFKILTENVICGNSGVTCSRAIKIFLGGLSVVLADRNYTVTGEEPHVQLGVTPGALSLVVDISIPGRYNLTLIWNRHMTILIRIARASQDPLCGLCGNFNGNMKDDFETRSRYVASSELELVNSWKESPLCGDVSFVTDPCSLNAFRRSWAERKCSVINSQTFATCHSKVYHLPYYEACVRDACGCDSGGDCECLCDAVAAYAQACLDKGVCVDWRTPAFCPIYCGFYNTHTQDGHGEYQYTQEANCTWHYQPCLCPSQPQSVPGSNIEGCYNCSQDEYFDHEEGVCVPCMPPTTPQPPTTPQLPTTGSRPTQVWPMTGTSTTIGLLSSTGPSPSSNHTPASPTQTPLLPATLTSSKPTASSGEPPRPTTAVTPQATSGLPPTATLRSTATKPTVTQATTRATASTASPATTSTAQSTTRTTMTLPTPATSGTSPTLPKSTNQELPGTTATQTTGPRPTPASTTGPTTPQPGQPTRPTATETTQTRTTTEYTTPQTPHTTHSPPTAGSPVPSTGPVTATSFHATTTYPTPSHPETTLPTHVPPFSTSLVTPSTHTVITPTHAQMASSASNHSAPTGTIPPPTTLKATGSTHTAPPITPTTSGTSQAHSSFSTNKTPTSLHSHTSSTHHPEVTPTSTTSITPNPTSTRTRTPMAHTNSATSSRPPPPFTTHSPPTGSSPFSSTGPMTATSFKTTTTYPTPSHPQTTLPTHVPPFSTSLVTPSTHTVITPTHAQMATSASIHSMPTGTIPPPTTLKATGSTHTAPTMTLTTSGTSQALSSLNTAKTSTSLHSHTSSTHHAEATSTSTTNITPNPTSTGTPPMTVTTSTRTPVAHTTSATSSRLPTPFTTHSPPTGTTPISSTGPVTATSFQTTTTYPTPSHPHTTLPTHVPSFSTSLVTPSTHTVIIPTHTQMATSASIHSMPTGTIPPPTTIKATGSTHTAPPMTPTTSGTSQSPSSFSTAKTSTSLPYHTSSTHHPEVTPTSTTNITPKHTSTGTRTPVAHTTSASSSRLPTPFTTHSPPTGSSPFSSTGPMTATSFQTTTTYPTPSHPQTTLPTHVPPFSTSLVTPSTHTVIITTHTQMATSASIHSTPTGTVPPPTTLKATGSTHTAPPMTVTTSGTSQTHSSFSTATASSSFISSSSWLPQNSSSRPPSSPITTQLPHLSSATTPVSTTNQLSSSFSPSPSAPSTVSSYVPSSHSSPQTSSPSVGTSSSFVSAPVHSTTLSSGSHSSLSTHPTTASVSASPLFPSSPAASTTIRATLPHTISSPFTLSALLPISTVTVSPTPSSHLASSTIAFPSTPRTTASTHTAPAFSSQSTTSRSTSLTTRVPTSGFVSLTSGVTGIPTSPVTNLTTRHPGPTLSPTTRFLTSSLTAHGSTPASAPVSSLGTPTPTSPGVCSVREQQEEITFKGCMANVTVTRCEGACISAASFNIITQQVDARCSCCRPLHSYEQQLELPCPDPSTPGRRLVLTLQVFSHCVCSSVACGD(MUC7, human)>sp|Q8TAX7|MUC7_HUMAN Mucin-7 OS =Homo sapiensGN = MUC7 PE = 1 SV = 1SEQ ID NO: 29MKTLPLFVCICALSACFSFSEGRERDHELRHRRHHHQSPKSHFELPHYPGLLAHQKPFIRKSYKCLHKRCRPKLPPSPNNPPKFPNPHQPPKHPDKNSSVVNPTLVATTQIPSVTFPSASTKITTLPNVTFLPQNATTISSRENVNTSSSVATLAPVNSPAPQDTTAAPPTPSATTPAPPSSSAPPETTAAPPTPSATTQAPPSSSAPPETTAAPPTPPATTPAPPSSSAPPETTAAPPTPSATTPAPLSSSAPPETTAVPPTPSATTLDPSSASAPPETTAAPPTPSATTPAPPSSPAPQETTAAPITTPNSSPTTLAPDTSETSAAPTHQTITSVTTQTTTTKQPTSAPGQNKISRFLLYMKNLLNRIIDDMVEQ(recMUC4)SEQ ID NO: 30PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDSHTT(recMUC1)SEQ ID NO: 31MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDPNSVTSAPDTRPAPGSTAPQAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDT(recMUC2)SEQ ID NO: 32MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDPNSSSVDKLDIEFLQPGGSVQCCECVTQPTTMTTTTTENPTPTPITTTTTVTPTPTPTSTQSTTPTPITTTNTVTPTPTPTGTQT(recMUC5AC)SEQ ID NO: 33MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPTSSTTSTPQTSTTSASTTSITSGPGTTPSPVPTTSTTSAPTTSTTSAATTSTISAPTTSTTSAPTTSTTSASTASKTSGLGTTPSPIPTTSTTSPPTTSTTSASTASKTSGPGTTPSPVPTTSTIFAPRTSTTSASTTSTTPGPGTTPSPVPTTSTASVSKTSTSHVSISKTTHSQAAALEHHHHHH(recMUC6)SEQ ID NO: 34MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIRSTSLVTPSTHTVITPTHAQMATSASNHSAPTGTIPPPTTLKATGSTHTAPPITPTTSGTSQAHSSFSTNKAAALEHHHHHH(recMUC7)SEQ ID NO: 35MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIPAPQDTTAAPPTPSATTPAPPSSSAPPETTAAPPTPSATTQAPPSSSAPPETTAAPPTPPATTPAPPSSSAPPETTAAPPTPSATTPAPLSSSAPPETTAVPPTPSATTLDPSSASAPPETTAAPPTPSATTPAPPSSPAPQETTAAPITTPNSSPTTLAPDTSETSAAPTHQTTTSVTTQTTTTKQPTSAPAAALEHHHHHH(hMUC3)SEQ ID NO: 36MQLLGLLGLLWMLKASPWATGTLSTATSISQVPFPRAEAASAVLSNSPHSRDLAGWPLGVPQLASPAPGHRENAPMTLTTSPHDTLISETLLNSPVSSNTSTTPTSKFAFKVETTPPTVLVYSATTECVYPTSFIITISHPTSICVTTTQVAFTSSYTSTPVTQKPVTTVTSTYSMTTTEKGTSAMTSSPSTTTARETPIVTVTPSSVSATDTTFHTTISSTTRTTERTPLPTGSIHTTTSPTPVFTTLKTAVTSTSSITSSITSTNTVTSMTTTASQPTATNTLSSPTRTILSSTPVLSTETITSGITNTTPLSTLVTTLPTTISRSTPTSETTYPTSPXXXXXXXXXXAMTSPPPVSSSITPTNTMTSMRTTTYWPTATNTLSPLTSSILSSTPVPSTEMITSHTTNTTPLSTLVTTLLTTITRSTPTSETTYPTSPTSIVSDSTTEITYSTSITGTLSTATTLPPTSSSLPTTETATMTPTTTLITTTPNTTSHSTPSFTSSTIYSTVSTSTTAISSASPTSGTMVTSTTMTPSSLSTDTPSTTPTTITYPSVGSTGFLTTATDLTSTFTVSSSSAMSKSVIPSSPSIQNTETSSLVSMTSATTPSLRPTITSTDSTLTSSLLTTFPSTYSFSSSMSASSAGTTHTETISSLPASTNTIHTTAESALAPTTTTSFTTSPTMEPPSTTVATTGTGQTTFPSSTATFLETTTLTPTTDFSTESLTTAMTSTPPITSSITPTDTMTSMRTTTSWPTATNTLSPLTSSILSSTPVPSTEVTTSHTTNTNPVSTLVTTLPITITRSTLTSETAYPSSPTSTVTESTTEITYPTTMTETSSTATSLPPTSSLVSTAETAKTPTTNLXXXXXXXXXXFTSSTSLLHSQHTTXLPSPSVPTTLGTMVTSTSXIPSSLSTDIPTSQPTTITPSSVGITGSLPMMTDLTSVYTVSSMSARPTSVIPSSPTVQNTETSIFVSMMSATTPSGGSTFTSTENTPTRSLLTSFPVTHSFSSSMSASSVGTTHTQSISSPPAITSTLHTTAESTPSPTTTMSFTTFTKMETPSSTVATTGTGQTTFTSSTATSPKTTTLTPTSDISTGSFKTAVSSTPPITSSITSTYTVTSMTTTTPLGPTATNTLPSFTSSVSSSTPVPSTEAITSGTTNTTPLSTLVTTFSNSDTSSTPTSETTYPTSLTSALTDSTTRTTYSTNMTGTLSTVTSLRPTSSSLLTTVTATVPTTNLVTTTTKITSHSTPSFTSSIATTETPSHSTPRFTSSITTTETPSHSTPRFTSSITNTKTTSHSSPSFTSSITTTDSIVXXXXXXXXXXITITETTSHSTPSYTTSITTTETPSHSTPSYTTSITTTETPSHSTPSFTSSITTTETTSHSTPSFTSSIRTTETTSYSTPSFTSSNTITETTSHSTPSYITSITTTETPSSSTPSFSSSITTTETTSHSTPGFTSSITTTETTSHSTPSFTSSITTTETTSHDTPSFTSSITTSETPSHSTPSSTSLITTTKTTSHSTPSFTSSITTTETTSHSARSFTSSITTTETTSHNTRSFTSSITTTETNSHSTTSFTSSITTTETTSHSTPSFSSSITTTETPLHSTPGLPSWVTTTKTTSHITPGLTSSITTTETTSHSTPGFTSSITTTETTSESTPSLSSSTIYSTVSTSTTAITSHFTTSETAVTPTPVTPSSLSTDIPTTSLRTLTPSSVGTSTSLTTTTDFPSIPTDISTLPTRTHIISSSPSIQSTETSSLVGTTSPTMSTVRMTLRITENTPISSFSTSIVVIPETPTQTPPVLTSATGTQTSPAPTTVTFGSTDSSTSTLHTLTPSTALSTIVSTSQVPIPSTHSSTLQTTPSTPSLQTSLTSTSEFTTESFTRGSTSTNAILTSFSTIIWSSTPTIIMSSSPSSASITPVFSTTIHSVPSSPYIFSTENVGSASITGFPSLSSSATTSTSSTSSSLTTALTEITPFSYISLPSTTPCPGTITITIVPASPTDPCVEMDPSTEATSPPTTPLTVFPFTTEMVTCPTSISIQTTLTTYMDTSSMMPESESSISPNASSSTGTGTVPTNTVFTSTRLPTSETWLSNSSVIPLPLPGVSTIPLTMKPSSSLPTILRTSSKSTHPSPPTTRTSETPVATTQTPTTLTSRRTTRITSQMTTQSTLTTTAGTCDNGGTWEQGQCACLPGFSGDRCQLQTRCQNGGQWDGLKCQCPSTFYGSSCEFAVEQVDLDVVETEVGMEVSVDQQFSPDLNDNTSQAYRDFNKTFWNQMQKIFADMQGFTFKGVEILSLRNGSIVVDYLVLLEMPFSPQLESEYEQVKTTLKEGLQNASQDANSCQDSQTLCFKPDSIKVNNNSKTELTPEAICRRAAPTGYEEFYFPLVEATRLRCVTKCTSGVDNAIDCHQGQCVLETSGPACRCYSTDTHWFSGPRCEVAVHWRALVGGLTAGAALLVLLLLALGVRAVRSGWWGGQRRGRSWDQDRKWFETWDEEVVGTFSNWGFEDDGTDKDTNFHVALENVDTTMKVHIKRPEMTSSSV(hMUC3B)SEQ ID NO: 37MQLLGLLSILWMLKSSPGATGTLSTATSTSHVTFPRAEATRTALSNSPHSRYLAEWPQGVPQLASPAPGHRENAPMTLTTSPHDTLISETLLSSLVSSNTSTTPTSKFAFKVETTPPTVLVYSATTECVYPTSFIITISHSTSICVTTTQVTFTSSYTPTPVTQKPVTTVTRTYPMTTTEKGTSAMISSPSTTTARETPIVTVTPSSSVSATDTTFHTTISSTTRTTERTPLPTGSIHTTMSPTPVFTTLKTAVTSTSPITSTITSTNTVTSMTTTTSRPTATNTLSSLTSSILSSTPAPNTEVITSHTTTTTPPSTLVTTLPTAIARSTPTSETTXXXXXXXXXXTIYSTVSSSTTAITSPFTTAETGVTSTPSSPSSLSTDIPTTSLRTLTPLSLSTSTSLTTTTDLPSIPTDISSLPTPIHIISSSPSIQSTETSSLVGTTSPTMSTVRATLRSTENTPISSFSTSIVVTPETPTTQAPPVLMSATGTQTSPVPTTVTFGSMDSSTSTLHTLTPSTALSKIMSTSQFPIPSTHSSTLQTTPSIPSLQTSLTSTSEFTTESFTRGSTSTNAILTSFSTIIWSSTPTIIMSSSPSSASITPVFATTIHSVPSSPYIFSTENVGSASITAFPSLSSSSTTSTSPTSSSLTTALTEITPFSYISLPSTTPCPGTITITIVPASPTDPCVEMDPSTEATSPPTTPLTVFPFTTEMVTCPSSISMQTTLATHMDTSSMTPESESSIIPNASSSTGTGTVPTNTVFTSTRLPTSETWLSNNSVIPTPLPGVSTIPLTMKPSSSLPTILRTSSKSTHPSPPTARTSETSVATTQTPTTLTTRRTTPITSWMTTQSTLTTTAGTCDNGGTWEQGQCACLPGFSGDRCQLQTRCQNGGQWDGLKCQCPSTFYGSSCEFAVEQVDLDVVETEVGMEVSVDQQFSPDLNDNTSQAYRDFNKTFWNQMQKIFADMQGFTFKGVEILSLRNGSIVVDYLVLLEMPFSPQLESEYEQVKTTLKEGLQNASQDANSCQDSQTLCFKPDSIKVNNNSKTELTPEAICRRAAPTGYEEFYFPLVEATRLRCVTKCTSGVDNAIDCHQGQCVLETSGPACRCYSTDTHWFSGPRCEVAVHWRALVGGLTAGAALLVLLLLALGVRAVRSGWWGGQRRGRSWDQDRKWFETWDEEVVGTFSNWGFEDDGTDKDTNFHVALENVDTTMKVHIKRPEMTSSSV(hMUC5B)SEQ ID NO: 38MGAPSACRTLVLALAAMLVVPQAETQGPVEPSWGNAGHTMDGGAPTSSPTRRVSFVPPVTVFPSLSPLNPAHNGRVCSTWGDFHYKTFDGDVFRFPGLCNYVFSEHCRAAYEDFNVQLRRGLVGSRPVVTRVVIKAQGLVLEASNGSVLINGQREELPYSRTGLLVEQSGDYIKVSIRLVLTFLWNGEDSALLELDPKYANQTCGLCGDFNGLPAFNEFYAHNARLTPLQFGNLQKLDGPTEQCPDPLPLPAGNCTDEEGICHRTLLGPAFAECHALVDSTAYLAACAQDLCRCPTCPCATFVEYSROCAHAGGQPRNWRCPELCPRTCPLNMQHQECGSPCTDTCSNPQRAQLCEDHCVDGCFCPPGSTVLDDITHSGCLPLGQCPCTHGGRTYSPGTSFNTTCSSCTCSGGLWQCQDLPCPGTCSVQGGAHISTYDEKLYDLHGDCSYVLSKKCADSSFTVLAELRKCGLTDNENCLKAVTLSLDGGDTAIRVQADGGVFLNSIYTQLPLSAANITLFTPSSFFIVVQTGLGLQLLVQLVPLMQVFVRLDPAHQGQMCGLCGNFNQNQADDFTALSGVVEATGAAFANTWKAQAACANARNSFEDPCSLSVENENYARHWCSRLTDPNSAFSRCHSIINPKPFHSNCMFDTCNCERSEDCLCAALSSYVHACAAKGVQLSDWRDGVCTKYMQNCPKSQRYAYVVDACQPTCRGLSEADVTCSVSFVPVDGCTCPAGTFLNDAGACVPAQECPCYAHGTVLAPGEVVHDEGAVCSCTGGKLSCLGASLQKSTGCAAPMVYLDCSNSSAGTPGAECLRSCHTLDVGCFSTHCVSGCVCPPGLVSDGSGGCIAEEDCPCVHNEATYKPGETIRVDCNTCTCRNRRWECSHRLCLGTCVAYGDGHFITFDGDRYSFEGSCEYILAQDYCGDNTTHGTFRIVTENIPCGTTGTTCSKAIKLFVESYELILQEGTFKAVARGPGGDPPYKIRYMGIFLVIETHGMAVSWDRKTSVFIRLHQDYKGRVCGLCGNFDDNAINDFATRSRSVVGDALEFGNSWKLSPSCPDALAPKDPCTANPFRKSWAQKQCSILHGPTFAACRSQVDSTKYYEACVNDACACDSGGDCECFCTAVAAYAQACHDAGLCVSWRTPDTCPLFCDFYNPHGGCEWHYQPCGAPCLKTCRNPSGHCLVDLPGLEGCYPKCPPSQPFFNEDQMKCVAQCGCYDKDGNYYDVGARVPTAENCQSCNCTPSGIQCAHSLEACTCTYEDRTYSYQDVIYNTTDGLGACLIAICGSNGTIIRKAVACPGTPATTPFTFTTAWVPHSTTSPALPVSTVCVREVCRWSSWYNGHRPEPGLGGGDFETFENLRQRGYQVCPVLADIECRAAQLPDMPLEELGQQVDCDRMRGLMCANSQQSPPLCHDYELRVLCCEYVPCGPSPAPGTSPQPSLSASTEPAVPTPTQTTATEKTTLWVTPSIRSTAALTSQTGSSSGPVTVTPSAPGTTTCQPRCQWTEWFDEDYPKSEQLGGDVESYDKIRAAGGHLCQQPKDIECQAESFPNWTLAQVGQKVHCDVHFGLVCRNWEQEGVFKMCYNYRIRVLCCSDDHCRGRATTPPPTTELETATTTTTQALFSTPQPTSSPGLTRAPPASTTAVPTLSEGLTSPRYTSTLGTATTGGPRQSAGSTEPTVPGVATSTLPTRSALPGTTGSLGTWRPSQPPTLAPTTMATSRARPTGTASTASKEPLTTSLAPTLTSELSTSQAETSTPRTETTMSPLTNTTTSQGTTRCQPKCEWTEWFDVDFPTSGVASGDMETFENIRAAGGKMCWAPKSIECRAENYPEVSIDQVGQVLTCSLETGLTCKNEDQTGRFNMCFNYNVRVLCCDDYSHCPSTLATSSTATPSSTPGTTWILTKPTTTATTTASTGSTATASSTQATAGTPHVSTTATTPTVTSSKATPFSSPGTATALPALRSTATTPTATSFTAIPSSSLGTTWTRLSQTTTPMATMSTATPSSTPETVHTSTVLTTTATTTGATGSVATPSSTPGTAHTTKVLTTTTTGFTATPSSSPGRARTLPVWISTTTTPTTRGSTVTPSSIPGTTHTPTVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTTPIPPVLTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLSPSSPHTVCTAWTSATSGILGTTHITEPSTGTSHTPAATTGTTQHSTPALSSPHPSSRTTESPPSPGTTTPGHTTATSRTTATATPSKTRTSTLLPSQPTSAPITTVVTMGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTELTTTATTTESTGSTATPTSTLRTAPPPKVLTTTATTPTVTSSKATPSSSPGTATALPALRSTATTPTATSVTPIPSSSLGTTWTRLSQTTTPTATMSTATPSSTPETAHTSTVLTATATTTGATGSVATPSSTPGTAHTTKVPTTTTTGFTATPSSSPGTALTPPVWISTTTTPTTRGSTVTPSSIPGTTHTATVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTRPIPPVLTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLSPSSPHTVRTAWTSATSGTLGTTHITEPSTGTSHTPAATTGTTQHSTPALSSPHPSSRTTESPPSPGTTTPGHTTATSRTTATATPSKTRTSTLLPSSPTSAPITTVVTMGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTEQTTAATTTATTGSTAIPSSTPGTAPPPKVLTSQATTPTATSSKATSSSSPRTATTLPVLTSTATKSTATSFTPIPSSTLGTTGTSQNRPPHPMATMSTIHPSSTPETTHTSTVLTTKATTTRATSSMSTPSSTPGTTWILTELTTAATTTAALPHGTPSSTPGTTWILTEPSTTATVTVPTGSTATASSTRATAGTLKVLTSTATTPTVISSRATPSSSPGTATALPALRSTATTPTATSVTAIPSSSLGTAWTRLSQTTTPTATMSTATPSSTPETVHTSTVLTTTATTTRTGSVATPSSTPGTAHTTKVPTTTTTGFTATPSSSPGTALTPPVWISTTTTPTTRGSTVTPSSIPGTTHTATVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTTPIPPVLTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLPPSSPHTVPTAWTSATSGILGTTHITEPSTGTSHTPAATTGTTQPSTPALSSPHPSSRTTESPPSPGTTTPGHTRGTSRTTATATPSKTRTSTLLPSSPTSAPITTVVTTGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLRELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTATPSSTPGTTWILTKLTTTATTTESTGSTATPSSTQGPPAGTPHVSTTATTPTVTSSKATPFSSPGTATALPALRSTATTPTATSFTAIPSSSLGTTWTRLSQTTTPMATMSTATPSSTPETVHTSTVLTTTATTTGATGSVATPSSTPGTAHTTKVPTTTTTGFTVTPSSSPGTARTPPVWISTTTTPTTSGSTVTPSSIPGTTHTPTVLTTTTQPVATGSMATPSSSTQTSGTPPSLITTATTITATGSTTNPSSTPGTTPIPPELTTTATTPAATSSTVTPSSALGTTHTPPVPNTTATTHGRSLSPSSPHTVRTAWTSATSGTLGTTHITEPSTGTSHTPAATTGTTTTSTPALSSPHPSSRTTESPPSPGTTTPGHTTATSRTTATATPSKTRTSTLLPSQPTSAPITTVVTTGCEPQCAWSEWLDYSYPMPGPSGGDFDTYSNIRAAGGAVCEQPLGLECRAQAQPGVPLGELGQVVECSLDFGLVCRNREQVGKFKMCFNYEIRVFCCNYGHCPSTPATSSTAMPSSTPGTTWILTELTTTATTTASTGSTATPSSTPGTAPPPKVLTSPATTPTATSSKATSSSSPRTATTLPVLTSTATKSTATSVTPIPSSTLGTTGTLPEQTTTPVATMSTIHPSSTPETTHTSTVLTTKATTRATSSTSTPSSTPGTTWILTELTTAATTTAGTGPTATPSSTPGTTWILTELTTTATTTASTGSTATLSSTPGTTWILTEPSTTATVTVPTGSTATASSTQATAGTPHVSTTATTPTVTSSKATPSSSPGTATALPALRSTATTPTATSFTAIPSSSLGTTWTRLSQTTTPTATMSTATPSSTPETVHTSTVLTTTATTTGATGSVATPSSTPGTAHTTKVPTTTTTGFTATPSSSPGTALTPPVWISTTTTPTTTTPTTSGSTVTPSSIPGTTHTARVLTTTTTTVATGSMATPSSSTQTSGTPPSLTTTATTITATGSTTNPSSTPGTTPIPPVLTSMATTPAATSSKATSSSSPRTATTLPVLTSTATKSTATSFTPIPSSTLWTTWTVPAQTTTPMSTMSTIHTSSTPETTHTSTVLTTTATMTRATNSTATPSSTLGTTRILTELTTTATTTAATGSTATLSSTPGTTWILTEPSTIATVMVPTGSTATTSSTLGTAHTPKVVTAMATMPTATASTVPSSSTVGTTRTPAVLPSSLPTFSVSTVSSSVLTTLRPTGFPSSHFSTPCFCRAFGQFFSPGEVIYNKTDRAGCHFYAVCNQHCDIDRFQGACPTSPPPVSSAPLSSPSPAPGCDNAIPLRQVNETWTLENCTVARCVGDNRVVLLDPKPVANVTCVNKHLPIKVSDPSQPCDFHYECECICSMWGGSHYSTFDGTSYTFRGNCTYVLMREIHARFGNLSLYLDNHYCTASATAAAARCPRALSIHYKSMDIVLTVTMVHGKEEGLILFDQIPVSSGFSKNGVLVSVLGTTTMRVDIPALGVTVTFNGQVFQARLPYSLFHNNTEGQCGTCTNNQRDDCLQRDGTTAASCKDMAKTWLVPDSRKDGCWAPTGTPPTASPAAPVSSTPTPTPCPPQPLCDLMLSQVFAECHNLVPPGPFFNACISDHCRGRLEVPCQSLEAYAELCRARGVCSDWRGATGGLCDLTCPPTKVYKPCGPIQPATCNSRNQSPQLEGMAEGCFCPEDQILFNAHMGICVQACPCVGPDGFPKFPGERWVSNCQSCVCDEGSVSVQCKPLPCDAQGQPPPCNRPGFVTVTRPRAENPCCPETVCVCNTTTCPQSLPVCPPGQESICTQEEGDCCPTFRCRPQLCSYNGTFYGVGATFPGALPCHMCTCLSGDTQDPTVQCQEDACNNTTCPQGFEYKRVAGQCCGECVQTACLTPDGQPVQLNETWVNSHVDNCTVYLCEAEGGVHLLTPQPASCPDVSSCRGSLRKTGCCYSCEEDSCQVRINTTILWHOGCETEVNITFCEGSCPGASKYSAEAQAMQHQCTCCQERRVHEETVPLHCPNGSAILHTYTHVDECGCTPFCVPAPMAPPHTRGFPAQEATAV(MUC8 [Homo sapiens])SEQ ID NO: 39MLEFWAQVLPRDQPPSLEFWAQVLPREGLLWEPSLTEESSWATCWMGSPWEGWWFWGWFWGWFLTDAYLPACGPGCVSPGARITVTGHPTQDSEPPWGLPGPWAELAARTPPPTLPLTSWCGAGWTSCACLVFQFVCSHICPLLPRAGWGALEGRDCSGKETSRPVACRARKWREWLGCPDGGRQPRVAQASAVWQVQLKNWIIHFTYRLQSKIQKPVGLVATMLDREVLDRLAWAPASAVPPTQNILGKGFNVLPSSRLQSGGGVAMRRPAVGGVKAPGCVRIGVRVNGGLRPTTGGRCEHTQLSPPQGSPPLTSEVLWGWSGTTLREPWVGMHHSQGPGTIRAVGGCSSSPPAPPHPLPQLLPAKTPFLSTLLEAHSSCPQLLPSPEGSALWPHHQPRSLSCSRHTPQLSSFQPARVPFLLTSHPRALILPTRPGQSPEWCYEHRWAPGWHISSSIVATGLCFRESFPAICKALCKVNGCEIVFKIQFGATDPNEALEISQAICPLGFKLACGVTAKKEKVCSISCSPQPHLRAGCQLQPSACTPLPSRNGTSVYFGCIVCSCGCRLVTQQVLRPHASLEKEQRNVSQLLLGERCVTNSPWALSDNALLTGLQGDKRGFASSFGLWLGPAIIHWPLSVGCRRSFSLSGSGGRAEPYKPRRSPCLKPHVVSVKEVSRLGPASTGQRSRAFCSGGVRWLSLSCRRPRRHQSGGLFLALQLECALSHMSRQLSVWSPQRAPFQSSCSRGCAVAWFRACCSSAGRPLPVHPGEQACSLRRGLRPAVLSPGPSLSPKPQPNATPSRGRWWLCVRWALLSSREFLRIFIARWLWVRADHRKPAGTLESRSGMQKAEGATRSSQVTLLPSCDCCRPPVPAPQRAWAVPGLQHRGLQPPPAIAPPSLCQEHWSLPVSSSGRYVTVMMSVSSHAPGSPRYHTYLSLLAATPWPSLESVSAGHAPYAVRECTSLHRCSHQKGTLQVGRRVLVPGPGPRVCPLQWGAQPGVGSGCGKEERPGTSSRDELGLFPLKVAEMQTQSSPYLWGSRQAPASGRAGFRQQPTPRGVPDRLRLQAGLDSGSTPYPVGFLTGPGFRQGWIQAAPRTRWGSRHALASGRAGFRQHPVPAGVPDMPRLQAGLDSGSTPYPVGFLTGPGFRQGWIQAAPRTRWGSRHALASAGLDSGTGVPAASALSGELRGSMSTGKIEFYEALSKVPSPLRMGSSGCQVPAARGCEAVTDRPRSTETTDRGGRGGPQNQMGTDTVGGQVPTRGPPASLDQHRGSSLHPTYPLPRSVPATPRSSLDIVSRLPHTSLRGPGSSLDTFLLLVPHCWFLGPSCSHLPCPAGPGYVPVMSGLRPGYVQVMSRLCSGYVLVRSRLCPGQVRVRFRLFLGYVWLGPGYVRVMSQLGPGYVQVVSGLGPGYFQVMSRLGPGYVRVTSRLRPGYVQVRSRLGPGYVALGPGQVRVMSGLCPGQVPVISGLCPGQVLLCSGYFQVRSQLGLGASVSHCCVPAMANLSVCGRERSHSSQLCRSAAGLPSTPGRSHARLEPGRLCFPMRDQGTGEPGLGGIRQCLQGRLGKMPLCDEERRTHKDVIRFSSCPLAPLLALNAALGAYAPESCGSLFALMRQRAPGGRANVLTQWGRKTETTEWGAGGLDQLPPDISSGKELSVQFELLFVGCSVTCILWIWPLAATGQHQFGETKTCLYSGREAFNVGTGTQMSEGLCGHRGHEGAGELGTLSVPTGLDTRGLGCSRANGTGHEGAGTLPGPMGLALVTCRAGFLAASVQVFPEETAVCVRVAEVKMTCPQCGWHLPAGVWREQKQKRGMCLSPRQLGCTLPLLPSDRTAGAPAFGLKDSHWQPRVSRLWPRAESRHQLPGFGCLLTWTEPHYRHPVLSSLQTACCGTSQPPSSGSPNRPCSLLSLCHVGSVSLETPDKHSCAGGEGSEEAVLWVTVCPQYHSLESQPPEPQNVTLLEIGSLQMKLQMLKGSSQIWLVSTSKHWPPCEKTHRPRGDGVWGREQRLQGRNHTGGKANTTRNRKRHGSSPGASGGSVAPITLVLDIRSLNLKGRHFCASSCTFFFFLTASYSVAQAGVQCRDLSSPPSLPPGFKRFSCLSLPSSWVYRCSPLYLVNFCIFSKDRVSPCWSGWSGTPDVKGSTRLGLSNCEDYRREPLHPTFTALCYGAWETTTGCSKKGSFYPTQMRAQQDSLREAHSAQGPGLLMGGGRWVPGGAASLVWRPNSFPSSWQGHLEAQLQGSLGHTGCGRPARSCTVPQGGGAARRKCQGPGASWTVWKFLFPECIISPSKIPQTQTKPTEADQRVGCSSSGGNGPGSQCLSGGHRGHKGCQPGRNQCTSTTSCPRPLQEGTRVHELPTSSPGRDPGPRAAHVLSRKGPGSTSCPRPLQEGTPGSRAAHALSRRGHRVHELPTSSPGGDTGFMSCPRPFQEGTPGSRAAHVLSRKGPRVHELPTSSPGRDPGFTSCPRPLQEGTRVTNCPRPLQEGTPGSRAAHVLSRRGHRVHELPTPSPGRDPGFMSCPRPLQEGTRVTNCPRPLQEGTRVTSCPRRLQEGTRVTSCPRPLQEGTRVTNCPRALQEGTPGSRAAHALSRKGPRVHELPTSSPGGDTGFTSCPRPLQEGTPGSRAAHALSRRGHRVHELPTSSPGRDPGHELPTSSPGGDTGFTSCPRTFQEGTPGSGLLPAHIVPLCKSEE(hMUC12)SEQ ID NO: 40MLVIWILTLALRLCASVTTVTPEGSAVHKAISQQGTLWTGEVLEKQTVEQGKSTLRRQKNHFHRSAGELRCRNALKDEGASAGWSVMFAGESVVVLVHLWMTGARVKNLGLVEFASPGDDGDGRAEGFSLGLPLSEQARAAGAREKERQETVINHSTFSGFSQITGSTVNTSIGGNTTSASTPSSSDPFTTFSDYGVSVTFITGSTATKHFLDSSTNSGHSEESTVSHSGPGATGTTLFPSHSATSVFVGEPKTSPITSASMETTALPGSTTTAGLSEKSTTFYSSPRSPDRTLSPARTTSSGVSEKSTTSHSRPGPTHTIAFPDSTTMPGVSQESTASHSIPGSTDTTLSPGTTTPSSLGPESTTFHSSPGYTKTTRLPDNTTTSGLLEASTPVHSSTGSPHTTLSPSSSTTHEGEPTTFQSWPSSKDTSPAPSGTTSAFVKLSTTYHSSPSSTPTTHFSASSTTLGHSEESTPVHSSPVATATTPPPARSATSGHVEESTAYHRSPGSTQTMHFPESSTTSGHSEESATFHGSTTHTKSSTPSTTAALAHTSYHSSLGSTETTHFRDSSTISGRSEESKASHSSPDAMATTVLPAGSTPSVLVGDSTPSPISSGSMETTALPGSTTKPGLSEKSTTFYSSPRSPDTTHLPASMTSSGVSEESTTSHSRPGSTHTTAFPGSTTMPGLSQESTASHSSPGPTDTTLSPGSTTASSLGPEYTTFHSRPGSTETTLLPDNTTASGLLEASMPVHSSTRSPHTTLSPAGSTTROGESTTFHSWPSSKDTRPAPPTTTSAFVEPSTTSHGSPSSIPTTHISARSTTSGLVEESTTYHSSPGSTQTMHFPESDTTSGRGEESTTSHSSTTHTISSAPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSERSTTFHSSPRSPATTLSPASTTSSGVSEESTTSRSRPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGPSQESTTSHSSSGSTDTALSPGSTTALSFGQESTTFHSNPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTAFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSRGSTETTVFPHSTTTSVHGEEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTKLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTSDLSQEPTTSHSSQGSTEATLSPGSTTASSLGQQSTTFHSSPGDTETTLLPDDTITSGLVEASTPTHSSTGSLHTTLTPASSTSAGLQEESTTFQSWPSSSDTTPSPPGTTAAPVEVSTTYHSRPSSTPTTHFSASSTTLGRSEESTTVHSSPGATGTALFPTRSATSVLVGEPTTSPISSGSTETTALPGSTTTAGLSEKSTTFYSSPRSPDTTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPGSTTMPGVSQESTASHSSPGSTDTTLSPGSTTASSLGPESTTFHSSPGSTETTLLPDNTTASGLLEASTPVHSSTGSPHTTLSPAGSTTROGESTTFQSWPSSKDTMPAPPTTTSAFVELSTTSHGSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTAYHSSPGSTQTMHFPESSTASGRSEESRTSHSSTTHTISSPPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSEKSTTFHSSPRSPATTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGPSQESTTSHSSPGSTDTALSPGSTTALSFGQESTTFHSSPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTAFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSPGSTETTVFPRTPTTSVRGEEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTTLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGPSQESTTSHSSPGSTDTALSPGSTTALSFGQESTTFHSSPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTTFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSPGSTETTVFPRSTTTSVRGEEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTTLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTSGLSQEPTASHSSQGSTEATLSPGSTTASSLGQQSTTFHSSPGDTETTLLPDDTITSGLVEASTPTHSSTGSLHTTLTPASSTSAGLQEESTTFQSWPSSSDTTPSPPGTTAAPVEVSTTYHSRPSSTPTTHFSASSTTLGRSEESTTVHSSPGATGTALFPTRSATSVLVGEPTTSPISSGSTETTALPGSTTTAGLSEKSTTFYSSPRSPDTTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPGSTTMPGVSQESTASHSSPGSTDTTLSPGSTTASSLGPESTTFHSGPGSTETTLLPDNTTASGLLEASTPVHSSTGSPHTTLSPAGSTTROGESTTFQSWPNSKDTTPAPPTTTSAFVELSTTSHGSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTTYHSSPGSTQTMHFPESDTTSGRGEESTTSHSSTTHTISSAPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSEKSTTFHSSPRSPATTLSPASTTSSGVSEESTTSHSRPGSTHTTAFPDSTTTPGLSRHSTTSHSSPGSTDTTLLPASTTTSGSSQESTTSHSSSGSTDTALSPGSTTALSFGQESTTFHSSPGSTHTTLFPDSTTSSGIVEASTRVHSSTGSPRTTLSPASSTSPGLQGESTAFQTHPASTHTTPSPPSTATAPVEESTTYHRSPGSTPTTHFPASSTTSGHSEKSTIFHSSPDASGTTPSSAHSTTSGRGESTTSRISPGSTEITTLPGSTTTPGLSEASTTFYSSPRSPTTTLSPASMTSLGVGEESTTSRSQPGSTHSTVSPASTTTPGLSEESTTVYSSSPGSTETTVFPRSTTTSVRREEPTTFHSRPASTHTTLFTEDSTTSGLTEESTAFPGSPASTQTGLPATLTTADLGEESTTFPSSSGSTGTKLSPARSTTSGLVGESTPSRLSPSSTETTTLPGSPTTPSLSEKSTTFYTSPRSPDATLSPATTTSSGVSEESSTSHSQPGSTHTTAFPDSTTTSGLSQEPTTSHSSQGSTEATLSPGSTTASSLGQQSTTFHSSPGDTETTLLPDDTITSGLVEASTPTHSSTGSLHTTLTPASSTSTGLQEESTTFQSWPSSSDTTPSPPSTTAVPVEVSTTYHSRPSSTPTTHFSASSTTLGRSEESTTVHSSPGATGTALFPTRSATSVLVGEPTTSPISSGSTETTALPGSTTTAGLSEKSTTFYSSPRSPDTTLSPASTTSSGVSEESTTSHSRPGSMHTTAFPSSTTMPGVSQESTASHSSPGSTDTTLSPGSTTASSLGPESTTFHSSPGSTETTLLPDNTTASGLLEASTPVHSSTGSPHTTLSPAGSTTROGESTTFQSWPNSKDTTPAPPTTTSAFVELSTTSHGSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTTYHSSPGSTQTMHFPESNTTSGRGEESTTSHSSTTHTISSAPSTTSALVEEPTSYHSSPGSTATTHFPDSSTTSGRSEESTASHSSQDATGTIVLPARSTTSVLLGESTTSPISSGSMETTALPGSTTTPGLSEKSTTFHSSPSSTPTTHFSASSTTLGRSEESTTVHSSPVATATTPSPARSTTSGLVEESTAYHSSPGSTQTMHFPESSTASGRSEESRTSHSSTTHTISSPPSTTSALVEEPTSYHSSPGSIATTHFPESSTTSGRSEESTASHSSPDTNGITPLPAHFTTSGRIAESTTFYISPGSMETTLASTATTPGLSAKSTILYSSSRSPDQTLSPASMTSSSISGEPTSLYSQAESTHTTAFPASTTTSGLSQESTTFHSKPGSTETTLSPGSITTSSFAQEFTTPHSQPGSALSTVSPASTTVPGLSEESTTFYSSPGSTETTAFSHSNTMSIHSQQSTPFPDSPGFTHTVLPATLTTTDIGQESTAFHSSSDATGTTPLPARSTASDLVGEPTTFYISPSPTYTTLFPASSSTSGLTEESTTFHTSPSFTSTIVSTESLETLAPGLCQEGQIWNGKQCVCPQGYVGYQCLSPLESFPVETPEKLNATLGMTVKVTYRNFTEKMNDASSQEYQNFSTLFKNRMDVVLKGDNLPQYRGVNIRRLLNGSIVVKNDVILEADYTLEYEELFENLAEIVKAKIMNETRTTLLDPDSCRKAILCYSEEDTFVDSSVTPGFDFQEQCTQKAAEGYTQFYYVDVLDGKLACVNKCTKGTKSQMNCNLGTCQLQRSGPRCLCPNTNTHWYWGETCEFNIAKSLVYGIVGAVMAVLLLALIILIILFSLSQRKRHREQYDVPQEWRKEGTPGIFQKTAIWEDQNLRESRFGLENAYNNFRPTLETVDSGTELHIQRPEMVASTV(hMUC13)SEQ ID NO: 41MKAIIHLTLLALLSVNTATNQGNSADAVTTTETATSGPTVAAADTTETNFPETASTTANTPSFPTATSPAPPIISTHSSSTIPTPAPPIISTHSSSTIPIPTAADSESTTNVNSLATSDIITASSPNDGLITMVPSETQSNNEMSPTTEDNQSSGPPTGTALLETSTLNSTGPSNPCQDDPCADNSLCVKLHNTSFCLCLEGYYYNSSTCKKGKVFPGKISVTVSETFDPEEKHSMAYQDLHSEITSLFKDVFGTSVYGQTVILTVSTSLSPRSEMRADDKFVNVTIVTILAETTSDNEKTVTEKINKAIRSSSSNFLNYDLTLRCDYYGCNQTADDCLNGLACDCKSDLQRPNPQSPFCVASSLKCPDACNAQHKQCLIKKSGGAPECACVPGYQEDANGNCQKCAFGYSGLDCKDKFQLILTIVGTIAGIVILSMIIALIVTARSNNKTKHIEEENLIDEDFQNLKLRSTGFTNLGAEGSVFPKVRITASRDSQMQNPYSRHSSMPRPDY(hMUC14)SEQ ID NO: 42MELLQVTILFLLPSICSSNSTGVLEAANNSLVVTTTKPSITTPNTESLQKNVVTPTTGTTPKGTITNELLKMSLMSTATFLTSKDEGLKATTTDVRKNDSIISNVTVTSVTLPNAVSTLQSSKPKTETQSSIKTTEIPGSVLQPDASPSKTGTLTSIPVTIPENTSQSQVIGTEGGKNASTSATSRSYSSIILPVVIALIVITLSVFVLVGLYRMCWKADPGTPENGNDQPQSDKESVKLLTVKTISHESGEHSAQGKTKN(hMUC15)SEQ ID NO: 43MLALAKILLISTLFYSLLSGSHGKENQDINTTQNIAEVFKTMENKPISLESEANLNSDKENITTSNLKASHSPPLNLPNNSHGITDFSSNSSAEHSLGSLKPTSTISTSPPLIHSFVSKVPWNAPIADEDLLPISAHPNATPALSSENFTWSLVNDTVKTPDNSSITVSILSSEPTSPSVTPLIVEPSGWLTTNSDSFTGFTPYQEKTTLQPTLKFTNNSKLFPNTSDPQKENRNTGIVFGAILGAILGVSLLTLVGYLLCGKRKTDSFSHRRLYDDRNEPVLRLDNAPEPYDVSFGNSSYYNPTLNDSAMPESEENARDGIPMDDIPPLRTSV(MUC16Homo sapiens)SEQ ID NO: 44MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIVVTEHTLPFTSPDKTLASPTSSVVGRTTQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSSTEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVTDSHTPGRTNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEPGSAGHSRISTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVPEARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMDIPSTHIAKTLASEWLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESEMTATLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGROSSSTAAHGSSDILRATTSSTSKASSWTSESTAQQFSEPQHTQWVETSPSMKTERPPASTSVAAPITTSVPSVVSGFTTLKTSSTKGIWLEETSADTLIGESTAGPTTHQFAVPTGISMTGGSSTRGSQGTTHLLTRATASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMVSSVIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKISTSIPLLSSASVLEDKVSATSTFSHHKATSSITTGTPEISTKTKPSSAVLSSMTLSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLSTSAETTDSPNIHPTGTLTSESSESPSTLSLPSVSGVKTTFSSSTPSTHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPTMDTWPTRSAQFSSSHLVSELRATSSTSVTNSTGSALPKISHLTGTATMSQTNRDTFNDSAAPQSTTWPETSPRFKTGLPSATTTVSTSATSLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTNGPGSMAVASTNIPIGKGYITEGRLDTSHLPIGTTASSETSMDFTMAKESVSMSVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGTSSALTPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVTMSSTFSTQRVTTSMIMDTVETSRWNMPNLPSTTSLTPSNIPTSGAIGKSTLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSESPSTIKLTMASVVKPGSYTPLTFPSIETHIHVSTARMAYSSGSSPEMTAPGETNTGSTWDPTTYITTTDPKDTSSAQVSTPHSVRTLRTTENHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTOLHYTKLAEKSSGFETQSAPGPVSVVIPTSPTIGSSTLELTSDVPGEPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSPSSPMSTFAIFPPMSTPSHELSKSEADTSAIRNTDSTTLDQHLGIRSLGRTGDLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLKDQTSIPASASPSHLTEVYPELGTOGRSSSEATTFWKPSTDTLSREIETGPTNIQSTPPMDNTTTGSSSSGVTLGIAHLPIGTSSPAETSTNMALERRSSTATVSMAGTMGLLVTSAPGRSISQSLGRVSSVLSESTTEGVTDSSKGSSPRLNTQGNTALSSSLEPSYAEGSQMSTSIPLTSSPTTPDVEFIGGSTFWTKEVTTVMTSDISKSSARTESSSATLMSTALGSTENTGKEKLRTASMDLPSPTPSMEVTPWISLTLSNAPNTTDSLDLSHGVHTSSAGTLATDRSLNTGVTRASRLENGSDTSSKSLSMGNSTHTSMTDTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLTLPFSSIPVEEVISTGITSGPDINSAPMTHSPITPPTIVWTSTGTIEQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSVSSGSSTSSPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLSEALSSGHSGVSNPSSTTTEFPLFSAASTSAAKQRNPETETHGPQNTAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLTSESTANPSLGTASSAGTKLTRTISLPTSESLVSFRMNKDPWTVSIPLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTVSFSPSPDTEVTTISHFPEKTTHSFRTISSLTHELTSRVTPIPGDWMSSAMSTKPTGASPSITLGERRTITSAAPTTSPIVLTASFTETSTVSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDVTKTASISPTSISGMTASSSPSLFSSDRPQVPTSTTETNTATSPSVSSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTFSTMVSTDTASGENPTSSNSVVTSVPAPGTWASVGSTTDLPAMGFLKTSPAGEAHSLLASTIEPATAFTPHLSAAVVTGSSATSEASLLTTSESKAIHSSPQTPTTPTSGANWETSATPESLLVVTETSDTTLTSKILVTDTILFSTVSTPPSKFPSTGTLSGASFPTLLPDTPAIPLTATEPTSSLATSFDSTPLVTIASDSLGTVPETTLTMSETSNGDALVLKTVSNPDRSIPGITIQGVTESPLHPSSTSPSKIVAPRNTTYEGSITVALSTLPAGTTGSLVFSQSSENSETTALVDSSAGLERASVMPLTTGSQGMASSGGIRSGSTHSTGTKTFSSLPLTMNPGEVTAMSEITTNRLTATQSTAPKGIPVKPTSAESGLLTPVSASSSPSKAFASLTTAPPSTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTASSSLSKSPEKNPRARMMTSTKAISASSFQSTGFTETPEGSASPSMAGHEPRVPTSGTGDPRYASESMSYPDPSKASSAMTSTSLASKLTTLFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPSMARQPNILVHLQTSALTLSPTSTLNMSQEEPPELTSSQTIAEEEGTTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISVPAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEETGTSPAGTSPGSPEVSTTLKIMSSKEPSISPEIRSTVRNSPWKTPETTVPMETTVEPVTLQSTALGSGSTSISHLPTGTTSPTKSPTENMLATERVSLSPSPPEAWTNLYSGTPGGTRQSLATMSSVSLESPTARSITGTGQQSSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTSPNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAEQQTSRSVDEAYSSTSSWSDQTSGSDITLGASPDVTNTLYITSTAQTTSLVSLPSGDQGITSLTNPSGGKTSSASSVTSPSIGLETLRANVSAVKSDIAPTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTPNPEVGMSTMDSTPATERRTTSTEHPSTWSSTAASDSWTVTDMTSNLKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIGTSLVTPSSDASAVKTETSTSERTLSPSDTTASTPISTFSRVQRMSISVPDILSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLSTLDWDTGRSLSSATATTSAPQGATTPQELTLETMISPATSQLPFSIGHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTOLPTTTSAHPGQVPRSAATTLDVIPHTAKTPDATFIPKFGKAAHMRELPLLSPPQDKEAIHPSTNTVETTGWVTSSEHASHSTIPAHSASSKLTSPVVTTSTREQAIVSMSTTTWPESTRARTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTEITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMILAMQTSPPGATSLSAPTLDTSATASWTGTPLATTQRFTYSEKTTLFSKGPEDTSQPSPPSVEETSSSSSLVPIHATTSPSNILLTSQGHSPSSTPPVTSVFLSETSGLGKTTDMSRISLEPGTSLPPNLSSTAGEALSTYEASRDTKAIHHSADTAVTNMEATSSEYSPIPGHTKPSKATSPLVTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVASSATETSTVITHVSSGDATTHVTKTQATFSSGTSISSPHQFITSTNTFTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMPYLTETPLAVTPDFMQSEKTTLISKGPKDVTWTSPPSVAETSYPSSLTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTSMEPVTNSPQNLNNPSNEILATLAATTDIETIHPSINKAVTNMGTASSAHVLHSTLPVSSEPSTATSPMVPASSMGDALASISIPGSETTDIEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATKMEVPSFDATFIPTPAQSTKFPDIFSVASSRLSNSPPMTISTHMTTTQTGSSGATSKIPLALDTSTLETSAGTPSVVTEGFAHSKITTAMNNDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVAFTPQWHSTSSPVSMSSVLTSSLVKTAGKVDTSLETVTSSPQSMSNTLDDISVTSAATTDIETTHPSINTVVTNVGTTGSAFESHSTVSAYPEPSKVTSPNVTTSTMEDTTISRSIPKSSKTTRTETETTSSLTPKLRETSISQEITSSTETSTVPYKELTGATTEVSRTDVTSSSSTSFPGPDQSTVSLDISTETNTRLSTSPIMTESAEITITTQTGPHGATSQDTFTMDPSNTTPQAGIHSAMTHGFSQLDVTTLMSRIPQDVSWTSPPSVDKTSSPSSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRTRLEPVTSSLPNFSSTSDKILATSKDSKDTKEIFPSINTEETNVKANNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSSETTNIKREPTYFLTPRLRETSTSQESSFPTDTSFLLSKVPTGTITEVSSTGVNSSSKISTPDHDKSTVPPDTFTGEIPRVFTSSIKTKSAEMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEVTTLMGMGPGNVSWMTTPPVEETSSVSSLMSSPAMTSPSPVSSTSPQSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITHERPATYKDTAHTEAAMHHSTNTAVTNVGTSGSGHKSQSSVLADSETSKATPLMSTTSTLGDTSVSTSTPNISQTNQIQTEPTASLSPRLRESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLDRPTIAPDISTGMITRLFTSPIMTKSAEMTVTTQTTTPGATSQGILPWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPVEETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALLTSVLVTTTNVLGTTSPETVTSSPPNLSSPTQERLTTYKDTAHTEAMHASMHTNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSVSTSTPAFFETRIQTESTSSLIPGLRDTRTSEEINTVTETSTVLSEVPTTTTTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTFPFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEGTHSPVTQRFPHSEETTTMSRSTKGVSWQSPPSVEETSSPSSPVPLPAITSHSSLYSAVSGSSPTSALPVTSLLTSGRRKTIDMLDTHSELVTSSLPSASSFSGEILTSEASTNTETIHFSENTAETNMGTTNSMHKLHSSVSIHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLTPELKEDSTALVMNSTTESNTVFSSVSLDAATEVSRAEVTYYDPTFMPASAQSTKSPDISPEASSSHSNSPPLTISTHKTIATQTGPSGVTSLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKTSPFSAEEANSLSSQAPLLVTTSPSPVTSTLQEHSTSSLVSVTSVPTPTLAKITDMDTNLEPVTRSPQNLRNTLATSEATTDTHTMHPSINTAMANVGTTSSPNEFYFTVSPDSDPYKATSAVVITSTSGDSIVSTSMPRSSAMKKIESETTFSLIFRLRETSTSQKIGSSSDTSTVFDKAFTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFAGLTKSEERTIATQTGPHRATSQGTLTWDTSITTSQAGTHSAMTHGFSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLLSLPAITSPSPVPTTLPESRPSSPVHLTSLPTSGLVKTTDMLASVASLPPNLGSTSHKIPTTSEDIKDTEKMYPSTNIAVTNVGTTTSEKESYSSVPAYSEPPKVTSPMVTSFNIRDTIVSTSMPGSSEITRIEMESTFSVAHGLKGTSTSQDPIVSTEKSAVLHKLTTGATETSRTEVASSRRTSIPGPDHSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGTFTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNKDPEILSWTIPPSIEKTSFSSSLMPSPAMTSPPVSSTLPKTIHTTPSPMTSLLTPSLVMTTDTLGTSPEPTTSSPPNLSSTSHVILTTDEDTTAIEAMHPSTSTAATNVETTCSGHGSQSSVLTDSEKTKATAPMDTTSTMGHTTVSTSMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEVPTGTTAEVSRTEVTSSGRTSIPGPSQSTVLPEISTRTMTRLFASPTMTESAEMTIPTQTGPSGSTSQDTLTLDTSTTKSQAKTHSTLTQRFPHSEMTTLMSRGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPPISSTLPVTISSSPLPVTSLLTSSPVTTTDMLHTSPELVTSSPPKLSHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSFGHESPSSALADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLSPKLRETGSSVETSSAIETSAVLSEVSIGATTEISRTEVTSSSRTSISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGSTSESTFTLDTSTTPSLVITHSTMTQRLPHSEITTLVSRGAGDVPRPSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSPLTPGQVKTTEVLDASAEPETSSPPSLSSTSVEILATSEVTTDTEKIHPFPNTAVTKVGTSSSGHESPSSVLPDSETTKATSAMGTISIMGDTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEANTVLSKVSTGATTEVSRTEAISFSRTSMSGPEQSTMSQDISIGTIPRISASSVLTESAKMTITTQTGPSESTLESTLNLNTATTPSWVETHSIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPAVTSPHPVSTTFLAHIPPSPLPVTSLLTSGPATTTDILGTSTEPGTSSSSSLSTTSHERLTTYKDTAHTEAVHPSTNTGGTNVATTSSGYKSQSSVLADSSPMCTTSTMGDTSVLTSTPAFLETRRIQTELASSLTPGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVTSIPGPAQSTISPDISTRTVSWFSTSPVMTESAEITMNTHTSPLGATTQGTSTLATSSTTSLTMTHSTISQGFSHSQMSTLMRRGPEDVSWMSPPLLEKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLAKTTDMLHKSSEPVTNSPANLSSTSVEILATSEVTTDTEKTHPSSNRTVTDVGTSSSGHESTSFVLADSQTSKVTSPMVITSTMEDTSVSTSTPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGVPTGATAEVSRTEVTSSSRTSISGFAQLTVSPETSTETITRLPTSSIMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQRFSHSEMTTLVSRSPGDMLWPSQSSVEETSSASSLLSLPATTSPSPVSSTLVEDFPSASLPVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEDMQPSTHTAVTNVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSALPESISSSPHPVTALLTLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRISTPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISSEVITRLSTSPIKAESTEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSHPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILATTEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPQSVVTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLATSKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMSSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQVTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVDDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYGVPTSATTEVSMTEIMSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSPFVEKTSSSSSLLSLPVTTSPSVSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGFRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDWPPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSTPILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASROMASTILTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVLNRESETTASLVSRSGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEVPGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVVTSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIPILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTSLVTSSRAVTSTTIPTLTISSDEPETTTSLVTHSEAKMISAIPTLAVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPSTVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTDTSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPGASKMLTSLVISSGTDSTTTFPTLTETPYEPETTAIQLIHPAETNTMVPKTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTVTWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALLSTHPRTGTSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLASESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSAMAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTAAGPLLMPFTLNFTITNLQYEEDMRYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTAAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMRHPGSRQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLOGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLOGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTETELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTERVLOGLLKPLFKNTSVGPLYSGCRLTLLRPEKHEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKHEAATGVDTICTHRVDPIGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLDREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLOGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLOGLLRPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAACTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRVSLYVNGFNPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSAMAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRRFNTTERVLOGLLTPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITDLHYEENMQHPGSRKFNTTERVLOGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTTLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTQDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTEIELGPYLLDRGSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPAXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICTHRVDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVXXGTSGTPSSXPXXTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLOGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGATTGMDAICTHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSAMAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLIPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLOGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLOGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTQDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETTGTPSSFPGHTEPGPLLIPFTFNFTITNLRYEENMQHPGSRKFNTTERVLOGLLTPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVDGFNPWSSVPTTSTPGTSTVHLATSGTPSPLPGHTAPVPLLIPFTLNFTITDLHYEENMQHPGSRKFNTTERVLOGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTPGTSTVHLATSGTPSSLPGHTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLHGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICHXXXPKXPGLXXEXLYWELSXLTNSITELGPYTLDRDSLYVNGFTHRSSMPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEEDMLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFHPRSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHRSSVPTTSSPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICXHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHRTSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLOGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYCELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTXXXPLLXPFTXNXTITNLXXXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHWSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHRSFGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLTPLFRNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLOGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTEXVLQGLLXPXFKNXSVGXLYSGCRLTXLRXEKXGAATGXDAICHXXXPKXPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHQTFAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATRVDAVCTHRPDPKSPGLXXEXLYWELSXLTXXIXELGPYTLDRXSLYVNGFTHXXSXPTTSTPGTSTVXXGTSGTPSSXPXXTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLOGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSVTELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEVDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGFTHRNFVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPGSRKFNTTERVLOGLLRPLFKNTSIGPLYSSCRLTLLRPEKDKAATRVDAICTHHPDPQSPGLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGIPPSLPETTXXXPLLXPFTXNXTITNLXXXXXMXXPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTITNLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITEGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLOGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDFQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLOGLLRSLFKSTSVGPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGHYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGSRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMKHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGLDEPPTTPKFATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQILSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDEAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGLITCLICGVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ(hMUC17)SEQ ID NO: 45MPRPGTMALCLLTLVLSLLPPQAAAEQDLSVNRAVWDGGGCISQGDVLNRQCQQLSQHVRTGSAANTATGTTSTNVVEPRMYLSCSTNPEMTSIESSVTSDTPGVSSTRMTPTESRTTSESTSDSTTLFPSSTEDTSSPTTPEGTDVPMSTPSEESISSTMAFVSTAPLPSFEAYTSLTYKVDMSTPLTTSTQASSSPTTPESTTIPKSTNSEGSTPLTSMPASTMKVASSEAITLLTTPVEISTPVTISAQASSSPTTAEGPSLSNSAPSGGSTPLTRMPLSVMLVVSSEASTLSTTPAATNIPVITSTEASSSPTTAEGTSIPTSTYTEGSTPLTSTPASTMPVATSEMSTLSITPVDTSTLVTTSTEPSSLPTTAEATSMLTSTLSEGSTPLTNMPVSTILVASSEASTTSTIPVDSKTFVTTASEASSSPTTAEDTSIATSTPSEGSTPLTSMPVSTTPVASSEASNLSTTPVDSKTQVTTSTEASSSPPTAEVNSMPTSTPSEGSTPLTSMSVSTMPVASSEASTLSTTPVDTSTPVTTSSEASSSSTTPEGTSIPTSTPSEGSTPLTNMPVSTRLVVSSEASTTSTTPADSNTFVTTSSEASSSSTTAEGTSMPTSTYSERGTTITSMSVSTTLVASSEASTLSTTPVDSNTPVTTSTEATSSSTTAEGTSMPTSTYTEGSTPLTSMPVNTTLVASSEASTLSTTPVDTSTPVTTSTEASSSPTTADGASMPTSTPSEGSTPLTSMPVSKTLLTSSEASTLSTTPLDTSTHITTSTEASCSPTTTEGTSMPISTPSEGSPLLTSIPVSITPVTSPEASTLSTTPVDSNSPVTTSTEVSSSPTPAEGTSMPTSTYSEGRTPLTSMPVSTTLVATSAISTLSTTPVDTSTPVTNSTEARSSPTTSEGTSMPTSTPGEGSTPLTSMPDSTTPVVSSEARTLSATPVDTSTPVTTSTEATSSPTTAEGTSIPTSTPSEGTTPLTSTPVSHTLVANSEASTLSTTPVDSNTPLTTSTEASSPPPTAEGTSMPTSTPSEGSTPLTRMPVSTTMVASSETSTLSTTPADTSTPVTTYSQASSSSTTADGTSMPTSTYSEGSTPLTSVPVSTRLVVSSEASTLSTTPVDTSIPVTTSTEASSSPTTAEGTSIPTSPPSEGTTPLASMPVSTTLVVSSEANTLSTTPVDSKTQVATSTEASSPPPTAEVTSMPTSTPGERSTPLTSMPVRHTPVASSEASTLSTSPVDTSTPVTTSAETSSSPTTAEGTSLPTSTTSEGSTLLTSIPVSTTLVTSPEASTLLTTPVDTKGPVVTSNEVSSSPTPAEGTSMPTSTYSEGRTPLTSIPVNTTLVASSAISILSTTPVDNSTPVTTSTEACSSPTTSEGTSMPNSNPSEGTTPLTSIPVSTTPVVSSEASTLSATPVDTSTPGTTSAEATSSPTTAEGISIPTSTPSEGKTPLKSIPVSNTPVANSEASTLSTTPVDSNSPVVTSTAVSSSPTPAEGTSIAISTPSEGSTALTSIPVSTTTVASSEINSLSTTPAVTSTPVTTYSQASSSPTTADGTSMQTSTYSEGSTPLTSLPVSTMLVVSSEANTLSTTPIDSKTQVTASTEASSSTTAEGSSMTISTPSEGSPLLTSIPVSTTPVASPEASTLSTTPVDSNSPVITSTEVSSSPTPAEGTSMPTSTYTEGRTPLTSITVRTTPVASSAISTLSTTPVDNSTPVTTSTEARSSPTTSEGTSMPNSTPSEGTTPLTSIPVSTTPVLSSEASTLSATPIDTSTPVTTSTEATSSPTTAEGTSIPTSTLSEGMTPLTSTPVSHTLVANSEASTLSTTPVDSNSPVVTSTAVSSSPTPAEGTSIATSTPSEGSTALTSIPVSTTTVASSETNTLSTTPAVTSTPVTTYAQVSSSPTTADGSSMPTSTPREGRPPLTSIPVSTTTVASSEINTLSTTLADTRTPVTTYSQASSSPTTADGTSMPTPAYSEGSTPLTSMPLSTTLVVSSEASTLSTTPVDTSTPATTSTEGSSSPTTAGGTSIQTSTPSERTTPLAGMPVSTTLVVSSEGNTLSTTPVDSKTQVTNSTEASSSATAEGSSMTISAPSEGSPLLTSIPLSTTPVASPEASTLSTTPVDSNSPVITSTEVSSSPIPTEGTSMQTSTYSDRRTPLTSMPVSTTVVASSAISTLSTTPVDTSTPVTNSTEARSSPTTSEGTSMPTSTPSEGSTPFTSMPVSTMPVVTSEASTLSATPVDTSTPVTTSTEATSSPTTAEGTSIPTSTLSEGTTPLTSIPVSHTLVANSEVSTLSTTPVDSNTPFTTSTEASSPPPTAEGTSMPTSTSSEGNTPLTRMPVSTTMVASFETSTLSTTPADTSTPVTTYSQAGSSPTTADDTSMPTSTYSEGSTPLTSVPVSTMPVVSSEASTHSTTPVDTSTPVTTSTEASSSPTTAEGTSIPTSPPSEGTTPLASMPVSTTPVVSSEAGTLSTTPVDTSTPMTTSTEASSSPTTAEDIVVPISTASEGSTLLTSIPVSTTPVASPEASTLSTTPVDSNSPVVTSTEISSSATSAEGTSMPTSTYSEGSTPLRSMPVSTKPLASSEASTLSTTPVDTSIPVTTSTETSSSPTTAKDTSMPISTPSEVSTSLTSILVSTMPVASSEASTLSTTPVDTRTLVTTSTGTSSSPTTAEGSSMPTSTPGERSTPLTNILVSTTLLANSEASTLSTTPVDTSTPVTTSAEASSSPTTAEGTSMRISTPSDGSTPLTSILVSTLPVASSEASTVSTTAVDTSIPVTTSTEASSSPTTAEVTSMPTSTPSETSTPLTSMPVNHTPVASSEAGTLSTTPVDTSTPVTTSTKASSSPTTAEGIVVPISTASEGSTLLTSIPVSTTPVASSEASTLSTTPVDTSIPVTTSTEGSSSPTTAEGTSMPISTPSEVSTPLTSILVSTVPVAGSEASTLSTTPVDTRTPVTTSAEASSSPTTAEGTSMPISTPGERRTPLTSMSVSTMPVASSEASTLSRTPADTSTPVTTSTEASSSPTTAEGTGIPISTPSEGSTPLTSIPVSTTPVAIPEASTLSTTPVDSNSPVVTSTEVSSSPTPAEGTSMPISTYSEGSTPLTGVPVSTTPVTSSAISTLSTTPVDTSTPVTTSTEAHSSPTTSEGTSMPTSTPSEGSTPLTYMPVSTMLVVSSEDSTLSATPVDTSTPVTTSTEATSSTTAEGTSIPTSTPSEGMTPLTSVPVSNTPVASSEASILSTTPVDSNTPLTTSTEASSSPPTAEGTSMPTSTPSEGSTPLTSMPVSTTTVASSETSTLSTTPADTSTPVTTYSQASSSPPIADGTSMPTSTYSEGSTPLTNMSFSTTPVVSSEASTLSTTPVDTSTPVTTSTEASLSPTTAEGTSIPTSSPSEGTTPLASMPVSTTPVVSSEVNTLSTTPVDSNTLVTTSTEASSSPTIAEGTSLPTSTTSEGSTPLSIMPLSTTPVASSEASTLSTTPVDTSTPVTTSSPTNSSPTTAEVTSMPTSTAGEGSTPLTNMPVSTTPVASSEASTLSTTPVDSNTFVTSSSQASSSPATLQVTTMRMSTPSEGSSSLTTMLLSSTYVTSSEASTPSTPSVDRSTPVTTSTQSNSTPTPPEVITLPMSTPSEVSTPLTIMPVSTTSVTISEAGTASTLPVDTSTPVITSTQVSSSPVTPEGTTMPIWTPSEGSTPLTTMPVSTTRVTSSEGSTLSTPSVVTSTPVTTSTEAISSSATLDSTTMSVSMPMEISTLGTTILVSTTPVTRFPESSTPSIPSVYTSMSMTTASEGSSSPTTLEGTTTMPMSTTSERSTLLTTVLISPISVMSPSEASTLSTPPGDTSTPLLTSTKAGSFSIPAEVTTIRISITSERSTPLTTLLVSTTLPTSFPGASIASTPPLDTSTTFTPSTDTASTPTIPVATTISVSVITEGSTPGTTIFIPSTPVTSSTADVFPATTGAVSTPVITSTELNTPSTSSSSTTTSFSTTKEFTTPAMTTAAPLTYVTMSTAPSTPRTTSRGCTTSASTLSATSTPHTSTSVTTRPVTPSSESSRPSTITSHTIPPTFPPAHSSTPPTTSASSTTVNPEAVTTMTTRTKPSTRTTSFPTVTTTAVPTNTTIKSNPTSTPTVPRTTTCFGDGCQNTASRCKNGGTWDGLKCQCPNLYYGELCEEVVSSIDIGPPETISAQMELTVTVTSVKFTEELKNHSSQEFQEFKQTFTEQMNIVYSGIPEYVGVNITKLRLGSVVVEHDVLLRTKYTPEYKTVLDNATEVVKEKITKVTTQQIMINDICSDMMCFNTTGTQVQNITVTQYDPEEDCRKMAKEYGDYFVVEYRDQKPYCISPCEPGFSVSKNCNLGKCQMSLSGPQCLCVTTETHWYSGETCNQGTQKSLVYGLVGAGVVLMLIILVALLMLVFRSKREVKROKYRLSQLYKWQEEDSGPAPGTFQNIGFDICQDDDSIHLESIYSNFQPSLRHIDPETKIRIQRPQVMTTSF(hMUC19)SEQ ID NO: 46XXXXXXXXXXSGSTGVSAGSITASPGASATSSESSKSGSTEGSVEATTSAGSGNTAGTSGTGDTGPGNTAGATGSSTGQTDTSGPSAKVTGNYGQSSEIPGTIKSSSDVSGTMGQSDTTSGPSVAVTRTSEQSSGVTVASEPSVGVSGTTGPLAEISGTTRPLVSGLRTTGSSAEGSGTTGPSSRESVTTRPLAEGSGTSGQSVTGSRATGLSATELGTTVSFTGGLGTSRSSARETRTTGPSADGSGTTGPSVVRSGTTRLSVGVTRATESSPGVTGTTTPSAEESRTTGPSVLVTGTTGQSGQGSGTTGKSFIESGPSVVGSGTTGPTSAGLGTTAPSTRRSSTTKPSVGRTGTTGQSGAESGTTEPSARVAGVTGTSAEVSGRIEPSATESSTSRPLGETTGTTIPSMEGSEATGPSVIGSETTRLSVIGSGTTGTSSGGSGATRSSGGGMGTTGQSTARSETTGPLFGLTGTFGQSATVTGTSSNSAGVTTPEKSPGVAMTTGLLVEGSATTQPRILESETTESSAGVIVTSGQSARVTGATGPSAGETGTTEPSTEGSVAAVLFVIGSETTRPLDIGSGTTGTLSGGSSTTRSSDGTTGTTRKSTARSETTGLSGLTGTSGQLAGVTGTSSKSAGVTVTSEKSAGVAVITGSFVERPVTTGPPLLESETTRPSGGVTVTSGQSARVTETVGASAGVTGTTGPSTEGSGATGPSVVGSGTTRPLAGESGTTESSAGVTGTRPSSSRESATTGPSDEGSGTTGLSAGVTVTSGQSVRKTGTTGAPAGVTETTRPSVVKSGTTGPSVIGTRTTGTSSGGSGATRSSGGETETTGQSAVKSGTTESFTRLTRTSGQSAGMTGTSAQSAGVALTSPFVEGLVTTGSSTVGLETTRPSAVGSGKTGPPVVKAQTTGPSAGVTVTSGQSARMTGASGPSVGVTGTTGPASKGLGTIRPSVVGLETTELSAEGSGTTGPPIVGETTVPSAGVTVTSGYSDRVTGATEPLAGVTGTIKPSVAGSVTTGPSVTGVETTAKTTSGGLSTTISSVGGTGTTGQSPERSGTTGPFTGLTGTSAQSAGVTMTSIQSAGVLVTTGLNVDGLGTTGKALIGSGTTGLSAEATGTIGPSTEGLEKTGPSITGSGTTRPLVTESWTAGTSSGGHSTTSPSVRGTETTGQSAAESVTTGPVTGYTETSGPSAGVTVTPRQSPTVTQTTGSSAAVSGTTVQSLTVSGTTRPSSGQTEITGSSVKESGTTESSAVRSGTTGPTAGVTGTNGPSSAGVTGITGSSPGVTGTTGSSPGVTGTTGSSARSGTSIPSVGKTGTTRTSVEESRTTRPSAGITGTNGLSAEVTGTTGPLAGVTGTTGPSAGVTRTTGLSAGETGTTGLSPGVTRTTRSSAGLTGKTGLSAGVTGKTGLSAEVTGTTRLSAGVTGTTGPSPGVTGTTGTPAGVTGTTELSAGVTGKTGLSSEVTETTGLSYGVKRTIGLSAGSTGTSGQSAGVAGTTTLSAEVTGTTRPSAGVTGTTGLSAEVTEITGISAVVTGTTGPSAGVTETTGSSAGVAGTTRLSAGVTGITGLSAGVTGTTGLSTEVTGTTGPSAGATGTTGLSVGVTGITGLSDVVTETTGSSARSGTGIPSVGETRTTSTSVEESRTTRPSAGIMGTNGLPAEVTGTTEPLAGGTGTTGILAGVTGTTGLSAGETGKIGSSAGVTGKTGSSARVTGKTGPSAEVTGKTGLSAGVTGTTGLSPGVTGTSGLSAEVTGTTGPSAEATGLPGVSAGVTGTTGSLAGGTGTIGLSAGVTGTTGSSAGVTGTTGLSAGVTGIAGLSAGVTGITGPSAGVTGTTTVSAGVTGTTGLSAEATEITGLSAGVTGTTGLSAGVTETIRLSAGVTGTIRSSAGVTGITGLSAGVTGTTGPSAGVTGSTGLLAGVTETTGOSAKVTGTTGQSVGVTGTTRSSGGVTGITGLSAGVTGTNGLSAVTGMTGLSAEVTGTTGLSVGVTGIAGLSAGVTGITGPSAGITGTTTISAGVTGTSGLSAEATGITGLSAGVTGKTGLSAGVTETIGLSAEATGTIGSSPGVTGTTGSSTGVTGITGLSAGVTGTTGLSTEVTGTTGPSAGVTRTTGLSAGVTGITGLSAIVTETTGSSARSGTSIPSVGETGTTRTSVEESRTTRPSAGITGTTGLSAGVTGTVGSSAVVTGTTGLSAGVTGTTGPSAEETGATGPSAEVTETTGPSAGVTGTGRLSAEVTGTTGPSAEVTGLPGESAEVTGTIGSPAGVTGTTQLSAVVTGITGLSAEVTGTTGLSAGVTGITGLSAEVTRTTGLSAGVTGTIGLSAGVTGTTRPSAGVTGTTGQSAEVTGTTEPSAGLTETTGSSTGVTGATGPLAGVTGTTGISTEVTGTTGPSARVTGTTVLSAGVTGITGLSAIVTETTGSSARSGTSTPSVGETGTTRTSVEESRATRPSAGITGTNGOSAEVTWITGPLAGVTGTTGISAGVTGTTGLSAGVTGTIGSSAVVTGINGLSAGVTGTTGPSAEETGATGPSAEVTGTTGPSAEETGATGPSAEVTGTTGPSGGVTGTNGLSAEVTGTTGPSAEVTGLPGVSAGVTGTIGSPAAVTGTIRPSAVVTGITGLSAEVTGTTGLSAWVTGIAGLSAGVTETIGSSAGVTGTNGLSAEATGTTGPSAGVTGTTGLSAGVTGTAGLSARVTESTGLSAGVTGTTGLSAGVTGTTGPSAGITGTNGLSAEVTGTTGPLAGVTGTIGLSAGVTGIAGLSAGVTESTGLSAGVTGTIRSSAVVTGINGLSAGVTGTTGPSAEETGATGPSAEVTGTTGPSGGVTGTSGISAEVTGTTGPSAEVTGLPGVSAGVTGTIGSPAAVTGTTRPSAVVTGISGLSAEVTGTTGLSAGVTETIGSSAGVTGTNGLSAEATETTGPSAGVTGTTGLSAGVTGTTGPSAGIAGTNGLSAGVTGTTGLSARVTESTGLSAGVTGTIGSSAVVTETTRLSSGVTGTIGPSAEETGATGLSAEVTGTTGSLAEVTGTTGLSAGVTGTIGSSAVVTGTTGLSAGITGTNGLSAEVTGTAGPLAGVTGTTGLSAGVTGTTGLSAGVTETTGOSAGVTESTGLSPGVTGTIGSSAVVTGIKGLSAGVTGTTGPSAEETGATGPSAEVTGTTGPSGGVTGTSVLSVEVTGTTGPSAEVTGLPGVSAGLTGTIGSPAAVRGTTWPSAVVTGISGLSGEVTGTTGLSAGVTGIGGLSAGVTGTIGSSAGVTGTNALSAEATGTTGPSAGVTGTTGLSAGVTGTTGLSAGVTGTIRSSAVVTETTGLSAGVTGTTGPSAGIAGTNGLSAEVTGTTGLSAGMTGTTGLSARVTESTGLSAGVTGTIGSSAVVTETTRLSAGVTGTIGPSAEETGATGLSAEVTRTTGSLAGVTGTTGPSAVVTGKTELSAEVTGTTELSAEVTEKTGPSAEVTGKTGLSAGVMETTGPSAEVTGTTGSSAGVTGTTGPSAGVTGTTGPSAEATGLPGVSAGVTGTIGSPAGVTGTARLSAVVTGISGLSAEVTGTTGLSTGVTGIAGHSAAVTGITRPSAGVTGTTTVSAGVTGTIGLSAEATGITLPSAGVTETTGLSAGVTETIGLSAGVTGTIGSSAGVTEITGLSAGVTGTTGPSAGVTGSTVLSAGVTATTGQSVGVTGTTGPSAGVTGTTGLSAGVTGIAGLSAGVTGITGPSAGVTGTTTVSAGVTGTTGLSAEATEITGLSAGVTGTTGLSAGVTGIAGLSAGVTETIGSSAGVTGTNGLSAEATGKTGPSAGVTGTTGLSAGVTGTTGLSAGVTETIGLSAGVTGTIGSSAGVKGTTGQSAEVTGATGQSVGVTGTTRSSGGVTGITGLSAGLRGTTVSSAKAGTSIPLTGKTGTTRTSVEESTTTGPSAGITGTNGLSAEMTGTNELSAGVTGTIGSSAGVTGTTGLSVEATVTTGLSAGVTGTTVPLAGVTWTPGPSAGVTGIAALSAGVTGKSGLSAGVTGKTGLSAGVTGTTGPSAEATGKTGLSAGVTGITGPFAEVTGTTGLSAGVIGTTGSSAEVTGITGLSAGVTGKTRSSAGVTGTTGLSAKSGTSIPSAGKTGTTKTSVEESRTTRPSAGITGTNGLPARVTGTXXXXXXXXXXGTSGVAPGTTVAPGSFSTAATTSPGASGVTGTGPTAETTTFLGGSSTTGAEIKSGATTGAPGSKTGTAKVLSGTTVASGSSNSEATTFSGITEAVTVPSKNGSMTTALGSQLSSSQTVIPGSSGTISHTTVAPGSSVTGTTTGASDDQVTGSKTGTTGVALSTTVAPGSSSTEATTSTGVHRTTVVGQKTGATTRGSAKQGTRSTIEATTSFRGTGTTGSGMNTGTTGVVSGNTISPSSFNTEATSGTSERPNPGSEIGTTGIVSGTTVAPGSSNTEATTSLGNGGTTEAGSKIVTTGITTGTTIVPGSFNTKATTSTDVGVATGVGMATGITNIISGRSQPTGSKTGYTVTGSGTTALPGGFRTGNTPGSTGVTSSQEGTTVVSSGITGIPETSISGPSKEASDKTTAPGPPTTVTASTGVKETSETGVQTGSTLVTAGVPTRPQVSQPETTVVATREVETENKTECLASLPPAPVCHGPLGEEKSPGDIWTANCHRGTCTDAKTIDCKPEECPSPPTCKTGEKLVKFQSNDTCCEIGYCEPRTCLFNNTDYEIGASFDDPSNPCVSYSCKDTGFAAVVQDCPKQTWCAEANRIYDSKKCCYTCKNNCRSSLVNVTVIYSGCKKRVQMAKCTGECEKTAKYNHDILLLEHSCLCCREENYELRDIVLDCPDGSTIPYQYKHITTCSCLDICQLYTTFMYS(hMUC20)SEQ ID NO: 47MGCLWGLALPLFFFCWEVGVSGSSAGPSTRRADTAMTTDDTEVPAMTLAPGHAALETQTLSAETSSRASTPAGPIPEAETRGAKRISPARETRSFTKTSPNFMVLIATSVETSAASGSPEGAGMTTVQTITGSDPREAIFDTLCTDDSSEEAKTLTMDILTLAHTSTEAKGLSSESSASSDSPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSRASESSASSDGLHPVITPSRASESSASSDGPHPVITPSRASESSASSDGPHPVITPSWSPGSDVTLLAEALVTVTNIEVINCSITEIETTTSSIPGASDTDLIPTEGVKASSTSDPPALPDSTEAKPHITEVTASAETLSTAGTTESAAPDATVGTPLPTNSATEREVTAPGATTLSGALVTVSRNPLEETSALSVETPSYVKVSGAAPVSIEAGSAVGKTTSFAGSSASSYSPSEAALKNFTPSETPTMDIATKGPFPTSRDPLPSVPPTTTNSSRGTNSTLAKITTSAKTTMKPPTATPTTARTRPTTDVSAGENGGFLLLRLSVASPEDLTDPRVAERLMQQLHRELHAHAPHFQVSLLRVRRG(MUC 21)SEQ ID NO: 48MKMQKGNVLLMFGLLLHLEAATNSNETSTSANTGSSVISSGASTATNSGSSVTSSGVSTATISGSSVTSNGVSIVTNSEFHTTSSGISTATNSEFSTASSGISIATNSESSTTSSGASTATNSESSTPSSGASTATNSDSSTTSSGASTATNSDSSTTSSEASTATNSESSTTSSGASTATNSESSTVSSRASTATNSESSTTSSGASTATNSESRTTSNGAGTATNSESSTTSSGASTATNSESSTPSSGAGTATNSESSTTSSGAGTATNSESSTVSSGISTVTNSESSTPSSGANTATNSESSTTSSGANTATNSDSSTTSSGASTATNSESSTTSSGASTATNSESSTTSSGASTATNSGSSTTSSGTSTATNSESSTVSSGASTATTSESSTTSSGASTATNSESSTVSSGASTATNSESSTTSSGANTATNSGSSVTSAGSGTAALTGMHTTSHSASTAVSEAKPGGSLVPWEIFLITLVSVVAAVGLFAGLFFCVRNSLSLRNTFNTAVYHPHGLNHGLGPGPGGNHGAPHRPRWSPNWFWRRPVSSIAMEMSGRNSGP(MUC HEG)SEQ ID NO: 49MASPRASRWPPPLLLLLLPLLLLPPAAPGTRDPPPSPARRALSLAPLAGAGLELQLERRPEREPPPTPPRERRGPATPGPSYRAPEPGAATQRGPSGRAPRGGSAASESLHLPSSSSEFDERIAAFQTKSGTASEMGTERAMGLSEEWTVHSQEATTSAWSPSFLPALEMGELTTPSRKRNSSGPDLSWLHFYRTAASSPLLDLSSSSESTEKLNNSTGLQSSSVSQTKTMHVATVFTDGGPRTLRSLTVSLGPVSKTEGFPKDSRIATTSSSVLLSPSAVESRRNSRVTGNPGDEEFIEPSTENEFGLTSLRWQNDSPTFGEHQLASSSEVQNGSPMSQTETVSRSVAPMRGGEITAHWLLTNSTTSADVTGSSASYPEGVNASVLTQFSDSTVHSANAEDRTSGVPSLGTHTLATVTGNGERTLRSVTLTNTSMSTTSGEAGSPAAAMHQETEGASLHVNVTDDMGLVSRSLAASSALGVAGISYGQVRGTAIEQRTSSDHTDHTYLSSTFTKGERALLSITDNSSSSDIVESSTSYIKISNSSHSEYSSFFHAQTERSNISSYDGEYAQPSTESPVLHTSNLPSYTPTINMPNTSVVLDTDAEFVSDSSSSSSSSSSSSSSGPPLPLPSVSQSHHLFSSILPSTRASVHLLKSTSDASTPWSSSPSPLPVSLTTSTSAPLSVSQTTLPQSSSTPVLPRARETPVTSFQTSTMTSFMTMLHSSQTADLKSQSTPHQEKVITESKSPSLVSLPTESTKAVTTNSPLPPSLTESSTEQTLPATSTNLAQMSPTFTTTILKTSQPLMTTPGTLSSTASLVTGPIAVQTTAGKQLSLTHPEILVPQISTEGGISTERNRVIVDATTGLIPLTSVPTSAKEMTTKLGVTAEYSPASRSLGTSPSPQTTVVSTAEDLAPKSATFAVQSSTQSPTTVSSSASVNSCAVNPCLHNGECVADNTSRGYHCRCPPSWQGDDCSVDVNECLSNPCPSTAMCNNTQGSFICKCPVGYQLEKGICNLVRTFVTEFKLKRTFLNTTVEKHSDLQEVENEITKTLNMCFSALPSYIRSTVHASRESNAVVISLQTTFSLASNVTLFDLADRMQKCVNSCKSSAEVCQLLGSQRRIFRAGSLCKRKSPECDKDTSICTDLDGVALCOCKSGYFQFNKMDHSCRACEDGYRLENETCMSCPFGLGGLNCGNPYQLITVVIAAAGGGLLLILGIALIVTCCRKNKNDISKLIFKSGDFQMSPYAEYPKNPRSQEWGREAIEMHENGSTKNLLQMTDVYYSPTSVRNPELERNGLYPAYTGLPGSRHSCIFPGQYNPSFISDESRRRDYF(MUC9)SEQ ID NO: 50MWKLLLWVGL VLVLKHHDGA AHKLVCYFTN WAHSRPGPAS ILPHDLDPFL CTHLIFAFASMNNNQIVAKD LQDEKILYPE FNKLKERNRE LKTLLSIGGW NFGTSRFTTM LSTFANREKFIASVISLLRT HDFDGLDLFF LYPGLRGSPM HDRWTFLFLI EELLFAFRKE ALLTMRPRLLLSAAVSGVPH IVQTSYDVRF LGRLLDFINV LSYDLHGSWE RFTGHNSPLF SLPEDPKSSAYAMNYWRKLG APSEKLIMGI PTYGRTFRLL KASKNGLQAR AIGPASPGKY TKQEGFLAYFEICSFVWGAK KHWIDYQYVP YANKGKEWVG YDNAISFSYK AWFIRREHFG GAMVWTLDMDDVRGTFCGTG PFPLVYVLND ILVRAEFSST SLPQFWLSSA VNSSSTDPER LAVTTAWTTDSKILPPGGEA GVTEIHGKCE NMTITPRGTT VTPTKETVSL GKHTVALGEK TEITGAMTMTSVGHQSMTPG EKALTPVGHQ SVTTGQKTLT SVGYQSVTPG EKTLTPVGHQ SVTPVSHQSVSPGGTTMTPV HFQTETLRON TVAPRRKAVA REKVTVPSRN ISVTPEGQTM PLRGENLTSEVGTHPRMGNL GLQMEAENRM MLSSSPVIQL PEQTPLAFDN RFVPIYGNHS SVNSVTPQTSPLSLKKEIPE NSAVDEEA(MUC18)SEQ ID NO: 51MGLPRLVCAF LLAACCCCPR VAGVPGEAEQ PAPELVEVEV GSTALLKCGL SQSQGNLSHVDWFSVHKEKR TLIFRVRQGQ GOSEPGEYEQ RLSLQDRGAT LALTQVTPQD ERIFLCQGKRPRSQEYRIQL RVYKAPEEPN IQVNPLGIPV NSKEPEEVAT CVGRNGYPIP QVIWYKNGRPLKEEKNRVHI QSSQTVESSG LYTLOSILKA QLVKEDKDAQ FYCELNYRLP SGNHMKESREVTVPVFYPTE KVWLEVEPVG MLKEGDRVEI RCLADGNPPP HFSISKQNPS TREAEEETTNDNGVLVLEPA RKEHSGRYEC QGLDLDTMIS LLSEPQELLV NYVSDVRVSP AAPERQEGSSLTLTCEAESS QDLEFOWLRE ETGQVLERGP VLQLHDLKRE AGGGYRCVAS VPSIPGLNRTQLVNVAIFGP PWMAFKERKV WVKENMVLNL SCEASGHPRP TISWNVNGTA SEQDQDPQRVLSTLNVLVTP ELLETGVECT ASNDLGKNTS ILFLELVNLT TLTPDSNTTT GLSTSTASPHTRANSTSTER KLPEPESRGV VIVAVIVCIL VLAVLGAVLY FLYKKGKLPC RRSGKQEITLPPSRKSELVV EVKSDKLPEE MGLLQGSSGD KRAPGDQGEK YIDLRH(p53 peptide)SEQ ID NO: 52QETFSDLWKLLPENN(p53 peptide)SEQ ID NO: 53DLWKLLPENNVLSPL(p53 peptide)SEQ ID NO: 54DDLMLSPDDIEQWFT(p53 peptide)SEQ ID NO: 55SPDDIEQWFTEDPGP(p53 peptide)SEQ ID NO: 56MPEAAPRVAPAPAAP(p53 peptide)SEQ ID NO: 57SVTCTYSPALNKMFC(p53 peptide)SEQ ID NO: 58YSPALNKMFCQLAKT(p53 peptide)SEQ ID NO: 59NKMFCQLAKTCPVQL(p53 peptide)SEQ ID NO: 60SQHMTEVVRRCPHHE(p53 peptide)SEQ ID NO: 61PQHLIRVEGNLRVEY(p53 peptide)SEQ ID NO: 62LRVEYLDDRNTFRHS(p53 peptide)SEQ ID NO: 63LDDRNTFRHSVVVPY(p53 peptide)SEQ ID NO: 64TFRHSVVVPYEPPEV(p53 peptide)SEQ ID NO: 65VVVPYEPPEVGSDCT(p53 peptide)SEQ ID NO: 66EPPEVGSDCTTIHYN(p53 peptide)SEQ ID NO: 67KKGEPHHELPPGSTK(p53 peptide)SEQ ID NO: 68HHELPPGSTKRALPN(p53 peptide)SEQ ID NO: 69KKLMFKTEGPDSD Example 2 Materials and Methods Synthesis of O-glycopeptides: Chemoenzymatic Synthesis of Library Synthetic peptides: A MUC1 60-mer peptide (VTSAPDTRPAPGSTAPPAHG)n=3representing three tandem repeats were kindly provided by Cancer Research UK. A 24-mer peptide derived from the C-terminal degenerate tandem repeats of MUC1(AHGVTSAPDNRPALGSTAPPVHNV) was synthesized by Schafer-N. A MUC2 33-mer peptide (PTTTPITTTTTVTPTPTPTGTQTPTTTPISTTC) corresponding to 1.4 tandem repeat was synthesized as previously described (Sabbatini, Ragupathi et al. 2007). Eight chemically synthesized 21-mer MUC1 tandem repeat glycopeptides with single Tn or T glycans were available. MUC4 peptides included: PMTDTKTVTTPGSSFTA),(PGSSFTASGHSPSEIVPQD), (SEIVPQDAPTISAATTFAPA),(TTFAPAPTGNGHTTQAPTTA), (TTQAPTTALQAAPSSHD),(APSSHDATLGPSGGTSLSKT), (SLSKTGALTLANSVVSTP),(NSVVSTPGGPEGQWTSASAS), (TSASASTSPRTAAAMTHT),(AAAMTHTHQAESTEASGQT), (EASGQTQTSEPASSGSRTT),(PASSGSRTTSAGTATPSSS), (TATPSSSGASGTTPSGSEGI),(SGSEGISTSGETTRFSSN), (GETTRFSSNPSRDSHTT),'PVTSPSSASTGHTTPLPVTDTSSASTGDTTP),(LPVTSLSSVSTGDTTPLPVTSPSSASTGH),(LPVTSPSSASTGHASPLLVTDASSASTGQ),(PLPVTSPSSASTGHASPLLVTDASSASTGQ),(STGDTLPLPVTDTSSV),(PVTYASSASTGDTTPLPVTDTSSVSTGHAT). Peptides were synthesized at Peptides and Elephants, Gmbh, Germany or Schaefer-N, Copenhagen, Denmark. Synthesis of recombinant mucin glycoprotein fragments inE. coli: N- or C-terminally 6×His and T7 tagged recombinant fragments of MUC2, MUC4, MUC5AC, MUC6, and MUC7 were produced inE. coli(SeeFIG.1B). Gene sequences were inserted into the bacterial expression vectors pET22 (Novagen), pET28 (Novagen) or pET28 (minus), a modified pET 28 vector without N-terminal tags. More than 10 bacterial strains were tested for expression fidelity and efficacy of recombinant mucin protein fragments. Robustness of the system was tested using different expression strategies for mucin-like gene sequences. Expression yields varied from 6 mg/l to 50 mg/l. Based on this, Rosetta2 (Novagen) was selected as the expression host. Overnight cultures were diluted 1:100, and induced 4 h at 37° C. by the addition of IPTG to a final concentration of 0.1 mM. Cell lysates were nickel purified using NiNTA agarose (Qiagen) as described by the manufacturer, and the eluted fractions were analyzed by SDS-PAGE on NuPAGE Bis-Tris 8-12% acryl amide gels (Novex) stained with Coomassie. Eluted fractions of recombinant mucin fragments were HPLC purified before and after in vitro O-glycosylation (see below). Samples were diluted in 0.1% TFA (triflouroacetic acid), loaded onto a Zorbax 300SB-C18 column mounted on an Agilent 1100 HPLC system, and eluted in a 40-minute linear gradient from O-90% acetonitrile. Eluted fractions were lyophilized and resuspended in water and mass confirmed by MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometry was performed on a Voyager-DE™ PRO workstation (Applied Biosystems) using 2,5-dihydroxybenzoic acid (Sigma) as matrix (Mirgorodskaya, Hassan et al. 1999). Synthesis of O-glycopeptides: Peptides and recombinant fragments were O-glycosylated in vitro using recombinant glycosyltransferases as previously described (Tarp, Sorensen et al. 2007) (Wandall, Irazoqui et al. 2007) Briefly, different polypeptide GalNAc-transferase isoforms were used to direct GalNAc O-glycan occupancies on peptides and theDrosophilaCore-1 β3GalT, human Core-3 β3GlcNAc-T and human ST6GalNAc-I were used to produce T, Core-3 and STn glycoforms. SeeFIG.1Afor structures of glycopeptides. All glycopeptides were HPLC purified and characterized by MALDI-TOF. Mucin O-glycopeptide array print and analysis: (Glyco)peptides and control structures were printed on Schott Nexterion® Slide H or Schott Nexterion® Slide H MPX 16 (Schott AG, Mainz, Germany). Quadruplicates of all compounds were printed at 20, 5, and 1 μM in 150 mM sodium phosphate pH 8.5 with 0.005% CHAPS and printed on a BioRobotics MicroGrid II spotter (Genomics Solution) with a 0.21 mm pitch using Stealth 3B Micro Spotting Pins (Telechem International ArrayIt Division). After printing, slides were incubated for 1 h in a humidified hybridization chamber with 75% relative humidity and stored until use at −20° C. Prior to use the microarrays were blocked for 1 h with 25 mM ethanolamine in 100 mM sodium borate pH 8.5. Human sera serially diluted from 1:25-1:400 or monoclonal antibodies (1 μg/ml or hybridoma supernatants) were incubated in a closed container with gentle agitation for 1 h, washed three times in PBS with 0.05% Tween-20 (PBS-T), followed by 1 h incubation with appropriate secondary antibodies. Human IgM and IgG antibodies were detected with Cy3-conjugated goat anti-human IgG (Fc specific) and goat anti-human IgM (Sigma) diluted 1:5000 in PBS-T. For subclass characterization, biotinylated mouse anti-human IgM, IgG1, IgG2, IgG3and IgG4(Sigma) were used and subsequently labelled with Streptavidin-DyLight547 (Pierce), both diluted 1:5,000 in PBS-T. Murine monoclonal antibodies were detected with Cy3-conjugated goat anti-mouse IgM (μ chain specific) and goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:5000 in PBS-T. After incubation with secondary antibodies the slides were washed 3 times in PBS-T, and after the final wash, slides were rinsed shortly in H2O, dried by centrifugation (200×g) and scanned in a ProScanArray HT Microarray Scanner (PerkinElmer) followed by image analysis with ProScanArray Express 4.0 software (PerkinElmer). Each spot were done in 4 replicates and the mean value of relative fluorescence intensity (RFU) was used. A positive sample from the colorectal cancer group was included on each experiment to confirm the reproducible of the assay allowing inter-experimental comparison. For comparison, slides were scanned with identical scanning parameters. Data were analyzed and plotted using Microsoft Excel or Graph Pad Prism software. Monoclonal antibodies: Printing of the MUC1 glycopeptides was confirmed using monoclonal antibodies against MUC1 (HMFG2)(5E10), T-MUC1 (1B9) and 5E5 (Tn-MUC1). Additional mucin fragments were detected with anti-His (AD.1.10; Santa Cruz Biotechnology, USA; Sc-53073) and anti-T7 antibodies (Novagen, USA; 69522) as well as carbohydrate-specific antibodies targeting Tn (1 E3) and STn (3F1). Human sera: Human colorectal cancer sera were obtained from Asterand, Inc., USA and from Hillerod Hospital. Sera from IBD patients were collected from Herlev University Hospital, control sera were obtained from healthy blood donors. Quantification of serum MUC1: MUC1 capture ELISA was performed as previously described (Wandall 2009). Briefly, Immuno MaxiSorp F96 plates (Nunc) were coated with 1 μg/mL mAb HMFG2, blocked, and incubation with serially diluted sera. Amount of bound MUC1 was detected using biotinylated MUC1 specific antibody HMFG2. Generation of monoclonal antibodies: Female Balb/c mice were immunized with 15Core-3-MUC1 60-mer glycopeptide, Tn-MUC4 20mer peptide PVTYASSASTGDTTPLPVTDTSSVSTGHAT and recombinant Tn-MUC4 all conjugated to KLH. Eye bleeds were collected 7 days after the third immunisation and sera tested by ELISA, with the Core-3/Tn-MUC2 glycopeptide serving as negative control or by immunocytochemistry. Three days after the fourth immunisation, spleen cells from one mouse were fused with NS1 myeloma cells (Kohler and Milstein 1975). Hybridomas specific to the antigens of interest were cloned by limiting dilution at least three times. Immunocytochemistry and immunohistochemistry: 6-10 μm tissue sections were fixed for 5 min in ice-cold acetone or 4% paraformaldehyde. Fixed cells were incubated 2 hours at room temperature or overnight at 4° C. with undiluted mAb supernatants, followed by incubation for 45 min at room temperature with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulins in 1:100 dilution with 0.1% BSA (Dako). Slides were mounted in glycerol-containing p-phenylenediamine and examined in a Zeiss fluorescence microscope. Cellular immune response: Peripheral blood mononuclear cells (PBMCs) were isolated from heparin anti-coagulated blood of patients with late stage (III-IV) colorectal cancer using Lymphoprep (Nycomed Pharma AS, Oslo, Norway). Cells were then cultured in X-VIVO 15 medium (Invitrogen) together with 2% heat inactivated human AB serum (Valley Biomedical, Winchester, USA) in round bottom microculture plates (Nunc, Roskilde, Denmark). Cells were cultured as 2*105cells/well and stimulated with peptide pools with 10 peptides in each pool (all from Schafer-N, Copenhagen, Denmark). The concentration of each peptide was 10 mg/ml and the peptide pools were 15-mer peptides with 10 amino acid overlapping, which allowed to test for reactivity against peptides representing the whole MUC1 peptide backbone. 50 IU/ml IL-2 (Proleukin, Chiron, The Netherlands) was added at day 1 and cells were harvested at day 9 and restimulated with the same peptide pools or without peptide as a negative control in an IFN-g ELISPOT assay (Svane, Pedersen et al. 2004) in order to identify MUC-1 specific CD4+ or CD8+ T cells. Results Assembly of the Mucin O-glycopeptide Array The employed strategy was based on chemo-enzymatic synthesis of large glycopeptides, either based on synthetic peptides or large mucin fragments produced inE-coli, and use micro-array technology to identify the most promising targets, followed by their deconvolution by overlapping glycopeptides. We first generated a comprehensive glycopeptide mucin array covering domains from MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7 carrying cancer associated glycans Tn, Sialyl-Tn and truncated Core3. Next targets selective identified The MUC1 tandem repeat was represented by 60mer synthetic peptides as well as recombinant protein. In order to cover multiple potential epitopes in the large mucin proteins we produced recombinant fragments of MUC2, MUC4, MUC5AC, MUC6, and MUC7 by a simple and robustE. coliexpression system. The mucin fragments were glycosylated with a combination of recombinant GalNAc-transferases (GalNAc-T1-4) yielding glycosylation products with high density of GalNAc addition comparable to what is expected in cancer cells. Further elongation of the Tn-glycoforms with sialyltransferase ST6GalNAc-T1 (to synthesize STn) and β3GlcNAc-T6 (to synthesize Core-3) yielded near complete glycosylation of all fragments as verified by SDS-PAGE and MALDI-TOF spectrometry (FIG.7). The integrity of the printed mucin structures was verified by consistent labelling with mAbs to MUC1 (HMFG2), T7-tag, Tn (GalNAc-α-S/T)(1E3/5F4), and STn (NeuAcα2,6GalNAc) (FIG.1D. Some residual Tn reactivity was detected in sialylated and Core-3 elongated glycosylation products, which was expected due to the very high density of Tn structures on the recombinant proteins. Purification of each glycosylation product followed by re-glycosylation with ST6GalNAc-1 or Core-3 synthase did not prevent such Tn exposure, excluding that the incomplete Tn-elongation was due to impurities and accumulation of by-products during in vitro glycosylation. Quality control of the microarrays was ensured by the inclusion a positive cancer sample as standard in each array analysis. The CV (coefficient of the variation) value differed between target compounds. For the targets of interest, however, it was less than 10%. IgG Auto-Antibodies Recognizing Cancer Associated Glycans in Combination with MUC1 Peptide Backbone as Biomarkers in Serum The development of the glycopeptide mucin array allowed us to test for the presence of auto-antibodies in newly diagnosed patients with colorectal cancer (n=58) and healthy controls n=50 (FIG.2). To our surprise we did not detect any significant IgG signature unambiguously identifying colorectal cancer patients from healthy individuals with the either non-glycosylated or glycosylated (Tn-, STn-, and truncated Core-3) recombinant mucin fragments (MUC2, MUC4, MUC5AC, MUC6, and MUC7) (FIG.8). In contrast specific IgG auto-antibodies against Tn-MUC1, STn-MUC1 or truncated Core-3-MUC1 glycopeptide epitopes were identified in colorectal cancer patients (FIGS.2and3A), with none or very low level of reactivity in healthy individuals. The reactivity with Tn-MUC1, STn-MUC1 or truncated Core-3-MUC1 glycopeptide epitopes were very homogeneous and the mean IgG reactivity expressed as fluorescence intensity was used to define the cut-off level for each glyco-peptide target. A serum sample was determined as positive if the fluorescent reactivity with a given glyco-peptide target was higher than three times the SD above the mean of the values obtained with sera from the healthy group. By this definition 74.1% of colorectal cancer patients had circulating antibodies towards either non-glycosylated, Tn, STn-, or Core3-glycosylated MUC1 (FIGS.2and3, Table I). The most selective of the three antibody targets was STn-MUC1, which detected 56.7% (33/58) cancer patients, while Tn-MUC1 detected 39.7 (23/58) and Core3-MUC1 antibodies 44.8% (26/58). For comparison 0-2% healthy individuals tested positive on the different glycoforms of MUC1. An extra set of controls (n=50) were analyzed using these parameters. 2% (1/50) of these controls tested positive for STn-MUC1 antibodies, (1/50) for Tn-MUC1, and (1/50) for Core-3-MUC1 antibodies (FIG.2,3A, Table I). Importantly these results were confirmed with both synthetic peptides covering the MUC1 repeat as well as recombinant MUC1. The induced antibodies did not cross-react with Tn or STn haptens as evidenced by lack of reactivity with other Tn and STn-glycopeptides. In sera from a few cancer patients (4/58; 6,9%) minimal reactivity with non-glycosylated MUC1 was detected, similar to the levels previously reported in breast cancer (von Mensdorff-Pouilly, Petrakou et al. 2000). Tn- and STn-MUC1 glycopeptides with at least two O-glycans in the immunodominant -GSTAP- epitope, i.e. 9 and 15 Tn -MUC1 (with two O-glycans in the -GSTAP- epitope) detected substantially more cancer patients than 6Tn-MUC1 (with one O-glycan in the -GSTAP- epitope). In particular glycopeptides with five glycans per repeat (VTSAPDTRPAPGSTAPPAHG) detected a higher number of colorectal cancer patients than glycoforms with three glycans per repeat (VTSAPDTRPAPGSTAPPAHG) (FIG.2, Table I). This indicates that a considerable number of patients had auto-antibodies directed against the glycosylated PDTR epitope and/or the additional epitope formed by two GalNAc in the -VTS- region of the MUC1 repeat. To define the specificity of the cancer induced auto-antibodies we analyzed serum reactivity with MUC1 peptides carrying GalNAc, STn, and Core-3 at specific glycosylation sites (Table I). The majority of the cancer patients demonstrated their main reactivity with -GST- epitope (82% n/n), which corresponds with the finding that 15 STn/Tn/Core-3-MUC1 60mer peptide was the best target antigen (Table I). Some patients demonstrated polyclonal responses with additional reactivity against the glycosylated -PDTR- epitope (14%) and -VST- epitope (4%). Glycoform Specificity of MUC1 Auto-Antibodies Discriminates Between Colorectal Cancer and Inflammatory Bowel Disease An inherent problem with many cancer markers identified to date is their lack of discrimination between cancer disease and inflammatory lesions causing benign diseases to be identified as cancer. Based on variations in glycan expression by cancer and inflammatory lesions we hypothesized that glycoform specificities of MUC1 auto-antibodies could distinguish between patients with colorectal cancer and chronic inflammatory bowel disease (IBD). Sera from patients diagnosed with Ulcerative Colitis or Crohn's disease in either active or remission state were therefore included in the study for comparison. In accordance with our hypothesis the cancer specific STn-MUC1 antibody response detected in 56.9% (33/58) of the colorectal cancer patients was only detected in 5.9% of IBD patients (FIG.2,3and Table I). This is in agreement with previous findings that tissue expression of the STn epitope is solely associated with dysplasia and colorectal cancer in IBD patients (Itzkowitz, Bloom et al. 1990). In contrast we found significant signals for auto-antibodies to Core-3-MUC1 28.2%) (FIG.2Aand Table I). When searching for the existence of auto-antibodies for the remaining mucin fragments we observed a higher reactivity to the MUC4, MUC5AC and MUC6 in the IBD population compared with healthy and cancer patients, although this was not-significant. Characterization of MUC1 Glyco-Peptide Auto-Antibodies In order to verify the selective nature of the identified auto-antibodies and eliminate cross-reactivity of the glyco-peptide antibodies inhibition studies were performed. The cancer specific serum reactivity to STn-MUC1 and Core-3-MUC1 was selectively inhibited by 40 μg/mL of the respective MUC1 glycopeptides STn-MUC1 and Core3-MUC1 (FIG.3B). Importantly, the reactivity of other mucins and glycoforms was left uninhibited. Only slight inhibition of the Core3-MUC1 reactivity was seen with free carbohydrate GlcNAc confirming that the peptide context of the carbohydrate structure plays an essential role. The selective nature of the cancer generated glycopeptides-MUC1 antibodies was additionally verified by pull-down assays with recombinant Tn-, STn, Core-3-MUC1 coupled to Dynabeads® (FIG.9). The affinity purified serum IgG antibodies selectively reacted with the respective glycoform of MUC1, while the reactivity was diminished in the depleted serum. In accordance with previous findings, IgM antibodies purified on Core-3-MUC1-beads revealed hapten specificity reacting with all mucins carrying Core3. We finally determined the subclass of a subset of the circulating auto-antibodies in cancer patients reactive with STn- and Core3-MUC1. Interestingly these were mainly (68%) of the IgG2subclass along with 23% of the patients having IgG3and 9% having IgG1/3/4. To explain the large interpersonal variance in auto-antibody levels we next examined the presence of measurable cellular response against MUC1 in a separate cohort of patients. For this purpose we used peptide pools that represented T cell epitopes from the whole peptide backbone of MUC1. However, no MUC1 specific CD4+ or CD8+ T cells could be identified. Another possibility for interpersonal variations could be variable presence of circulating antigen. To correlate the presence of circulating MUC1 in cancer patients with the presence of auto-antibodies we next employed a capture ELISA strategy using the mAb HMFG2 recognizing all-glycoforms of MUC1. Although, various MUC1 glycoforms secreted from cancer cell lines (T47D) were readily detected by this method (Wandall 2009), the inventors could not detect any circulating MUC1 in colorectal cancer patients. This is in accordance with the lack of MUC1 detection using the commercial available CA 15-3 assay (Gebauer, Jager et al. 1998), and suggests that most secreted MUC1 is selectively cleared from the circulation. IgA Autoantibody Signatures Against Tn-MUC4 Aid Selective Detection of Colorectal Cancer Because of the large quantities of IgA produced in the colon we next extended our analysis to test for the presence of auto-antibodies of the IgA subclass. The rationale for this approach was the known down-regulation of epithelial transcytosis of polymeric IgA in colon carcinomas mediated by the polymeric immunoglobulin receptor (pIgR) and loss of cellular polarity in the carcinoma cells. The combined effect of these events would be expected to increase circulating IgA specific for relevant cancer targets (Kaetzel 2005) (Baseler, Maxim et al. 1987). In accordance with the hypothesis, we detected increased levels of IgA auto-antibodies targeting the GalNAc glycosylated recombinant MUC4 fragment (Tn-MUC4) or non-glycosylated MUC4 in 75% of colorectal cancer. This finding was MUC4 specific. In contrast IgA auto-antibody reactivity to membrane mucin MUC1 and secreted mucins MUC2, MUC5AC, MUC6, and MUC7 could not be used to discriminate between colon cancer patients and healthy individuals. To evaluate if the IgA auto-antibody responses to MUC4 were due to a single immunodominant epitope or the collective reactivity of a polyclonal immune-responses, we analyzed overlapping glycopeptides covering the recombinant fragment of MUC4 with the inclusion of an additional set of 20mer MUC4 peptides covering the MUC4 tandem repeat area. The immune response against MUC4 had a polyclonal nature. However, up to 30% of the colorectal cancer patients had IgA autoantibodies to a single MUC4 tandem repeat peptide (TRM4-5; PVTYASSASTGDTTPLPVTDTSSVSTGHAT), with the majority of these patients having glycopeptide specific responses to GalNAc-glycosylated MUC4 tandem repeat peptide (FIG.4, C). Apart from the immunodominant Tn-TRM4-5 glycopeptide, each cancer patient recognised different peptide and glyco-peptide epitopes among the remaining MUC4 targets. In order to compile results obtained with all MUC4 peptides, the values obtained for each target were expressed as the number of SD above the mean values from healthy patients. Thereby, a multiplex result was constructed, which demonstrated that 79.3% of cancer patients had detectable IgA MUC4 antibodies with cut-off values elected to be five times the SD of the mean of values from healthy patients to ensure high specificity. Importantly, the level of IgA reactivity was much lower in patients with inflammatory bowel disease (FIG.4). We next tested IgA levels against MUC4 in serum from patients with other cancer than colorectal (prostate, ovarian, breast). A significantly portion of the patients had IgA auto-antibodies against epitopes outside the tandem repeat, but only little reactivity to the tandem-repeat region (7.1%) compared with the colorectal cancer group (55.1%). Generation of Glycopeptide Specific Antibodies to MUC1 and MUC4 Glycopeptides Two of the identified immodominant MUC1 glycopeptides (Tn-MUCI and STn-MUC1) has previously been shown to override tolerance in humans and humanized mice with the generation of potent monoclonal antibodies (5E5 and 2D9) specific for combined glycopeptide epitopes (Sorensen, Reis et al. 2006) (Tarp, Sorensen et al. 2007). In the present study we tested if the novel identified immunodominant glycopeptides Core3-MUC1 and the two different Tn-MUC4 targets (Tn-recMUC4 and Tn-MUC4TR5) induced immune-responses in wild type mice enabling generation of glycopeptides specific monoclonal antibodies. 60mer MUC1-Core3 glycopeptide with complete O-glycan occupancy and conjugated to KLH elicited strong polyclonal antibody response in Balb/c mice reacting with fully glycosylated Core3-MUC1 as demonstrated by ELISA (FIG.5). A monoclonal antibody was produced with specificity for MUC1 carrying the Core-3 structure in T in -PDTR- in the MUC1 tandem repeat as analyzed by ELISA and array presenting mucins with cancer associated glycans. Reactivity was detected with Sialylated-Core3 as demonstrated by array analysis (FIG.5C-E). No cross reactivity was detected with non-Core3-based structures or with other mucins carrying Core-3. In the same way, we tested the immunogenicity of Tn-MUC4rec and Tn-MUC4TR5-KLH. In both cases a prominent immune response specific for Tn-MUC4 was generated in Balb/c mice (FIG.5, Panel I). A monoclonal antibody 6E3 was generated using Tn-MUC4rec as immunogen. Elisa and array analysis demonstrated strong reactivity with Tn-MUC4 (FIG.5I-L). No reactivity was seen with non-glycosylated peptide, while some cross-reactivity was noted with Tn-MUC1. Both the monoclonal antibody 6E3 and the polyclonal sera from mice immunized with Tn-MUC4TR stained HT29 and LSC cells known to express Tn-MUC4 due to disrupted cosmc and hence T-synthase function. No reaction was seen with cells not expressing MUC4. These findings confirm the immunogenic nature of the glycopeptides identified as targets in the screen for autologous antibodies and provide valuable tools for studying the expression of such glycopeptide epitopes in human cancer and inflammatory lesions. Tissue Expression of Immunodominant MUC1 and MUC4 Glycopeptide Epitopes in Colorectal Cancer and Inflammatory Bowel Disease Using the developed glycopeptide specific antibodies we next tested the expression of Tn/STn-MUC1, Core3-MUC1, Tn-MUC4 in healthy tissue, tissue from inflammatory bowel patients and cancer patients. Examination of 25 cases of colorectal adenocarcinomas for the expression of Tn-, STn, and Core-3 MUC1 was performed with the well-characterized mAbs HMFG2, 5E5 and 2D9 with specificity for non-glycosylated MUC1, Tn- and STn-MUC1 respectively (Sorensen, Reis et al. 2006; Tarp, Sorensen et al. 2007). Core-3-MUC1 and Tn-MUC4 expression was analyzed with the novel Core3-MUC1, Tn-MUC4 mAbs (5C10, 6E3) respectively. 92% (23/25) of the cancer cases were positive, with between 20-90% of the cancer cells staining bright positive. Intensive labelling of intracellular structures as well as the luminal surface of cancer cells were seen with mAbs HMFG2, 5E5, 5C10, and 2D9 verifying the presentation of large amounts of Tn-, STn, and Core3-MUC1 on cancer cells. Importantly, apparent healthy neighbouring tissue had substantial lower expression levels, although a supranuclear staining pattern were seen in most cells. ADCC 6E3 Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy donors by density centrifugation with Lymphoprep (Nycomed Pharma Diagnostics, Oslo, Norway). The blood was diluted 1:2 in PBS with 0.5% FBS and was gently poured down the side of 50 ml tubes containing 15 ml Lymphoprep. After centrifugation at 2000 rpm in 20 min, the layer containing PBMCs was transferred to a new tube, washed, and resuspended in RPMI 1640 before eosin staining for evaluation of viability. ADCC were tested using a standard 51Cr release assay. Target cells were loaded with 100 μCi 51Cr (PerkinElmer) for 1 h at 37° C., and washed in culture media×5, and plated at 1×104 per well in a 96-well flat-bottom plate before incubation for 1 hour with antibody before addition of effector cells. After 4 hours of incubation at 37° C., 30 μl of supernatant was removed from each well and plated on a LumaPlate-96, dried down, and counted on a Packard's TopCount®. The results demonstrated inFIG.13are expressed as the percentage of specific release calculated by following equation: (experimental release−spontaneous release×100)/(maximal release−spontaneous release), where the spontaneous release represents the mean cpm for target cells incubated without antibody and immune effectors, and the maximal release represents the mean cpm for target cells incubated with 30% ethanol. P53 Array Print and Analysis. 15 mer peptides with 10 amino acid overlap representing the whole p53 protein backbone and control structures were printed on Schott Nexterion® Slide H or Schott Nexterion® Slide H MPX 16 (Schott AG, Mainz, Germany). Quadruplicates of all compounds were printed at 200 and 50 μM in 150 mM sodium phosphate pH 8.5 with 0.005% CHAPS and printed on a BioRobotics MicroGrid II spotter (Genomics Solution) with a 0.21 mm pitch using Stealth 3B Micro Spotting Pins (Telechem International ArrayIt Division). After printing, slides were incubated for 1 h in a humidified hybridization chamber with 75% relative humidity and stored until use at −20° C. Prior to use unspotted slide areas were blocked for 1 h with 25 mM ethanolamine in 100 mM sodium borate pH 8.5. Human sera serially diluted 1:25 and were incubated in a closed container with gentle agitation for 1 h, washed three times in PBS with 0.05% Tween-(PBS-T), followed by 1 h incubation with appropriate secondary antibodies. Human IgG antibodies were detected with Cy3-conjugated goat anti-human IgG (Fc specific) diluted 1:5000 in PBS-T. After incubation with secondary antibodies the slides were washed 3 times in PBS-T, and after the final wash, slides were rinsed shortly in H2O, dried by centrifugation (200×g) and scanned in a ProScanArray HT Microarray Scanner (PerkinElmer) followed by image analysis with ProScanArray Express 4.0 software (PerkinElmer). Each spot were done in 4 replicates and the mean value of relative fluorescence intensity (RFU) was used. For comparison, slides were scanned with identical scanning parameters. Data were analyzed and plotted using Microsoft Excel or GraphPad Prism software. A total of 78 15mer peptides were printed. 18 peptides p53 peptides (number: 4, 5, 9, 10, 14, 25, 26, 27, 34, 39, 41, 42, 43, 44, 45, 58, 59, and 78) had had sensitivity over 10% with specificity 95%. 19 out of 58 colorectal cancer patients had autoantibodies to peptide 34. Combining the p53-34 with MUC1STn increased the sensitivity from 57% to 72%; a combination of p53-34, p53-44, and MUC1 STn increased the sensitivity to 79% with a specificity of 94%. The results are demonstrated inFIG.15. TABLE IIgG Auto-antibodies to selected MUC1 glycopeptidesPercentage of healthy controls, IBD patients and colorectal cancerpatients with auto-antibodies to MUC1 glycopeptides. A positive testis defined as 3 standard deviation over the mean of the healthy controls.Combined results with two or three glycopeptides are also shown.GlycopeptideControlsIBDCRCMUC14% (2/50)2.6% (1/39)6.9% (4/58)MUC1 6Tn2% (1/50)5.1% (2/39)20.7% (12/58)MUC1 9Tn2% (1/50)5.1% (2/39)20.7% (12/58)MUC1 15Tn2% (1/50)15.4% (6/39)39.7% (23/58)MUC1 9Core32% (1/50)17.9% (7/39)27.6% (16/58)MUC1 15Core30% (1/50)20.5% (8/39)44.8% (26/58)MUC1 9STn2% (1/50)7.7% (3/39)41.4% (23/58)MUC1 15STn2% (1/50)10.3% (4/39)56.9% (33/58)MUC1 9C3 or 15C32% (1/50)28.2% (11/39)44.8% (26/58)MUC1 STn or C34% (2/50)33% (13/39)63.8% (37/58)MUC1 Tn or STn or8% (4/50)38.5% (15/39)74.1% (43/58)C3 TABLE IIIgA Auto-antibodies to selected MUC4 glycopeptidesPercentage of the healthy controls, IBD patients, colorectal cancer patients, and acombined group of prostate, ovarian and breast cancer patients with IgA auto-antibodies to different MUC4 glycopeptides. A positive test is defined as 5 standarddeviation over the mean of the healthy controls. Combined results withtwo or more glycopeptides are also shown.GlycopeptideProstate,Ovarian andControlsIBDCRCBreast cancerMUC40%2.6% (1/39)29.3% (17/58)3.6% (1/28)MUC4 Tn0%7.7% (3/39)37.9% (22/58)7.1% (2/28)MUC4 non0%7.7% (3/39)55.1% (32/58)7.1% (2/28)glycosylated or TnMUC4s fragments0%17.9% (7/39)41.4% (24/58)35.7% (10/28)MUC4s fragments Tn0%7.7% (3/39)29.3% (17/58)57.1% (16/28)All Tn-MUC40%7.7% (3/39)53.4% (31/58)57.1% (16/28)recMUC4s0%2.6% (3/39)3.4% (2/58)7.1% (2/28)recMUC4s Tn0%2.6% (3/39)13.8% (8/58)32.1% (9/28)MUC1Tn, STn, C38%43.6% (17/39)87.9% (51/58)MUC4 Tn, TR2%15.4% (6/39)82.8% (48/58)MUC1STn+ MUC4 TABLE IIILegend to glycopeptide sequences of Table IIMUC4(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP)(LPVTSLSSVSTGDTTPLPVTSPSSASTGH)(LPVTSPSSASTGHASPLLVTDASSASTGQ)(STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT)MUC4 Tn(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ)(S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)MUC4 non(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP)glycosylated or(LPVTSLSSVSTGDTTPLPVTSPSSASTGH)Tn(LPVTSPSSASTGHASPLLVTDASSASTGQ)(STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ)(S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)MUC4sPMTDTKTVTTPGSSFTA (SEQ ID NO: 3),fragmentsPGSSFTASGHSPSEIVPQD (SEQ ID NO: 4),SEIVPQDAPTISAATTFAPA (SEQ IDNO: 5),TTFAPAPTGNGHTTQAPTTA (SEQ ID NO: 6),TTQAPTTALQAAPSSHD (SEQ ID NO: 7),APSSHDATLGPSGGTSLSKT (SEQ ID NO: 8),SLSKTGALTLANSVVSTP (SEQ ID NO: 9),NSVVSTPGGPEGQWTSASAS (SEQ IDNO: 10),TSASASTSPRTAAAMTHT (SEQ ID NO: 11),AAAMTHTHQAESTEASGQT(SEQ ID NO: 12),EASGQTQTSEPASSGSRTT (SEQ ID NO: 13),PASSGSRTTSAGTATPSSS (SEQ ID NO: 14),TATPSSSGASGTTPSGSEGI (SEQ ID15 NO: 15),GSEGISTSGETTRFSSN (SEQ ID NO: 16),GETTRFSSNPSRDSHTT (SEQ ID NO: 17)MUC4sPMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO: 3),fragments TnPGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),All Tn-MUC4S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO: 7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO: 8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16),GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(PLPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ)(S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)(PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*)PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO: 3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO: 7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO: 8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16),GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17)Recombinant(PMTDTKTVTTPGSSFTASGHSPSEIVPQDAPTISAATZFAPAPTGNMUC4sGHTTQAPTTALQAAPSSHDATLGPSGGTSLSKTGALTLANSVVSTPGGPEGQWTSASASTSPDTAAAMTHTHQAESTEASGQTQTSEPASSGSRTTSAGTATPSSSGASGTTPSGSEGISTSGETTRFSSNPSRDSHTT)Recombinant(PMT*DT*KT*VT*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZMUC4s TnFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*)MUC1Tn,STn,C3(MUC1STn)MUC4 TnVT(Tn)S(Tn)APDT(Tn)RPAPGS(Tn)T(Tn)APPAHGVT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHGVT(Core3)S(Core3)APDT(Core3)RPAPGS(Core3)T(Core3)APPAHGMUC4(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP)(LPVTSLSSVSTGDTTPLPVTSPSSASTGH)(LPVTSPSSASTGHASPLLVTDASSASTGQ)(STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ)(S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*)(PMT*DT*KTV*T*T*PGS*S*FT*AS*GHS*PS*EIVPQDAPT*IS*AAT*ZFAPAPT*GNGHT*T*QAPT*T*ALQAAPS*S*HDAT*LGPS*GGT*S*LS*KT*GALT*LANS*VVS*T*PGGPEGQWT*S*AS*AS*T*S*PDT*AAAMT*HT*HQAES*T*EAS*GQT*QT*S*EPAS*S*GS*RT*T*S*AGT*AT*PS*S*S*GAS*GT*T*PS*GS*EGIS*T*S*GET*T*RFS*S*NPS*RDS*HT*T*)PMT*DT*KT*VT*T*PGS*S*FT*A (SEQ ID NO: 3),PGS*S*FT*AS*GHS*PS*EIVPQD (SEQ ID NO: 4),S*EIVPQDAPT*IS*AAT*T*FAPA (SEQ IDNO: 5),T*T*FAPAPT*GNGHT*T*QAPT*T*A (SEQ ID NO: 6),T*T*QAPT*T*ALQAAPS*S*HD(SEQ ID NO:7),APS*S*HDAT*LGPS*GGT*S*LS*KT* (SEQ ID NO:8),S*LS*KT*GALT*LANS*VVS*T*P (SEQ ID NO: 9),NS*VVS*T*PGGPEGQWT*S*AS*AS* (SEQ IDNO: 10),T*S*AS*AS*T*S*PRT*AAAMT*HT* (SEQ ID NO: 11),AAAMT*HT*HQAES*T*EAS*GQT*(SEQ ID NO: 12),EAS*GQT*QT*S*EPAS*S*GS*RT*T* (SEQ ID NO: 13),PAS*S*GS*RT*T*S*AGT*AT*PS*S*S* (SEQ ID NO: 14),T*AT*PS*S*S*GAS*GT*T*PS*GS*EGI (SEQ ID15 NO: 15),GS*EGIS*T*S*GET*T*RFS*S*N (SEQ ID NO: 16),GET*T*RFS*S*NPS*RDS*HT*T* (SEQ ID NO: 17)MUC1STn+MUC1STnMUC4VT(STn)S(STn)APDT(STn)RPAPGS(STn)T(STn)APPAHGMUC4(PVTSPSSASTGHTTPLPVTDTSSASTGDTTP)(LPVTSLSSVSTGDTTPLPVTSPSSASTGH)(LPVTSPSSASTGHASPLLVTDASSASTGQ)(STGDTLPLPVTDTSSV)(PVTYASSASTGDTTPLPVTDTSSVSTGHAT)(PVT*S*PS*S*AS*T*GHT*T*PLPVT*DT*S*S*AS*T*GDT*T*P)(LPVT*S*LS*S*VS*T*GDT*T*PLPVT*S*PS*S*AS*T*GH)(LPVT*S*PS*S*AS*T*GHAS*PLLVT*DAS*S*AS*T*GQ)(S*T*GDT*LPLPVT*DT*S*S*V)(PVT*YAS*S*AS*T*GDT*T*PLPVT*DT*S*S*VS*T*GHAT*) | 334,048 |
11860166 | DETAILED DESCRIPTION Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list. Unless defined otherwise, all technical terms used herein have the same meanings as those generally understood by one of ordinary skill in the art to which the present disclosure belongs. Further, although methods or samples are described herein, those similar or equivalent thereto are also incorporated in the scope of the present disclosure. Processes described below are only one example according to the present disclosure. In the present disclosure, part of each process may be omitted or repeatedly performed, and in some cases, the order of the processes may be appropriately changed. The numerical values described herein are considered to include the meaning of “about”, unless otherwise specified. The contents of all the publications disclosed as references herein are incorporated in the present disclosure. An aspect provides a red fluorescent protein-based biosensor for measuring the activity of dopamine receptor D1, the red fluorescent protein-based biosensor including: a recombinant gene including a dopamine receptor D1 gene, and a gene encoding a red fluorescent protein or a modified red fluorescent protein; a recombinant expression vector including the recombinant gene; or a transformant transformed with the recombinant expression vector. FIG.1Ais a schematic diagram illustrating a working principle and a mechanism of a red fluorescent biosensor for measuring the activity of dopamine receptor D1 (DRD1) and detecting a ligand (e.g., dopamine, DA) binding to DRD1. As used herein, “dopamine (DA or 3,4-dihydroxyphenethylamine, C8H11NO2)”, a catecholamine-based organic compound, refers to a hormone or neurotransmitter found in the central nervous system of various animals. Dopamine is one of the neurotransmitters secreted to transmit certain signals between cranial nerve cells, and activates dopamine receptors. As used herein, the “dopamine receptor” is a type of G-protein coupled receptor (GPCR), and five subtypes (D1, D2, D3, D4, and D5 types) are known. The subtypes of the dopamine receptors may be further classified into D1-like receptors (D1 and D5) and D2-like receptors (D2, D3, and D4). The receptors are each unique in terms of intimacy with dopamine, binding to G proteins, signaling methods, distribution of specific neurons, etc. For example, D1-like receptors transduce signals to the cAMP pathway through separation of Gas in the G protein, whereas D2-like receptors inhibit adenylate cyclase (AC) activity through separation of Gαi, resulting in reduction of cAMP levels. In one specific embodiment, the dopamine receptor may be dopamine receptor D1. In another specific embodiment, the dopamine receptor D1 gene may encode an amino acid represented by SEQ ID NO: 1 (FIG.6A). As used herein, the “fluorescent protein” refers to a protein that exhibits fluorescence when exposed to light. Examples of the fluorescent protein may include a red fluorescent protein (RFP), a blue fluorescent protein (BFP), an enhanced blue fluorescent protein (EBFP), a cyan fluorescent protein (CFP), an enhanced cyan fluorescent protein (ECFP), a yellow fluorescent protein (YFP), an enhanced yellow fluorescent protein (EYFP), a green fluorescent protein (GFP), a modified green fluorescent protein, an enhanced green fluorescent protein (EGFP), etc. In one specific embodiment, the fluorescent protein may be a red fluorescent protein or a modified red fluorescent protein. In one specific embodiment, the red fluorescent protein may be circularly permuted red fluorescent protein (cpRFP). As used herein, the term “circular permutation” means a modification whereby new N- and C-termini are created in a protein (i.e., the protein is split into two parts), and original N- and C-termini of the protein are linked through an appropriate linker sequence. In one specific embodiment, the circular permutation modification is induced in the fluorescent protein to change wavelength characteristics (e.g., absorption wavelength and/or fluorescence wavelength). In the circularly permuted fluorescent protein according to one specific embodiment, new N- and C-termini are located in close proximity to a chromophore which is necessary for fluorescence, and arbitrary proteins x and y are linked thereto such that fluorescence intensity is changed in response to their interaction. Based on this, a biosensor was developed. FIG.1Bis a schematic diagram illustrating modification of red fluorescent protein according to the kind and position change of a linker peptide (linker amino acids) introduced to optimize the biosensor for measuring the activity of dopamine receptor D1. LSSxx represents a linker peptide composed of amino acids of leucine(L)-serine(S)-serine(S) linked to the N-terminus of cpRFP and any two amino acids (x)(x) linked thereto. xxDDL represents a linker peptide composed of amino acids of aspartic acid (D)-aspartic acid (D)-leucine(L) linked to the C-terminus of cpRFP and any two amino acids (x)(x) linked thereto. In one specific embodiment, the gene encoding the modified red fluorescent protein may include a gene encoding an amino acid sequence according to the following Formula 1: L1-cpRFP-L2[Formula 1] in Formula 1, L1includes a linker peptide having LSS and 1 to 5 amino acid residues at the N-terminus of cpRFP, and L2includes a linker peptide having 1 to 5 amino acid residues and DDL at the C-terminus of cpRFP. The amino acid residues may be any amino acid residues each independently selected. The amino acid residue may be selected from naturally occurring amino acids. In one specific embodiment, in Formula 1, L1includes a linker peptide having LSS and any 2 independently selected amino acid residues at the N-terminus of cpRFP, and L2includes a linker peptide having any 2 independently selected amino acid residues and DDL at the C-terminus of cpRFP. In one specific embodiment, L1includes LSSX1X2, and L2includes X3X4DDL, wherein X1, X2, X3, and X4may be each independently any amino acid. For example, X1, X2, X3, and X4may be each independently any one amino acid selected from the group consisting of alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), pyrrolysine (O), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), selenocysteine (U), valine (V), tryptophan (W), and tyrosine (Y). In one specific embodiment, X1X2may be any one selected from the group consisting of glutamic acid-arginine (ER), glutamine-arginine (QR), and arginine-arginine (RR), and X3X4may be any one selected from the group consisting of tyrosine-aspartic acid (YD), threonine-serine (TS), and histidine-proline (HP). In one specific embodiment, X1X2may be glutamic acid-arginine (ER), and X3X4may be tyrosine-aspartic acid (YD). The biosensor according to one specific embodiment may include a sequence of “LSS-ER-cpRFP-YD-DDL”. The receptor D1 biosensor including cpRFP having the substituted sequence exhibits high fluorescence intensity by reacting with a ligand binding to dopamine receptor D1, and has the excellent effect of measuring the activity of dopamine receptor D1. In one specific embodiment, the gene encoding the modified red fluorescent protein may include a gene encoding an amino acid sequence represented by SEQ ID NO: 2 (FIG.6(B)). It is known that when G protein binding receptor (GPCR) is activated by ligands, its structural changes are most frequently found in Intracellular loop 3 (ICL3). In one specific embodiment, the amino acid sequence of the red fluorescent protein or the modified red fluorescent protein may bind to ICL3 of the dopamine receptor D1. In one specific embodiment, the amino acid sequence of the red fluorescent protein or the modified red fluorescent protein may bind immediately after any one of the 1stto 54thamino acids from the N-terminus of ICL3 of dopamine receptor D1. In one specific embodiment, the amino acid sequence of the red fluorescent protein or the modified red fluorescent protein may bind immediately after the 9thamino acid from the N-terminus of ICL3 of dopamine receptor D1. In one specific embodiment, the gene of the dopamine receptor D1 may encode a sequence in which any one amino acid of 120thto 140thamino acids of the amino acid sequence represented by SEQ ID NO: 1 is substituted with another amino acid. In one specific embodiment, the gene of the dopamine receptor D1 may encode an amino acid sequence in which phenylalanine (F) at the 129thposition of the amino acid sequence represented by SEQ ID NO: 1 is substituted with alanine (A). The receptor D1 biosensor including the amino acid sequence in which the amino acid at the 129thposition in SEQ ID NO: 1 is substituted with alanine (A) exhibits high fluorescence intensity by reacting with the ligand binding to dopamine receptor D1, and has the excellent effect of measuring the activity of dopamine receptor D1. The fluorescent biosensor for measuring the activity of dopamine receptor D1 according to one specific embodiment may measure the activity of dopamine receptor ligand at concentration of 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 5 μM, 6.25 μM, 10 μM, 12.5 μM, 50 μM, or 100 μM. The fluorescent biosensor for measuring the activity of dopamine receptor D1 according to one specific embodiment may effectively detect the dopamine receptor ligand at a concentration of 10 μM or more. In one specific embodiment, the recombinant gene may further include a gene encoding a signal peptide. In one specific embodiment, the signal peptide may include an amino acid sequence represented by SEQ ID NO: 3 (FIG.6C). In one specific embodiment, the recombinant gene may include an amino acid sequence represented by SEQ ID NO: 4 (FIG.6D). FIG.2is a schematic diagram illustrating a procedure of constructing candidate plasmids for preparing the biosensor for measuring the activity of dopamine receptor D1. To insert the nucleotide sequence of cpRFP into ICL3 of the dopamine receptor D1, based on cpRFP, any two independent linker amino acids were added after LSS linked to the N-terminus of cpRFP and before DDL linked to C-terminus of cpRFP. Random mutations were induced in two amino acids each immediately before and after cpRFP to construct candidate plasmids for the dopamine receptor D1 biosensor (FIG.2). Another aspect provides a method of measuring the activity of dopamine receptor D1 using the red fluorescent protein-based biosensor, the method including: introducing the biosensor into cells; adding a test sample to the biosensor-introduced cells; and measuring the activity of the dopamine receptor D1 according to the addition of the test sample to the biosensor. Still another aspect provides a method of detecting a ligand binding to the dopamine receptor D1 using the red fluorescent protein-based biosensor, the method including: introducing the biosensor into cells; adding a test sample to the biosensor-introduced cells; and detecting the ligand binding to the dopamine receptor D1 according to the addition of the test sample to the biosensor. In one specific embodiment, provided is a method of detecting dopamine or dopamine agonists using the red fluorescent protein-based biosensor. As used herein, the term “gene” refers to any nucleotide sequence or part thereof that has a functional role in protein coding or transcription or regulation of other gene expression. The gene may consist of the entire of nucleotides encoding a functional protein or only a part of nucleotides encoding or expressing a protein. The nucleotide sequence may include exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or gene abnormalities within a unique sequence adjacent to the gene. As used herein, the “protein” also includes fragments, analogs, and derivatives thereof that retain biological activity or function substantially identical to that of a reference protein. As used herein, the “transfection” refers to a process whereby extracellular DNA enters a host cell in the presence or absence of an accompanying substance. Transfected cells refer to cells having extracellular DNA by introducing the extracellular DNA to the cells. DNA may be introduced into cells so that it may be inserted into chromosome or it may replicate as an extrachromosomal material. As used herein, the “transduction” is a phenomenon in which DNA of a bacterium is transferred to another bacterium via a virus. Transduction, even when a foreign DNA is introduced into another cell via a viral vector, is often used by molecular biologists to introduce a foreign gene into the host cell's genome. As used herein, the “transformation” refers to a molecular biological phenomenon in which a DNA chain fragment or plasmid containing a gene of a different kind from that of original cells penetrate the cells and binds to DNA present in the original cells, and as a result, genetic characteristics of the cell are changed. Cells introduced with a foreign DNA, etc. are called ‘transformant’. As used herein, the term “vector” refers to any nucleic acid including a competent nucleotide sequence that is inserted into a host cell to be incorporated into the genome of the host cell by recombination, or to autonomously replicate as an episome. Such a vector may include a linear nucleic acid, a plasmid, a phagemid, a cosmid, an RNA vector, a viral vector, etc. As used herein, the expression “about” or “approximately” means that a mentioned value may vary to some degree. For example, the value may vary to 10%, 5%, 2%, or 1%. As used herein, the term “have”, “may have”, “include”, or “may include” indicates existence of corresponding features (e.g., elements such as numeric values or components) but do not exclude presence of additional features. As used herein, the term “including” does not limit the present disclosure to exclude any modification or addition. Although described using the term “including”, the method, substance, and composition described herein may be described using “consisting substantially of” or “consisting of”. As used herein, the singular forms include plural forms unless the context clearly dictates otherwise. EXAMPLES Hereinafter, the present disclosure will be described in more detail with reference to the following Examples. However, the following Examples are only for illustrating the present disclosure, and the scope of the present disclosure is not limited by these Examples. As used herein, the term “to” includes the endpoint of the range and all midpoints therebetween. Those skilled in the art will understand that the numerical amount of deviation is possible. Therefore, it is understood that whenever a numerical value is mentioned in the specification or claims, the numerical value or any additional value relating to such a numerical value is also within the scope of the present disclosure. Example 1. Preparation of Red Fluorescent Protein-Based Biosensor for Measuring Activity of Dopamine Receptor D1 (1) Construction of Genetic Recombinant Plasmid cDNA of human dopamine receptor D1 amplified by PCR was obtained (SEQ ID NO: 5). The gene of human dopamine receptor D1 encodes an amino acid represented by SEQ ID NO: 1. The amplified cDNA of dopamine receptor D1 thus obtained was fused into a pcDNA5/FRT plasmid vector digested with Cla1/Xho1 restriction enzymes using an infusion technique. Further, cDNA of cpRFP which is circularly permuted red fluorescent protein was amplified by PCR (cDNA of cpRFP, SEQ ID NO: 6). To optimize the biosensor for measuring the activity of dopamine receptor D1, a genetic recombinant plasmid was prepared, in which the amplified cpRFP was inserted into a specific site of ICL3 (immediately after the 9thamino acid from the N-terminus of ICL3) which is an intracellular loop 3 of dopamine receptor D1 (FIG.1B). To insert the nucleotide sequence of cpRFP into ICL3 of the dopamine receptor D1, 5 linker peptides (linker amino acids) were added before and after cpRFP, respectively such that LSSPV-cpRFP-TDDDL was prepared. (2) Random Mutagenesis and Transfection In the recombinant amino acid sequence (LSSXaXb-cpRFP-XcXdDDL) including the linker peptides each linked to the N- and C-termini of cpRFP, random mutation was induced in two amino acids each immediately before and after cpRFP to prepare candidate plasmids for the dopamine receptor D1 biosensor (FIG.2). A variety of genetic recombinant dopamine receptor D1 plasmids were constructed using a principle of the cpRFP-based biosensor, in which the brightness of the sensor varies depending on the linker peptide arrangement. In the linker peptides (LSSXaXband XcXdDDL) each linked to the N- and C-termini of cpRFP, random mutation was induced in two amino acids (XaXband XcXd) each immediately before and after cpRFP to substitute XaXband XcXdwith glutamic acid-arginine(ER) and tyrosine-aspartic acid (YD), respectively. (3) Selection of Candidate Plasmids for Dopamine Receptor D1 Biosensor and Cell Culture After transformation intoEscherichia colias a competent cell, a large amount of genetic recombinant plasmids were obtained through cloning. Subsequently, isolation and purification were performed, and then plasmids of the sensor for measuring the activity of dopamine receptor D1 were obtained through sequencing. Each plasmid was transfected into an animal cell HEK293A, followed by cell culture. The HEK293A cell line was seeded on a cell culture dish at an equal density, and cultured at a temperature of 37° C. and 5% CO2for about 16 hours. Thereafter, 2 μl of Lipofectamine 2000 and the prepared dopamine receptor D1 biosensor (1 μg) were stabilized in an Opti-MEM medium (ThermoFisher Scientific) for 20 minutes, and then added to subcultured cells. 6 hours after transfection, cells were subcultured in a mini-confocal dish coated with fibronectin, and DMEM (Hyclone, SH30604.01) containing 10% (v/v) fetal bovine serum (FBS) (Hyclone, SH30084.03) and 0.5% penicillin/streptomycin (Corning, 30-002-CI) was added, followed by culturing overnight. Next day, observation was performed using a live fluorescence microscope. Through the live fluorescence microscope, HEK293A single cells transfected with the biosensor for measuring the activity of dopamine receptor D1 were observed. To measure the activity of dopamine receptor D1, the dopamine receptor D1 biosensor is required to locate in the cell membrane of animal cell HEK293A. Thus, whether the biosensor located in the cell membrane of animal cell HEK293A was examined through a fluorescence microscope. Further, according to the principle of the cpRFP-based biosensor, in which the brightness of the sensor varies depending on the linker peptide arrangement, sensor candidates with high expression efficiency were imaged using the fluorescence microscope, after treatment of the medium with dopamine. (4) Measurement of Activity of Dopamine Receptor D1 Using Dopamine Receptor D1 Biosensor The prepared dopamine receptor D1 biosensor was treated with a ligand of dopamine receptor D1 (agonist), and fluorescence intensity before and after the treatment was measured in real time to determine intensity change. First, dopamine which is a ligand of dopamine receptor D1 was prepared at a final concentration of 10 μM and added to a system. To minimize a photobleaching phenomenon, the live fluorescence microscope was set. When stimulated with light at a wavelength of 560 nm in the live fluorescence microscope, intensity at a cpRFP fluorescence wavelength of 610 nm was measured. To induce fluorescence intensity suitable for experiments, 50 msec exposure and ND16 intensity were maintained. The fluorescence cycle was performed for total 10 minutes by measuring cpRFP images for 1 minute using the fluorescence microscope. The saved images were analyzed using an NIS program (Nikon). cpRFP fluorescence intensity of the entire single cells in the regions of interest (ROI) was analyzed in the NIS program. Change of the corresponding fluorescence intensity means efficiency of the biosensor for measuring the activity of dopamine receptor D1 in the single cell. After acquiring the change of fluorescence intensity over time, the fluorescence intensity before adding the ligand dopamine was averaged and set as Fo, and the change of fluorescence intensity over time was set as F, and then F/Fo data were made and normalized. FIG.3Ais a graph showing changes (F/F0) in fluorescence intensity of the biosensor candidates for measuring the activity of dopamine receptor D1 with respect to dopamine as an agonist. The X-axis represents the number of the biosensor candidates (number of variants) tested. The fluorescence intensity of the biosensor candidate corresponding to each number was measured, and their effects on measuring the activity of dopamine receptor D1 were evaluated.FIG.3Bis a graph showing results of measuring the degree of increase of the maximum fluorescence intensity (y axis: normalized maximum intensity) according to the addition of 10 μM dopamine by using three candidates (DRD1 Red 1.1, 1.2, and 1.3) having excellent efficiency among the biosensor candidates for measuring the activity of dopamine receptor D1. DRD1 Red 1.1 represents a prototype sensor prepared by cloning using the dopamine receptor D1 gene and the original red fluorescent protein. As shown inFIG.2, DRD1 Red 1.2 and DRD1 Red 1.3 represent two candidates that exhibited high fluorescence intensity at the time of measuring the effects of the biosensors using the candidate plasmids for the dopamine receptor D1 biosensor which were prepared by inducing random mutation in two amino acids each immediately before and after cpRFP. DRD1 Red 1.2 and DRD1 Red 1.3 include, in common, “LSS-ER-cpRFP-YD-DDL” sequence, which was prepared by substituting two amino acids each immediately before and after cpRFP in the linker peptides (LSSXaXband XcXdDDL) linked to each of the N- and C-termini of cpRFP with glutamic acid-arginine(ER) and tyrosine-aspartic acid (YD), respectively. As shown inFIGS.3B and3C, DRD1 Red 1.2 and DRD1 Red 1.3 exhibited higher fluorescence intensity than DRD1 Red 1.1, indicating that they are excellent red fluorescent biosensors for measuring the activity of dopamine receptor D1. DRD1 Red 1.3 includes a substitution of alanine (A) for phenylalanine (F) at position 129 of the amino acid sequence (SEQ ID NO: 1) of dopamine receptor D1 in DRD1 Red 1.2. As shown inFIGS.3B and3C, DRD1 Red 1.3 exhibited the highest fluorescence intensity, indicating that it is the most effective red fluorescent biosensor for measuring the activity of dopamine receptor D1 in terms of measuring the activity of dopamine receptor D1. FIG.3Cis a graph showing results of measuring the change in the fluorescence intensity (y axis: normalized intensity) over time by using three candidates (DRD1 Red 1.1, 1.2, and 1.3) having excellent efficiency among the biosensor candidates for measuring the activity of dopamine receptor D1. As shown inFIG.3C, the change of fluorescence intensity was measured in real time, and as a result, candidate exhibiting a larger change in the fluorescence intensity and a high reaction rate were selected by inducing mutation and testing a larger number of candidates. Example 2. Characterization of Red Fluorescent Biosensor for Measuring Activity of Dopamine Receptor D1 The red fluorescent biosensor for dopamine receptor D1 prepared in Example 1 was transfected into HEK293A cell line as in Example 1, and expressed therein, and expression of the dopamine receptor D1 biosensor on the membrane of HEK293A was examined using the live fluorescence microscope. FIG.4Ashows images of a cell in which activation of the biosensor exhibited a color change when the biosensor was treated with dopamine which is a dopamine receptor D1 ligand at a concentration of 10 μM (left: before dopamine treatment, right: after dopamine treatment). FIG.4Bis a graph showing results of measuring normalized intensity when the fluorescent biosensor for measuring the activity of dopamine receptor D1 was treated with dopamine which is a dopamine receptor D1 agonist and quinpirole which is a dopamine D2 receptor selective ligand.FIG.4Bshows that the dopamine receptor D1 fluorescent biosensor did not response to quinpirole which is a dopamine D2 receptor selective ligand. FIG.4Cis a graph showing fluorescence intensity (y-axis: normalized intensity) of the biosensor for measuring the activity of dopamine receptor D1, measured after adding dopamine which is a dopamine receptor D1 ligand at a final concentration of 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 5 μM, 6.25 μM, 10 μM, 12.5 μM, 50 μM, or 100 μM in cell media. The intensity of the red channel that was specified by ROI through the NIS program (Nikon) performed in Example 1 was measured and analyzed. As shown inFIG.4C, the fluorescent biosensor for measuring the activity of dopamine receptor D1 according to one specific embodiment may detect activity for the dopamine receptor ligand at a concentration of 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 5 μM, 6.25 μM, 10 μM, 12.5 μM, 50 μM, or 100 μM. Further, the biosensor may effectively detect the dopamine receptor ligand at a concentration of 10 μM or more. FIG.4Dis a graph showing fluorescence intensity (y-axis: normalized intensity) of the biosensor for measuring the activity of dopamine receptor D1 over time, when the fluorescent biosensor for measuring the activity of dopamine receptor D1 was co-treated with dopamine (DA) which is a dopamine receptor D1 ligand and an inhibitor (Haloperidol) (red plot) or treated with only dopamine (DA) (blue plot). FIG.5Ashows results of performing co-fluorescent imaging using a green fluorescent biosensor (GRAB-DA1m) for measuring the activity of dopamine receptor D2 which was separately prepared for comparison with the red fluorescent biosensor for measuring activity of dopamine receptor D1 (DRD1 Red 1.3). The graph shows the increase in fluorescence by selective response of each fluorescent biosensor to each ligand, when first treated with quinpirole which is a D2 selective ligand, and then serially treated with SKF38393 which is a D1 selective ligand. FIG.5Bshows results of performing fluorescent imaging before adding the ligand (Before), 5 minutes after treatment with quinpirole which a D2 selective ligand (+Quinpirole, 5 min), and 5 minutes after treatment with SKF38393 which is a D1 selective ligand (+SKF38393, 5 min) in the experiment explained inFIG.5A. The fluorescent images of cells each expressing the red fluorescent biosensor for measuring the activity of dopamine receptor D1 (DRD1 Red 1.3) or the green fluorescent biosensor for measuring the activity of dopamine receptor D2 (GRAB-DA1m) are shown. The red fluorescent protein-based biosensor according to an aspect may have selectivity depending on the dopamine receptor subtype, and may detect the activity of dopamine receptor D1 with high accuracy and resolution. Unlike the green fluorescent protein-based biosensor, the red fluorescent protein-based biosensor may detect the activity of dopamine receptor D1 with high accuracy without overlapping with excitation wavelength. Further, when the red fluorescent protein-based biosensor and another fluorescent biosensor of different wavelength (e.g., green fluorescent protein biosensor) are used in combination, the dopamine activity may be measured by changes and interaction of two kinds of signals. A method of measuring the activity of dopamine receptor D1 according to an aspect may sensitively measure activity of dopamine receptor D1 in live cells. A method of detecting a ligand binding to dopamine receptor D1 according to another aspect may effectively and reversibly detect the ligand binding to dopamine receptor D1, e.g., dopamine or dopamine agonist. It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims. | 29,345 |
11860167 | DETAILED DESCRIPTION In general, the present disclosure is directed to click conjugates, as well as methods of employing click conjugates for detecting one or more targets present in a biological sample. In some embodiments, the click conjugates (or kits comprising one or more click conjugates) are used in a multiplex assay to detect multiple targets within a tissue sample, either simultaneously or sequentially. These and other aspects of the present disclosure are detailed more fully herein. Definitions As used herein, the singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “of” is intended to include “and” unless the context clearly indicates otherwise. The term “includes” is defined inclusively, such that “includes A or B” means including A, B, or A and B. The terms “comprising,” “including,” “having,” and the like are used interchangeably and have the same meaning. Similarly, “comprises,” “includes,” “has,” and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a device having components a, b, and c” means that the device includes at least components a, b and c. Similarly, the phrase: “a method involving steps a, b, and c” means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary. As used herein, alkaline phosphatase (AP) is an enzyme that removes (by hydrolysis) and transfers phosphate group organic esters by breaking the phosphate-oxygen bond, and temporarily forming an intermediate enzyme-substrate bond. For example, AP hydrolyzes naphthol phosphate esters (a substrate) to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes. As used herein, the term “antibody,” occasionally abbreviated “Ab,” refers to immunoglobulins or immunoglobulin-like molecules, including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, (e.g., in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. Antibody further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies may be composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins and the variants and portions of them well known in the art. As used herein, the phrase “antibody conjugates,” refers to those antibodies conjugated (either directly or indirectly) to one or more labels, where the antibody conjugate is specific to a particular target and where the label is capable of being detected (directly or indirectly), such as with a secondary antibody (an anti-label antibody). For example, an antibody conjugate may be coupled to a hapten such as through a polymeric linker and/or spacer, and the antibody conjugate, by means of the hapten, may be indirectly detected. As an alternative example, an antibody conjugate may be coupled to a fluorophore, such as through a polymeric linker and/or spacer, and the antibody conjugate may be detected directly. Antibody conjugates are described further in US Publication No. 2014/0147906 and U.S. Pat. Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191. By way of a further example, the term “antibody conjugates” includes those antibodies conjugated to an enzyme, e.g. HRP or AP. As used herein, the term “antigen” refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins. As used herein, the term a “biological sample” can be any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. In some examples, a biological sample is a nuclear extract. In certain examples, a sample is a quality control sample, such as one of the disclosed cell pellet section samples. In other examples, a sample is a test sample. Samples can be prepared using any method known in the art by of one of ordinary skill. The samples can be obtained from a subject for routine screening or from a subject that is suspected of having a disorder, such as a genetic abnormality, infection, or a neoplasia. The described embodiments of the disclosed method can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as “normal” samples. Samples can include multiple targets that can be specifically bound by one or more detection probes. As used herein, the term “chromophore” refers to a molecule or a part of a molecule responsible for its color. Color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. A molecule having an energy difference between two different molecular orbitals falling within the range of the visible spectrum may absorb visible light and thus be aptly characterized as a chromophore. Visible light incident on a chromophore may be absorbed thus exciting an electron from a ground state molecular orbital into an excited state molecular orbital. As used herein, the term “conjugate” refers to two or more molecules or moieties (including macromolecules or supra-molecular molecules) that are covalently linked into a larger construct. In some embodiments, a conjugate includes one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecules moieties. As used herein, the terms “couple” or “coupling” refers to the joining, bonding (e.g. covalent bonding), or linking of one molecule or atom to another molecule or atom. As used herein, “haptens” are small molecules that can combine specifically with an antibody, but typically are substantially incapable of being immunogenic except in combination with a carrier molecule. In some embodiments, haptens include, but are not limited to, pyrazoles (e.g. nitropyrazoles); nitrophenyl compounds; benzofurazans; triterpenes; ureas (e.g. phenyl ureas); thioureas (e.g. phenyl thioureas); rotenone and rotenone derivatives; oxazole (e.g. oxazole sulfonamides); thiazoles (e.g. thiazole sulfonamides); coumarin and coumarin derivatives; and cyclolignans. Additional non-limiting examples of haptens include thiazoles; nitroaryls; benzofurans; triperpenes; and cyclolignans. Specific examples of haptens include di-nitrophenyl, biotin, digoxigenin, and fluorescein, and any derivatives or analogs thereof. Other haptens are described in U.S. Pat. Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of which are incorporated herein by reference in their entirety. The haptens themselves may be suitable for direct detection, i.e. they may give off a suitable signal for detection. As used herein, horseradish peroxidase (HRP) is an enzyme that can be conjugated to a labeled molecule. It produces a colored, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP acts in the presence of an electron donor to first form an enzyme substrate complex and then subsequently acts to oxidize an electronic donor. For example, HRP may act on 3,3′-diaminobenzidinetrahydrochloride (DAB) to produce a detectable color. HRP may also act upon a labeled tyramide conjugate, or tyramide like reactive conjugates (i.e. ferulate, coumaric, caffeic, cinnamate, dopamine, etc.), to deposit a colored or fluorescent or colorless reporter moiety for tyramide signal amplification (TSA). As used herein, the terms “multiplex,” “multiplexed,” or “multiplexing” refer to detecting multiple targets in a sample concurrently, substantially simultaneously, or sequentially. Multiplexing can include identifying and/or quantifying multiple distinct nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) both individually and in any and all combinations. As used herein, the term “primary antibody” refers to an antibody which binds specifically to the target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in an immunohistochemical procedure. As used herein, a “quinone methide” is a quinone analog where one of the carbonyl oxygens on the corresponding quinone is replaced by a methylene group (CH2) to form an alkene. As used herein, the term “secondary antibody” herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent (e.g. a label, an enzyme, etc.), if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure. As used herein, the term “specific binding entity” refers to a member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10-3 M greater, 10-4 M greater or 10-5 M greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Particular examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins. As used herein, the term “target” refers to any molecule for which the presence, location and/or concentration is or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens, such as haptens covalently bonded to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label. “Click” Conjugates The present disclosure provides two general subsets of click conjugates. A first subset of click conjugates comprises a tissue reactive moiety coupled to a reactive functional group through an optional linker. In some embodiments, this first subset of click conjugates is used as first members of pairs of click conjugates. A second subset of click conjugates comprises one or more reporter moieties coupled to a reactive functional group through an optional linker. In some embodiments, this second subset of click conjugates are used as second members of pairs of click conjugates. It will be appreciated that the different subsets of click conjugates disclosed herein may serve as modular “building blocks” such that when any two conjugates having appropriate reactive function groups are combined (a “pair of click conjugates”), they may undergo a reaction and form a covalent bond, thereby coupling the two conjugates to form a “click adduct” having the desired structure or component parts. As will be described further herein, the click adducts formed may serve as species suitable for detecting targets in a biological assay. Without wishing to be bound by any particular theory, it is believed that the click conjugates disclosed herein are stable in aqueous media, and thus suitable for use in certain biological assays, including in IHC and ISH. Moreover, it is believed that the click conjugates have a large thermodynamic driving force that favors a fast reaction providing a single product. In addition, the solubility of any of the click conjugates described herein may be “tuned” to meet the requirements of any particular assay, and such “tuning” may be accomplished by, for example, introducing a water soluble linker or water soluble linker components into the conjugates. Moreover, the reactions comprising the click conjugates described herein may be carried out in a wide variety of buffers and thus at a wide variety of pHs, allowing the skilled artisan to choose the ideal conditions for reporter stability. In one aspect of the present disclosure are click conjugates of Formula (I): ALinkerB (I),wherein A is a reactive functional group, “Linker” is an optional linking group, B is selected from a “tissue reactive moiety” or a reporter moiety. As used herein, the term “tissue reactive” refers to a moiety that is capable of reacting with an enzyme. As such, when a click conjugate comprising a tissue reactive moiety is reacted with an appropriate enzyme, the tissue reactive moiety portion of the click conjugate undergoes a structural, conformational, and/or electronic change, thereby providing a tissue reactive species (an intermediate, including radical intermediates) suitable for bonding directly or indirectly onto (or, to the extent possible, within) a biological sample. For example, where the tissue reactive moiety is a tyramide or derivative thereof, when the tyramide reacts with an appropriate enzyme (e.g. an HRP), a tyramide radical species (an intermediate) is formed. This highly reactive tyramide radical species is capable of bonding to tyrosine residues in biological samples. In a similar manner, a quinone methide precursory moiety, upon reaction with an appropriate enzyme (e.g. AP), is converted to a quinone methide, which is believed to be highly reactive with nucleophiles in a biological sample. The role of the tissue reactive moiety portion of any click conjugate, its interaction with a suitable enzyme, and the formation of an immobilized tissue-click conjugate complex is described further herein. In some embodiments, A is selected from the group consisting of dibenzocyclooctyne (“DBCO”), trans-cyclooctene (“TCO”), azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. In some embodiments, A is selected from a group capable of undergoing a photo-initiated reaction. The click conjugates optionally comprise a “Linker.” In some embodiments, a ‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, the “Linker” comprises one or more groups selected from amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine groups. The linker chain may also comprise an aromatic group, including heteroaromatic groups, wherein the heteroaromatic groups comprise 1 to 4 heteroatoms selected from O, N, or S. In some embodiments, the ‘Linker’ has the structure depicted in Formula (Ia): wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(Rc)(Rd); Raand Rbare independently H, a C1-C4alkyl group, F, Cl, or N(Rc)(Rd); Rcand Rdare independently CH3or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, X and Y include carbonyl groups, amide groups, ester groups, ester groups, substituted or unsubstituted aryl groups, or any combination thereof. In other embodiments, d and e are integers ranging from 2 to 10. In yet other embodiments, d and e are integers ranging from 2 to 6. In some embodiments, the “Linker” has the structure depicted in Formula (Ib): whereind and e are integers each independently ranging from 2 to 20;t and u are independently either 0 or 1;Q is a bond, O, S, or N(Rc)(Rd);Rcand Rdare independently CH3or H; andX and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 8 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, the “Linker” has the structure depicted in Formula (Ic): whereind and e are integers each independently ranging from 2 to 20;t and u are independently either 0 or 1; andX and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. In other embodiments, d and e are integers ranging from 2 to 10. In yet other embodiments, d and e are integers ranging from 2 to 6. The alkylene oxide based “Linker” of Formulas (Ia), (Ib), and (Ic), are represented herein by reference to glycols, such as ethylene glycols. In some embodiments, the incorporation of such alkylene oxide linkers is believed to increase the hydrophilicity of the click conjugate. A person of ordinary skill in the art will appreciate that, as the number alkylene oxide repeat units in the linker increases, the hydrophilicity of the conjugate also may increase. Additional heterobifunctional polyalkyleneglycol spacers useful for practicing certain disclosed embodiments of the present disclosure are described in assignee's co-pending applications, including “Nanoparticle Conjugates,” U.S. patent application Ser. No. 11/413,778, filed Apr. 28, 2006; “Antibody Conjugates,” U.S. application Ser. No. 11/413,415, filed Apr. 27, 2006; and “Molecular Conjugate,” U.S. Provisional Patent Application No. 60/739,794, filed Nov. 23, 2005; all of which applications are incorporated herein by reference. Tissue Reactive Precursor Moiety “Click” Conjugates In some embodiments, the click conjugates of the present disclosure have the structure of Formula (Id): wherein the “Tissue Reactive Precursor Moiety” is (i) a tyramide or a derivative or analog thereof, or (ii) a quinone methide precursor; and where A and Linker are as defined above. Exemplary quinone methide precursor derivatives suitable for incorporation into the disclosed click conjugates of Formula (I) include those recited in PCT/EP2015/053556, entitled “Quinone Methide Analog Signal Amplification,” having an international filing date of Feb. 20, 2015, the disclosure of which is hereby incorporated by reference herein in its entirety. Quinone Methide “Click” Conjugates In some embodiments, the compounds have Formula (II): ALinkerU (II)whereinA is as defined above;“Linker” is an optional linking group as defined above; andU is a quinone methide precursor, derivative, or analog thereof. In some embodiments, the compounds of Formula (II) are a first member of a pair of click conjugates. As used herein, “quinone methide precursors” are a class of conjugated compounds that, when reacted with an appropriate enzyme (e.g. AP), are converted to a highly reactive quinone methide. As noted above, quinone methide precursors and their conversion to quinone methides are described in PCT/EP2015/053556, the disclosure of which is hereby incorporated by reference herein in its entirety. In some embodiments, the quinone methide precursor portion of the conjugate of Formula (II) is derived from one of the following quinone methide precursor derivatives: In some embodiments, the conjugates of Formula (II) have the structure of Formula (IIa): wherein “Linker” and A are as defined herein,R1is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar;R2is a halide;R3, R5, and R6are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms;R4is a hydrogen, an aliphatic group having between 1 and 4 carbon atoms, or the group —CH(R2)—R7-[Linker]-A;R7is —(CH2)wNH—, —O(CH2)wNH—, —N(H)C(O)(CH2)wNH—, —C(O)N(H)(CH2)wNH—, —(CH2)wO—, —O(CH2)wO—, —O(CH2CH2O)w—, —N(H)C(O)(CH2)wO—, —C(O)N(H)(CH2)wO—, —C(O)N(H)(CH2CH2O)w—, —(CH2)wS—, —O(CH2)wS—, —N(H)C(O)(CH2)wS—, —C(O)N(H)(CH2)wS—, —(CH2)wNH—, —C(O)N(H)(CH2CH2O)wCH2CH2NH, —C(O)(CH2CH2O)wCH2CH2NH—, —C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2)wNH—, or —N(H)(CH2)wNH—, where w is an integer ranging from 1 to 12. When R1 is a sugar, the sugar may be selected from glucose, β-glucose, a-galactoside, β-galactoside, a-glucuronose, neuraminide, or β-glucuronose. In other embodiments, the conjugates of Formula (II) have the structure of Formula (IIb): where R1is selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar; andwhere R7is —(CH2)wNH—, —O(CH2)wNH—, —N(H)C(O)(CH2)wNH—, —C(O)N(H)(CH2)wNH—, —(CH2)wO—, —O(CH2)wO—, —O(CH2CH2O)w—, —N(H)C(O)(CH2)wO—, —C(O)N(H)(CH2)wO—, —C(O)N(H)(CH2CH2O)w—, —(CH2)wS—, —O(CH2)wS—, —N(H)C(O)(CH2)wS—, —C(O)N(H)(CH2)wS—, —(CH2)wNH—, —C(O)N(H)(CH2CH2O)wCH2CH2NH, —C(O)(CH2CH2O), CH2CH2NH—, —C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2)wNH—, or —N(H)(CH2)wNH—, where w is an integer ranging from 1 to 12. In some embodiments of the conjugates of Formula (IIb), R1is a phosphate and R7is —C(O)N(H)(CH2)wNH—, and w ranges from 2 to 10. In yet other embodiments, the conjugates of Formula (II) have the structure of Formula (IIc): where R7is —(CH2)wNH—, —O(CH2)wNH—, —N(H)C(O)(CH2)wNH—, —C(O)N(H)(CH2)wNH—, —(CH2)wO—, —O(CH2)wO—, —O(CH2CH2O)w—, —N(H)C(O)(CH2)wO—, —C(O)N(H)(CH2)wO—, —C(O)N(H)(CH2CH2O)w—, —(CH2)wS—, —O(CH2)wS—, —N(H)C(O)(CH2)wS—, —C(O)N(H)(CH2)wS—, —(CH2)wNH—, —C(O)N(H)(CH2CH2O)wCH2CH2NH, —C(O)(CH2CH2O)wCH2CH2NH—, —C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2)wNH—, or —N(H)(CH2)wNH—, where w is an integer ranging from 1 to 12. In some embodiments, R7is C(O)N(H)(CH2)wNH and w is as defined above. In other embodiments, R7is C(O)N(H)(CH2)wNH and w ranges from 2 to 6. In yet further embodiments, the conjugates of Formula (II) have the structure of Formula (IId): wherew ranges from 1 to 12, and“Linker” and A are as defined above. In some embodiments w ranges from 1 to 8. In other embodiments, w ranges from 2 to 8. In yet other embodiments, w ranges from 2 to 6. In further embodiments, w is 6. Specific examples of the compounds of Formulas (II) include the following: The quinone methide precursor click conjugates of Formula (II) may be synthesized according to any method as known to those of ordinary skill in the art. In some embodiments, a reagent comprising the desired reactive functional group and linker are merely coupled with a quinone methide precursor or derivative or analog thereof as illustrated in the reaction schemes which follow. For example, a quinone methide precursor having a terminal amine group may be coupled to a compound comprising an amine reactive group (e.g. active esters such as N-Hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like). In some of the specific examples below, a Click partner having an NHS-ester group is coupled with a quinone methide precursor having a terminal amine. In some embodiments, the reaction takes place in DMSO and is allowed to react for 60 minutes. The reaction is then diluted with methanol and directly purified by preparative HPLC. Tyramide “Click” Conjugates In other embodiments, the compounds have Formula (III): ALinkerM (III)wherein A is as defined above; “Linker” is an optional linking group as defined above; and M is tyramide or a derivative or analog thereof. In some embodiments, the compounds of Formula (III) are a first member of a pair of click partners. In some embodiments, the conjugates of Formula (III) comprise a tyramide derived from a compound having the structure of Formula (IIIa) wherein each R group is independently selected from hydrogen or lower alkyl group (which may be straight chain or branched) having between 1 and 4 carbon atoms, and where Linker and A are as defined herein. In some embodiments, the compounds of Formula (III) comprise a tyramide derived from a compound having the structure of Formula (IIIb) where A is selected from an azide, a thiol, a 1,3-nitrone, a hydrazine, or a hydroxylamine, and where Linker is as defined herein. In some embodiments, the compounds of Formula (III) comprise a moiety from a compound having the structure of Formulas (IIIc) or (IIId): Non-limiting examples of particular tyramide click conjugates include the following: The tyramide click conjugates of Formula (III) may be synthesized according to any method as known to those of ordinary skill in the art. In some embodiments, a reagent comprising the desired reactive functional group and linker are merely coupled with a tyramide or derivative or analog thereof as illustrated in the reaction schemes below. For example, a tyramide (having a terminal amine group) may be coupled to a compound comprising an amine reactive group (e.g. active esters such as N-Hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like). In some of the specific examples below, a Click partner having an NHS-ester group is coupled with a tyramide. In some embodiments, the reaction takes place in DMSO and is allowed to react for 60 minutes. The reaction is then diluted with methanol and directly purified by preparative HPLC. Reporter Moiety “Click” Conjugates In other embodiments, the click conjugates of the present disclosure have the structure of Formula (IV): ALinkerZ (IV),wherein A is as defined above; “Linker” is an optional linking group as defined above; and Z comprises at least one reporter moiety (the terms “reporter moiety” and “reporter” are used interchangeably herein). In some embodiments, the compounds of Formula (IV) are a second member of a pair of click partners. In some embodiments, the conjugates of Formula (IV) comprise one reporter moiety, and thus the group Z is a reporter moiety coupled directly, or indirectly through a Linker, to the reactive functional group A. In other embodiments, the conjugates of Formula (IV) comprise a plurality of reporter moieties, and thus Z represents a group having two or more reporter moieties. In embodiments where Z represents a group having two or more reporter moieties, the group Z is coupled directly, or indirectly through a Linker, to the reactive functional group A. In some embodiments, Z comprises two reporters. In other embodiments, Z comprises four reporters. In other embodiments, Z comprises six reporters. In yet other embodiments, Z comprises greater than 6 reporters. In embodiments where Z comprises more than one reporter, the reporters may be the same or different. For example, Z may comprise two of the same chromogens (e.g. two TAMRA chromogens). Alternatively, Z may comprise two different chromogens (e.g. TAMRA and cy5). In some embodiments, Z comprises at least two reporter moieties, and the at least two reporter moieties are linked to each other via a straight chain or branched aliphatic group, optionally comprising one or more heteroatoms. In other embodiments, Z comprises at least two reporter moieties, and the at least two reporter moieties are linked to each other via a dendrimer or branched polymer. In some embodiments, the compounds of Formula (IV) have the structure of Formula (V): ALinkerScaffoldZ]v(V),wherein“Scaffold” is a group capable of coupling multiple reporter moieties, andv is an integer ranging from between 1 and 20. In some embodiments, “Scaffold” is a polyamine (e.g. norspermidine, spermine, and derivatives or analogs thereof; or a polyamine comprising between 2 and 10 amine groups); a heterobifunctional linker (e.g. lysine or lysine derivative); a dendrimer (e.g. polyamidoamine (PAMAM) dendrimers, Janus dendrimers (i.e. dendrimers constituted of two dendrimeric wedges and terminated by two different functionalities), and bis-MPA dendrimers and derivatives thereof); or a polymer. In some embodiments, ‘Scaffold’ is a bond. In some embodiments, the click conjugates have the following formulas: where Linker and Z are as defined herein, In some embodiments, the reporter moiety is selected from a chromophore, a fluorophore, an enzyme, a hapten, or a chelator. Non-limiting examples of suitable haptens include pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenyl ureas, and even more particularly phenyl thioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans, exemplified by Podophyllotoxin and Podophyllotoxin derivatives; and combinations thereof. Further examples of haptens and methods for their synthesis and use are disclosed in U.S. Pat. No. 7,695,929, the disclosure of which is hereby incorporated in its entirety herein by reference. In some embodiments, suitable haptens include BD (benzodiazepine), BF (benzofurazan), DABSYL (4-(dimethylamino)azobenzene-4′-sulfonamide, which has a max of about 436 nm), DCC (7-(diethylamino)coumarin-3-carboxylic acid), DIG (digoxigenin), DNP (dinitrophenyl), HQ (3-hydroxy-2-quinoxalinecarbamide) NCA (nitrocinnamic acid), NP (nitropyrazole), PPT (Podophyllotoxin), Rhod (rhodamine), ROT (rotenone), and TS (thiazolesulfonamide). Other suitable haptens include biotin and fluorescein derivatives (FITC (fluoresceinisothiocyanate), TAMRA (tetramethylrhodamine), Texas Red), and Rhodamine 110 (Rhodamine). Non-limiting examples of suitable chromophores include coumarin and coumarin derivatives. Examples of coumarin-based chromophores include DCC and 2,3,6,7-tetrahydro-11-oxo-1H,5H,1 1H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylic acid. Other suitable chromophores include diazo-containing chromogens, such as tartrazine. Yet other suitable chromophores include triarylmethane, including those provided below Other non-limiting examples of suitable chromophores include those provided below: Other suitable chromophores include annulated chromophores, such as those provided below: Fluorophores belong to several common chemical classes including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores and cyanines. Additional examples of fluorescent molecules can be found in Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, TheroFisher Scientific, 11th Edition. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaraine derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent moiety is selected from a CF dye (available from Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY (available from Invitrogen), Alexa Fluor (available from Invitrogen), DyLight Fluor (e.g. DyLight 649) (available from Thermo Scientific, Pierce), Atto and Tracy (available from Sigma Aldrich), FluoProbes (available from Interchim), Abberior Dyes (available from Abberior), DY and MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (available from Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau and Square Dyes (available from SETA BioMedicals), Quasar and Cal Fluor dyes (available from Biosearch Technologies), SureLight Dyes (available from APC, RPEPerCP, Phycobilisomes)(Columbia Biosciences), and APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen). Suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, neuraminidase, β-galactosidase, β-glucuronidase or β-lactamase. In other embodiments, enzymes include oxidoreductases or peroxidases (e.g. HRP, AP). Use of an enzyme as a reporter moiety is further illustrated with reference toFIG.14herein. There, the second member of a pair of click conjugates comprises a compound of Formula (IV), where Z is an enzyme, in particular an alkaline phosphatase. As will be appreciated further herein, the resulting click adduct may be detected by introducing further alkaline phosphatase reporters (chromogens, fluorophores). In some embodiments, the reporter moiety is a chelator or chelating agent, which may become chelated in the presence of a lanthanide (e.g. europium). Without wishing to be bound by any particular theory, it is believed that the lanthanide atoms may be detected using Inductively Coupled Plasma Mass Spectral Imaging (ICP-MSI). In addition, the lanthanides may be detected using time-resolved fluorescence microscopy, which take advantage of the relatively long lifetimes of the lanthanide luminescence compared to conventional fluorophores. In order to visualize the lanthanides, an antenna ligand must be present to absorb and transfer energy to the normally poorly-absorbing lanthanides. The reaction product of the DBCO-azide Click reaction may be able to act as the antenna ligand, greatly simplifying the design of these systems. An example of a compound of Formula (IV) comprising an azide reactive group conjugated to a chelator moiety is shown below: In some specific embodiments, the reporter moiety portion (Z) of the click conjugates of Formula (IV) are selected from: In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVa) wherein A is as recited above. While Formula (IVa) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted. In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVb) wherein A is as recited above. While Formula (IVb) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted. In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVc) wherein A is as recited above. While Formula (IVc) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted. In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVd) wherein A is as recited above. While Formula (IVd) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted. Specific non-limiting examples of conjugates of the Formula (IV) include the following: A non-limiting example of a conjugate of Formula (V) is illustrated below: The reporter moiety compounds of Formula (IV) may be synthesized according to methods known to those of ordinary skill in the art. Example synthetic procedure for the coupling of an NHS ester to an amine. This procedure can be applied to the reaction of any tyramine or quinone methide precursor containing an amine or NHS functionality with a Click partner containing an amine or NHS ester functionality. It can also be applied to the reaction of a reporter group (chromogen, hapten, etc.) containing an amine or NHS functionality with a Click partner containing an amine or NHS ester functionality. Tyramide-peg5-DBCO. Tyramine (1.1 eq, 110 mg, 0.79 mmol) was dissolved in DMSO (3 mL) followed by addition of triethylamine (5.0 eq, 360 mg, 3.6 mmol). DBCO-peg5-DBCO (1.0 eq, 500 mg, 0.72 mmol) was then added, and the resulting reaction mixture was stirred for 1 hour at room temperature. The reaction mixture was diluted with MeOH (2 mL) and the resulting mixture was purified by preparative RP-HPLC (C18; 40 mL/min; 0.05% TFA in H2O:MeCN 95:5 to 5:95 over 40 minutes) to give Tyramide-peg5-DBCO (450 mg, 87% yield) as a colorless glass after removal of solvents under high vacuum. MS (ESI) m/z (M+H)+ calcd for C40H50N3O9+ 716.4, found 716.6. Coupling of Click Conjugate Pairs The skilled artisan will recognize that the click conjugates disclosed herein are suitable for coupling to each other to form “click adducts.” The skilled artisan will also recognize that for one member of a pair of click conjugates to react with another member of the pair of click conjugates, and thus form a covalent bond, the two members of the pair of click conjugates must have reactive functional groups capable of reacting with each other. The table which follows exemplifies different pairs of reactive functional groups that will react with each other to form a covalent bond. Reactive Functional GroupReactive Functional Groupon a First Member of aon a Second Member of aPair of Click ConjugatesPair of Click ConjugatesDBCOAzideAlkeneTetrazineTCOTetrazineMaleimideThiolDBCO1,3-NitroneAldehyde or ketoneHydrazineAldehyde or ketoneHydroxylamineAzideDBCOTetrazineTCOThiolMaleimide1,3-NitroneDBCOHydrazineAldehyde or ketoneHydroxylamineAldehyde or ketoneTetrazineAlkene Specific non-limiting examples of pairs of click conjugates possessing these reactive function groups are illustrated inFIGS.3and4. In particular,FIG.3provides examples of pairs of click conjugates, where one member of each pair of click conjugates comprises a compound of Formula (II).FIG.4also provides examples of pairs of click conjugates, where one member of each pair of click conjugates comprises a compound of Formula (III). In some embodiments, the click conjugates are coupled via “strain-promoted azide-alkyne cycloaddition” (SPAAC), or “TCO-tetrazine ligation” (TTL). SPAAC involves the reaction between azides and strained alkynes, whose high energy allows the 1,3-dipolar cycloaddition to occur in the absence of a Cu(I) catalyst (required for traditional azide-alkyne “click” chemistry). In some embodiments, dibenzocyclooctynes are utilized as the strained cyclooctyne due to their commercial availability and literature precedent. TTL utilizes the reaction between trans-cyclooctene and tetrazine to form a dihydropyridazine bond. These reagents are also commercially available and have been shown to react orthogonally to the SPAAC system. The schematics which follow further illustrate the coupling of pairs of click conjugates comprising difference reactive functional groups. In the schemes which follow, one member of the pair of click conjugates is provided as an immobilized, tissue-click conjugate complex. As will be described further herein, the immobilized, tissue-click conjugate complex is formed through the reaction of a click conjugate having either Formula (II) or (III) with an appropriate enzyme, and the subsequent coupling of a reactive intermediate generated therefrom with tissue. For example, Scheme 2 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive azide group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine. Likewise, Scheme 3 illustrates the reaction between an immobilized tissue-click conjugate complex having a TCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive tetrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine. Scheme 4 again illustrates the reaction between an immobilized tissue-click conjugate complex having a malemide reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive thiol group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine. Scheme 5 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive 1,3-nitrone group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine. Scheme 6 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde reactive functional group, and a second click conjugate of Formula (IV) a comprising reactive hydrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine. Scheme 7 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive hydroxylamine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine. Scheme 8 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive azide group and a chelator as the reporter Z. In some embodiments, a resulting intermediate adduct comprises a chelator which, when a lanthanide is introduced, forms a chelated adduct complex, suitable for detection with MSI. Scheme 9A illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) or Formula (V) comprising a reactive azide group coupled to a dendrimer, the dendrimer coupled to two, four, or eight reporter moieties, as shown. Without wishing to be bound by any particular theory, it is believed that the use of dendrimers allows for the incorporation of a plurality of reporters (which may be the same or different), thus providing significant signal amplification. Scheme 9B illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (V) comprising a reactive azide group coupled to a dendrimer (PAMAM), the dendrimer coupled to four reporter moieties Z. Scheme 10 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula Formula (V) comprising a reactive azide group coupled to a Z group comprising two chromophores, where the two chromophores are linked via a lysine group. While the chromogens are depicted as being the same, the skilled artisan will recognize that the chromogens linked via the lysine group may be different. Specific examples of immobilized, tissue-click conjugate complexes and their reaction with click conjugates comprising specific reporter moieties are represented inFIGS.5and6. Methods of Detecting Targets in a Sample Using Click Conjugates The present disclosure also provides methods of detecting one or more targets within a tissue sample using pairs of any of the click conjugates. While certain disclosed embodiments, examples, or figures herein may refer to the use of the click conjugates in conjunction in an IHC assay, the skilled artisan will appreciate that the click conjugates may also be used in situ hybridization (ISH) assays, or any combination of IHC and ISH assays. The skilled artisan will also appreciate that the click conjugates may be used in both simplex assays and multiplex assays. The methods described herein refer to pairs of click conjugates suitable for use in biological assays. In those assays, one member (or “partner”) of a particular pair of click conjugates comprises a conjugate of either Formula (II) or (III), and another member of the pair of click conjugates comprises a conjugate of Formula (IV) or Formula (V). In general, a first member of a pair of click conjugates is covalently deposited onto tissue using QMSA or TSA. Then, a second member of the pair of click conjugates comprising a reporter molecule (i.e. chromophore, fluorophore, enzyme, hapten) is applied to the tissue. The “click” reaction between the two “click” partners occurs rapidly, covalently binding the reporter molecules to tissue in the locations dictated by the QMSA or TSA chemistries. Moreover, and as noted herein, the presently disclosed amplification methodology allows for the reporter moiety to be separated from the QMSA or TSA assay conditions, which is believed to enhance signal intensity. For example,FIGS.1A,1B,2A, and2Billustrate the reaction between a first member of a pair of click conjugates having a tissue reactive moiety (10,20) and a target-bound enzyme (11,21) to form an immobilized tissue-click conjugate complex (13,23). This first part of the amplification process is similar to that used in QMSA and TSA amplification processes.FIGS.1A,1B,2A, and2Balso illustrate the subsequent reaction between the immobilized tissue-click conjugate (13,23) complex and a second member of the pair of click conjugates (14,24), to provide an immobilized tissue-click adduct complex (15,25) comprising a detectable reporter moiety. With reference toFIG.1A, a compound of Formula (II) comprising a reactive functional group (10) is brought into contact with a target-bound enzyme (11) to produce a reactive intermediate (12). In this example, the reactive intermediate, a quinone methide, forms a covalent bond to a nucleophile on or within a tissue sample, thus providing an immobilized tissue-click conjugate complex (13). The immobilized tissue-click conjugate complex may then react with a compound of Formula (IV) (14), provided that the click conjugate10and click conjugate14possess reactive functional groups that may react with each other to form a covalent bond. The reaction product of immobilized tissue-click conjugate complex13and click conjugate14produces the immobilized tissue-click adduct complex15. The tissue-click adduct complex15may be detected by virtue of signals transmitted from the linked reporter moiety. In some embodiments, the reporter moiety is at least one chromophore. FIG.1Billustrates the reaction between a specific compound of Formula (II) having a quinone methide precursor moiety linked to a DBCO reactive functional group and a target-bound enzyme to produce the reactive quinone methide intermediate, followed by coupling of that reactive intermediate with a nucleophile on or within the biological sample. More specifically, alkaline phosphatase recognizes and cleaves the phosphate group from the illustrated quinone methide precursor portion of the click conjugate, resulting in ejection of the leaving group, and formation of the respective quinone methide intermediate. The immobilized tissue-click conjugate may then react with a compound of Formula (IV), such as one comprising an azide group and a chromophore, as illustrated. The resulting product is a tissue-click adduct complex having the depicted detectable chromophore. Similarly, and with reference toFIG.2A, a compound of Formula (III) comprising a reactive functional group (20) is brought into contact with a target-bound enzyme (21), to produce a reactive intermediate (22), namely a tyramide radical species (or derivative thereof). The tyramide radical intermediate may then form a covalent bond to a tissue sample, thus providing an immobilized tissue-click conjugate complex (23). The immobilized tissue-click conjugate complex may then react with a compound of Formula (IV) (24), provided that click conjugates20and24possess reactive functional groups that may react with each other to form a covalent bond. The reaction product of immobilized tissue-click conjugate complex23and click conjugate24produces the tissue-click adduct complex25. FIG.2Billustrates the reaction between a specific compound of Formula (III), namely a compound having tyramide moiety linked to a DBCO reactive functional group.FIG.4also illustrates a target-bound enzyme to produce the reactive tyramide radical intermediate, followed by coupling of that intermediate with the biological sample to form an immobilized tissue-click conjugate complex. The immobilized tissue-click conjugate complex may then react with a compound of Formula (IV), such as one comprising an azide group and a chromophore, as illustrated. The resulting product is a tissue-click adduct complex (25) having a detectable chromophore. In some embodiments, the methods of detecting targets in a biological sample comprise the following steps. First, the biological sample is contact with a first detection probe specific to a first target. The first detection probe may be a primary antibody or a nucleic acid probe. Subsequently, the sample is contacted with a first labeling conjugate, the first labeling conjugate comprising a first enzyme. In some embodiments, the first labeling conjugate is a secondary antibody specific for either the primary antibody or to a label conjugated to the nucleic acid probe. Next, the biological sample is contacted with a first member of a pair of click conjugates, the first member of the pair of click conjugates having the structure of any of the compounds of Formulas (II) or (III). As described herein, the first enzyme cleaves the first member of the pair of click conjugates, thereby converting the first member into a reactive intermediate which covalently binds to the biological sample proximally to or directly on the first target. Next, a second member of the pair of click conjugates is introduced, the second member of the pair of click conjugates comprising a first reporter moiety and a second reactive functional group, where the second reactive functional group of the second member of the first pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the pair of click conjugates. The second member of the pair of click conjugates may have the structure as provided in Formula (IV). Finally, signals from the first reporter moiety are detected. With reference toFIG.15, the method of detecting one or more targets within a tissue sample using the click conjugates described herein can generally be divided into two stages. In a first stage, each target within the tissue sample is labeled with an enzyme (see block155and the steps contained therein). In a second stage, a reporter moiety is deposited directly on or proximal to each of the targets (see block165and the steps contained therein), wherein the reporter moiety is deposited using a pair of the click conjugates enumerated herein (e.g. a first conjugate comprising a tissue reactive moiety portion and having the structure of any of Formulas (II) and (III), and a second conjugate comprising a reporter moiety and having the structure of Formula (IV)). The skilled artisan will appreciate that each of these general steps may be repeated in a multiplex assay (step170) to detect a plurality of different targets within the tissue sample. Each of these steps will be described in further detail herein. In some embodiments, and prior to the introduction of any detection reagents, the tissue samples are pre-treated with an enzyme inactivation composition to substantially or completely inactivate endogenous peroxidase activity. For example, where cells or tissues contain endogenous peroxidase, use of a HRP conjugated antibody may result in high, non-specific background staining. This non-specific background can be reduced by pre-treatment of the sample with an enzyme inactivation composition as disclosed herein. In some embodiments, the samples are pre-treated with hydrogen peroxide only (about 1% to about 3% by weight of an appropriate pre-treatment solution) to reduce endogenous peroxidase activity. Referring again toFIG.15, a tissue sample containing one or more targets is contacted with a first specific binding moiety specific to a first target to provide a first specific binding moiety-target complex (step100). In some embodiments, the first specific binding moiety is a primary antibody or antibody conjugate (e.g. an unmodified antibody or an antibody conjugated to a detectable label, such as a hapten). In other embodiments, the first specific binding moiety is a nucleic acid probe conjugated to a detectable label, such as a hapten. The first specific binding moiety-target complex is subsequently labeled with a first enzyme through the first specific binding moiety (step110). In some embodiments, the labeling of the target complex may be achieved with a secondary antibody, the secondary antibody being an anti-antibody antibody (e.g. one that is specific to a primary antibody, namely an anti-antibody antibody) or an anti-label antibody (e.g. an anti-label antibody or an anti-hapten antibody), the secondary antibody being conjugated to an enzyme (e.g. HRP, AP, etc.). The tissue sample is then contacted with a first member of a first pair of click conjugates, where the first member of the first pair of click conjugates comprises a tissue reactive moiety and a first reactive functional group (step120). The first member of the first pair of click conjugates may have the formula as provided in any of Formulas (II) or (III). The first member of the first pair of click conjugates interacts/reacts with the first enzyme to form a reactive species or intermediate, where the reactive species or intermediate is capable of forming a covalent bond directly or indirectly with the tissue sample either directly on or proximal to the first target. Next, a second member of the first pair of click conjugates is introduced (step130), the second member of the first pair of click conjugates comprising a first reporter moiety and a second reactive functional group, where the second reactive functional group of the second member of the first pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the first pair of click conjugates. The second member of the first pair of click conjugates may have the structure as provided in Formula (IV). Finally, signals from the first reporter moiety are detected (e.g. brightfield microscopy) (step140). In some embodiments, the first reporter moiety is a chromophore. In some embodiments, the second member of the first pair of click conjugates is conjugated to at least two chromophores, and where the second member of the first pair of click conjugates has the structure of Formula (V). The aforementioned process may be repeated for any number of targets within the sample (step170). In some embodiments, an enzyme inactivation composition may be introduced to substantially or completely inactivate any enzymes from any upstream steps. Then, the tissue sample may be contacted with a second specific binding moiety specific to a second target to provide a second specific binding moiety target complex (step100). The second specific binding moiety target complex is subsequently labeled with a second enzyme through the second specific binding moiety (step110). The tissue sample is then contacted with a first member of a second pair of click conjugates, where the first member of the second pair of click conjugates comprises either a quinone methide precursor or a tyramide moiety and a first reactive functional group (step120). The first member of the second pair of click conjugates interacts with the second enzyme to form a reactive species, where the reactive species is capable of forming a covalent bond either directly on or proximal to the second target. The first member of the first pair of click conjugates may have the formula as provided in any of Formulas (II) or (III). Next, a second member of the second pair of click conjugates is introduced (step130), the second member of the second pair of click conjugates comprising a second reporter moiety and a second reactive functional group, where the second reactive functional group of the second member of the second pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the second pair of click conjugates. The second member of the second pair of click conjugates may have the structure as provided in Formula (IV). The second reporter moiety is then detected (step140). The process may be repeated for third, fourth, or nth targets within the tissue sample (step170). The skilled artisan will appreciate that the steps illustrated inFIG.15may be performed sequentially (or serially) or substantially simultaneously. For example, the tissue sample may be contacted simultaneously at step100with two specific binding moieties (where each specific binding moiety is specific to a particular target); and then each specific binding moiety-target complex simultaneously labeled with different enzymes at step110. In these embodiments, either the reagents used at either step100or110may be supplied as a “pool” or “cocktail” of reagents. Alternatively, a first specific binding moiety may be deposited (step100) followed by labeling of that first specific binding moiety-target complex (step110). Steps100and110may be serially repeated any number of times (step150) prior to the introduction of any click conjugates. Subsequently, a tissue sample having a plurality of enzyme labeled target complexes (steps100,110, and150) may then be contacted with a plurality of click conjugates. First members of pairs of click conjugates may be added simultaneously at step120prior to the simultaneous introduction of second members of pairs of click conjugates at step130. Alternatively, a first member of a first pair of click conjugates may be introduced followed by the introduction of a second member of a first pair of click conjugates, and the sequential introduction of first and second members of pairs of click conjugates may be repeated any number of times (step160) to introduce a reporter moiety for each of the labeled target complexes. Advantageously, for the methods just described, the first enzyme and the second enzyme are different enzymes. For example, the first enzyme can be a phosphatase or phosphodiesterase, and the second enzyme can be a peroxidase. In certain embodiments, the first enzyme is alkaline phosphatase and the second enzyme is horseradish peroxidase. Also advantageously, the first enzyme does not interact with the first member of a second pair of click conjugates to deposit a reactive intermediate derived from first member of the second pair of click conjugates proximally to the first target. Automation The assays and methods of the present disclosure may be automated and may be combined with a specimen processing apparatus. The specimen processing apparatus can be an automated apparatus, such as the BENCHMARK XT instrument, the SYMPHONY instrument, the BENCHMARK ULTRA instrument sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively, specimens can be manually processed. The specimen processing apparatus can apply fixatives to the specimen. Fixatives can include cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation). If the specimen is a sample embedded in paraffin, the sample can be deparaffinized with the specimen processing apparatus using appropriate deparaffinizing fluid(s). After the waste remover removes the deparaffinizing fluid(s), any number of substances can be successively applied to the specimen. The substances can be for pretreatment (e.g., protein-crosslinking, expose nucleic acids, etc.), denaturation, hybridization, washing (e.g., stringency wash), detection (e.g., link a visual or marker molecule to a probe), amplifying (e.g., amplifying proteins, genes, etc.), counterstaining, coverslipping, or the like. The specimen processing apparatus can apply a wide range of substances to the specimen. The substances include, without limitation, stains, probes, reagents, rinses, and/or conditioners. The substances can be fluids (e.g., gases, liquids, or gas/liquid mixtures), or the like. The fluids can be solvents (e.g., polar solvents, non-polar solvents, etc.), solutions (e.g., aqueous solutions or other types of solutions), or the like. Reagents can include, without limitation, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen recovering fluids (e.g., aqueous- or non-aqueous-based antigen retrieval solutions, antigen recovering buffers, etc.), or the like. Probes can be an isolated nucleic acid or an isolated synthetic oligonucleotide, attached to a detectable label. Labels can include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes. After the specimens are processed, a user can transport specimen-bearing slides to the imaging apparatus. The imaging apparatus used here is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo™ brightfield scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the imaging apparatus is a digital pathology device as disclosed in International Patent Application No.: PCT/US2010/002772 (Patent Publication No.: WO/2011/049608) entitled IMAGING SYSTEM AND TECHNIQUES or disclosed in U.S. Patent Application Publication No. 2014/0178169, filed on Feb. 3, 2014, entitled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International Patent Application No. PCT/US2010/002772 and U.S. Patent Application Publication No. 2014/0178169 are incorporated by reference in their entities. In other embodiments, the imaging apparatus includes a digital camera coupled to a microscope. Counterstaining Counterstaining is a method of post-treating the samples after they have already been stained with agents to detect one or more targets, such that their structures can be more readily visualized under a microscope. For example, a counterstain is optionally used prior to coverslipping to render the immunohistochemical stain more distinct. Counterstains differ in color from a primary stain. Numerous counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain that may be used. In some examples, more than one stain can be mixed together to produce the counterstain. This provides flexibility and the ability to choose stains. For example, a first stain, can be selected for the mixture that has a particular attribute, but yet does not have a different desired attribute. A second stain can be added to the mixture that displays the missing desired attribute. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain. Detection and/or Imaging Certain aspects, or all, of the disclosed embodiments can be automated, and facilitated by computer analysis and/or image analysis system. In some applications, precise color or fluorescence ratios are measured. In some embodiments, light microscopy is utilized for image analysis. Certain disclosed embodiments involve acquiring digital images. This can be done by coupling a digital camera to a microscope. Digital images obtained of stained samples are analyzed using image analysis software. Color or fluorescence can be measured in several different ways. For example, color can be measured as red, blue, and green values; hue, saturation, and intensity values; and/or by measuring a specific wavelength or range of wavelengths using a spectral imaging camera. The samples also can be evaluated qualitatively and semi-quantitatively. Qualitative assessment includes assessing the staining intensity, identifying the positively-staining cells and the intracellular compartments involved in staining, and evaluating the overall sample or slide quality. Separate evaluations are performed on the test samples and this analysis can include a comparison to known average values to determine if the samples represent an abnormal state. Kits In some embodiments, the click conjugates may be utilized as part of a “detection kit.” In some embodiments, the detection kits comprise at least a first click conjugate in a first container and a second click conjugate in a second container. The first click conjugate is a first member of a pair of click conjugates having a first reactive functional group; and the second click conjugate is a second member of a pair of click conjugates having a second reactive functional group, wherein the first and second reactive functional groups are capable of reacting with each other to form a covalent bond. In some embodiments, the first click conjugate is selected from a compound having the structure of any of Formulas (II) or (III). In some embodiments, the second click conjugate is selected from a compound having the structure of Formula (IV) or Formula (V). The detection kits may also comprise other reagents including specific binding moieties and secondary antibodies specific to the specific binding moieties, the secondary antibodies conjugated to a detectable label. Of course, any kit may include other agents, including buffers; counterstaining agents; enzyme inactivation compositions; deparaffinization solutions, etc. as needed for manual or automated target detection. The kit may also include instructions for using any of the components of the kit, including methods of applying the kit components to a tissue sample to effect detection of one or more targets therein. Samples and Targets Samples include biological components and generally are suspected of including one or more target molecules of interest. Target molecules can be on the surface of cells and the cells can be in a suspension, or in a tissue section. Target molecules can also be intracellular and detected upon cell lysis or penetration of the cell by a probe. One of ordinary skill in the art will appreciate that the method of detecting target molecules in a sample will vary depending upon the type of sample and probe being used. Methods of collecting and preparing samples are known in the art. Samples for use in the embodiments of the method and with the composition disclosed herein, such as a tissue or other biological sample, can be prepared using any method known in the art by of one of ordinary skill. The samples can be obtained from a subject for routine screening or from a subject that is suspected of having a disorder, such as a genetic abnormality, infection, or a neoplasia. The described embodiments of the disclosed method can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as “normal” samples. Such normal samples are useful, among other things, as controls for comparison to other samples. The samples can be analyzed for many different purposes. For example, the samples can be used in a scientific study or for the diagnosis of a suspected malady, or as prognostic indicators for treatment success, survival, etc. Samples can include multiple targets that can be specifically bound by a probe or reporter molecule. The targets can be nucleic acid sequences or proteins. Throughout this disclosure when reference is made to a target protein it is understood that the nucleic acid sequences associated with that protein can also be used as a target. In some examples, the target is a protein or nucleic acid molecule from a pathogen, such as a virus, bacteria, or intracellular parasite, such as from a viral genome. For example, a target protein may be produced from a target nucleic acid sequence associated with (e.g., correlated with, causally implicated in, etc.) a disease. A target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example, a target nucleic acid sequence can have at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues. Similarly, a target polypeptide can vary substantially in size. Without limitation, the target polypeptide will include at least one epitope that binds to a peptide specific antibody, or fragment thereof. In some embodiments that polypeptide can include at least two epitopes that bind to a peptide specific antibody, or fragment thereof. In specific, non-limiting examples, a target protein is produced by a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) associated with a neoplasm (for example, a cancer). Numerous chromosome abnormalities (including translocations and other rearrangements, amplification or deletion) have been identified in neoplastic cells, especially in cancer cells, such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like. Therefore, in some examples, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., genomic target nucleic acid sequence) amplified or deleted in at least a subset of cells in a sample. Oncogenes are known to be responsible for several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18q11.2 are common among synovial sarcoma soft tissue tumors. The t(18q11.2) translocation can be identified, for example, using probes with different labels: the first probe includes FPC nucleic acid molecules generated from a target nucleic acid sequence that extends distally from the SYT gene, and the second probe includes FPC nucleic acid generated from a target nucleic acid sequence that extends 3′ or proximal to the SYT gene. When probes corresponding to these target nucleic acid sequences (e.g., genomic target nucleic acid sequences) are used in an in situ hybridization procedure, normal cells, which lack a t(18q11.2) in the SYT gene region, exhibit two fusion (generated by the two labels in close proximity) signals, reflecting the two intact copies of SYT. Abnormal cells with a t(18q11.2) exhibit a single fusion signal. In other examples, a target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is selected that is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), and the pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (that encompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system. The aforementioned examples are provided solely for purpose of illustration and are not intended to be limiting. Numerous other cytogenetic abnormalities that correlate with neoplastic transformation and/or growth are known to those of ordinary skill in the art. Target proteins that are produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have been correlated with neoplastic transformation and which are useful in the disclosed methods, also include the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC 000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC 000008, nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. NC-000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC 000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK™ Accession No. NC 000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC 000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession No. NC 000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK™ Accession No. NC-000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCND1 (1113; e.g., GENBANK™ Accession No. NC-000011, nucleotides 69165054.69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No. NC-000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™ Accession No. NC-000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK™ Accession No. NC 000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No. NC 000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™ Accession No. NC 000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK™ Accession No. NC-000007, complement, nucleotides 13897379-13995289), EWS (22q12.2; e.g., GENBANK™ Accession No. NC 000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No. NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC-000002, complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™ Accession No. NC 000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™ Accession No. NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK™ Accession No. NC 000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ Accession No. NC 000001, complement, nucleotides 40133685-40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC-000002, nucleotides 60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC-000005, complement, nucleotides 149413051-149473128). In other examples, a target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of the virus- or microorganism-derived target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in a cell or tissue sample is indicative of the presence of the organism. For example, the target peptide, polypeptide or protein can be selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such asPlasmodium falciparumand otherPlasmodiumspecies,Leishmania(sp.),Cryptosporidium parvum, Entamoeba histolytica, andGiardia lamblia, as well asToxoplasma, Eimeria, Theileria, andBabesiaspecies). In some examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from a viral genome. Exemplary viruses and corresponding genomic sequences (GENBANK™ RefSeq Accession No. in parentheses) include human adenovirus A (NC-001460), human adenovirus B (NC-004001), human adenovirus C(NC 001405), human adenovirus D (NC-002067), human adenovirus E (NC-003266), human adenovirus F (NC-001454), human astrovirus (NC-001943), human BK polyomavirus (V01109; GI:60851) human bocavirus (NC-007455), human coronavirus 229E (NC-002645), human coronavirus HKU1 (NC-006577), human coronavirus NL63 (NC-005831), human coronavirus OC43 (NC-005147), human enterovirus A (NC-001612), human enterovirus B (NC-001472), human enterovirus C(NC-001428), human enterovirus D (NC-001430), human erythrovirus V9 (NC-004295), human foamy virus (NC-001736), human herpesvirus 1 (Herpes simplex virus type 1) (NC-001806), human herpesvirus 2 (Herpes simplex virus type 2) (NC 001798), human herpesvirus 3 (Varicella zoster virus) (NC-001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC-007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC-009334), human herpesvirus 5 strain AD 169 (NC-001347), human herpesvirus 5 strain Merlin Strain (NC-006273), human herpesvirus 6A (NC-001664), human herpesvirus 6B (NC-000898), human herpesvirus 7 (NC-001716), human herpesvirus 8 type M (NC 003409), human herpesvirus 8 type P (NC-009333), human immunodeficiency virus 1 (NC 001802), human immunodeficiency virus 2 (NC-001722), human metapneumovirus (NC 004148), human papillomavirus-1 (NC-001356), human papillomavirus-18 (NC-001357), human papillomavirus-2 (NC-001352), human papillomavirus-54 (NC-001676), human papillomavirus-61 (NC-001694), human papillomavirus-cand90 (NC-004104), human papillomavirus RTRX7 (NC-004761), human papillomavirus type 10 (NC-001576), human papillomavirus type 101 (NC-008189), human papillomavirus type 103 (NC-008188), human papillomavirus type 107 (NC-009239), human papillomavirus type 16 (NC-001526), human papillomavirus type 24 (NC-001683), human papillomavirus type 26 (NC-001583), human papillomavirus type 32 (NC-001586), human papillomavirus type 34 (NC-001587), human papillomavirus type 4 (NC-001457), human papillomavirus type 41 (NC-001354), human papillomavirus type 48 (NC-001690), human papillomavirus type 49 (NC-001591), human papillomavirus type 5 (NC-001531), human papillomavirus type 50 (NC-001691), human papillomavirus type 53 (NC-001593), human papillomavirus type 60 (NC-001693), human papillomavirus type 63 (NC-001458), human papillomavirus type 6b (NC-001355), human papillomavirus type 7 (NC-001595), human papillomavirus type 71 (NC-002644), human papillomavirus type 9 (NC-001596), human papillomavirus type 92 (NC-004500), human papillomavirus type 96 (NC-005134), human parainfluenza virus 1 (NC-003461), human parainfluenza virus 2 (NC-003443), human parainfluenza virus 3 (NC-001796), human parechovirus (NC-001897), human parvovirus 4 (NC-007018), human parvovirus B19 (NC 000883), human respiratory syncytial virus (NC-001781), human rhinovirus A (NC-001617), human rhinovirus B (NC-001490), human spumaretrovirus (NC-001795), human T-lymphotropic virus 1 (NC-001436), human T-lymphotropic virus 2 (NC-001488). In certain examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV18). In other examples, the target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV). EXAMPLES The non-limiting examples presented herein each incorporate the use of at least one pair of click conjugates. Applicants submit that the click conjugates disclosed herein are suitable for use in IHC assays, including multiplex IHC assays, and ISH assays, as demonstrated in the following examples. General Immunohistochemistry (IHC) Protocol(s) All IHC staining experiments were carried out on a BenchMark XT automated tissue staining platform and the reagents used in these protocols were from Ventana Medical Systems, Inc. (Tucson, AZ, USA; “Ventana”) unless otherwise specified. Polyclonal goat anti-rabbit antibodies, polyclonal goat anti-mouse antibodies, horseradish peroxidase (HRP) and alkaline phosphatase (AP) were obtained from Roche Diagnostics (Mannheim, Germany). The following common steps were performed: (1) deparaffinization with EZ Prep detergent solution (Ventana Medical Systems, Inc. (VMSI), #950-101) (75° C.; 20 minutes); (2) washing with Reaction Buffer (VMSI, #950-300); (3) antigen retrieval in Cell Conditioning 1 (VMSI #950-124) (100° C.; time dependent on antigen of interest); (4) washing (same as step2); (5) for protocols with subsequent HRP detection steps endogenous peroxidase was inactivated using iVIEW inhibitor (VMSI, E253-2187) (37° C.; 4 minutes); (6) washing (same as step2); (7) primary antibody incubation (anti-target antibody) was performed at 37° C. for a time dependent on primary antibody ranging from 8-32 minutes; (8) washing (same as step2); and (9) secondary antibody incubation with a goat polyclonal anti-species antibody conjugated to an enzyme (HRP or AP, 37° C.; 8-12 minutes). All subsequent reagent incubation steps were separated by washing as in step (2). The targets were detected as described in examples 1-6. Example 1: “Click” Amplification with Compounds of Formula (II) Three examples of IHC “click” amplification with different compounds of Formula (II) are illustrated inFIGS.7A,7B, and7C. In general, each IHC assay was conducted according to the methods disclosed herein. InFIGS.7A,7B, and7C, each tissue sample was first contacted with a primary antibody specific to a particular target (FIG.7ACD8;FIG.7BBcl6; andFIG.7CKi67). Following introduction of the respective primary antibodies, each of the antibody-target complexes was labeled with an enzyme, such as by introducing a secondary antibody coupled to an alkaline phosphatase (AP) enzyme (e.g. a goat-anti-rabbit antibody-AP conjugate or a goat-anti-mouse antibody-AP conjugate). Next, a first member of a pair of click conjugates was introduced and reacted with each AP labeled target. InFIG.7Aa compound of Formula (II) comprising a quinone methide precursor linked to a DBCO reactive function group was introduced and a quinone methide-DBCO tissue conjugate complex was formed after reaction with the target bound alkaline phosphatase. Subsequently, a conjugate of Formula (IV) comprising the chromogen TAMRA and an azide reactive functional group were introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex.FIG.7Aclearly shows staining of the CD8 glycoprotein within a tonsil tissue sample. InFIG.7Ba compound of Formula (II) comprising a quinone methide precursor linked to an azide reactive function group was introduced and a quinone methide-azide tissue conjugate complex was formed after reaction with the target bound alkaline phosphatase. Subsequently, a conjugate of Formula (IV) comprising the chromogen TAMRA and a DBCO reactive functional group were introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex.FIG.7Bclearly shows staining of B-cell lymphoma 6 protein within a tonsil tissue sample. InFIG.7Ca compound of Formula (II) comprising a quinone methide precursor linked to a TCO reactive function group was introduced and a quinone methide-TCO tissue conjugate complex was formed after reaction with the target bound alkaline phosphatase. Subsequently, a conjugate of Formula (IV) comprising the chromogen TAMRA and a tetrazine reactive functional group were introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex.FIG.7Cclearly shows staining of the Ki67 protein within a tonsil tissue sample. Example 2: “Click” Amplification with Compounds of Formula (III) Three examples of IHC “click” amplification with different compounds of Formula (III) are illustrated inFIGS.8A,8B, and8C. In general, each IHC assay was conducted according to the methods disclosed herein. InFIGS.8A,8B, and8C, each tissue sample was first contacted with a primary antibody specific to a particular target (FIG.8ACD8;FIG.8BBcl6; andFIG.8CKi67). Following introduction of the respective primary antibodies, each of the antibody-target complexes was labeled with an enzyme, such as by introducing a secondary antibody coupled to a horseradish peroxidase (HRP) enzyme (e.g. a goat-anti-rabbit antibody-HRP conjugate or a goat-anti-mouse antibody-HRP conjugate). Next, a first member of a pair of click conjugates was introduced and reacted with each HRP labeled target. InFIG.8Aa compound of Formula (III) comprising a tyramide linked to an azide reactive function group was introduced and a tyramide-azide tissue conjugate complex was formed after reaction with the target bound HRP. Subsequently, a compound of Formula (IV) comprising the chromogen TAMRA and a DBCO reactive functional group were introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex.FIG.8Aclearly shows staining of the CD8 glycoprotein within a tonsil tissue sample. InFIG.8Ba compound of Formula (III) comprising a tyramide linked to a DBCO reactive function group was introduced and a tyramide-DBCO tissue conjugate complex was formed after reaction with the target bound HRP. Subsequently, a compound of Formula (IV) comprising the chromogen TAMRA and an azide reactive functional group were introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex.FIG.8Bclearly shows staining of the Ki67 protein within a tonsil tissue sample. InFIG.8Ca compound of Formula (III) comprising a tyramide linked to a TCO reactive function group was introduced and a tyramide-TCO tissue conjugate complex was formed after reaction with the target bound HRP. Subsequently, a compound of Formula (IV) comprising the chromogen TAMRA and a tetrazine reactive functional group were introduced and reacted with the tissue conjugate complex to form a detectable tissue-click adduct complex.FIG.8Cclearly shows staining of the CD8 glycoprotein within a tonsil tissue sample. Example 3 FIGS.9A,9B,9C, and9Dillustrate the results from four different IHC assays. Each assay was conducted using the general procedures described herein, and exemplified in Example 1. As applied here, each assay utilized the same click conjugate of Formula (II), namely one comprising a tyramine moiety conjugated to a DBCO reactive functional group (“Tyramide-DBCO”). However, four different click conjugates of Formula (IV) were used for coupling with Tyramide-DBCO, each having a different chromogen or chromogenic system coupled to an azide reactive functional group. As depicted inFIG.9A, the Tyramide-DBCO conjugate was reacted with a click conjugate of Formula (IV) where the click conjugate of Formula (IV) comprised a coupled Cy5 chromogen. As depicted inFIG.9B, the Tyramide-DBCO conjugate was reacted with a click conjugate of Formula (IV) where the click conjugate of Formula (IV) comprised a coupled Dabsyl chromogen. As depicted inFIG.9C, the Tyramide-DBCO conjugate reacted with a click conjugate of Formula (IV) where the click conjugate of Formula (IV) comprised both a TAMRA chromogen and a Dabcyl chromogen, where the two chromogens were coupled via lysine scaffold. As depicted inFIG.9D, the Tyramide-DBCO conjugate was reacted with a click conjugate of Formula (IV) where the click conjugate of Formula (IV) comprised a coupled TAMRA chromogen. Each ofFIGS.9A to9Dthus illustrate that a click conjugate species comprising a tissue reactive precursor moiety and a particular reactive functional group may be reacted with different compounds of Formula (IV) having different chromogens to stain tissue different colors. Example 4: Comparison of “Traditional” TSA to “Click” Amplification in an IHC Assay FIG.10comparatively illustrates staining with a DAB control, various TSA chromogens (TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl), and the TSA “click” conjugates of the present disclosure (tyramide-DBCO:TAMRA-Azide; tyramide-DBCO:Cy5-Azide; and tyramide-DBCO:Dabsyl-Azide). The tissue sample labeled “DAB control” was stained in an IHC assay utilizing a primary antibody specific for Ki67 and a goat-anti-rabbit antibody conjugated to HRP. The antigen was visualized via a brown precipitate produced by HRP upon the addition of hydrogen peroxide and 3,3′-diaminobenzidine (DAB). The DAB hue was toned by the addition of copper sulfate. The tissue samples identified as being stained with TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl inFIG.10were stained in an IHC assay using a traditional tyramide signal amplification technique. First, a primary antibody specific to Ki67 was introduced to form a primary antibody-Ki67 complex. The primary antibody-Ki67 complex was then labeled with a horseradish peroxidase enzyme, through a secondary antibody, namely a goat-anti-rabbit antibody-HRP conjugate. Subsequently, a tyramide coupled to a chromogen, namely TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl, were each independently introduced and each was subsequently deposited on or adjacent the target following reaction with horseradish peroxidase. The tissue samples identified as being stained with TSA-DBCO:TAMRA-Azide, tyramide-DBCO:Cy5-Azide; and tyramide-DBCO:Dabsyl-Azide inFIG.10were stained in an IHC assay using the general techniques described herein and those provided in Example 2. As compared with the samples stained in the traditional TSA assays, the tissues stained using Cy5 or Dabsyl in “click” amplification according to the methods described herein showed significant increases in staining intensity, as clearly shown inFIG.10. Example 5: Comparison of “Traditional” TSA to “Click” Amplification in an ISH Assay FIG.11comparatively illustrates staining of various TSA chromogens (TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl), and the TSA “click” conjugates of the present disclosure (tyramide-DBCO:TAMRA-Azide; tyramide-DBCO:Cy5-Azide; and tyramide-DBCO:Dabsyl-Azide). The tissue samples identified as being stained with TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl inFIG.11were stained in an ISH assay using a traditional tyramide signal amplification technique. First, a nucleic acid probe specific to Her2 was introduced to a tissue sample, the Her2 probe conjugated to a detectable label, namely a DNP hapten. The DNP was bound by a rabbit-anti-DNP antibody, which was then labeled with a goat-anti-rabbit antibody conjugated to HRP. Subsequently, TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl were each independently introduced and each was subsequently deposited on or adjacent the target following reaction with horseradish peroxidase. The tissue samples identified as being stained with TSA-DBCO:TAMRA-Azide, tyramide-DBCO:Cy5-Azide; and tyramide-DBCO:Dabsyl-Azide inFIG.11were stained according to the general techniques described herein (see, e.g.,FIG.15). As compared with the samples stained in the traditional TSA assays, the tissues stained using Cy5 or Dabsyl in “click” amplification according to the methods described herein showed significant increases in staining intensity, as clearly shown inFIG.11. Example 6 FIG.12illustrates the differences in staining using a click conjugate of Formula (IV) comprising a single reporter moiety versus another click conjugate of Formula (IV) comprising multiple reporter moieties. The tissue sample on the left was stained using a click conjugate of Formula (IV) comprising a single TAMRA chromogen. The tissue sample on the right was staining using a click conjugate of Formula (V) comprising at least two TAMRA chromogens, the TAMRA chromogens coupled together using a dendrimer. Example 7 FIG.13illustrates the staining of tissue with an enzyme-tissue click adduct. The IHC assay was conducted according to the methods disclosed herein. Following introduction of the rabbit-anti-Ki67 primary antibody, each of the antibody-target complexes was labeled with a secondary goat-anti-rabbit antibody coupled to a horseradish peroxidase (HRP) enzyme. Next, a compound of Formula (III) comprising a tyramide linked to a DBCO reactive function group (first member of a pair of click conjugates), was introduced along with hydrogen peroxide and reacted with each HRP labeled target. A tyramide-DBCO tissue conjugate complex was formed after reaction with the target bound HRP. Subsequently, a compound of Formula (IV) comprising the enzyme AP and an azide reactive functional group were introduced (second member of a pair of click conjugates) and reacted with the tissue conjugate complex to form a detectable tissue-click adduct. The AP-tissue click adduct was then detected with QMSA-TAMRA chromogenic detection.FIG.13clearly shows the increase in staining of the Ki67 protein within a tonsil tissue sample corresponding to increasing concentration of AP-azide. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments. ADDITIONAL EXEMPLARY EMBODIMENTS The following embodiments are also explicitly disclosed. This is not intended to be an exhaustive list.Embodiment 1. A conjugate having Formula (IIa): whereinA is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine;‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S;R1is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar;R2is a halide;R3, R5, and R6are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms;R4is a hydrogen, an aliphatic group having between 1 and 4 carbon atoms, or the group —CH(R2)—R7-[Linker]-A; andR7is selected from the group consisting of —(CH2)wNH—, —O(CH2)wNH—, —N(H)C(O)(CH2)wNH—, —C(O)N(H)(CH2)wNH—, —(CH2)wO—, —O(CH2)wO—, —O(CH2CH2O)w—, —N(H)C(O)(CH2)wO—, —C(O)N(H)(CH2)wO—, —C(O)N(H)(CH2CH2O)w—, —(CH2)wS—, —O(CH2)wS—, —N(H)C(O)(CH2)wS—, —C(O)N(H)(CH2)wS—, —(CH2)wNH—, —C(O)N(H)(CH2CH2O)wCH2CH2NH, —C(O)(CH2CH2O)wCH2CH2NH—, —C(O)N(H)(CH2)NHC(O)CH(CH3)(CH2)wNH—, or —N(H)(CH2)wNH—, where w is an integer ranging from 1 to 12.Embodiment 2. The conjugate of embodiment 1, wherein R6, R5, R4, and R3are each hydrogen.Embodiment 3. The conjugate of embodiment 1 or 2, wherein R1is a phosphate.Embodiment 4. The conjugate of any of embodiments 1 to 3, wherein R2is fluorine.Embodiment 5. The conjugate of embodiment 1, wherein R1is a phosphate; R2is fluorine; and R6, R5, R4, and R3are each hydrogen.Embodiment 6. The conjugate of any of embodiments 1 to 5, wherein ‘Linker’ has the Formula (Ia): whereind and e are integers each independently ranging from 2 to 20;t and u are independently 0 or 1;Q is a bond, O, S, or N(Rc)(Rd);Raand Rbare independently H, a C1-C4alkyl group, F, Cl, or N(Rc)(Rd);Rcand Rdare independently CH3or H; andX and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.Embodiment 7. The conjugate of claim6, wherein Raand Rbare each hydrogen.Embodiment 8. The conjugate of claim7, wherein Q is oxygen.Embodiment 9. The conjugate of any of claims1to8, wherein R7is —C(O)N(H)(CH2)wNH—.Embodiment 10. The conjugate of claim1or2, wherein R1is a phosphate and R7is —C(O)N(H)(CH2)wNH—, and w ranges from 2 to 10.Embodiment 11. The conjugate of claim10, wherein R2is fluorine; and R6, R5, R4, and R3are each hydrogen.Embodiment 12. The conjugate of claim11, wherein ‘Linker’ comprises a PEG group.Embodiment 13. A conjugate having Formula (IId): whereinA is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine;‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; andw ranges from 1 to 12.Embodiment 14. The conjugate of claim13, wherein ‘Linker’ has the Formula (Ia): whereind and e are integers each independently ranging from 2 to 20;t and u are independently 0 or 1;Q is a bond, O, S, or N(Rc)(Rd);Raand Rbare independently H, a C1-C4alkyl group, F, Cl, or N(Rc)(Rd);Rcand Rdare independently CH3or H; andX and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.Embodiment 15. The conjugate of embodiment 14, wherein w ranges from 1 to 8; and wherein Raand Rbare each hydrogen.Embodiment 16. The conjugate of embodiment 15, wherein w ranges from 2 to 8, and wherein Q is oxygen.Embodiment 17. The conjugate of embodiment 16, wherein d and e are independently an integer ranging from 2 to 10.Embodiment 18. The conjugate of any of embodiments 13 to 17, wherein A is dibenzocyclooctyne.Embodiment 19. The conjugate of embodiment 18, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group.Embodiment 20. The conjugate of any of embodiments 13 to 17, wherein A is trans-cyclooctene.Embodiment 21. The conjugate of embodiment 20, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group.Embodiment 22. The conjugate of any of embodiments 13 to 17, wherein A is azide.Embodiment 23. The conjugate of embodiment 23, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group.Embodiment 24. The conjugate of any of embodiments 13 to 17, wherein A is tetrazine.Embodiment 25. The conjugate of embodiment 24, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group.Embodiment 26. A conjugate having Formula (III): ALinkerM (III),whereinM is derived from a propionic acid, a cinnamic acid, or a compound of Formula (Ilia), wherein each R group is independently selected from hydrogen or lower alkyl group having between 1 and 4 carbon atoms;A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; and‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S;provided that when each R is hydrogen, A is selected from the group consisting of an azide, a thiol, a 1,3-nitrone, a hydrazine, or a hydroxylamine.Embodiment 27. The conjugate of embodiment 26, wherein ‘Linker’ has the formula (Ia) whereind and e are integers each independently ranging from 2 to 20;t and u are independently 0 or 1;Q is a bond, O, S, or N(Rc)(Rd);Raand Rbare independently H, a C1-C4alkyl group, F, Cl, or N(Rc)(Rd); Rcand Rdare independently CH3or H; andX and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.Embodiment 28. The conjugate of embodiment 27, wherein Raand Rbare each hydrogen.Embodiment 29. The conjugate of embodiment 27 or 28, wherein Q is oxygen.Embodiment 30. The conjugate of embodiment 27, wherein Raand Rbare each hydrogen, Q is oxygen, and e ranges from 2 to 10.Embodiment 31. A conjugate having Formula (Id): whereinA is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine;‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and‘Tissue Reactive Moiety’ is derived from a compound selected from the group consisting of: Embodiment 32. The conjugate of embodiment 31, wherein ‘Linker’ has the Formula (Ia): whereind and e are integers each independently ranging from 2 to 20;t and u are independently 0 or 1;Q is a bond, O, S, or N(Rc)(Rd);Raand Rbare independently H, a C1-C4alkyl group, F, Cl, or N(Rc)(Rd);Rcand Rdare independently CH3or H; andX and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.Embodiment 33. The conjugate of embodiment 32, wherein Raand Rbare each hydrogen.Embodiment 34. The conjugate of embodiment 32 or 33, wherein Q is oxygen.Embodiment 35. The conjugate of embodiment 32, wherein Raand Rbare each hydrogen, Q is oxygen, and e ranges from 2 to 10.Embodiment 36. A conjugate having Formula (IV): ALinkerZ (IV),whereinA is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine;‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; andZ is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator.Embodiment 37. The conjugate of embodiment 36, Z is a chromophore selected from the group consisting of tetramethylrhodamine, Cyanine 5, and Dabsyl.Embodiment 38. The conjugate of embodiment 36, where Z is selected from the group consisting of: Embodiment 39. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVa): Embodiment 40. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVb): Embodiment 41. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVc): Embodiment 42. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVd): Embodiment 43. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 44. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 45. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 46. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 47. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 48. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 49. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 50. The conjugate of embodiment 36, wherein the conjugate is: Embodiment 51. A method of detecting a first target in a biological sample, comprising:(i) contacting the biological sample with a first detection probe specific to the first target to form a first detection probe-target complex;(ii) contacting the biological sample with a first labeling conjugate specific for the first detection probe, the first labeling conjugate comprising a first enzyme such that the first-detection probe-target complex becomes labeled with the first enzyme;(iii) contacting the biological sample with a first member of a first pair of click conjugates, the first member of the first pair of click conjugates comprising a tissue reactive moiety, wherein the first enzyme converts the first member of the first pair of click conjugates to a first reactive intermediate which covalently bonds to the biological sample proximally to or directly on the first target to form a first immobilized tissue-click conjugate complex;(iv) contacting the biological sample with a second member of a first pair of click conjugates, the second member of the first pair of click conjugates comprising a second reactive moiety capable of reacting with a first reactive moiety of the first immobilized tissue-click conjugate complex such that a covalent bond is formed between the first immobilized tissue-click conjugate complex and the second member of the first pair of click conjugates to form a first tissue-click conjugate adduct; and(v) detecting signals from a first reporter moiety of first tissue-click conjugate adduct Embodiment 52. The method of embodiment 51, wherein the first member of the first pair of click conjugates comprises the conjugate of any of embodiments 1 to 35.Embodiment 53. The method of embodiment 51 or 52, wherein the second member of the first pair of click conjugates comprises the conjugate of any of embodiments 36 to 50.Embodiment 54. The method of embodiment 51 or 52, wherein the second member of the first pair of click conjugates comprises at least one chromophore.Embodiment 55. The method of embodiment 51, wherein the first member of the first pair of click conjugates comprises a quinone methide precursor moiety; and wherein the second member of the first pair of click conjugates comprises a chromophore.Embodiment 56. The method of embodiment 51, wherein the first member of the first pair of click conjugates comprises a tyramide moiety; and wherein the second member of the first pair of click conjugates comprises a chromophore.Embodiment 57. The method of any of embodiments 51 to 56, wherein the first detection probe is a primary antibody, and wherein the first labeling conjugate comprises an anti-antibody antibody.Embodiment 58. The method of any of embodiments 51 to 57, wherein the first enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, neuraminidase, alpha-lactase and beta-lactase.Embodiment 59. The method of any of embodiments 51 to 58, further comprising detecting a second target in the biological sample, wherein the second target is detected by(i) contacting the biological sample with a second detection probe specific to the second target to form a second detection probe-target complex;(ii) contacting the biological sample with a second labeling conjugate specific for the second detection probe, the second labeling conjugate comprising a second enzyme such that the second-detection probe-target complex becomes labeled with the second enzyme;(iii) contacting the biological sample with a first member of a second pair of click conjugates, the first member of the second pair of click conjugates comprising a tissue reactive moiety, wherein the second enzyme converts the first member of the second pair of click conjugates to a second reactive intermediate which covalently bonds to the biological sample proximally to or directly on the second target to form a second immobilized tissue-click conjugate complex.(iv) contacting the biological sample with a second member of a second pair of click conjugates, the second member of the second pair of click conjugates comprising a second reactive moiety capable of reacting with a first reactive moiety of the second immobilized tissue-click conjugate complex such that a covalent bond is formed between second immobilized tissue-click conjugate complex and the second member of the second pair of click conjugates; and(v) detecting signals from a second reporter moiety of the second tissue-click conjugate adduct, wherein the second reporter moiety is different than the first reporter moiety.Embodiment 60. The method of embodiment 59, wherein the first member of the second pair of click conjugates comprises the conjugate of any of embodiments 1 to 35.Embodiment 61. The method of embodiment 59 or 60, wherein the second member of the second pair of click conjugates comprises the conjugate of any of embodiments 36 to 50.Embodiment 62. The method of embodiment 59 or 60, wherein the second member of the second pair of click conjugates comprises at least one chromophore.Embodiment 63. The method of embodiment 59, wherein the first member of the second pair of click conjugates comprises a quinone methide precursor moiety; and wherein the second member of the second pair of click conjugates comprises a chromophore.Embodiment 64. The method of embodiment 59, wherein the first member of the second pair of click conjugates comprises a tyramide moiety; and wherein the second member of the second pair of click conjugates comprises a chromophore.Embodiment 65. The method of any of embodiments 59 to 64, wherein the second detection probe is a primary antibody, and wherein second first labeling conjugate comprises an anti-antibody antibody.Embodiment 66. The method of any of embodiments 59 to 65, wherein the second enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, neuraminidase, alpha-lactase and beta-lactase.Embodiment 67. The method of any of embodiments 51 to 66, wherein one or more of the steps are performed by an automated system.Embodiment 68. An immobilized click-conjugate covalently bonded to a tissue sample, the immobilized click-conjugate comprising a first reactive functional group selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine.Embodiment 69. The immobilized click-conjugate of embodiment 68, wherein the click-conjugate is bonded to the tissue through a tyrosine residue or a nucleophilic species within or on the surface of the tissue sample.Embodiment 70. A detectable tissue-click adduct complex formed by reacting the immobilized click-conjugate of embodiments 68 and 69 with a conjugate having Formula (IV): ALinkerZ (IV),whereinA is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine;‘Linker’ is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; andZ is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator; andwherein the conjugate of Formula (IV) comprises an A group capable of reacting with the first reactive functional group of the immobilized click-conjugate.Embodiment 71. The detectable tissue-click adduct complex of embodiment 70, wherein Z is at least one chromophore.Embodiment 72. The detectable tissue-click adduct complex of embodiment 70 or 71, wherein the first reactive functional group is dibenzocyclooctyne and where A of Formula (IV) is selected from the group consisting of an azide or a 1,3-nitrone.Embodiment 73. The detectable tissue-click adduct complex of embodiment 70 or 71, wherein the first reactive functional group is trans-cyclooctene and where A of Formula (IV) is a tetrazine.Embodiment 74. The detectable tissue-click adduct complex of embodiment 70 or 71, wherein the first reactive functional group is an azide and where A of Formula (IV) is a dibenzocyclooctyne.Embodiment 75. The detectable tissue-click adduct complex of embodiment 70 or the detectable tissue-click adduct complex of any of embodiments 72 to 74, wherein Z is a chelator, and wherein a lanthanide is introduced to the formed detectable tissue-click adduct complex. Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims. | 117,205 |
11860168 | DETAILED DESCRIPTION OF THE INVENTION The term “mycobacterium,” as used herein, refers to a genus of actinobacteria given its own family, the mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Mycobacterium tuberculosiscomplex (MTBC) members are causative agents of human and animal tuberculosis. Species in this complex may includeM. tuberculosis, the major cause of human tuberculosis,M. bovis, M. bovisBCG,M. africanum, M. canetti, M. caprae, M. microti, andM. pinnipedii. Mycobacterium aviumcomplex (MAC) is a group of species that, in a disseminated infection but not lung infection, used to be a significant cause of death in AIDS patients. Species in this complex includeM. avium, M. avium paratuberculosis, which has been implicated in Crohn's disease in humans and is the causative agent of Johne's disease in cattle and sheep,M. avium silvaticum, M. avium “hominissuis,” M. colombiense, andM. indicus pranii. Mycobacterial infections are notoriously difficult to treat. The organisms are hardy due to their cell wall, which is neither truly Gram negative nor Gram positive. In addition, they are naturally resistant to a number of antibiotics that disrupt cell-wall biosynthesis, such as penicillin. Due to their unique cell wall, they can survive long exposure to acids, alkalis, detergents, oxidative bursts, lysis by complement, and many antibiotics. Most mycobacteria are susceptible to the antibiotics clarithromycin and rifamycin, but antibiotic-resistant strains have emerged. The term “biomolecule,” as used herein, refers to any organic molecule that is part of or from a living organism. Biomolecules may include nucleic acids, a nucleotide, a polynucleotide, an oligonucleotide, a peptide, a protein, a carbohydrate, a ligand, a receptor, among others. In one embodiment of the present invention, biomolecules may include genes and their expression products. The term “expression product,” as used herein, refers to any product produced during the process of gene expression. These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA. The terms “polypeptide,” “peptide,” and “protein,” as used herein, refer to a polymer comprising amino acid residues predominantly bound together by covalent amide bonds. The terms apply to amino acid polymers in which one or more amino acid residue may be an artificial chemical mimetic of a naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms may encompass amino acid chains of any length, including full length proteins, wherein the amino acids are linked by covalent peptide bonds. The protein or peptide may be isolated from a native organism, produced by recombinant techniques, or produced by synthetic production techniques known to one skilled in the art. The term “recombinant protein,” as used herein, refers to a polypeptide of the present disclosure which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is in turn used to transform a heterologous host cell (e.g., a microorganism or yeast cell) to produce the heterologous protein. The term “recombinant nucleic acid” or “recombinant DNA,” as used herein, refers to a nucleic acid or DNA of the present disclosure which is produced by recombinant DNA techniques, wherein generally, DNA encoding a polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein. The term “mammal,” as used herein, refers to any living species which can be identified by the presence of sweat glands, including those that are specialized to produce milk to nourish their young. In one embodiment, the mammal suitable for the present invention may include bubaline, elephantine, musteline, pardine, phocine, rhinocerine, caprine, hircine, leonine, leporine, lupine, lyncine, murine, rusine, tigrine, ursine, vulpine, zebrine, vespertilionine, porcine, bovine, equine, swine, elaphine, ovine, caprine, camelidae, feline, cervine, primate, human and canine mammals. In one preferred embodiment of the present invention, the mammal may be one of the ruminants such as cattle, goats, sheep, giraffes, yaks, deer, camels, llamas, antelope, and some macropods. In one specific embodiment of the present invention, the mammal may include any of the milk cattle species, such as cow, sheep and goat. The term “antibody,” as used herein, refers to a class of proteins that are generally known as immunoglobulins. The term “antibody” herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). The term “marker” or “biomarker,” as used herein, refers to a biomolecule (e.g., protein, nucleic acid, carbohydrate, or lipid) that is differentially expressed in the cell, differentially expressed on the surface of an infected cell, differentially phosphorylated, or differentially secreted by a infected cell in comparison to a normal cell or in a paracrine fashion by neighboring uninfected cells, and which is useful for the diagnosis of mycobacterial infection, differentiating between infected and vaccinated animals, and for preferential targeting of a pharmacological agent to an infected mammal. In some embodiments, the biomarker is differentially expressed in an infected subject in comparison to a normal subject. In some embodiments, the biomarker is differentially expressed in a vaccinated subject in comparison to a non-vaccinated subject. In some embodiments, the biomarker is differentially expressed in a vaccinated subject in comparison to an infected subject. The term “differentially expressed,” as used herein, refers to a change in expression of at least 2-fold. In some embodiments, differential expression indicates that a given biomarker is over-expressed in a first subject or cell in comparison to a second subject or cell, for instance, at least 2-fold over-expression, at least 3-fold over-expression, at least 4-fold over-expression or more in comparison to the second subject or cell. In some embodiments, differential expression indicates that a given biomarker has decreased expression in a first subject or cell in comparison to a second subject or cell, for instance, at least a 2-fold decease in expression, at least a 3-fold decrease in expression, at least a 4-fold decrease in expression or more. The term “mycobacterial-specific biomarkers,” as used herein, refers to biomarkers which are specifically related to mycobacterial infection. Some of these biomarkers are listed inFIG.1and Tables 4 and 5. The term “lyophilization,” as used herein, refers to freezing of a material at low temperature followed by dehydration by sublimation, usually under a high vacuum. Lyophilization is also known as freeze drying. Many techniques of freezing are known in the art of lyophilization such as tray freezing, shelf freezing, spray-freezing, shell-freezing and liquid nitrogen immersion. Each technique will result in a different rate of freezing. Shell freezing may be automated or manual. For example, flasks can be automatically rotated by motor driven rollers in a refrigerated bath containing alcohol, acetone, liquid nitrogen, or any other appropriate fluid. A thin coating of product is evenly frozen around the inside “shell” of a flask, permitting a greater volume of material to be safely processed during each freeze drying run. Tray freezing may be performed by, for example, placing the samples in lyophilizer, equilibrating 1 hr at a shelf temperature of 0° C., then cooling the shelves at 0.5° C./min to −40° C. Spray-freezing, for example, may be performed by spray freezing into liquid, dropping by ˜20 μl droplets into liquid N2, spray freezing into vapor over liquid, or by other techniques known in the art. The term “antigen,” as used herein, refers to any molecule that is capable of eliciting an immune response, whether a cell-mediated or humoral immune response, whether in the presence or absence of an adjuvant. An antigen can be any type of molecule, e.g., a peptide or protein, a nucleic acid, a carbohydrate, a lipid, and combinations thereof. A “vaccine antigen” is an antigen that can be used in a vaccine preparation. A “therapeutic antigen” is an antigen that can be used for therapeutic purposes. The term “vaccine,” as used herein, refers to an antigenic preparation used to produce active immunity to a disease, in order to prevent or ameliorate the effects of infection. The antigenic moiety making up the vaccine may be either a live or killed microorganism, or a natural product purified from a microorganism or other cell including, but not limited to tumor cells, a synthetic product, a genetically engineered protein, peptide, polysaccharide or similar product or an allergen. The term “immunologically active,” as used herein, refers to the ability to raise one or more of a humoral response or a cell mediated response specific to an antigen. The term “adjuvant,” as used herein, refer to compounds that, when used in combination with specific vaccine antigens in formulations, augment or otherwise alter or modify the resultant immune responses. An adjuvant combined with a vaccine antigen increases the immune response to the vaccine antigen over that induced by the vaccine antigen alone. An adjuvant may augment humoral immune responses or cell-mediated immune responses or both humoral and cell-mediated immune responses against vaccine antigens. The term “detecting,” as used herein, refers to confirming the presence of the biomarker or marker present in the sample. Quantifying the amount of the biomarker or marker present in a sample may include determining the concentration of the biomarker present in the sample. Detecting and/or quantifying may be performed directly on the sample, or indirectly on an extract therefrom, or on a dilution thereof. The term “homology,” as used herein, refers to the resemblance or similarity between two nucleotide or amino acid sequences. As applied to a gene, “homolog” may refer to a gene similar in structure and/or evolutionary origin to a gene in another organism or another species. As applied to nucleic acid molecules, the term “homolog” means that two nucleic acid sequences, when optimally aligned (see below), share at least 80 percent sequence homology, preferably at least 90 percent sequence homology, more preferably at least 95, 96, 97, 98 or 99 percent sequence homology. “Percentage nucleotide (or nucleic acid) homology” or “percentage nucleotide (or nucleic acid) sequence homology” refers to a comparison of the nucleotides of two nucleic acid molecules which, when optimally aligned, have approximately the designated percentage of the same nucleotides or nucleotides that are not identical but differ by redundant nucleotide substitutions (the nucleotide substitution does not change the amino acid encoded by the particular codon). For example, “95% nucleotide homology” refers to a comparison of the nucleotides of two nucleic acid molecules which, when optimally aligned, have 95% nucleotide homology. In one aspect, the present invention relates to a method for diagnosis of mycobacterial infection in a mammal. In one embodiment, the present invention discloses a method for early detection of mycobacterial infection. The term “early detection,” as used herein, refers to detection of mycobacterial infection during the early stage of infection, e.g., a stage before the development of chronic diarrhea. In another embodiment, the present invention discloses a method for differentiating a vaccinated mammal from a non-infected mammal or a mycobacterial infected mammal. The detection of mycobacterial infection and related diseases such as Johne's disease is very difficult because the disease generally takes many years to develop, and the organism is shed by the mammal periodically, so every mammal must be repeatedly tested over long time periods. Applicants have identified mycobacterial-specific biomarkers and host-specific biomarkers, such as genes and/or expression products derived thereof, useful for detection of mycobacterial infection. Mycobacterial-specific biomarkers, host-specific biomarkers or a combination of such biomarkers may also be used to differentiate a vaccinated mammal (e.g., from genetically engineered vaccines) from a non-infected mammal or a mycobacterial-infected mammal. Differentiating Vaccinated Mammals from Mycobacterial-Infected Mammals In one embodiment, the present invention discloses a method for differentiating a vaccinated mammal from a mycobacterial infected mammal. In one embodiment, the method for differentiating a vaccinated mammal from a mycobacterial-infected mammal comprises the steps of (a) obtaining a sample from the mammal; (b) testing the sample for the concentration level of at least one biomarker and comparing the level of the biomarker against the level detected in an infected mammalian sample; and (c) determining the infection or vaccination status of the mammal. A sample suitable for the present invention may include any biological sample from the mammal. The biological sample may include, without limitation, saliva, sputum, blood, plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue of the subject of mammal. In one specific embodiment, the biological sample is a blood sample. In some embodiments, when comparing the expression level of a biomarker between two subjects, it is desirable to compare expression levels in the same type of sample. A major problem in employing mass vaccination program for the control of Johne's disease in dairy herds is the inability to differentiate between infected and vaccinated animals with the current vaccine (DIVA principal). Applicants have previously proposed using genetically engineered vaccines (PCT patent application publication WO2014164055, U.S. Pat. Nos. 9,663,758, and 9,446,110, each of which is incorporated herein by reference). One would wish to consider the DIVA principal and wish to distinguish betweenM. paratuberculosisinfected and Johne's disease vaccinated animals that have been vaccinated with genetically engineered vaccines. In one embodiment, Applicants identify biomolecules as mycobacterial-specific biomarkers. For example, the biomolecules of mycobacterial-specific biomarkers may include genes and their expression products which are present in aM. apwild-type strain but not present or have a low expression level in genetically engineered vaccines and vaccinated animals. In one embodiment involving sigL and sigH mutants, the mycobacterial-specific biomarker may comprise at least one member selected from the group consisting of gene sequences Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZ0, Q740D1 and Q73UE0 and expression products derived thereof. In another embodiment, the mycobacterial-specific biomarker may comprise at least two, three, four, five, six, seven or eight members selected from the group consisting of gene sequences Q73SF4, Q73Y73, Q73ZE6, Q73SL7, Q73VK6, Q73XZ0, Q740D1 and Q73UE0 and expression products derived thereof. Preferably, the mycobacterial-specific biomarker may comprise at least two members selected from the group as discussed above. In one embodiment involving sigL and sigH mutants, the mycobacterial-specific biomarker comprises at least one member selected from the group consisting of gene sequences Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, and Q73SU6 and expression products derived thereof. In one embodiment, the mycobacterial-specific biomarker comprises at least two, three, four, five, six, seven, eight, nine or ten members selected from the group consisting of gene sequences Q73VL6, Q73YW9, Q741L4, Q744E5, Q73YP5, Q73WE5, Q73U21, Q73UH9, Q741M5, Q742F4, and Q73SU6 and expression products derived thereof. Applicants envision that the present invention may be applicable to any genetically engineered vaccines. In some embodiments, the vaccine may be an inactived vaccine (e.g., Mycopar™). In one specific embodiment, the present invention is applicable to live attenuated vaccines. The example of the live attenuated vaccines may include sigL, sigH, or LipN mutants. PCT patent application publication WO20141640055A1 discloses live attenuated vaccines, such as sigL and sigH mutants. PCT patent application publication WO20141640055A1 is incorporated herein by reference in its entirety. In one embodiment, the presence or absence of the biomarkers in a mammal may demonstrate the infection status of the mammal. In one specific embodiment, the biomarkers that are significantly over-expressed in the wild type strain and not in the mutant vaccine and could be used for the mutant vaccine-DIVA testing. For example, when the biomarkers are those significantly over-expressed in the wild type strain and not in the mutant vaccine, the presence of at least one biomarker in a mammal shows that the mammal may be infected and not merely vaccinated. On the other hand, the absence of at least one biomarker in a mammal shows that the mammal may be vaccinated. In one embodiment, Applicants envision that the present invention is also applicable when antigens are inoculated to a mammal and the infection status of the mammal needs to be identified. Specifically, the infection status may include whether a mammal is vaccinated or whether a mammal is infected withM. paratuberculosis. Table 7, which tabulates the result of one of the Examples drawn to host transcriptome analysis of goats, lists additional markers that will be useful for embodiments of the invention. The first part of Table 7 lists DNA markers that are useful for early diagnosis of John's disease in ruminants, as described above, because the markers differentiate infected from naïve animals. Table 7 lists the locus in goats and provides homologous locus in cows, if it is known. Table 7 also lists host markers that can differentiate live attenuated vaccine (LAV) vaccinated animals from naïve animals and markers that can differentiate inactivated-vaccine immunized from naïve animals. Table 7 also lists host markers to distinguish between infected and vaccinated animals. As used herein, “naïve” refers to animals that are not vaccinated nor infected. In addition to the biomarkers recited herein, additional biomarkers useful in the disclosed methods include those described in U.S. Pat. No. 10,054,586, which is incorporated herein in its entirety. In some embodiments, the present invention provides methods of distinguishing between infected and naïve animals by measuring expression of one or more biomarkers selected from the group of LOC108634521, LOC108637251, LOC108637252, LOC108634594, FAM198B, LOC108637671, CDCP1, TMTC1, BAIAP2L1, MEI1, SEPT10, IFNG, IL17F, FCER2, ADGRG1, APBB1, PIWIL2, AOAH, and homologs thereof (Table 1) In some embodiments, two, three, four, five, six or more of the recited biomarkers may be measured. In some embodiments, the present invention provides methods of distinguishing between live attenuated vaccine (LAV) vaccinated animals from naïve animals by measuring expression of one or more biomarker selected from the group of LOC108634521, NOS2, LOC108637251, TINAGL1, RETN, C1QL2, TDRD10, TGFB3, ADGRE2, LIPG, KCNJ2, AQP9, BPI, IL9, IL1R2, IL36B, IGF1, BGN, PIWIL2, RAET1E, CRABP2, AOAH, and homologs thereof (Table 2) In some embodiments, two, three, four, five, six or more of the recited biomarkers may be measured. In some embodiments, the present invention provides methods of distinguishing between LAV-vaccinated animals from infected animals by measuring expression of one or more biomarker selected from the group of LOC106503226, PMP22, ART5, LOC102169116, GNLY, ASAP3, LOC108633178, TBKBP1, SLC17A7 and homologs thereof (Table 3) In some embodiments, two, three, four, five, six or more of the recited biomarkers may be measured. In some embodiments, biomarkers may be used to distinguish between naïve, infected, and vaccinated animals. By measuring expression of two or more biomarkers recited herein, an animal may be identified as naïve, infected, or vaccinated. A selection of suitable biomarkers and their relative expression is outlined in Table 4A and Table 4B. For example, as demonstrated in Table 4A, when relative expression of FAM198B is higher than relative expression of AOAH, the subject is infected with a mycobacterial infection. When relative expression of AOAH is higher than relative expression of FAM198B, the subject has been vaccinated with an LAV vaccine. When relative expression of AOAH and FAM198B are equal, or within error of the method used to quantify relative expression, the subject is naïve. Similar comparisons and conclusions may be drawn using other biomarkers described herein, such as those outlined in Table 4B or any of Tables 1-3. TABLE 1List of host (goat and cow) genes that can differentiation infected from naïve animals.Log2Expression inExpression inFold Change inEntreza Naïvean InfectedExpressionSymbolGene IDDescriptionAnimalAnimalInfected vs NaïveLOC108634521108634521non-coding RNA0.66258.78.66LOC108637251108637251multidrug resistance-associated protein 4-like1.28137.446.74LOC108637252108637252multidrug resistance-associated protein 4-like5.3190.394.11LOC108634594108634594multidrug resistance-associated protein 4-like3.35110.845.06FAM198B102191727family with sequence similarity 198 member B71.81768.853.42LOC108637671108637671tripartite motif-containing protein 5-like46.57316.632.77CDCP1102187276CUB domain containing protein8.936.662.02TMTC1102185637transmembrane and tetratricopeptide repeat13.247.851.86BAIAP2L1102173150BAI1 associated protein 2 like8.1729.541.85MEI1102169168meiotic double-stranded break formation protein203.8609.181.58SEPT10102171885septin 1016.8340.161.27IFNG100860815interferon gamma63.7719.26−1.75IL17F102171111interleukin 17F625.76267.69−1.22FCER2102171507Fc fragment of IgE receptor II626.49243.68−1.36ADGRG1102171366G protein-coupled receptor G72.4321.2−1.78APBB1102179305amyloid beta precursor protein binding family B member97.3921.75−2.16PIWIL2102173845piwi like RNA-mediated gene silencing57.3210.67−2.41AOAH102189546acyloxyacyl hydrolase258.05268.210.06 TABLE 2List of host (goat and cow) genes that can differentiate LAV vaccinated from naïve animals.Log2Expression inExpression in anFold Change inEntreza NaïveLAV vaccinatedExpressionSymbolGene IDDescriptionAnimalAnimalVaccinated vs NaïveLOC108634521108634521non-coding RNA0.66238.298.54NOS2100860742nitric oxide synthase 20.78103.47.03LOC108637251108637251multidrug resistance-associated protein 4-like1.28150.286.87TINAGL1102169636tubulointerstitial nephritis antigen like4.6290.014.27RETN102170965resistin44.98708.353.96C1QL2102176742complement C1q like 22.2930.23.69TDRD10102174259tudor domain containing 1002.493.53TGFB3102189962transforming growth factor beta 340.44367.33.19ADGRE2102171592adhesion G protein-coupled receptor E2101.97601.22.56LIPG102191574lipase G endothelial type33.18175.342.40KCNJ2102168940potassium voltage-gated channel subfamily J member 278.28378.072.27AQP9102181396aquaporin 972.94342.392.24BPI102185756bactericidal/permeability-increasing protein13.6749.851.85IL9102179848interleukin 913.724.82−1.54IL1R2102186601interleukin 1 receptor type 2170.8647.04−1.86IL36B102182235interleukin 36 beta80.8214.69−2.45IGF1100860838insulin like growth factor109.3619.9−2.45BGN102183219biglycan227.5226.44−3.10PIWIL2102173845piwi like RNA-mediated gene silencing57.327−3.10RAET1E108636743retinoic acid early transcript832.2348.24−4.11CRABP2102174348cellular retinoic acid binding protein 2103.463.31−4.91AOAH102189546acyloxyacyl hydrolase258.051014.31.98 TABLE 3List of host (goat and cow) genes that can differentiate LAV vaccinated from infected animals.Log2Expression inExpression in anFold Change inEntrezan InfectedLAV vaccinatedExpressionSymbolGene IDDescriptionAnimalAnimalInfected vs VaccinatedLOC106503226106503226non-coding RNA37.385.832.74PMP22102184371peripheral myelin protein 22268.9636.552.87ART5102169686ADP-ribosyltransferase 516.98103.7−2.62LOC102169116102169116ecto-ADP-ribosyltransferase 543.11198.79−2.20GNLY102191341granulysin32.21149.78−2.21ASAP3102182646ArfGAP with SH3 domain15.6270.26−2.18ankyrin repeat and PH domain 3LOC108633178108633178granzyme B-like6.650.4−2.98TBKBP1102172659TBK1 binding protein74.03499.27−2.76SLC17A7102169042solute carrier family 17 member 73.153.98−4.10FAM198B102191727family with sequence similarity 198 member B768.85311.611.30AOAH102189546acyloxyacyl hydrolase268.211014.3−1.92 TABLE 4AUse of host genes, for example, as measured by quantitative PCR, todifferentiate between naïve, infected, and vaccinated animalsRelativeRelativeexpressionexpressionStatus of the Animalof FAM198Bof AOAHNaïve11Infected6.891.10Vaccinated with LAV vaccine3.4910.47 TABLE 4BSummary of biomarkers for use in differentiationof infected and vaccinated animals.FAM198BAOAHMEI1IL-22CDCP1Infected with MAP vs.6.891.1−1.58−3.84−1.05Naïve animasVaccinated with3.4910.47−3.17−1.291.64LAV vaccine vs.Naïve Animals Applicants envision that the biomarker may include genes or the polynucleotides containing less than an entire gene sequence of the above genes. The biomarker of genes or the polynucleotides may be either single- or double-stranded nucleic acids. A polynucleotide may be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or combinations thereof. The polynucleotides can be purified free of other components, such as proteins, lipids and other polynucleotides. For example, the polynucleotide may be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% purified. The purified polynucleotides may comprise additional heterologous nucleotides. The purified polynucleotides of the invention can also comprise other nucleotide sequences, such as sequences coding for linkers, primer, signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands. The gene or the polynucleotides of the invention may also comprise fragments that encode immunogenic polypeptides. Polynucleotides of the invention may encode full-length polypeptides, polypeptide fragments, and variant or fusion polypeptides. Polynucleotides of the invention may comprise coding sequences for naturally occurring polypeptides or may encode altered sequences that do not occur in nature. If desired, polynucleotides may be cloned into an expression vector comprising expression control elements, including for example, origins of replication, promoters, enhancers, or other regulatory elements that drive expression of the polynucleotides of the invention in host cells. Detection of Biomarkers or Markers The present biomarkers or markers may be detected by any suitable method. In one embodiment, the testing is via ELISA assay for antibodies formed against the biomarkers or markers. The biomarker or marker in the present invention may be directly detected, e.g., by SELDI or MALDI-TOF. Alternatively, the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, or oligonucleotide, capable of specifically binding the biomarker. The ligand may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag. For example, detecting and/or quantifying may be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spectrometry (MS), reverse phase (RP) LC, size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques. Appropriate LC MS techniques may include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (e.g., high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin-layer chromatography, NMR (nuclear magnetic resonance) spectroscopy may also be used. Methods of diagnosing and/or monitoring according to the invention may comprise analyzing a plasma, serum or whole blood sample by a sandwich immunoassay to detect the presence or level of the biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment. Detecting and/or quantifying the biomarkers or markers may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the analyte biomarkers is performed using two antibodies which recognize different epitopes on a analyte biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay (e.g., using gold, silver, or latex particles, magnetic particles, or Q-dots). Immunological methods may be performed, for example, in microtiter plate or strip format. The gene or the polynucleotides of the invention may be detected by, for example, a probe or primer or a PCR primer. The gene or the polynucleotides of the invention may be the basis for designing a complimentary probe or primer, to detect the presence and/or quantity of biomarker in a subject, such as a biological sample. Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example, through hybridization. Primers are a subset of probes that can support specific enzymatic manipulation and that can hybridize with a target nucleic acid such that the enzymatic manipulation occurs. A primer may be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with the enzymatic manipulation. “Specific” means that a gene sequence recognizes or matches another gene of the invention with greater affinity than to other non-specific molecules. Preferably, “specifically binds” or “specific to” also means a gene sequence recognizes and matches a gene sequence comprised in a biomarker described herein, with greater affinity than to other non-specific molecules. The hybridization of nucleic acids is well understood in the art. Typically a primer may be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art. The ability of such primers to specifically hybridize to polynucleotide sequences of the recited biomarkers will enable them to be of use in detecting the presence of complementary sequences in a given subject. The primers of the invention may hybridize to complementary sequences in a subject such as a biological sample, including, without limitation, saliva, sputum, blood, plasma, serum, urine, feces, cerebrospinal fluid, amniotic fluid, wound exudate, or tissue of the subject. Polynucleotides from the sample can be, for example, subjected to gel electrophoresis or other size separation techniques or can be immobilized without size separation. The probes or the primers may also be labeled for the detection. Suitable labels, and methods for labeling primers are known in the art. For example, the label may include, without limitation, radioactive labels, biotin labels, fluorescent labels, chemiluminescent labels, bioluminescent labels, metal chelator labels and enzyme labels. The polynucleotides from the sample are contacted with the probes or primers under hybridization conditions of suitable stringencies. Preferably, the primer is fluorescently labeled. Also, the detection of the presence or quality of the gene sequence of interest can be accomplished by any method known in the art. For instance, the detection can be made by a DNA amplification reaction. In some embodiments, “amplification” of DNA denotes the use of polymerase chain reaction (PCR) to increase the concentration of a particular DNA sequence within a mixtures of DNA sequences. In some embodiments, quantitative polymerase chain reaction (qPCR) or real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure expression levels of nucleotide biomarkers. These methods detect and quantify the products generated during each cycle of the PCR process which are directly proportionate to the amount of the messenger RNA, DNA, or cDNA prior to the start of the PCR process. Some qPCR and RT-qPCR methods may use non-specific fluorescent dyes that intercalate with any double stranded DNA or sequence specific DNA probes with fluorescently labeled oligonucleotides to permit detection only after hybridization of the probe with the complementary sequence. Suitable RT-qPCR and qPCR methods, probes and dyes are known in the art. In another embodiment, the amplification of DNA may be done by the loop-mediated isothermal amplification (LAMP). Similar to PCR, LAMP utilizes a polymerization-based reaction to amplify DNA from examined samples, but the enzyme for LAMP, Bst DNA polymerase large fragment, possesses a DNA strand displacement activity. This makes the DNA extension step possible without having to fully denature DNA templates. Moreover, the primers are designed in a way that a hairpin loop structure is formed in the first cycle of amplification, and the following products are further amplified in an auto-cycling manner. Therefore, in about an hour, the repeated reactions can amplify by ˜109copies of DNA molecules and can be done at a constant temperature in a single heat block, instead of at various cycles of temperature in a relatively expensive thermal cycler. The detection of LAMP has been described in PCT patent application publication WO20141640055A1, which is incorporated herein in its entirety. In one embodiment, the detection of the presence of the gene or the specific binding between the gene inmycobacteriummutant and a gene that is not a component of a subject's immune response to a particular vaccine may indicate a natural or experimentalmycobacteriuminfection. For example, the absence of such binding or presence may indicate the absence ofmycobacteriuminfection. Or, a second, separate gene, such as a mutatedmycobacteriumgene that is specific to a component of a mammal's immune response to a particularmycobacteriumvaccine, may be used to detect corresponding antibodies produced in response to vaccination. Thus, if an antibody specific to a gene inmycobacteriumvaccine is detected, then the mammal has been vaccinated and/or infected. The detection of neither genes indicates no infection and no vaccination. As such, various combinations can lead to a determination of the vaccination and/or infection status of the mammal. Kits of the Present Invention In another aspect, the present invention discloses a diagnostic kit suitable for carrying out the diagnostic method of the first aspect of the invention. In one embodiment, the kit may be a “one-day” kit, meaning that it is capable of providing the diagnostic result within one day of sample collection. In another embodiment, the kit may be able to provide a diagnostic result within 12, 10, 8, 6, 4, 2, 1 or 0.5 hours of sample collection. In one embodiment, the diagnostic kit may be used for early detection of mycobacterial infection in a mammal. The diagnostic kit may also be used to differentiate a vaccinated mammal from an infected mammal. In one embodiment, the diagnostic kit may be portable. The portable diagnostic kit may specifically suitable for field testing. Applicants envision that the present diagnostic kit may be used in a farm field such as a milk farm, where farmers/veterinarians may collect samples and run the assay on the field (point of care assay) to identify early stages of Johne's disease infection and to differentiate infected from vaccinated mammals. The kit may include a substrate. In one embodiment, the substrate may be coated with biomolecules such as antibodies, which are specifically binding to the specific biomarkers as discussed above. The biomolecules may further possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag. In one embodiment of the present invention, the substrate may be used as a sample holder. Exemplary substrates may include microtiter strips or plates. In one specific embodiment, a sample such as a diluted serum may be pipetted into the wells of the microtiter plate or strip. A binding between the biomarkers in the serum and the biomolecules takes place. The presence or absence of the specific biomarkers or a combination of biomarkers as discussed above may indicate the infection status of the mammal. The kit may further include a means of detection. The means of detection may include any detection method as discussed above. In one embodiment, the means of detection may be a spectroscopic technique, such as UV-Vis or MS. In one specific embodiment, the means of detection may be ELISA. In one embodiment, the kit may include standard data for specific biomarker or a combination of biomarkers as discussed. One may compare the test result of a mammalian sample with the standard data for specific biomarker or a combination of biomarkers to determine the infection status of the mammal. For example, specific biomarkers or a combination of biomarkers may be visualized by a simple means of detection such as different colors. The detection result (e.g., showing one specific color) of a mammalian sample may be compared with the standard data (e.g., different colors for different biomarkers) to determine the infection status of the mammals. In one embodiment, the kit may also be in the form of reagents (e.g. protein extract) that can be inoculated into animals to estimate the level of cell-mediate immunity (e.g. single intradermal comparative skin test, SICST). The reagents may include any of the biomarkers as discussed above. In one embodiment, the reagents may also include any genetically engineered vaccines. Suitable genetically engineered vaccines may include those Applicants previously proposed in PCT patent application publication WO20141640055A1, which is incorporated herein in its entirety. The diagnostic kit may also include one or more of the following: instructions for use (detailing the method of the first aspect of the invention); sample collection apparatus (such as a needle and syringe); a chart for interpretation of the results; an electronic readout system; software providing a database for accurate data management. The present invention has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the invention. EXAMPLES Example 1—Proteomic Analysis ofM. aviumSubsp.ParatuberculosisVaccine Candidates Johne's disease (JD) is a worldwide health problem for dairy herds that carries a heavy economic burden for producing safe food. Infected cattle suffer from chronic diarrhea, weight loss, low milk yield and low, but persistent mortality (1). For the dairy industry alone, the economic losses caused by JD are estimated to range between $200-$500 million annually, in the USA alone (2, 3). Identifying protective vaccine candidates against JD could be the cornerstone of controlling this widespread infection. In our group, we deciphered genomic information available forM. apto identify key gene regulators that could control the expression of large number of genes. Throughout the genome ofM. apthere are 19 sigma factors that act as global gene regulators that could contribute to the ability ofM. apto grow in many environments (4). Through previous funding from USDA, we examined severalM. apsigma (σ) factors that were important for growth in murine macrophages. Using transcriptional profiling, we compared mid-log phaseM. aptoM. apthat had infected IFN-γ activated macrophages for 2 and 24 hours. Of the 19 sigma factors monitored, 6 sigma factor transcripts were up-regulated and one sigma factor transcript was down-regulated during the 24 hour time frame. Of the up-regulated transcripts, the sigL transcript was the only transcript up-regulated 2 hours after infection while sigH was up-regulated at 24 hrs (5). SigL is implicated in cell membrane protein biosynthesis as well as virulence inM. tuberculosis(6) while SigH was shown to be involved in combating the host intracellular responses such as oxidative stress (7). To assess the role of sigL and sigH inM. apvirulence, we replaced the target sigma factors gene coding regions with a hygromycin-resistant gene cassette inM. apK-10 using a specialized transduction protocol that was adapted forM. ap. Both genes were shown to be necessary forM. apvirulence in different stages of murine infection as detailed before (5, 8). Interestingly, the same mutants were shown to provide protective immunity against challenge with the virulent strain ofM. apwhen they were used as vaccine candidates in mice. To better analyze proteins expressed in each mutant, we grow cultures ofM. apΔIsigL,M. apΔsigH mutants and the wild type parent strain,M. apK10 to mid-log phase. All cultures were washed twice in PBS, resuspended in buffer cocktail with endonuclease before proteomic analysis using nano-Liquid Chromatography-Mass Spectroscopy-MS (nano-LC MS/MS) at the University of Wisconsin Biotechnology Center. From 3 biological replicates, a total of −900 proteins were identified in this analysis comparing sigL and sigH mutant toM. apK10 proteome. Diagnostic Markers for JD-Vaccinated Animals. A major problem in employing mass vaccination program for the control of JD in dairy herds is the inability to differentiate between infected and vaccinated animals with the current vaccine (DIVA principal). In addition, vaccinated animals could not be differentiated from positive reaction to the infection withM. bovis, a significant health problem for domesticated and wildlife animals. However, the DIVA principal and ability to distinguish betweenM. bovisand JD vaccinated animals could be achieved in genetically engineered vaccines (such as live attenuated vaccines based on sigL and sigH mutant) using a novel approach designed by the Applicant. In this approach, a simple blood test targeting proteins or sequences present inM. apwild type strain and with lower expression level in the vaccine strain or even not encoded in theM. bovisgenome would be developed. The target proteins include the following list of genes that could be used for the sigL-based vaccines. TABLE 5M. approteins that are significantly over-expressedin the wild type strain and not in the sigL-vaccineand could be used for sigL-DIVA testing.AccessionFold ChangeNumberNumber(K10/sigH)Name/Function1Q73SF41.75hypothetical protein2Q73Y732.66aldehyde dehydrogenase (NAD+)3Q73ZE62.13nucleotide-sugar epimerase EpiA4Q73SL72.69hypothetical protein Mb0574c5Q73VK61.14oxidoreductase6Q73XZ01.88antigen CFP27Q740D14.71peptide synthetase Nrp8Q73UE01.99cutinase TABLE 6M. approteins that are significantly over-expressedin the wild type strain and not in the sigH-vaccineand could be used for sigH-DIVA testing.AccessionFold ChangeNumberNumber(WT/sigH)Name/Function1Q73VL63.05diguanylate cyclase (GGDEF)domain-containing protein2Q73YW91.64PE family protein, partial3Q741L41.88hypothetical protein4Q744E52.67ABC transporter ATPase5Q73YP52.47Pup--protein ligase6Q73WE51.78arginine decarboxylase7Q73U211.88PE family protein PE178Q73UH92.16XRE family transcriptionalregulator9Q741M52.11nitroreductase10Q742F42.72metallo-beta-lactamase11Q73SU62.473-ketoacyl-ACP reductase In addition, another vaccine candidate is based on lipN mutant. In this case, epitopes that are different in theM. approtein compared to those inM. boviswill be the target for DIVA diagnostic test. FIG.1shows the alignment plot of amino acids deduced from the protein sequence in LipN of bothM. paratuberculosisandM. bovis. Peptides conserved inM. paratuberculosissequence but absent fromM. boviswould be the target for developing the DIVA test. Example 2—Biomarkers for Early Diagnosis and Differentiation of Mycobacterial Infections Johne's disease, caused byMycobacterium aviumsubspeciesparatuberculosis(MAP) is a chronic gastroenteritis of ruminants. Although infection often occurs within the first few months of life, clinical signs do not appear until 2-5 years of age. Current diagnostic tests, such as fecal culture and ELISA, have poor sensitivity for detection of the sub-clinical phase of disease. Therefore, biomarkers have been increasingly investigated as a method for sub-clinical detection. In this project, we set out to develop rapid assays (e.g. PCR or field skin test) for early detection of presence of Johne's disease and for the differentiation of Johne's disease vaccinated vs. infected animals (with MAP orM. bovis). To speed up the project outcome, we capitalized on ongoing vaccine study in goats (Capra hircus) and collected Peripheral blood mononuclear cells (PBMC's) for transcriptional profiling followed by gene prediction for disease initiation and progression. The PBMC's have been shown to be a predictor of infection and inflammatory disease. The PBMC transcriptomes of the goats were profiled using RNA-sequencing (RNA-Seq) to evaluate differential gene expression between a subset of samples from either 30 days post-vaccination, 30 days post-infection, or a naive, non-infected control group (3-4 biological replicates per group). Preliminary results on differential gene expression indicated the presence of 88 significantly differentially expressed genes out of 11,009 genes between goats at 30 days post-infection and the naïve, non-infected controls. The 30 days post-vaccination group had 720 out of 10,985 and 746 out of 11,099 significantly differentially expressed genes compared to the naïve, non-infected control group and the 30 days post-infection group, respectively. However, preliminary evaluation of the expressed genes indicated a large number of genes with immunological and inflammatory functions, including IL-18 binding protein, IFN-γ, IL-17A, and IL-22. Because of this inquiry, Table 7 summarizes selected genes/targets suitable to use in the present invention. TABLE 7List of DNA markers that are derived from the host transcriptome analysis and can be usedfor early diagnosis of Johne's disease in ruminants (cattle, goats, sheep and camels).Homolog inLocus in goatsSymbolProteinDescriptionBos taurus (cows)Homolog descriptionSelected list of host (goat and cow) markers that can differentiateinfected from naïve animals.NW_005125111.1: 0-184unplaced genomicN/AN/AscaffoldNW_005101181.1:unplaced genomicN/AN/A1703-1858scaffoldNW_005101181.1:LOC102180841XP_005701370.1PREDICTED: multidrugXP_005199610multidrug resistance-168292-168418resistance-associatedassociated protein 4-protein 4-likelike isoform X1NC_022320.1:Non-codingN/AN/A39973839-39974080regionNC_022297.1:IL-22XP_005680263.1interleukin 22NP_001091849.1interleukin 2244037534-44043184NC_022296.1:Non-codingN/AN/A81262820-81263390regionNW_005101844.1:ABCC4XP_005701761.1PREDICTED: multidrugXP_010820300.1PREDICTED: multidrug141791-142987resistance-associatedresistance-associatedprotein 4-like, partialprotein 4 isoform X1NW_005101711.1:LOC102185556XP_005701708.1PREDICTED: multidrugXP_003585348.3PREDICTED: multidrug48628-48757resistance-associatedresistance-associatedprotein 4-likeprotein 4 isoform X1NW_005132660.1: 0-240unplaced genomicN/AN/AscaffoldNW_005109943.1: 2-224unplaced genomicN/AN/AscaffoldNW_005149706.1: 0-366unplaced genomicN/AN/AscaffoldNW_005153011.1: 2-407unplaced genomicN/AN/AscaffoldNW_017189548.1:LOC108634521N/AncRNAN/AN/A2899 . . . 17746NC_030819.1:LOC108637251N/AN/AN/Amultidrug resistance-complementassociated protein 4-like(13836329 . . . 13914672)NC_030819.1:LOC108637252N/AN/AN/Amultidrug resistance-13926013 . . . 14000960associated protein 4-likeNW_017189646.1:LOC108634594N/AN/AN/Amultidrug resistance-complementassociated protein 4-like(5337 . . . 40350)NC_030824.1:FAM198BN/AN/AN/Afamily with sequencecomplementsimilarity 198 member B(30222726 . . . 30294233)NC_030822.1:LOC108637671N/AN/AN/Atripartite34764774 . . . 34772382motif-containingprotein 5-likeNC_030829.1:CDCP1102187276CUB domain containingXP_002697164;54084869 . . . 54144689proteinXP_612363NC_030812.1:TMTC1102185637transmembrane andN/A78337529 . . . 78655742tetratricopeptide repeatNC_030832.1:BAIAP2L1102173150BAI1 associatedXP_003584109;38530654 . . . 38607511protein 2 likeXM_003584061;XP_003587892;XM_003587844NC_030812.1:MEI1102169168meiotic double-strandedNP_001295589111693222 . . . 111748937break formation proteinNC_030818.1:SEPT10102171885septin 10NP_00103964143871666 . . . 43923217NC_030812.1:IFNG100860815interferon gammaNP_77651144984285 . . . 44988400NC_030830.1:IL17F102171111interleukin 17FNP_00117901124511444 . . . 24519042NC_030814.1:FCER2102171507Fc fragment of IgEN/A93891943 . . . 93902156receptor IINC_030825.1:ADGRG1102171366G protein-coupledNP_00107712526945415 . . . 26990225receptor GNC_030822.1:APBB1102179305amyloid beta precursorNP_001068654complementprotein binding family(35694810 . . . 35718058)B memberNC_030815.1:PIWIL2102173845piwi like RNA-mediatedXP_015320079;69261357 . . . 69324811gene silencingXM_015464593;XP_015328077;XM_015472591Selected list of host (goat and cow) markers that can differentiatelive attenuated vaccinated (LAV) from naïve animals.NC_022296.1:Non-codingN/AN/A32351255-32351413regionNC_022307.1:Non-codingN/AN/A44045143-44403012regionNC_022295.1:Non-codingN/AN/A13176472-13182094regionNC_022321.1:Non-codingN/AN/A6000551-6000875regionNW_005126018.1: 16-203unplaced genomicN/AN/AscaffoldNW_005101711.1:LOC102185556XP_005701708.1PREDICTED: multidrugXP_003585348.3PREDICTED: multidrug48628-48757resistance-associatedresistance-associatedprotein 4-likeprotein 4 isoform X1NW_005101844.1:ABCC4XP_005701761.1PREDICTED: multidrugXP_010820300.1PREDICTED: multidrug141790-142987resistance-associatedresistance-associatedprotein 4-like, partialprotein 4 isoform X1NW_005101645.1:unplaced genomicN/AN/A16151-23647scaffoldNW_017189548.1:LOC108634521108634521ncRNA2899 . . . 17746NC_030826.1:NOS2100860742nitric oxide synthase 2NP_001070267complement(19203362 . . . 19245850)NC_030819.1:LOC108637251108637251multidrug resistance-complementassociated protein 4-like(13836329 . . . 13914672)NC_030809.1:TINAGL1102169636tubulointerstitialXP_015315454;13806906 . . . 13817913nephritis antigen likeXP_015317919;XP_015315453;XM_015459967;XP_015317918;XM_015462432NW_017189666.1:RETN102170965resistinNP_899206complement(10281 . . . 11678)NC_030809.1:C1QL2102176742complement C1q like 2NP_00119276565236017 . . . 65240282NC_030810.1:TDRD10102174259tudor domain containing 10XP_005197751;104057279 . . . 104111513XM_005197694;XP_005203865;XM_005203808NC_030817.1:TGFB3102189962transforming growthNP_001094653;15809952 . . . 15835252factor beta 3XP_005212263;XP_005212264NC_030814.1:ADGRE2102171592adhesion G protein-coupled96350745 . . . 96400437receptor E2NC_030831.1:LIPG102191574lipase G endothelial type49397175 . . . 49422345NC_030826.1:KCNJ2102168940potassium voltage-gatedNP_776798complementchannel subfamily J member 2(59803404 . . . 59814242)NC_030817.1:AQP9102181396aquaporin 9XP_015328629;51022717 . . . 51074079XM_015473143;XP_015328630;XM_015473144NC_030820.1:BPI102185756bactericidal/permeability-NP_77632066719462 . . . 66768979increasing proteinNC_030814.1:IL9102179848interleukin 9XP_015319783;63194434 . . . 63197324XM_015464297;XP_015327708;XM_015472222NC_030818.1:IL1R2102186601interleukin 1 receptorNP_001039675;6611732 . . . 6648492type 2XP_010808117;XP_010808118NC_030818.1:IL36B102182235interleukin 36 betaXP_002691396;46359268 . . . 46368303XM_002691350;XP_002700827;XM_002700781NC_030812.1:IGF1100860838insulin like growth factorXP_005206547;complementXP_015326547;(64862983 . . . 64943172)XP_015326549NW_017190169.1:BGN102183219biglycanNP_847888;complementXP_005227715(56783 . . . 70690)NC_030815.1:PIWIL2102173845piwi like RNA-mediatedXP_015320079;69261357 . . . 69324811gene silencingXM_015464593;XP_015328077;XM_015472591NC_030816.1:RAET1E108636743retinoic acid early74364855 . . . 74372073transcriptNC_030810.1:CRABP2102174348cellular retinoic acidNP_001008670complementbinding protein 2(105983749 . . . 105989432)Selected list of host (goat and cow) markers that can differentiateinactivated-vaccine immunized from naïve animals.NW_005125111.1: 0-184unplaced genomicN/AN/AscaffoldNC_022320.1:Non-codingN/AN/A39973839-39974080regionNC_022303.1:Non-codingN/AN/A46207878-46237242regionNC_022296.1:Non-codingN/AN/A81262820-81263390regionNW_005101711.1:LOC102185556XP_005701708.1PREDICTED: multidrugXP_003585348.3PREDICTED: multidrug48628-48757resistance-associatedresistance-associatedprotein 4-likeprotein 4 isoform X1NW_005102056.1:LOC102190036XP_005701827.1PREDICTED: tyrosine-proteinNP_786982.1tyrosine-protein2049-9786phosphatase non-receptorphosphatase non-receptortype substrate 1-like, partialtype substrate 1precursorNC_022309.1:—XP_005691363.1PREDICTED: protein FAM198BNP_001077247.1protein FAM198B40520580-40588889NW_005101931.1:LOC102180487XP_005701808.1PREDICTED: interferonNP_001069925.2uncharacterized protein48185-54192alpha-inducible proteinLOC61742027-like protein 2-likeNW_005164924.1: 1-636unplaced genomicN/AN/AscaffoldSelected list of host (goat and cow) markers that can differentiateLAV-vaccine immunized from infected animals.NC_030826.1:LOC10650322610650322639149778 . . . 39151507NC_030826.1:PMP22102184371peripheral myelinNP_001094626;complementprotein 22XP_005220437;(32435859 . . . 32463764)XP_010814341NC_030822. 1:ART5102169686ADP-ribosyl-complementtransferase 5(31912716 . . . 31916296)NC_030822.1:LOC102169116102169116ecto-ADP-ribosyl-complementtransferase 5(31947901 . . . 31951735)NC_030818.1:GNLY102191341granulysinNP_001068611complement(48786756 . . . 48789216)NC_030809.1:ASAP3102182646ArfGAP with SH3 domainNP_001076915complementankyrin repeat and PH(6533315 . . . 6583406)domain 3NC_030828.1:LOC108633178108633178granzyme B-likecomplement(69374237 . . . 69377734)NC_030826.1:TBKBP1102172659TBK1 binding proteinXP_001253301;complementXP_005195770;(38379103 . . . 38396803)XP_005220704;XP_010814497;XP_010822429;XP_015314281NC_030825.1:SLC17A7102169042solute carrier family 17NP_001091515complementmember 7(56899764 . . . 56911150)NC_030826.1:LOC10863819210863819253112179 . . . 53116348NC_030812.1:IFNG100860815interferon gammaNP_77651144984285 . . . 44988400 Materials and Methods Animals—Approximately one week-old kids were purchased from a farm with no previous history of Johne's disease. All study kids, and their dams, tested negative forM. paratuberculosisby ELISA for serum antibody (Paracheck®, Biocor Animal Health, Omaha, NE). Additionally, fecal samples collected from the originating farm environment were negative forM. paratuberculosisby culture. All kids were housed in a restricted biosafety animal facility (BSL-2). All animal care was handled in accordance to the standards of the University of Wisconsin-Madison Animal Care and Use Committee. The kids were randomly assigned to one of four groups as shown in Table 8. One group of kids (n=6 but only 4 used for transcriptome analysis) were vaccinated with a live-attenuated vaccine (LAV) construct (M. paratuberculosisΔlipN mutant (Wu et al., 2007)) at a dose of 1×109CFU/animal. The second groups of kids (n=4) were vaccinated with the USDA-licensed inactivated vaccine (Mycopar®). A third group inoculated with PBS served as the vaccine control. Both vaccines and PBS were given subcutaneously. At 60 days post-vaccination, kids in these three groups were inoculated withM. paratuberculosisstrain JTC1285 at a dose of 1×108CFU administered orally in the milk replacer for three consecutive days. A fourth group (n=4), inoculated with PBS and not challenged withM. paratuberculosisserved as a naïve control. Goat kids were monitored daily for signs of clinical disease and evaluated monthly for potential weight loss. A detailed report on the outcome of this vaccine/challenge study was previously published (Shippy et al., 2017). TABLE 8Experimental GroupsVaccineGroupNo.Vaccine*DoseChallenge Strain/Dose**Infected4PBS0.5 mlM. paratuberculosisJTC1285/1 × 108CFULAV-4M. ap1 × 109M. paratuberculosisJTC1285/vaccinatedΔlipNCFU1× 108CFUMycopar-3Mycopar ®0.5 mlM. paratuberculosisJTC1285/vaccinated1 × 108CFUNaïve4PBS0.5 mlNoneControl*All vaccines were given subcutaneously.**Challenge dose was given orally in milk replacer for three consecutive days and was performed at 60 days post-vaccination in LAV- and Mycopar- vaccinated groups. Isolation of blood cells—Blood samples (10 ml) were collected from the jugular vein of goats into EDTA vacutainer tubes pre-vaccination, 1 week, 30 days, 60 days post-vaccination and 1 week post-challenge (for 3 groups), and then monthly for 12 months. Peripheral blood mononuclear cells (PBMC) were isolated using Histopaque®-1077 (Sigma-Aldrich®) with the following modifications. Anti-coagulated blood was diluted with an equal volume of RPMI-1640 medium (Sigma Aldrich®), layered over 10 ml of Histopaque®-1077, and centrifuged at 400×g for 30 minutes at room temperature. Following centrifugation, PBMC's were aspirated from the interface and washed twice with RPMI-1640 medium. Residual red blood cells were lysed with 0.83% NH4Cl2. The PBMC's were then resuspended in complete culture medium (RPMI-1640 containing 10% fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin (final concentration 100 IU/ml), and 1% nonessential amino acids). Cell density was determined by use of 0.4% Trypan blue stain and a hemocytometer. PBMC stimulation and RNA extraction—PBMC's were plated at a density of 1×106/well in 96 well plates with either medium alone (non-stimulated) orM. paratuberculosiswhole cell lysate (WCL). The WCL was prepared by resuspending the centrifuged cell pellet of actively grownM. paratuberculosis(O.D.˜1.0) in protein lysis buffer (100 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl2, 1 mM PMSF, complete ultra-protease inhibitor cocktail (Roche, Indianapolis, IN; pH 7.5) and bead-beating to homogenize (maximum pulse for 45 sec for a total of 4 pulses; with cooling on on ice for 30 sec between pulses). The supernatant was then transferred to a new 1.5 ml tube and non-soluble material was removed by centrifugation at 10,000×g for 5 min at 4° C. The protein content of the supernatant was measured via the Pierce™ BCA protein assay (Thermo Fisher Scientific), aliquoted and stored at −80° C. until used. Final concentrations of WCL was 10 μg/ml. IL-2 was added to all wells at a concentration of 100 U/ml. Plates were incubated at 37° C. with 5% CO2for 24 hours. Supernatants were then removed and cell pellets were stored in 100 μl TRIzol® and frozen at −80° C. until used for RNA extraction. RNA was extracted from stimulated PBMC's using TRIzol® and RNeasy® Mini Kit (Qiagen®) according to manufacturer's directions for the remainder of the extraction. TURBO DNA-Free™ DNase Treatment (Ambion®) was used to eliminate residual genomic DNA. RNA quantity and quality was assessed using the RNA Pico Series Chip on the Bioanalyzer 2100 (Agilent). RNA integrity numbers (RINs)>8 were obtained for all total RNA samples purified. RNA Sequence Analysis—RNA-Sequencing (RNA-Seq) was performed by the University of Wisconsin-Madison Biotechnology Center on RNA extracted from WCL-stimulated PBMC's from goats at 30 days post-vaccination, 30 days post-challenge (PBS vaccinated), or at the same time for the naïve control group (4 goats/biological replicates per group). A total of 1 μg of RNA was used as input for TruSeq® RNA Sample Prep Rev.F (March 2014; Illumina®). Paired-end RNA Sequencing was performed on the Illumina HiSeq 2000 sequencer according to manufacturer's instructions. Raw RNA-Seq reads were uploaded to CLC Genomics Workbench 8.5 (Qiagen, Redwood City, CA) for processing. Two read files from one RNA sample were paired and trimmed. The ambiguous trim limit was set at 1 and quality trim limit was at 0.05. Reads shorter than 25 nucleotides were excluded. The trimmed sequences were then mapped to the reference genome sequence ofCapra hircusassembly ARS1 (Bickhart et al., 2017) and read counts against the reference genome annotation tracks, generated with files, available at ncbi.nlm.nih.gov/genomes/Capra_hircus, were compiled and tabulated using the CLC Genomics Workbench NGS tools. The mapping parameters were set as follows: mismatch cost, 2; insertion and deletion cost, 3; length and similarity fraction, 0.8. Unique gene reads from each sample were exported from CLC Genomics Workbench and used for normalization and differential gene expression analysis with an R package, DESeq2 version 1.16.1 (Love et al., 2014). Transcripts that had an average of normalized read count<3 in all three tested groups were excluded from the analysis (N=11,541). Differentially expressed transcripts are defined as transcripts with fold changes≥2.0 or ≤−2.0 (or Loge-transformed fold changes≥1.0 or ≤−1.0), and p-value<0.05 when compared to the naïve control group. Gene ontology (GO) analysis was performed for the differentially expressed genes with agriGO, an automated tool to identify enriched GO terms, which is specially focused on agricultural species (Du et al., 2010). The gene products are categorized with respect to biological processes, cellular components, and molecular functions. Because the gene ontology in the goat genome is poorly annotated, we chose theBos taurusENSEMBL genome B2G list (2010 version) as the reference genome. Goat genes (assembly ARS1) with an Entrez gene name were mapped to the counterparts in the bovine genome, resulting in a total gene list of 9,115 GO-annotated genes. Goat DE genes identified in the RNA-Seq analysis were also mapped to the bovine genome and used as query lists against the 9,115-gene reference. FDR was calculated using the Fisher test. Network analysis was performed using the STRING database (Szklarczyk et al., 2015) with DE transcripts identified in this study. The input DE transcripts were treated as homologues ofBos taurusbecause of availability in the database. Quantitative RT-PCR—cDNA was synthesized from each RNA sample using SuperScript III Reverse Transcriptase (Invitrogen, Waltham, MA) and oligo(dT)12-18Primer according to manufacturer's instructions. Quantitative PCR (qPCR) assays were performed in triplicates for each cDNA sample. Primers were designed across adjacent exons in order to differentiate products from genomic DNA and cDNA. The GAPDH gene served as an internal control to normalize the data for the ΔΔCt relative quantitation method. The assays were performed on an Applied Biosystems StepOne Plus Real-Time PCR System (Foster City, CA), and the cDNA amplifications were monitored by the measurement of SYBR Green fluorescence at a specific cycle threshold. Each reaction was carried out in a 20 μl volume that contained 10 μl of 2× GoTaq qPCR Master Mix (Promega, Madison, WI), 5.0 μl of ddH2O, 0.5 μl of each primer (10 μm) and 4.0 μl of the template (100-150 ng/ul). The qPCR amplification process began with the temperature at 95° C. for 2 min, followed by 40 cycles of the amplification process (95° C. for 3 s, 60° C. for 30 s). Subsequent to the cycling process, melting curves were generated by inclining the temperature from 60° C. to 95° C. at 0.3° C./s increments. With the exception of the infected group at 1 month post-challenge where two samples were used, cDNA samples from three animals in each group were included in the qPCR analysis. Average ΔΔCt values and standard errors of the mean (SEM) of the three measurements were calculated and transformed to linear fold change. qRT-PCR primersSEQPrimerIDPrimer IDnamePrimer sequenceNO:AMT2341SEPT10_ Fggtgagcgccagaggaa4AMT2342SEPT10_Rcagcttctcctcttggtggac5AMT2343IL18BP_Faactggatcccagacccc6AMT2344IL18BP_Rgtagctgctgggagcgc7AMT2351IL17A_Fggaacacgaactccagaaggc8AMT2352IL17A_Racagagttcatgtgatggtccac9AMT2353CRABP2_Faccaccgtgcgtaccac10AMT2354CRABP2_Rggaggtcttgggaccctctc11AMT2355IL36_Fcgttaatagcagttccttctagcaac12AMT2356IL36_Rggatagccctggatttctgtgc13AMT2361RETN_Ftgaggcagtaaggaacattggc14AMT2362RETN_Ragtccatgcctgcgcac15AMT2363IFNG_Fgcagctctgagaaactggagg16AMT2364IFNG_Rtccggcctcgaaagagattct17AMT2365GAPDH_Fggcgtgaaccacgagaagtataa18AMT2366GAPDH_Rggcagtgatggcgtggac19AMT 2899ABCC4_Fcttggatcgccatacccctc20AMT 2900ABCC4_Rgggctccgggttgtagattc21AMT 2914IL 17F_Fgaggaccacattgtgagggt22AMT 2915IL 17F_Rcgggtgatgttgtaatcccag23AMT 2918TINAGL1_Fcgacgaggggttgtgtctg24AMT 2919TINAGL1_Racatagctattggggcagcg25AMT 2971FAM198B_Ftcatccaagatggccgcc26AMT 2972FAM198B_Rgccagcacttctgtttcagc27AMT 2973AOAH_Fgaaatcacggaggagtggca28AMT 2974AOAH_Raacagctgtgaaaccacctca29AMT 2988IL 22_Fcagggaatcaatcaggtgacga30AMT 2989IL 22_Ratgggggtggaattcatcgg31AMT 2992MEI1_Fcagtgaagtgctcgtctggt32AMT 2993MEI1_Rcgactcaatcccatacaccgt33AMT 2994CDCP1_Faagccaagcttccgctatca34AMT 2995CDCP1_Rcgatgacagtcaggtccgtg35 Results Transcriptome analysis of goat groups. The transcriptome analysis of goats infected withM. paratuberculosisand/or vaccinated LAV vaccine strainM. apΔlipN is a proportion of a larger study that examined the performance of this vaccine published earlier (Shippy et al., 2017). The transcriptome analysis is the focus of this report. The summary statistics of the RNA-Seq data for each replicate are shown in Table 9. Mean values of 58.88 million raw reads were generated per library (each RNA sample). Following trimming of reads based on read length, quality score and adapter sequences, an average of 20.04 million paired reads remained. Alignment of the trimmed RNA-Seq reads to theCapra hircusreference genome yielded mean values per library of 18.71 million paired reads (93.32%) mapped to unique locations. TABLE 9Summary statistics for Illumina RNA sequencing dataTotal% TotalNumber ofPairedPaired%TotalRead PairsReadsReadsUniquelyUniquelyGroup/ReplicateNumber ofBeingAfterAftermappedmappedNumber*ReadsTrimmedmappingTrimmingreadsreadsInfected 173,105,63422,810,29225,147,67168.823,533,31393.58Infected 272,155,10823,491,13224,331,98867.4422,701,37893.30Infected 368,005,38221,295,31623,355,03368.6921,941,42093.95Infected 448,108,72615,062,19416,523,26668.6915,433,81793.41LAV-vaccinated 173,973,05824,360,88224,806,08867.0723,253,48693.74LAV-vaccinated 263,076,12620,765,85021,155,13867.0819,668,16792.97LAV-vaccinated 334,967,37011,169,55811,898,90668.0611,062,09392.97LAV-vaccinated 466,996,26021,387,97222,804,14468.0821,282,72793.33Mycopar-60,074,72620,141,68219,966,52266.4718,707,05993.69vaccinated 1Mycopar-70,284,03623,645,79223,319,12266.3621,978,59694.25vaccinated 2Mycopar-64,746,83221,633,92021,556,45666.5920,141,99393.44vaccinated 3Naïve 173,575,07622,859,18225,357,94768.9323,681,55893.39Naïve 250,851,25016,107,62017,371,81568.3216,180,97793.14Naïve 346,913,01215,083,74615,914,63367.8514,815,44393.09Naïve 434,829,12411,209,87011,809,62767.8110,972,71292.91*Time when blood samples were taken: Infected: 30 days post-infection; LAV-vaccinated: 30 days post-LAV vaccination; Mycopar-vaccinated: 30 days post-Mycopar ® vaccination; Naïve:: 30 days post-PBS vaccination. Changes in the goat transcriptomes related to infection or vaccination—Transcriptomes of different animal groups were analyzed to identify differentially expressed (DE) genes with significant change using ap-value threshold of >0.05 and >2-fold change. A summary of comparative numbers of differentially expressed genes is presented in Table 10. MA-plots inFIGS.3A-3Cdepict the distributions of the DE transcripts PI and post-vaccination groups compared to naïve control group. Generally, the infected goat group had 226 significantly DE transcripts out of 17,380 (total goat transcripts identified by RNA-Seq) at 30 days PI in comparison to the naïve, non-infected controls. Of the 226 significantly DE transcripts, 113 were up-regulated in the PI group, while the other 113 were down-regulated. A total of 106 out of the 226 DE transcripts had more than a 2.8 fold change (or 1.5 loge fold change) with a selected group of known function listed in Table 11. On the other hand, the LAV-vaccinated goat group had 1018 significantly DE transcripts out of 17,380 compared to the naïve, non-infected control group. A total of 628 and 390 transcripts were up- and down-regulated, respectively. A total of 517 out of the 1018 had >2.8 fold change with a selected group of known function listed in Table 11. Additionally, when the transcripts of both LAV-vaccinated and infected groups were compared, at total of 1133 transcripts were significantly DE out of 17,380 (Table 10). Of these transcripts, 629 and 504 transcripts were up- and down-regulated, respectively. A total of 575 out of the 1133 DE transcripts were greater than a 2.8 fold change. Interestingly, the immunization with the inactivated, oil-based vaccine (Mycopar) triggered significant changes in a large number of goat genes (N=1714) including key genes involved in immune responses (Table 10). TABLE 10Differentially Expressed (DE) Genes for each comparison groupComparisonTotal analyzed GenesDE Genes*Infected vs Naïve17,380226LAV-vaccinated vs Naïve1018Mycopar-vaccinated vs Naïve1714LAV-vaccinated vs Infected1133*DE genes were identified as those with a p value threshold of ≤ 0.05 TABLE 11Selected differentially up- or down-regulated genes by fold change,between 30 days post-infection and naïve groupsGene symbolGene IDFold changeP valueDescriptionFAM198B10219172710.700.0016family with sequence similarity 198member BCDCP11021872764.060.0143CUB domain containing protein 1TMTC11021856373.630.0217transmembrane and tetratricopeptiderepeat containing 1BAIAP2L11021731503.610.0196BAI1 associated protein 2 like 1MEI11021691682.990.0155meiotic double-stranded breakformation protein 1SEPT101021718852.410.0239septin 10IFNG100860815−3.360.0047interferon, gammaIL17F102171111−2.330.0098interleukin 17FFCER2102171507−2.570.0001Fc fragment of IgE receptor IIADGRG1102171366−3.430.0037adhesion G protein-coupled receptorG1APBB1102179305−4.470.0002amyloid beta precursor proteinbinding family B member 1PIWIL2102173845−5.310.0400piwi like RNA-mediated genesilencing 2 TABLE 12Selected differentially up- or down-regulated genes by fold change,between 30 days post-LAV-vaccination and naïve groupsGene symbolGene IDFold changep-valueDescriptionNOS2100860742130.422.3E−09nitric oxide synthase 2TINAGL110216963619.311.2E−05tubulointerstitial nephritis antigen likeRETN10217674212.914.4E−13resistinC1QL210217674212.890.002complement C1q like 2TDRD1010217425911.540.019tudor domain containing 10TGFB31021899629.130.0020transforming growth factor beta 3ADGRE21021715925.900.0135adhesion G protein-coupled receptor E2LIPG1021915745.280.0001lipase G, endothelial typeKCNJ21021689404.820.0003potassium voltage-gated channel subfamily J member 2AQP91021813964.720.0007aquaporin 9BPI1021857563.610.0140bactericidal/permeability-increasing, proteinIL9102179848−2.910.0083interleukin 9IL1R2102186601−3.630.0055interleukin 1 receptor type 2IL36B102182235−5.460.0013interleukin 36 betaIGF1100860838−5.460.0463insulin, like, growth, factor, 1BGN102183219−8.570.0045biglycanPIWIL2102173845−8.570.009piwi like RNA-mediated gene silencing 2RAET1E108636743−17.270.0008retinoic acid early transcript 1ECRABP2102174348−30.122.0E−20cellular retinoic acid binding protein 2 TABLE 13Selected differentially up- or down-regulated genes by fold change, between 30days post-Mycopar ®-vaccination and naïve groupsGene symbolGene IDFold changep-valueDescriptionNOS2100860742269.2003.7E−11nitric oxide synthase 2BMP1010218557782.7460.0003bone morphogenetic protein 10TDRD1010217425918.4380.0061tudor domain containing 10RETN10217096516.9014.2E−12resistinAMOTL210216970814.3890.0065angiomotin like 2KLRG210217740712.7332.9E−10killer cell lectin like receptor G2IL211008612488.1244.6E−05interleukin 21C21021760857.952.4E−7complement C2C31008608266.4950.0002complement C3MCEMP11021723486.4367.4E−08mast cell expressed membraneprotein 1IL341021731155.4340.0084interleukin 34IL12A1008612933.9070.0035interleukin 12ATLR41008609553.4233.8E−07toll like receptor 4TNF1008612323.3990.0003tumor necrosis factorIL18100861190−4.4413.6E−06interleukin 18IL9102179848−4.8020.0012interleukin 9IL9R102191479−4.9619.4E−08interleukin 9 receptorIL5102188034−4.9640.0396interleukin 5IL36B102182235−9.5570.0001interleukin 36 betaIL13102187477−9.6753.4E−07interleukin 13PIWIL2102173845−22.1520.0009piwi like RNA-mediated genesilencing 2IL11102184367−46.8231.6E−07interleukin 11 Several genes involved in immune responses were significantly regulated in all goat groups. For example, leukemia inhibitory factor (LIF), interferon-gamma (IFN-γ), and interleukin 22 (IL-22), were found to be DE genes in the infected group when compared to both the control and the LAV-vaccinated groups. More gene lists are provided in the Tables included in Appendices A-G. In the infected group, LIF was down-regulated by −2.51 fold change when compared to the control group and by 3.84 fold when compared to the vaccinated group. IL-22, a Th17-related cytokine, was also down-regulated by a −5.78 fold in the infected group vs the control group and by −33.82 fold when compared to the LAV-vaccinated group. Interestingly, NOS2 gene involved in controlling infection of a closely related mycobacteria,M. tuberculosis(Kutsch et al., 1999; Velez et al., 2009), was significantly induced (>100 fold) in both vaccine groups, suggesting an important role of this gene in adaptive immune responses following immunization with LAV (Table 12) or inactivated (Table 13) vaccine. A group of genes with unique diphasic regulatory responses in both LAV and infected goats included immune response genes (e.g. IFN-γ, Granulysin) as well as basic cell metabolic process (e.g. ART5). This list of genes (Table 14) could expand gene categories utilized as targets for developing a sensitive assay to differentiate infected from vaccinated animals (DIVA). TABLE 14Common differentially expressed genes regulated in opposite directionbetween 30 days post-infection and 30 days post-LAV-vaccinatedgroups, each compared to the naïve groupFoldFoldchange inchange inGene symbolGene IDInfected groupVaccinated groupDescriptionLOC1065032261065032262.62−2.53non-coding RNAPMP221021843712.11−3.46peripheral myelin protein 22ART5102169686−2.013.05ADP-ribosyltransferase 5LOC102169116102169116−2.032.27ecto-ADP-ribosyltransferase5GNLY102191341−2.132.19granulysinASAP3102182646−2.162.10ArfGAP with SH3 domainankyrin repeat and PHdomain 3LOC108633178108633178−2.682.95granzyme B-likeTBKBP1102172659−3.032.23TBK1 binding proteintranscriptSLC17A7102169042−3.125.50solute carrier family 17member 7LOC108638192108638192−3.275.28non-coding RNAIFNG100860815−3.363.89interferon gamma Among those identified DE transcripts in the infected and LAV-vaccinated groups (each referenced against the naïve group), there were 68 transcripts in common (FIG.4A). The majority of those transcripts were regulated in the same direction in both groups, but 11 transcripts were regulated in the opposite direction. A non-coding RNA transcript, LOC106503226 and a gene, PMP22, were the only two that were up-regulated 30 days PI and down-regulated 30 days post-vaccination. The remaining 9 transcripts (e.g. ART5 and IFNG) were down-regulated 30 days PI and up-regulated 30 days post-vaccination (Table 14). More comparative analysis of transcript profiles identified 76 transcripts commonly up- or down-regulated shared between the lists of genes from comparing infected vs. naïve control andM. paratuberculosis-infected vs. LAV-vaccinated transcripts (FIG.4B). Those common genes could be considered the core responsive genes forM. paratuberculosisinfection or vaccination with an LAV vaccine. For the inactivated vaccine, a total of 667 core genes were also regulated when compared to the LAV-vaccine group (Appendix F). Such core genes included those with potential rules in immunity (e.g. NOS2, RETN and IL21), another indication of core genes responsive to anyM. paratuberculosis-specific vaccines whether live-attenuated or inactivated were used. Pathways and networks of differentially expressed genes—To better define gene pathways involved inM. paratuberculosisinfection, genes with significant differential expression were evaluated through gene ontology (GO) analysis using agriGO. This analysis provides categories of genes involved in different biological or molecular functions and those integral for different cellular components. Interestingly, the most abundant significant terms for the GO analysis for the infected vs naïve control group included genes involved in protein binding, regulation of cellular process and response to stimulus, which includes significant subcategories immune responses (GO:0006955) and inflammatory response (GO:0006954) (FIG.3), suggesting the importance of controlling immune genes byM. paratuberculosisfollowing infection. On the other hand, the largest gene groups with significant GO terms for the Mycopar®- or LAV-vaccinated vs infected groups included genes involved in binding, cellular process and metabolic process while those for the LAV-vaccinated vs infected group included genes involved in cellular process and biological regulation (FIGS.8A-8C). To better characterize gene networks activated during infection and vaccination, gene transcripts were further analyzed to identify co-regulated genes.FIGS.6A-6Bdisplay gene network analysis in the post-infection group. Several in the up-regulated group of genes (FIG.6A), such as ACER3, SYNJ2, CORO6 and PLS1, showed physical associations and co-expression among transcripts of theM. paratuberculosis-infected group. In addition, homologs of PDE4C and TSKU were also found associated (Halls and Cooper, 2010; Schlecht et al., 2012; Costanzo et al., 2016) and suggested to be involved in signaling and relaxin regulation (Halls and Cooper, 2010). InFIG.6B, a co-down-regulation of ATP7B, ATP12A and ATP2B3 suggests a possible reduced activity of calcium transport in infected cells. This analysis also highlighted the negative regulation byM. paratuberculosisof host cytokines such as IFN-γ, IL-13, IL-17A, IL-17F and IL-22. Prolonged Changes of Key Host Genes. To further analyze the utility of transcriptome analysis for prediction of unique transcripts associated with infection or vaccination, we used real-time, quantitative PCR to compare transcript levels among animal groups over 12 months post-challenge (MPC) (FIGS.7A-7D). Interestingly, IL-17 cytokine was repressed in the challenged and Mycopar® and LAV-vaccinated goats compared to the naïve control group for all examined times, except for the infected group at 2 MPC. Similarly, the Sept10 gene was induced, only at 2 MPC. On the other hand, IL-36 was activated soon after vaccination (1 and 2 MPC) but then repressed for the rest of the examined time points, i.e. 6 and 12 MPC. More interestingly, the IFN-γ expression profile was refractive to elicited immune responses. IFN-γ was induced soon in the LAV-vaccine group (1 MPC) but then continued to be expressed in the Mycopar®-vaccinated andM. paratuberculosis-challenged groups starting from 2 MPC until the end of the experiment. At all of these sampling times, the IFN-γ was consistently higher in the LAV-vaccine group compared to the challenged group. Differential Expression in LAV vaccinated animals, Mycopar™ vaccinated animals, and infected animals. TABLE 15List of host genes (goat and cow) differentially expressed in both LAVand Mycopar vaccinated animals compared to infected animalsMycoparLAV vs. Infectedvs InfectedFoldGeneFoldFoldchangesymbolGene IDchangep-valuechangep-valuedifferenceDescriptionSTC1102179386−4.6681.4E−07−8.9375.6E−174.268stanniocalcin 1NGF100862660−1.7782.8E−02−5.0011.2E−043.223nerve growthfactorFAM150B102183516−1.4064.8E−02−4.3552.0E−052.949family withsequencesimilarity 150member BFOXE1106502413−1.6791.4E−02−4.2904.5E−072.611forkhead box E1C28H10orf71102181364−2.8705.2E−03−5.4631.3E−042.593chromosome 28C10orf71homologHEBP2102174123−1.5844.2E−02−4.1331.9E−032.549heme bindingprotein 2IL11102184367−2.2188.9E−03−4.6591.1E−052.440interleukin 11KRT82102183763−1.6305.6E−03−3.9514.0E−042.320keratin 82NTNG1102190191−1.7048.1E−06−3.7267.9E−162.022netrin GSORCS2102176511−1.3574.9E−03−3.3655.8E−092.008sortilin relatedVPS10 domaincontainingreceptor 2HS3ST2102183286−3.6381.0E−07−1.4323.4E−02−2.206heparan sulfate-glucosamine 3-sulfotransferase 2TGFB31021899622.7068.5E−035.1034.2E−06−2.397transforminggrowth factorbeta 3 TABLE 16List of host genes (goat and cow) differentially expressed in LAVvaccinated animals compared to infected animals but not differentially expressed in Mycopar ®vaccinated animals compared to infected animals.LAVMycoparvs. Infectedvs InfectedGeneFoldFoldsymbolGene IDchangep-valuechangep-valueDescriptionLOC1021764391021764393.8650.0030.7630.603misc_RNALOC1021871301021871303.3410.002−0.3000.800protein ARMCX6-likeLOC1086331781086331782.9760.0000.8950.129granzyme B-likeCPNE61021805002.7550.0000.8410.289copine 6IL131021874772.7520.000−0.2200.752interleukin 13CCR101021840012.5810.0070.7000.502C-C motif chemokine receptor10C1QL21021767422.4270.032−0.5200.684complement C1q like 2MGAT31021854452.4180.005−0.5700.553mannosyl (beta-4-)-glycoprotein beta-4-N-acetylglucosaminyltransferaseGNLY1021913412.2120.0000.8070.146granulysin transcriptKY102169426−3.0130.003−0.1440.884kyphoscoliosis peptidaseRAET1E108636743−3.7840.002−0.8950.497retinoic acid early transcript1E TABLE 17List of host genes (goats and cow) differentially expressed in Mycopar ®vaccinated animals compared to infected animals but not differentially expressed in LAVvaccinated animals compared to infected animals.LAV vs.MycoparInfectedvs InfectedGeneFoldFoldsymbolGene IDchangep-valuechangep-valueDescriptionLOC1021748951021748951.7620.1415.2780.000vascular cell adhesion proteinBMP101021855770.5570.7214.1920.012bone morphogenetic protein10CXCL12102169556−0.7550.3023.6130.000C-X-C motif chemokineligand 12LOC108633303108633303−0.2720.7692.6590.008platelet glycoprotein 4-likePPARG1008613090.9040.0532.3390.000peroxisome proliferatoractivated receptor gammaF13A1102169238−0.1320.8832.2390.021coagulation factor XIII AchainLOC108634012108634012−0.1730.804−2.7360.000homeobox protein MSX-3-likeLRRC31021889020.3570.786−2.9070.042leucine rich repeat containing 3MYO10102175716−0.9670.023−3.1830.000myosin XLOC102179419102179419−0.4710.759−3.4350.040myeloid-associateddifferentiation marker Discussion Infection withM. paratuberculosisis costing the dairy industry significant economic losses (Cho et al., 2012) and is difficult to detect its presence, especially during early disease stages (Li et al., 2017). In this project, the goat PBMC transcriptome was profiled using RNA-Sequencing (RNA-Seq) to compare the early gene expression, 30 days post-infection and post-vaccination, compared to healthy, naïve controls. In addition to better understanding of disease progression, such analysis is expected to yield targets for further development into a diagnostic assay for early stages of Johne's disease. Many transcriptomic analyzing tools largely depend on information from an annotated genome. In this study, our quality of transcriptomic analyses improved as the goat genome assembly was significantly refined (Bickhart et al., 2017). According to NCBICapra hircusAnnotation Release 102, of 20,593 predicted coding genes, 20,256 had a protein aligned 50% or more of the query against the UniProtKB/Swiss-Prot curated proteins (NCBI, 2016). The updated annotation thus provides a much more reliable reference to our analysis. The generated RNA-Seq dataset could also benefit further improvement of goat genome annotation. As expected, a large number of differentially expressed (DE) transcripts were found between the vaccinated and infected groups (1133 genes) and between the vaccinated and naïve control group (1018 genes). In contrast, there was a relatively small number (226) of DE transcripts when comparing the infected and naïve control group. This large difference in the number of DE transcripts is most likely associated with the route of administration since both vaccines were administered subcutaneously (contrary to oral infection), allowing for increased contact with PBMCs in the bloodstream, while challenge dose ofM. paratuberculosiscould reach PBMC following intestinal invasion (Stabel et al., 2009). Our analysis, further illustrated the importance of route of infection and/or vaccination for the type and magnitude of the generated host responses. Although the comparison between the infected and naïve control group produced a relatively small number of DE transcripts, preliminary evaluation of these genes indicated a large number of genes with immunological and inflammatory functions, including interferon gamma (IFN-γ), IL-18 binding protein, IL-17A, and IL-22. IFN-γ is an important player in the defense against intracellular pathogens including mycobacteria (Arsenault et al., 2012). A previous study in cattle showed that in the subclinical stages of infection, IFN-γ expression increased at the site of infection (Sweeney et al., 1998). Other studies indicate thatM. paratuberculosis-infected animals produce IFN-γ but are unresponsive to it (Arsenault et al., 2012). In that study, IFN-γ was secreted significantly less (−3.36 fold change) in subclinically infected goats compared with the naïve, control goats. This IFN-γ profile was also evident in subclinically infected goats vs vaccinated goats (−13.0 fold change). Previously, IFN-γ was reported to be induced in PBMC's stimulated withM. paratuberculosiswhole-cell sonicate from subclinically infected cows (Stabel, 2000). However, these cows ranged from 2-10 years of age and therefore were much further along in the infection pathogenesis than in the current study, which tested goats 30 days PI. The host response clearly changes over time and this data may demonstrate that. Potentially linked to the identified repression of IFN-γ, is the moderate up-regulation (+1.30 fold change) of interleukin 18 binding protein (IL-18 bp) in the infected vs naïve control group. IL-18 bp binds to IL-18 to block its biological activity (Novick et al., 1999). IL-18 is a pro-inflammatory cytokine that functions in the early Th1 cytokine response and induces IFN-γ production. A major source of IL-18 bp is from intestinal endothelial cells and macrophages (Corbaz et al., 2002). Therefore, IL-18 bp serves to modulate the early Th1 immune response in the intestine, the site ofM. paratuberculosisinfection. Interestingly, IL-18 bp has been found to be up-regulated during active Crohn's disease, an inflammatory bowel disease in humans with potential association toM. paratuberculosisinfection (Corbaz et al., 2002). As expected, genes involved in immune responses (e.g. LIF, IFN-γ and IL-22), were found to be DE among examined goat groups. LIF is a pleiotropic cytokine belonging to the IL-6 cytokine family with receptors primarily on monocytes/macrophages (Nicola and Babon, 2015). In the infected group, both LIF and IL-22, a Th17-related cytokine, were down-regulated in the infected group vs the control or the vaccinated groups. These three genes, along with IL-13 and IL-17, were also found having associations in the protein network analysis. IL-17 was also down-regulated in the infected vs control group. Down-regulation of IFN-γ, IL-22 and IL-17 genes may suggest overall down-regulation of Th1 and Th17 cell activities and reduced cellular immunity against infections. Several studies inMycobacterium tuberculosisandMycobacterium bovishave shown significant IL-17 responses (Blanco et al., 2011; Jurado et al., 2012). A recent study on RNA-Seq analysis in cattle infected withM. bovisshowed an up-regulation of IL-17, IL-22, and IFN-γ at one-month PI (Waters et al., 2015). This is in contrast to some of our findings in the present study (in case of IL-17) which was further confirmed by prolonged analysis of key genes up to 12 months post infection (FIGS.7A-7D). Such difference could be attributed to the difference in host response toM. bovisvs.M. paratuberculosis. Further investigation into these key immune regulated genes as will aid in understanding how the host is dynamically responding toM. paratuberculosisinfection or vaccination. Our gene network analysis also shows associations among genes that were up-regulated in the infected group (FIG.7A). Interestingly, homologs of ACER3, SYNJ2, CORO6 and PLS1 in animal species other than goats (mainly bovine,Bos Taurus) were also shown to have physical associations (Schlecht et al., 2012; Hein et al., 2015) and co-expression (Clancy et al., 2003; Janji et al., 2010) as well. Particularly, homologs of CORO6, an actin binding protein, was suggested to be involved in cytokinesis. InM. tuberculosis-infected macrophages, CORO6 homolog coronin-la was suggested to inhibit auto-phagosome formation and facilitateM. tuberculosissurvival (Seto et al., 2012). In addition, homologs of PDE4C and TSKU were also found associated (Halls and Cooper, 2010; Schlecht et al., 2012; Costanzo et al., 2016) and suggested to be involved in signaling and relaxin regulation (Halls and Cooper, 2010). It may thus imply a status of progression of anM. paratuberculosisinfection in hosts as observed inM. tuberculosisinfection (Seto et al., 2012). This observation, along with the likely reduced cellular immunity discussed above, is consistent with the infection status of the host. It is unclear, however, how bacterial or host factors regulate the expression of those genes. Understanding the host-pathogen interaction early in infection will allow for the identification of genes upregulated during initial infection. A useful biomarker for infection must be specific, detectable over the course of the disease with varying inoculation doses, and easily measurable. Moreover, it would improve interpretation of early disease detection if the biomarkers could differentiate infected and vaccinated animals. In our analyses, we identified 9 transcripts (out of 11 in Table 14) that were down-regulated 30 days PI and up-regulated 30 days post-vaccination. This biphasic regulation of those genes or transcripts might make them specific markers for differentiating vaccinated animals that are healthy or those infected withM. paratuberculosis. The RNA-Seq analysis was performed only on samples taken one month post-infection or post-vaccination to identify early gene regulations in tested groups, notably, between one month after vaccinated only and infected only groups. This comparison differentiates host gene regulating responses after exposure to vaccine strains or virulent strains ofM. paratuberculosis. The vaccinated animals were then challenged two months after the vaccination and several key gene expressions were profiled with quantitative PCR (FIGS.7A-7D). The temporal expression patterns within the tested one year period could reflect unique characteristics of host responses after exposure to virulentM. paratuberculosiswith or without prior vaccinations and could also benefit development of diagnostics. For example, IL-17 expressions in the vaccinated animals remained highly repressed at all time while peaking at 2 month post-challenge in the infected only group. | 92,638 |
11860169 | DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Applicants have assessed whether circulating OPN levels have the same effect with regards to Gi-mediated response and risk of developing scoliosis among the three functional groups and have undertaken a retrospective study with IS subjects to determine whether patient bracing outcome could be differentiated based on their Gi functional status (FG1, FG2 and FG3). The present invention is based on the findings that i) OPN has a differential (opposite) effect on the response to Gi stimulation among IS functional groups (it decreases the Gi-mediated response in control, FG2 and FG3 subjects while it increases the Gi-mediated cellular response in FG1 subjects); ii) inhibition of the expression or activity of CD44 (a receptor for OPN) potentiates the effect of OPN; iii) Hyaluronic acid (HA) (which binds to CD44 receptor with higher affinity than OPN) also has a differential effect on Gi-mediated cellular response (it decreases the Gi-mediated response in control, FG2 and FG3 subjects while it increases the Gi-mediated cellular response in FG1 subjects; iv) inhibition of the expression or activity of integrins (which bind OPN) reduce the effect of OPN on the Gi-mediated cellular response in FG2 and FG3 subjects; v) high circulating OPN level in FG1 subjects has a protective effect while it is a risk factor in FG2 and FG3 subjects; vi) brace treatment outcome is most favorable in FG2 and FG3 subjects (mainly in FG3); vii) bracing is less effective in FG1 subjects with significant increased likelihood to progress over 45° and to have surgery than the other 2 groups; and viii) bracing generally decreases circulating OPN levels in all AIS subjects. Taken together, these results enable to improve IS treatment, to more accurately predict brace treatment outcome and to select the most appropriate treatment method and follow up schedule for each patient according to their biological endophenotype (FG1, FG2 and FG3) and/or circulating OPN level. IS patient bracing outcome was evaluated in regard to curve progression leading up to surgery between the 3 functional groups (FG1, FG2 and FG3). Each patient had been previously classified in one of the 3 functional groups (FG1, FG2 or FG3) using a cell-based assay measuring cAMP (Moreau et al., 2004) variation and/or CDS response (Akoume et al., 2010) following Gi stimulation. Outcome of brace treatment in terms of curve progression over 45° and occurrence of corrective surgery was determined for each functional group. It was found that bracing is less effective in FG1, with an increased likelihood to progress over 45° and to have surgery than the other 2 groups. Outcomes of bracing were most favorable for patients presenting the FG3 endophenotype. Applicants have determined that subjects classified in the FG1 functional group are less likely to benefit from bracing. Furthermore, FG1 subjects having high level of OPN (e.g., above 1000 ng/ml) are less likely to progress than FG1 subjects having low levels of OPN (≤500 ng/ml). Results suggest that in FG1 subjects, when the level of OPN decreases around 500 ng/ml or below, scoliosis tend to progress (i.e., increase in Cobb's angle). These results are consistent with applicant's findings that in the FG1 functional group, OPN reduces the Gi-mediated signaling defect (i.e., increases Gi-mediated cell signaling) generally present in scoliosis subjects. Applicants have also determined that brace treatment generally decreases OPN levels by way of a retroinhibition mechanism and that effect of brace treatment may further be distinguished based on initial circulating OPN levels prior to beginning of treatment. Indeed, it was found that in certain subjects having initial low level of circulating OPN, brace treatment first induces a sharp rise in OPN levels (within the first 6 months) while it induces a sharp decrease in OPN levels in subjects having high initial OPN levels, thereby supporting a retroinhibition mechanism controlling circulating OPN levels in vivo. Furthermore, Applicants have found that high circulating OPN levels have a protective effect on patients of functional group FG1 and have a detrimental effect (i.e., increasing their risk of developing a scoliosis) in subjects classified into the FG2 and FG3 functional groups. Accordingly, FG1 subjects (especially having a high level of circulating OPN) should generally not be prescribed brace treatment even if very short. Subjects of the FG2 and FG3 functional groups are more likely to benefit from brace treatment (e.g., long term brace treatment) possibly because it generally decreases OPN levels and an elevated OPN level is a risk factor for these subjects. Hence, further combining endophenotype classification with OPN circulating levels allows to further distinguish among functional groups which subjects should be treated with a brace, which subjects should have their level or activity of OPN lowered (e.g., FG2 and FG3 subjects), which subject should have their level or activity of OPN increased (FG1 subjects), which subjects should have their level of CD44 (e.g., sCD44) increased (FG2 and FG3); which subjects should have their level of CD44 (e.g., sCD44) decreased (e.g., FG1); which subjects should have their level of HA increased (FG1); which subjects should have their level of HA decreased (FG2 and FG3); which subjects should have their level or activity of integrins (e.g., α5, β1, β3and β5) decreased (FG2 and FG3); as well as the optimal duration of treatment. Other treatment regimens known to have an effect on OPN, HA, CD44 or integrins level or activity may also be adapted according to each specific functional group (e.g., specific exercises or massages (e.g., application of compressive pressure for 15 to 90 minutes—See for example U.S. Ser. No. 13/822,982, and low intensity pulsed ultrasounds (LIPUS), for FG1 patients, because such approaches can increase OPN expression level (e.g., OPN plasma level)), acupoint heat sensitive moxibustion or heat therapy with pad, thermal bath, electroacupuncture (for FG2 and FG3 subjects because such approaches are known to decrease OPN levels in serum of subjects). These findings enable personalized treatment prescription according to each patient Gi-endophenotype and/or OPN level, early on following diagnosis thereby avoiding unnecessary delay in finding best treatment options which will ultimately improve IS treatment outcome. Accordingly, the present invention provides a method of predicting brace treatment outcome in a subject in need thereof comprising; i) classifying the subject into functional group FG1, FG2 or FG3, wherein the classification enables the prediction of brace treatment outcome. Specifically, according to the above method, classification of the subject into the FG1 functional group is indicative that the subject: i) is less likely to benefit from brace treatment (e.g., is less likely to have brace treatment success); ii) is more likely to require surgery; iii) is more likely to show a curve progression >6° in Cobb's angle; iv) is less likely to have a Cobb angle ≤ to 45°; and v) is more likely to aggravate his/her condition (e.g., increase speed of curve progression or increased final Cobb angle) by brace treatment as compared to FG2 and FG3 functional groups. According to the above brace treatment outcome prediction method, classification of the subject into the FG3 functional group is indicative that the subject: i) is more likely to benefit from brace treatment (e.g., is more likely to have brace treatment success); ii) is less likely to require surgery; iii) is less likely to show a curve progression >6° in Cobb's angle; iv) is more likely to have a Cobb angle to 45°; and v) that the subject is less likely (or unlikely) to aggravate his/her condition (e.g., increase speed of curve progression or increased final Cobb angle) by brace treatment as compared to FG1 and FG2 functional groups. Finally, classification of the subject into the FG2 functional group according to the above brace treatment outcome prediction method is indicative that the subject: i) has moderate chances of benefiting from brace treatment (e.g., the subject has moderate chances of brace treatment success); ii) has moderate risk of requiring surgery; iii) has moderate risk to show a curve progression >6° in Cobb's angle; iv) has moderate risk of having a Cobb angle ≤ to 45°; and v) has low risk of aggravating his/her condition (e.g., increase speed of curve progression or increased final Cobb angle) by brace treatment as compared to FG1 and FG3 functional groups. Under certain circumstances, certain rare FG1 subjects could nevertheless benefit from a short brace treatment if, in such patients bracing increases OPN level. It was found that subjects in each functional group may further be distinguished based on their level of circulating OPN (low or high level of OPN). In order to further distinguish among each groups which subjects could benefit from brace treatment, the present prediction method can advantageously further comprise measuring the level of circulating OPN prior to the beginning of brace treatment. According to this method, certain subjects classified into the FG1 functional group and having a low level of circulating OPN (e.g., below 500 ng/ml) may benefit from a short brace treatment (e.g., 6 months or less) and are less likely to aggravate their condition by short treatment than FG1 subjects having high levels of circulating OPN because brace treatment can induce an increase in circulating OPN in these subjects at the beginning of treatment and OPN has a protective effect in these subjects. The short brace treatment may be for 18 months or less, preferably 12 months or less and more preferably, 6 months or less or until OPN concentration is at its maximal concentration or close to its maximal concentration (i.e., below the retroinhibition concentration). It should be noted that if an FG1 subject is treated with a brace, his/her OPN level should be monitored closely in order to detect any drop in circulating OPN. Preferably, brace treatment would be pursued only if and while bracing induces an increase in OPN level. If a drop in circulating OPN level is detected, then brace treatment should be stopped. According to the above method, subjects classified into the FG2 or FG3 functional group and having a high level of circulating OPN may more rapidly benefit from brace treatment than FG2 or FG3 subjects having low levels of circulating OPN because OPN is a risk factor in these subjects and brace treatment reduces the level of circulating OPN in these subjects. In subjects of the FG2 and FG3 functional groups having a low level of circulating OPN, brace treatment is nevertheless beneficial but is preferably maintained for a sufficient time so that the level of OPN level is decreased (e.g., 12-18 months and preferably more than 18 months). In a related aspect, the present invention also encompasses selecting the most efficient and least invasive known preventive action, treatment or follow-up schedule in view of the determined classification and concentration of circulating OPN level. Accordingly, the present invention provides a method of treating or preventing IS in a subject comprising, classifying the subject into functional group FG1, FG2 or FG3, wherein when the subject is classified into the FG1 functional group: i) the subject is treated with OPN; ii) the subject is treated with an OPN agonist (e.g., HA supplements or treatment or preventive measures which increase HA level such as a HA rich diet); iii) the subject is treated with a CD44 antagonist (e.g., an antibody against CD44); iv) the subject is treated with an integrin agonist (or the subject is prescribed treatment or preventive measures which increase integrin level or activity); iv) the subject is prescribed treatment or preventive measures which increase circulating OPN levels (e.g., massages such as by compressive pressure as described in U.S. Ser. No. 13/822,982; low intensity pulsed ultrasound (LIPUS), etc.); v) the subject is prescribed treatment or preventive measures which decrease CD44 level or activity (e.g., siRNA specific for CD44 or antibody which blocks CD44 binding to OPN); and vi) any combinations of i) to v); and wherein when the subject is classified into functional group FG2 or FG3, the subject is vii) treated with an OPN antagonist (e.g., OPN antibody, OPN siRNA, melatonin, vitamin D, PROTANDIM™ (nutraceutical cocktail known to reduce plasma or serum OPN levels and used as a natural anti-oxidant mix), an inactive OPN derivative or analog blocking one or more OPN receptors (e.g., α5β1, α4β1, α9β1, and α9β4)); viii) the subject is treated with sCD44 or a CD44 agonist; ix) the subject is treated with an integrin antagonist (e.g., RGD peptide or derivative thereof, a synthetic peptide acting as specific αvintegrin inhibitor (e.g. cilengitide™) or monoclonal antibodies targeting specifically integrin (volociximab™ (α5β1); etaratuzumab™ (αvβ3), etaracizzumab™ (αvβ3), vitaxin (αvβ3), MEDI-522 (αvβ3)) or anti-αvintegrin (CNT095); or x is prescribed treatment or preventive measures which reduce the level of circulating OPN (e.g., brace treatment, acupoint heat sensitive moxibustion, heat therapy with pad, thermal bath, electroacupuncture, etc.); xi) the subject is prescribed treatment or preventive measures which increase CD44/sCD44 level; xii) the subject is prescribed treatment or preventive measures which decrease HA level (e.g., HA-poor diet); xiii) the subject is prescribed an integrin antagonist (e.g., an antibody or siRNA specific for integrin α5, β1, β3 and β5 or treatment or preventive measures which decrease integrin level or activity); and xiv) any combinations of vii) to xiii). In addition to the above, non-limiting treatments or preventive measures include: exercises (physiotherapy), orthodontic treatment, and administration of other natural substances increasing or reducing OPN, CD44 and HA levels. Once a subject is classified into a specific functional group, his/her OPN levels are preferably monitored periodically. When a new treatment or preventive measure is prescribed OPN levels should be monitored in order to maintain the optimal level of OPN (e.g., below or above the OPN retroinhibition/retroactivation concentrations) for this subject and detect any variation that could potentially accelerate the development of IS (including curve progression). Accordingly, the above treatment or prevention method may further be improved by measuring the level of circulating OPN in the subject and determining whether the subject has a high or low level of circulating OPN. Determination of the level of circulating OPN (and of its variation with time) enables to more appropriately select the best treatment option and follow-up schedule.FIG.1summarizes treatment options in view of the functional status of the subject and his/her level of circulating OPN. For example, an FG1 subject could be prescribed OPN or an OPN agonist. For FG1 subjects, brace treatment should generally be avoided. However, FG1 subjects having low levels of circulating OPN could under specific circumstances be prescribed brace treatment for a short period of time (e.g., about 6 months or until OPN concentration has been sufficiently increased i.e., at or near the retroinhibition concentration) so as to maintain his/her level of OPN high. Brace treatment could be stopped completely or temporarily when the maximal concentration of OPN is reached (i.e., near (but below) the retroinhibition concentration for a given patient e.g., for example between about 600 ng/ml and 1200 ng/ml, preferably between about 600 ng/ml and 1000 ng/ml. Generally, for FG1 subjects, preventive and treatment measures should aim at maintaining their level of OPN as high as possible. For FG1 subjects already having high levels of OPN (i.e., close to the maximal OPN concentration where retroinhibition is induced), brace treatment should be avoided. If brace treatment is nevertheless prescribed, OPN levels and curve progression should be monitored closely so as to make sure that OPN levels do not drop significantly and that the rate or curve progression is not increased. OPN or an OPN agonist could also be prescribed to maintain OPN concentration high (as OPN has a protective effect in FG1 subjects as indicated above). In general, any treatment or preventive measure which will help maintaining the level or activity of OPN as high as possible is desirable for FG1 subjects. In an embodiment, massages which increase OPN's level can be performed on a regular basis. For example, in U.S. Ser. No. 13/822,962 Applicants show that the local application of pressure (e.g., pulsative compressive pressure) on at least one body part of the subject (e.g., arm or leg) for 15-90 minutes increases circulating OPN blood level. Hence, such treatment could be applied to the subject periodically (e.g., every day, every two days, every 3 days, twice a week, once a week or once every two weeks) to increase or maintain the level of circulating OPN. Furthermore, as disclosed herein, HA increases (i.e., compensate in part) the Gi-mediated signaling defect present in FG1 subjects. Without being bound to any particular theory, HA could act by increasing OPN's bioavailability by competing with OPN for binding to CD44 (and thus act as an OPN agonist). By doing so, more OPN could be available for increasing the Gi-mediated cellular response. Accordingly, one way of increasing the level or desired activity of OPN is by increasing the amount of Hyaluronic Acid (HA) in subjects. This can be done for example by taking HA supplements or by increasing HA intake or HA synthesis by favoring certain food. Non-limiting examples of food with high HA content or which stimulates/support HA production include, meat and meat organs (e.g., veal, lamb, beef and gizzards, livers, hearts and kidneys), fish, poultry (including meat fish and poultry broths), soy (including soy milk), root vegetables containing starch including potatoes and sweet potatoes, satoimo (Japanese sweet potato), imoji (Japanese sweet potato), Konyaku concoction (root vegetable concoction. Fruits and vegetables rich in vitamin C, magnesium or zinc are also useful as they support the synthesis of HA by the body. Non-limiting examples of food rich in vitamin C include lemons, oranges, limes, grapefruit, guava, mango, cherries, kiwi, blueberries, raspberries, all varieties of grapes, parsley, and thyme. Fruits and vegetables rich in magnesium include apples, bananas, tomatoes, avocados, pineapples, melons, peaches, pears, spinach, cauliflower, broccoli, asparagus, green lettuce, Brussels sprouts, and green beans. Non-limiting examples of food rich in zinc include pumpkins, yeast, peanuts, whole grains, beans, and brown rice. Other possible treatments of preventive measures include the administration of agents which increase OPN expression or secretion (e.g., angiotensin, tumour necrosis factor α (TNFα), infterleukin-1β (IL-1β)), angiotensin II, transforming growth factor β (TGFβ) and parathyroid hormone (PTH)), low intensity pulsed ultrasounds (LIPUS), and treatment and preventive measure which decrease CD44 expression or binding to OPN (e.g., an antibody or siRNA specific for CD44/sCD44). Also, FG1 subjects should avoid diets rich in selenium since selenium is a powerful inhibitor of OPN or any other nutraceutical that decreases OPN level. As indicated above, as opposed to the FG1 group, FG2 and FG3 subjects are particularly sensitive to OPN. High OPN levels in these subjects increase the risk of scoliosis development and progression. Generally, for FG2 and FG3 subjects, preventive and treatment measures should aim at maintaining their level of OPN as low as possible, especially since these subjects are sensitive to OPN (especially FG2 subjects, which are the most sensitive to OPN i.e., hypersensitive). Accordingly, any treatment or preventive measure which will help decreasing or maintaining the level or activity of OPN as low as possible is desirable for FG2 and FG3 subjects. Non-limiting examples of such treatment or preventive measure include, acupoint heat sensitive moxibustion, heat therapies with pad, thermal baths, electroacupuncture, which are known to decrease OPN in serum of subjects. For FG2 and FG3 subjects, possible treatment and preventive measures also includes administration of an OPN antagonist to reduce OPN levels (administration of OPN antagonists (e.g., melatonin, selenium supplements or selenium from the diet (e.g., Brazil nuts), the use of nutraceutical like PROTANDIM) and/or brace treatment as it is likely to be beneficial to these subjects, especially to FG3 subjects. In FG2 and FG3 subjects having low levels of OPN, brace treatment could be postponed or not prescribed at all depending on the skeletal maturity, age and sex of the subject but if prescribed, it will be for preferably be at least 12-18 months, more preferably 24-36 months and even more preferably for 36 months or more, or for a sufficient time to induce a significant reduction in OPN levels. In a specific embodiment brace treatment will last at least 12, 18, 24, 30 or 36 months. Since HA exacerbates the effect of OPN, FG2 and FG3 subjects should avoid taking HA supplements and preferably avoid taking food with high HA content or which stimulates/support HA production (e.g., comply to a HA-poor or HA-low diet). Similarly, any compound (synthetic or natural) or activity which are known to increase the level of OPN, or HA should preferably be avoided (e.g., angiotensin, tumour necrosis factor α (TNFα), infterleukin-1β (IL-1β)), angiotensin II, transforming growth factor β (TGFβ) and parathyroid hormone (PTH, regular application of compressive pressure (e.g., pulsative compressive pressure), LIPUS, etc.). As disclosed herein, CD44 inhibition further decreases the Gi-mediated cellular response in FG2 and FG3 subjects. Accordingly, FG2 and FG3 subjects could also be treated with soluble CD44 or any compound which will increase its level. Furthermore, as the effect of OPN on the Gi-mediated response is dependent on the binding of OPN to integrins (e.g., α5β1), molecules that specifically block the binding of OPN to integrins are also considered useful. For example, one known molecule that specifically blocks the binding of OPN to integrin (e.g., α5β1) is a RGD peptide or derivative thereof. Other useful molecules include a peptide fragment of OPN comprising a RGD motif (e.g., GRGDSVVYGLRS (SEQ ID NO: 13); an siRNA specific for an integrin (e.g., α5, β1, β3, or β5) or an antibody against an integrin (e.g., as, α5, β1, β3, and/or β5and/or volociximab™; etaratuzumab™, etaracizzumab™, Vitaxin™, MEDI-522 or CNT095). Preferably, the level of OPN in the subject should be monitored periodically (e.g., every 6 months, every 5 months, every 4 months, preferably every 2 or 3 months, even more preferably every month) prior to and during any form of treatment or preventive measures and the frequency of OPN level monitoring increased when the level approaches retroinhibition concentration (e.g., 580-1000 ng/ml of OPN) in order to adapt treatment. For Example, for FG1 subjects having low levels of OPN, brace treatment could be performed, stopped when the level of OPN approaches retroinhibition concentration and restarted later (e.g., 6-18 months later) so as to induce another surge in OPN level. This cycle could be repeated as necessary. The present invention also provides a method of predicting the risk of developing IS in a subject comprising: a) classifying the subject into functional group FG1, FG2 or FG3; and b) determining the level of circulating OPN in a blood sample from the subject, wherein when the subject is classified into the FG1 functional group and the level of circulating OPN in the blood sample of the subject is low, the subject has an increased risk of curve progression (as compared to FG1 subjects having high circulating level of OPN); and wherein when the subject is classified into the FG2 or FG3 functional group and the level of circulating OPN in the blood sample of the subject is high, the subject has an increased risk of curve progression (as compared to FG2 or FG3 subjects having low circulating level of OPN). The present invention also provides kits for predicting the risk of developing scoliosis, for predicting brace treatment outcome and for selecting the best treatment or preventive measures. Such kits may comprise one or more reagents for classifying subjects into functional group FG1, FG2, or FG3 such as (a) one or more ligands (e.g., agonists) for stimulating GiPCRs; (b) ligands (e.g., antibodies) for detecting Giα proteins (Giα1, Giα2 and Giα3) and their phosphorylation pattern (e.g., antibodies for detecting serine phosphorylation); and/or (c) reagents for determining cellular proliferation; and optionally (d) (i) one or more ligands for stimulating GsPCRs (e.g., agonists) and (ii) instructions for using the kit. The kit may further comprise reagents for determining the level of circulating OPN in a blood sample such as primary antibodies (labeled or not) against OPN and optionally secondary antibodies to detect the binding of primary antibodies. Definitions For clarity, definitions of the following terms in the context of the present invention are provided. Methods of classifying subjects into a functional group (FG1, FG2 or FG3) according to the degree of their imbalance in Gi-mediated cellular signaling are known in the art and have been described previously (see for example, Moreau at al. (2004), Akoume et al., (2010), Akoume et al., (2013), Azeddine et al., 2007; Letellier et al., 2008; WO2003/073102, WO2010/040234, International Publication No. WO2014/201557, and International Publication No. WO2015/032005 to Moreau, which are incorporated herein by reference in their entirety). Hence, in accordance with the present invention, any method or combination of methods of classifying a subject into the FG1, FG2 or FG3 group can be used. Non-limiting examples of classifying subjects following Gi-stimulation include i) detection of changes in cAMP concentration (Moreau et al., 2004), ii) change in cellular impedance (e.g., by cellular dielectric spectroscopy (CDS), Akoume et al., 2010 and Akoume et al., 2013b), detection of Gi phosphorylation pattern (Akoume et al. 2013), and cellular proliferation rate (WO03073102 and U.S. application No. 61/875,162). Classification may also be effected by determining the degree of imbalance between Gi and Gs as described in Akoume et al., 2013; Akoume et al., 2013b; and International Publication No. WO2015/032005). As used herein, the terms “brace treatment outcome” refers to a genetic or metabolic predisposition of a subject to benefit or not from brace treatment. Non-limiting examples of brace treatment outcome includes: i) a final Cobb angle ≤5 to 45°; ii) a final Cobb angle to 45 (severe scoliosis); iii) curve progression; iv) absence of curve progression; and v) need for surgery or any other benefit that may be measured following brace treatment. A curve progression is defined as a progression of Cobb's angle ≥ to 6°. A “successful brace treatment” or “brace treatment success” is a brace treatment following which the Cobb's angle is ≤ to 45° or no surgery is required. As used herein, the term “benefit” in for example, “benefit from brace treatment” means that brace treatment has a positive effect on the prevention and/or treatment of IS. For example, a “benefit” of brace treatment can be one or more of: i) a reduction in the speed of curve progression; ii) a complete prevention of curve progression (i.e., a curve progression ≤6°; ii) a reduction of Cobb's angle in a preexisting spinal deformity; iii) improvement of column mobility; iv) preservation/maintenance of column mobility; v) improvement of equilibrium and balance in a specific plan; vi) maintenance/preservation of equilibrium and balance in a specific plan; vii) improvement of functionality in a specific plan; viii) preservation/maintenance of functionality in a specific plan; ix) cosmetic improvement; x) avoidance of corrective surgery; and xi) combination of at least two of any of i) to x). As used herein, the term “likely” in for example, “likely to have a successful brace treatment” refers to an increased chance of having a Cobb's angle ≤ to 45° or of not requiring surgery as compared to IS subjects in general, following brace treatment. In an embodiment, the increased chance of having successful brace treatment refers to a 50% chance or more (e.g., 60%, 65%, 70%, 75%, 80%, 85% . . . etc.) of having a Cobb's angle ≤ to 45° or of not requiring surgery following brace treatment. Similarly, the term “unlikely” (or less likely) in for example “unlikely to have a successful brace treatment” refers to a decreased chance of having a Cobb's angle ≤ to 45° or of not requiring surgery as compared to IS subjects in general, following brace treatment. In an embodiment, the decreased chance of having successful brace treatment refers to less than 50% chance (e.g., 49%, 45% 40%, 35%, 30%, 25%, 20% . . . etc.) of having a Cobb's angle ≤ to 45° or of not requiring surgery following brace treatment. As used herein the term “subject” is meant to refer to any mammal including human, mouse, rat, dog, chicken, cat, pig, monkey, horse, etc. In a particular embodiment, it refers to a human. In a specific embodiment, the subject is a pediatric subject. In an embodiment, the subject is skeletally immature. As used herein, the terms “subject in need thereof” refer to a subject already diagnosed with IS or at risk of developing IS (i.e., a likely candidate for developing scoliosis). In an embodiment, the subject in need thereof is a subject already diagnosed with idiopathic scoliosis. In an embodiment, the subject in need thereof is an asymptomatic subject having at least one family member having been diagnosed with idiopathic scoliosis. In an embodiment, the subject in need thereof is a pediatric subject. In an embodiment, the above-mentioned subject is a likely candidate for developing a scoliosis, such as idiopathic scoliosis (e.g., Infantile Idiopathic Scoliosis, Juvenile Idiopathic Scoliosis or Adolescent Idiopathic Scoliosis (AIS)). As used herein the terms “likely candidate for developing scoliosis” include subjects (e.g., children) of which at least one parent has a scoliosis (e.g., adolescent idiopathic scoliosis). Among other factors, age (adolescence), gender and other family antecedent are factors that are known to contribute to the risk of developing a scoliosis and are used to a certain degree to assess the risk of developing a scoliosis. In certain subjects, scoliosis develops rapidly over a short period of time to the point of requiring a corrective surgery (often when the deformity reaches a Cobb's angle ≥50°. Current courses of action available from the moment a scoliosis such as AIS is diagnosed (when scoliosis is apparent) include observation (when Cobb's angle is around)10-25°, orthopedic devices (when Cobb's angle is around)25-30°, and surgery (over)45°. A more reliable determination of the risk of progression could enable to 1) select an appropriate diet to remove certain food products identified as contributors to scoliosis; 2) select the best therapeutic agent; and/or 3) select the least invasive available treatment such as postural exercises, orthopedic device, or less invasive surgeries or surgeries without fusions (a surgery that does not fuse vertebra and preserves column mobility). The present invention encompasses selecting the most efficient and least invasive known preventive actions or treatments in view of the determined risk of developing scoliosis. As used herein, the terms “severe scoliosis”, “severe IS” or “severe progression” is an increase of the Cobb's angle to 45° or more, potentially at a younger age. As used herein the term “treating” or “treatment” in reference to idiopathic scoliosis (e.g., Infantile Idiopathic scoliosis (0-2 years old at the time of onset), Juvenile Idiopathic scoliosis (from 4 to 9 years old at the time of onset) and Adolescent Idiopathic scoliosis (from 10 to 17 years old at the time of onset) is meant to refer to e.g., at least one of a reduction of Cobb's angle in a preexisting spinal deformity, improvement of column mobility, preservation/maintenance of column mobility, improvement of equilibrium and balance in a specific plan; maintenance/preservation of equilibrium and balance in a specific plan; improvement of functionality in a specific plan, preservation/maintenance of functionality in a specific plan, cosmetic improvement, and combination of at least two of any of the above. As used herein the term “preventing” or “prevention” in reference to scoliosis is meant to refer to a at least one of a reduction in the progression of a Cobb's angle in a patient having a scoliosis, a reduction in the speed of curve progression; or, in an asymptomatic patient, a complete prevention of apparition of a spinal deformity, including changes affecting the rib cage and pelvis in 3D, or a combination of any of the above. As used herein the terms “at risk of developing a scoliosis” or “at risk of developing IS” refer to a genetic or metabolic predisposition of a subject to develop a scoliosis (i.e., spinal deformity) and/or a more severe scoliosis at a future time (i.e., curve progression of the spine). For instance, an increase of the Cobb's angle of a subject (e.g., from 40° to 50° or from 18° to 25°) is a “development” of a scoliosis. The terminology “a subject at risk of developing a scoliosis” includes asymptomatic subjects which are more likely than the general population to suffer in a future time of a scoliosis such as subjects (e.g., children) having at least one parent, sibling, or family member suffering from a scoliosis. Among others, age (adolescence), gender and other family antecedent are factors that are known to contribute to the risk of developing a scoliosis and are used to evaluate the risk of developing a scoliosis. Also included in the terminology “a subject at risk of developing a scoliosis” are subjects already diagnosed with IS but which are at risk to develop a more severe scoliosis (i.e., curve progression). As used herein, a “low” level of OPN (e.g., Gene ID 6696, NP_001035147.1 (SEQ ID NO: 1) and NM_001040058 (SEQ ID NO: 2) SPP1-Gene ID: 6696, OPNa: NP_001035147.1, OPNb: NP_000573.1, OPNc: NP_001035149.1, OPN Isoform 4: NP_001238758.1, OPN Isoform 5: NP_001238759.1, NM_001251829.1, GI_352962173); is a level of OPN that is lower than the average level of OPN in IS (e.g., AIS) subjects. In an embodiment, the IS subjects are matched for age and/or sex. In another embodiment, the IS subjects are matched to a specific functional group (FG1, FG2 or FG3). In a specific embodiment, a low level of OPN is a level of OPN <than about 600 ng/ml, 580 ng/ml; 575 ng/ml, 560 ng/ml, 550 ng/ml, 520 ng/ml, 500 ng/ml, 450 ng ml, 400 ng/ml or 300 ng/ml. In specific embodiment, a low level of OPN is a level of OPN <600 ng/ml in a blood sample from the subject. In another specific embodiment, a low level of OPN is a level of OPN ≤500 ng/ml in a blood sample from the subject. In another specific embodiment, a low level of OPN is a level of OPN ≤250 ng/ml in a blood sample from the subject. In another specific embodiment, a low level of OPN is a level of OPN that is about that of healthy subjects. In a specific embodiment, for FG2 subjects (which are hypersensitive to OPN), in the context of the treatment method of the present invention, the level of OPN is maintained as low as possible, preferably below 400 ng/ml, more preferably below 300 ng/ml and even more preferably below 200 ng/ml. As used herein, a “high” level of OPN (e.g., Gene ID 6696, NP_001035147.1 (SEQ ID NO: 1) and NM_001040058 (SEQ ID NO: 2) is a level of OPN that is higher than the average level of OPN in IS (e.g., AIS) subjects. In an embodiment, the IS subjects are matched for age and/or sex. In another embodiment, the IS subjects are matched to a specific functional group (FG1, FG2 or FG3). In a specific embodiment, a high level of OPN is a level of OPN than about 1200 ng/ml, 1000 ng/ml, 900, ng/ml, 850 ng/ml, 800 ng/ml, 750 ng/ml, 700 ng/ml, 550 ng/ml, 580 ng/ml, 600 ng/ml; 610 ng/ml, 620 ng/ml, 630 ng/ml, 650 ng/ml, 675 ng/ml, 700 ng/ml or 750 ng/ml. In a specific embodiment, a high level of OPN is a level of OPN between about 650-1000 ng/ml in a blood sample from the subject. In another specific embodiment, a high level of OPN is a level of OPN 600 ng/ml in a blood sample from the subject. In another embodiment, a high level of OPN is a level of OPN that is close to but below the retroinhibition concentration (e.g., 80%, 85%, 90%, 95% of the retroinhibition concentration). In another specific embodiment, for FG1 subjects, in the context of the treatment method of the present invention, the level of OPN is maintained as high as possible, preferably above 500 ng/ml, above 800 ng/ml or above 900 ng/ml and even more preferably above 1000 ng/ml or above 1200 ng/ml. As used herein, the term “retroinhibition concentration” refers to the in vivo concentration at which OPN level reaches its maximum and the retroinhibition mechanism is induced so as to decrease the level of circulating OPN in the blood endogenously. As used herein, the term “retroactivation concentration” refers to the in vivo concentration at which OPN level reaches its minimum and the retroactivation mechanism is induced so as to increase the level of circulating OPN in the blood endogenously. In a specific embodiment, it refers the concentrations of OPN at which brace treatment first induces an increase in OPN level. In an embodiment, the retroactivation concentration is 600 ng/ml or less, preferably 500 ng/ml or less and even more preferably, 400 ng/ml or less. As used herein the terms “follow-up schedule” is meant to refer to future medical visits a subject diagnosed with a scoliosis or at risk of developing a scoliosis is prescribed once the diagnosis or risk evaluation is made. For example, when a subject is identified as belonging to the FG1 functional group and as having a low level of OPN (and the subject is prescribed OPN, an OPN agonist or treatment and preventive measures which increase OPN levels), the number of medical visits is increased to make sure that OPN levels are stable, preferably increase and remain as high as possible. In addition, in the rare case where an FG1 subject is prescribed a brace treatment, the number of medical visits is increased to make sure that brace treatment lasts for an optimal time and the level of OPN does not decrease. For example, OPN levels could be monitored every 2 months, preferably every month and the treatment adjusted in view of the detected OPN level. For example, when OPN level reached or approached retroinhibition concentration treatment would be stopped completely or temporarily until OPN level decrease sufficiently and the treatment could be started again. In addition, or alternatively, curve progression could be monitored, and the treatment maintained until curve progression is detected. Another limiting example include when a subject being at risk of developing a severe scoliosis or at risk of rapid curve progression (e.g., a subject classified as belonging to the FG2 functional group and having a high level or circulating OPN), the number of medical visits (e.g., to the orthopedist) is increased, the frequency of OPN monitoring is increased and/or the number of x-rays in a given period (e.g., 1, 2, 3, 6 or 12 months) is increased. On the other hand, when a subject is identified as having a lower risk of curve progression or rapid curve progression (e.g., subject being classified as belonging to the FG1 functional group and having high levels of OPN) the number of medical visits, OPN level monitoring or x-rays may be decreased to less than the average (e.g., less than 22 x-rays over a 3-year period or less than 1 visit every month, every 3 months, 6 months, or 12 months). The follow-up schedule and OPN monitoring frequency is adapted in view of several factor including sex, age, Cobb's angle, skeletal maturity (Risser of 5), menarche, functional classification (FG1, FG2 or FG3) and OPN level. As used herein, the term “brace treatment” refers to the use of a brace for reducing (i.e., slowing or stopping) curve progression of the scoliosis or for improving scoliosis (i.e., reversing completely or partially the scoliosis, e.g., a reduction of a Cobb's angle from 30 to)24°. There are a number of bracing options known in the art. Non-limiting examples of braces used in the treatment of scoliosis include the Thrombo-Lumbar-Sacral Orthosis (TLSO) brace, the Milwaukee brace, the Charleston brace and the SpineCor™ brace. Other examples include the Dynamic scoliosis orthosis brace (DSO) (U.S. Pat. No. 7,967,767); scoliosis braces with angle adjustment (U.S. Pat. No. 8,066,653) and braces with adjustable inflatable air bags (US2009/0275871). The physician will recommend a particular back brace and bracing schedule based on factors such as the location of the curve, degree of curvature (Cobb's angle), age, growth status of the IS subject (e.g., pre- or post-menarche, and skeletal maturity (Risser of 5), endophenotype (IS functional group) and lifestyle (e.g., for subjects involved in sports, a more flexible brace (e.g., SpineCor™ or Charleston may be favored). Moreover, a combination of braces may also be prescribed (e.g., a TLSO brace for daytime and a Charleston brace for night time). The most common form of TLSO brace is called the “Boston brace”, and it may be referred to as an “underarm” brace. This brace is fitted to the child's body and custom molded from plastic. It works by applying three-point pressure to the curvature to prevent its progression. The TLSO brace is usually worn 23 hours/day, and it can be taken off to swim, play sports or participate in gym class during the day. This type of brace is usually prescribed for curves in the lumbar or thoraco-lumbar part of the spine. The Cervico-Thoraco-Lumbo-Scacral-Orthosis brace (Milwaukee brace) is similar to the TLSO described above, but also includes a neck ring held in place by vertical bars attached to the body of the brace. It is usually worn 23 hours a day, and can be taken off to swim, play sports or participate in gym class during the day. This type of brace is often prescribed for curves in the Thoracic spine. The Charleston brace, also called nighttime brace is a back brace which is molded to the patient while they are bent to the side, and thus applies more pressure and bends the child against the curve. This pressure improves the corrective action of the brace. This type of brace is worn only at night while the child is asleep. Curves must be in the 20- to 40-degree range and the apex of the curve needs to be below the level of the shoulder blade for the Charleston brace to be effective. In accordance with the present invention, the skilled practitioner (e.g., the treating physician) can select the most appropriate treatment regimen based on the subject's classification. The particular choice of treatment or combination of treatment will be adapted based on the subject's classification and optionally based on his/her level of circulating OPN. For example, brace treatment may be delayed, shortened/lengthened, the choice of a particular brace or braces adapted (in view of age, sex, and Cobb's angle) and the time at which surgery is performed (if needed) modified in view of the subject's classification and optionally, circulating OPN level. In the context of treating FG1 subjects with a brace, a “short” brace treatment or “short term” brace treatment includes brace treatment for 18 months or less, preferably 12 months or less and more preferably, 6 months or less (e.g., 1, 2, 3, 4, 5 or 6 months). Preferably, if brace treatment is prescribed for FG1 subjects, the brace treatment may be continued until the OPN concentration reaches its maximal concentration or close to its maximal concentration (retroinhibition concentration). In an embodiment, brace treatment will be continued until OPN concentration starts declining in the subject. In a specific embodiment, brace treatment is continued until OPN concentration reaches 700, 800, 1000, 1100 or 1200 or more ng/ml. In the context of treating FG2 and FG3 subjects with a brace, a “long” brace treatment or “long-term” brace treatment includes brace treatment for at least 18 months (e.g., 18, 19, 20, 21, 22, 23 months), preferably at least 24 months (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 months) and more preferably, at least 36 months. Preferably, for FG2 and FG3 subjects brace treatment will be continued until OPN concentration is significantly reduced or until skeletal maturity is reached. In a specific embodiment, brace treatment is maintained until the OPN concentration reaches its minimum or until the OPN concentration begins increasing. In a specific embodiment, brace treatment is maintained up to two years after menarche in a female subject. In a particular embodiment, brace treatment is maintained until the concentration of OPN reaches less than 600 ng/ml, preferably less than 500 ng/ml or until the OPN concentration reaches its minimum or starts increasing. In a particular embodiment for FG3 subjects, brace treatment is maintained until the concentration of OPN reaches less than 600 ng/ml, preferably less than 500 ng/ml. In another particular embodiment, for FG2 subjects brace treatment is maintained until the concentration of OPN reaches less than 400 ng/ml, preferably less than 300 ng/ml more preferably less than 200 ng/ml (due to their hypersensitivity toward OPN). The terms “activator” or “agonist” are well known in the art and are used herein interchangeably. Similarly, the terms “suppressor”, “inhibitor” and “antagonist” are well known in the art and are used herein interchangeably As used herein, the expression “OPN agonist” or “OPN activator” is used to refer to any compound capable to increase, at least partially, the level and/or desired biological activity of OPN (e.g., Gene ID 6696, NP_001035147.1 (SEQ ID NO: 1) and NM_001040058 (SEQ ID NO: 2) SPP1-Gene ID: 6696, OPNa: NP_001035147.1, OPNb: NP_000573.1, OPNc: NP_001035149.1, OPN Isoform 4: NP_001238758.1, OPN Isoform 5: NP_001238759.1, NM_001251829.1, GI_352962173). Without being so limited it includes OPN functional fragment or derivative thereof and activators of OPN expression such as (but not limited to) transcriptional and translational activators of the OPN gene (e.g., tumour necrosis factor α (TNFα), infterleukin-1β (IL-1β)), angiotensin II (Ang II), transforming growth factor β (TGFβ) and parathyroid hormone (PTH)). Activator of OPN activity includes compounds that are able to bind to OPN receptors in order to increase the desired biological activity of OPN, peptidomimetics, OPN fragments and the like. In a specific embodiment, the OPN biological activity is an increase in Gi-mediated cellular response in FG1 subjects and the OPN activator or agonist is HA. As used herein, the term “functional fragment” of OPN refers to a molecule (e.g., polypeptide) which retains substantially the same desired activity as the original molecule, but which differs by any modifications, and/or amino acid/nucleotide substitutions, deletions, or additions (e.g., fusion with another polypeptide). Modifications can occur anywhere including the polypeptide/polynucleotide backbone (e.g., the amino acid sequence, the amino acid side chains and the amino or carboxy termini). Such substitutions, deletions or additions may involve one or more amino acids or in the case of polynucleotide, one or more nucleotide. The substitutions are preferably conservative, i.e., an amino acid is replaced by another amino acid having similar physico-chemical properties (size, hydrophobicity, charge/polarity, etc.) as well known by those of ordinary skill in the art. Functional fragments of OPN (SEQ ID NO: 1) include a fragment or a portion of OPN polypeptide or a fragment or a portion of a homologue or allelic variant of OPN which retains activity, i.e., binds to integrins (e.g., α5β1) and/or CD44. In an embodiment, the OPN functional fragment is at least 80, 85, 88, 90, 95, 98 or 99% identical to the polypeptide sequence of (SEQ ID NO: 1). In an embodiment, the OPN functional fragment is a functional variant which includes variations in amino acids which are not conserved between rat, mouse and human OPN. Preferably, the OPN functional fragment is human. A “functional derivative” refers to a molecule derived from the OPN polypeptide or polynucleotide and which is substantially similar in structure and biological activity to the OPN protein or nucleic acid of the present invention. An OPN polypeptide derivative may for example include modifications to increase its bioavailability, its stability, to simplify its purification or to preferentially target the OPN derivative to a particular tissue or cell. As used herein, the expression “OPN antagonist” or “OPN inhibitor” is used to refer to any compound capable to block completely or partially (i.e., negatively affect) the expression (at the transcriptional (mRNA) and/or translational (protein)) level or targeted biological activity of OPN (e.g., binding to one or more of its integrin receptors) in cells. In an embodiment, the biological activity of OPN in cells is a reduction in GiPCR signaling. OPN inhibitors include intracellular as well as extracellular suppressors. Without being so limited, such suppressors include RNA interference agents (siRNA, shRNA, miRNA), antisense molecules, ribozymes, proteins (e.g., dominant negative, inactive variants), peptides, small molecules, antibodies, antibody fragments, etc. In an embodiment, the OPN antagonist is a neutralizing antibody against human OPN. In an embodiment, the OPN antagonist is melatonin. In an embodiment, the OPN antagonist is selenium. In an embodiment, the OPN antagonist is PROTANDIM™. In an embodiment, the OPN antagonist is soluble CD44 (sCD44) or a stimulator or enhancer of sCD44/CD44 expression. As used herein, the expression “integrin antagonist” or “integrin inhibitor” is used to refer to any compound capable to block completely or partially (i.e., negatively affect) the expression (at the transcriptional (mRNA) and/or translational (protein)) level or targeted biological activity of integrins (e.g., binding to OPN) in cells. In an embodiment, the biological activity of integrins in cells is a reduction in GiPCR signaling. Integrin inhibitors include intracellular as well as extracellular suppressors. Without being so limited, such suppressors include RNA interference agents (siRNA, shRNA, miRNA), antisense molecules, ribozymes, proteins (e.g., dominant negative, inactive variants), peptides, small molecules, antibodies, antibody fragments, etc. In an embodiment, the integrin antagonist is a neutralizing antibody against human integrin (volociximab™; etaratuzumab™, etaracizzumab™, Vitaxin™, MEDI-522, CNT095, cilengitide™). The terms “inhibitor of OPN expression” or “inhibitor of integrin expression” (e.g., α5, β1, β3, and/or β5) expression” include any compound able to negatively affect OPN's or integrin's (e.g., α5's, β1's, β3's, and/or β5's) expression (i.e., at the transcriptional and/or translational level), i.e. the level of OPN/integrin mRNA and/or protein or the stability of the protein. Without being so limited, such inhibitors include agents which negatively affect the expression of OPN (e.g., vitamin D, melatonin, selenium, PROTANDIM™) or integrin, RNA interference agents (siRNA, shRNA, miRNA), antisense molecules, and ribozymes. Such RNA interference agents are design to specifically hybridize with their target nucleic acid under suitable conditions and are thus substantially complementary their target nucleic acid. The terms “inhibitor of OPN activity” or “inhibitor of integrin activity” (e.g., (e.g., α5, β1, β3, and/or β5) refers to any molecules that is able to reduce or block the effect of OPN or integrins (e.g., 531) on Gi-mediated signaling. These molecules increase GiPCR signaling in cells (i.e., in FG2 and FG3 subjects) by blocking/reducing totally or partially the inhibitory effect induced by OPN and/or integrins activity. Non-limiting examples of inhibitors of OPN's activity include proteins (e.g., dominant negative, inactive variants), peptides, small molecules, anti-OPN antibodies (neutralizing antibodies), antibody fragments, inactive fragments of α5 and/or β1 integrins etc. Non-limiting examples of inhibitors of integrin (e.g., α5β1) activity include proteins (e.g., dominant negative, inactive variants), peptides (RGD peptides or RGD peptide-derivatives), small molecules, anti α5and/or β1antibodies (e.g., neutralizing antibodies such as Volociximab™ M200, etaratuzumab™, etaracizzumab™, Vitaxin™, MEDI-522, CNT095, cilengitide™), antibody fragments, etc. In an embodiment, the RGD peptide is a peptide fragment of OPN comprising a RGD motif comprising the amino acid sequence GRGDSVVYGLRS corresponding to amino acid 158 to 169 of OPN (SEQ ID NO: 1). In an embodiment, the OPN fragment comprising the RGD motif comprises amino acids 158 to 162, 158 to 165, 158 to 167, 158 to 170, 158 to 175, 158 to 180, 158 to 185, 158 to 190, 158 to 195, or 158 to 200 of OPN (e.g., SEQ ID NO: 1). In an embodiment, peptide fragment of OPN comprising a RGD motif comprises amino acids 158 to 161, 156 to 161, 154 to 161, 152 to 162, 150 to 162, 148 to 162, 146 to 162, 144 to 162, 140 to 162, 159 to 163, 159 to 164, 159 to 162, 159 to 166, 159 to 167, or 159 to 169 of OPN (e.g., SEQ ID NO: 1). In an embodiment, the “inhibitor of OPN's activity” is a neutralizing antibody directed against (or specifically binding to) a human OPN polypeptide which inhibits its binding to integrins such as α5β1(i.e., binding to α5and/or β1integrin) In an embodiment, the “inhibitor of integrin activity” is a neutralizing antibody directed against (or specifically binding to) a human integrin (α5, β1, β3, and/or β5) polypeptide which inhibits the binding of OPN to integrins (i.e., binding to α5, β1, β3, and/or β5integrin). In an embodiment, the antibody binds to the RGD domain of OPN. In an embodiment, the antibody is directed against amino acids 159 to 162, 158 to 162, 158 to 165, 158 to 167, 158 to 170, 158 to 175, 158 to 180, 158 to 185, 158 to 190, 158 to 195, or 158 to 200 of OPN (e.g., SEQ ID NO: 1). In an embodiment, the antibody is directed against amino acids 158 to 161, 156 to 161, 154 to 161, 152 to 162, 150 to 162, 148 to 162, 146 to 162, 144 to 162, 140 to 162, 159 to 163, 159 to 164, 159 to 162, 159 to 166, 159 to 167, or 159 to 169 of OPN (e.g., SEQ ID NO: 1). Similarly, the terms “inhibitor of integrin's activity”, “inhibitor of α5β1's activity”, “inhibitor of Q5's activity” or “inhibitor of β1's activity”, “inhibitor of β3's activity”, “inhibitor of β5's activity” and the like include any compound able to negatively affect the expression and/or activity of α5(e.g., Gene ID 3678, NP_002196.2 (SEQ ID NO: 5) and NM_002205.2 (SEQ ID NO: 6)), β1(Gene ID 3688, NP_002202.2 (SEQ ID NO: 7) and NM_002211.3 (SEQ ID NO: 8)), β3(Gene ID 3690, NP_000203.2 (SEQ ID NO: 9) and NM_000212 (SEQ ID NO: 10) and/or β5(Gene ID 3693, NP_002204.2 (SEQ ID NO: 11) and NM_002213.3 (SEQ ID NO: 12)) in cells. In a particular embodiment, the “activity” of α5and/or β1in cells is the transduction of the signal leading to the OPN-dependent inhibition of GiPCR signaling. In a particular embodiment, the inhibitor is Volociximab™ M200, etaratuzumab™, etaracizzumab™, Vitaxin™, MEDI-522, CNT095 or cilengitide™. The term “inhibitor” of sCD44/CD44 expression (e.g., Gene ID 960, NP_000601.3, (SEQ ID NO: 3), NM_000610 (SEQ ID NO: 4)) refers to an agent able to decrease the level of expression of CD44 and an agent able to decrease CD44 secretion. In an embodiment, the inhibitor of sCD44/CD44 is an agent able to decrease CD44 binding with OPN. Without being so limited, the agent can be a protein (e.g., an antibody specific to CD44), a peptide, a small molecule, or a nucleotide. Inhibitors of sCD44 or CD44 generally increase OPN's bioavailability for other receptor of OPN (e.g., integrins) and may be particularly useful for treating and preventing scoliosis development in FG1 subjects. The term “stimulator” or “enhancer” of sCD44/CD44 expression (e.g., Gene ID 960, NP_000601.3, (SEQ ID NO: 3), NM_000610 (SEQ ID NO: 4)) refers to an agent able to increase the level or expression of CD44 and an agent able to increase CD44 secretion. In an embodiment, the stimulator of sCD44/CD44 is an agent able to increase CD44 affinity toward OPN. Without being so limited, the agent can be a protein, a peptide, a small molecule, or a nucleotide. “Stimulators” or “enhancers” of sCD44/CD44 expression generally decrease OPN's bioavailability for other receptor of OPN (e.g., integrins) and may be particularly useful for treating and preventing scoliosis development in FG2 and FG3 subjects. Antibodies In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 2000, In “Monoclonal Antibody Technology: The production and characterization of Rodent and Human Hybridomas”, Elsevier Science Publisher, Amsterdam, The Netherlands) and Recombinant Monoclonal Antibodies (Mariel Donzeau and Achim Knappik; Methods in Molecular Biology; Volume 378, 2007, pp 15-31). As used herein, the term “anti-OPN antibody”, refers to an antibody that specifically binds to (interacts with) OPN and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as OPN. Similarly, the expression “anti-CD44 antibody”, “anti-β1antibody” and the like (anti-α5, anti-β3, anti-β5. . . ) refers to an antibody that specifically binds to (interacts with) CD44 or β1and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as CD44/β1, The term “antibody” or “immunoglobulin” is used in the broadest sense, and covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies™, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention. In an embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a humanized or CDR-grafted antibody. TABLE 1commercially available human OPN Elisa kits.CatalogueCompanyKit namenumberSensitivityIBL HambourgHuman Osteopontin ELISAJP 171 583.33ng/mlIBL AmericaHuman Osteopontin N-Half272583.90pmol/LAssay Kit-IBLIBL-AmericaHuman Osteopontin Assay271583.33ng/mlKit-IBLAssay designsOsteopontin (human) EIA Kit900-1420.11ng/mlAmerican ResearchOsteopontin, human kit17158?Products, Inc.R&D SystemsHuman Osteopontin (OPN)DOST000.024ng/mLELISA KitPromokineHuman Osteopontin ELISAPK-EL-KA42313.6ng/mlUscnlifeHuman Osteopontin, OPNE0899h?ELISA Kit TABLE 2Non-limiting examples of commercially available antibodies for OPN(Human, Unconjugated)CatalogueCompany NameNumberHostEMD MilliporeAB10910rabbitBoster ImmunoleaderPA1431LifeSpan BioSciencesLS-C63082-mouse100LifeSpan BioSciencesLS-B5940-50mouseLifeSpan BioSciencesLS-C137501-mouse100LifeSpan BioSciencesLS-C31763-rabbit100LifeSpan BioSciencesLS-C99283-rabbit400LifeSpan BioSciencesLS-C9410-rabbit100LifeSpan BioSciencesLS-C122259-rabbit20LifeSpan BioSciencesLS-C88774-rabbit0.1LifeSpan BioSciencesLS-C136850-rabbit100LifeSpan BioSciencesLS-C96393-rabbit500LifeSpan BioSciencesLS-C193595-mouse200LifeSpan BioSciencesLS-C193596-mouse100LifeSpan BioSciencesLS-C63081-mouse100LifeSpan BioSciencesLS-C193597-mouse100LifeSpan BioSciencesLS-C169155-mouse100LifeSpan BioSciencesLS-C189569-mouse1000LifeSpan BioSciencesLS-C189635-mouse1000LifeSpan BioSciencesLS-C189636-mouse1000LifeSpan BioSciencesLS-C189634-mouse1000LifeSpan BioSciencesLS-C73947-mouse500LifeSpan BioSciencesLS-C189134-rabbit50LifeSpan BioSciencesLS-B5272-rabbit250LifeSpan BioSciencesLS-C176152-rabbit100LifeSpan BioSciencesLS-C194024-rabbit100LifeSpan BioSciencesLS-B5626-50rabbitLifeSpan BioSciencesLS-C131159-rabbit20LifeSpan BioSciencesLS-B9287-rabbit200LifeSpan BioSciencesLS-C73949-rabbit200LifeSpan BioSciencesLS-C182368-rabbit50LifeSpan BioSciencesLS-B2411-50goatLifeSpan BioSciencesLS-B8326-mouse100LifeSpan BioSciencesLS-B7193-50rabbitLifeSpan BioSciencesLS-B425-50rabbitLifeSpan BioSciencesLS-C9413-rabbit100LifeSpan BioSciencesLS-B7193-50rabbitLifeSpan BioSciencesLS-C9413-rabbit100LifeSpan BioSciencesLS-B9080-rabbit100LifeSpan BioSciencesLS-C201116-rabbit100Boster ImmunoleaderPA1431antibodies-onlineABIN933617mouseantibodies-onlineABIN1381708ChickenBACHEMT-4816.0400RabbitBACHEMT-4815.0050RabbitBiorbytorb12414mouseBiorbytorb128774RabbitBiorbytorb12506mouseBiorbytorb94522RabbitBiorbytorb13123RabbitBiorbytorb88187goatBiorbytorb94961mouseBiorbytorb86662rabbitBiorbytorb170816mouseBiorbytorb175965mouseBiorbytorb19047goatBiorbytorb43142rabbitBiorbytorb120032rabbitBiorbytorb11192rabbitBiorbytorb11191rabbitantibodies-onlineABIN933617mouseBioVision5426-100mouseBioVision5422-100mouseBioVision5424-100mouseBioVision5423-100mouseBioVision5425-100mouseBioVision5421-100mouseMerck Millipore04-970mouseMerck MilliporeMAB3055rabbitMerck MilliporeAB1870RabbitMerck MilliporeAB10910RabbitGenWay Biotech, Inc.GWB-T00561mouseGenWay Biotech, Inc.GWB-T00557mouseGenWay Biotech, Inc.GWB-T00558mouseGenWay Biotech, Inc.GWB-T00559mouseGenWay Biotech, Inc.GWB-T00560mouseGenWay Biotech, Inc.GWB-goat3A2E99GenWay Biotech, Inc.GWB-rabbit23C38DGenWay Biotech, Inc.GWB-295359RabbitGenWay Biotech, Inc.GWB-806785GoatEnzo Life Sciences,ADI-905-629-mouseInc.100Enzo Life Sciences,ADI-905-630-mouseInc.100Enzo Life Sciences,ADI-905-500-1RabbitInc.Enzo Life Sciences,ALX-210-RabbitInc.309-R100GeneTexGTX28448RabbitGeneTexGTX37500rabbitGeneTexGTX15489rabbitGeneTexGTX89519goatSpring BioscienceE3282rabbitSpring BioscienceE3280rabbitSpring BioscienceE3281rabbitSpring BioscienceE3284rabbitAbbiotec251924rabbitAbbiotec250801rabbitMBL InternationalCY-P1035Rockland100-401-404RabbitImmunochemicals, Inc.Bioss Inc.bs-0026RRabbitBioss Inc.bs-0019RRabbitProteintech Group Inc22952-1-APRabbit TABLE 4Non-limiting examples of commercially available ELISAKits for integrin α5(ITGA5, Human)CatalogueCompany NamenumberRangeSensitivityantibodies-onlineABIN4176120.156-10 ng/mL0.054 ng/mLantibodies-onlineABIN365741nanaDLdevelopDL-ITGa5-Hu0.156-10 ng/mL0.054 ng/mLMyBioSource.-MBS814027nanacomR&D SystemsDYC3230-2312-20,000 pg/mLnaBiomatikE91287Hu0.156-10 ng/mL0.054 ng/mL TABLE 5Non-limiting examples of commercially availableAntibodies for α5(ITGA5, human)Company NameCatalogue NumberHostNovus BiologicalsNBP1-84576rabbitBiorbytorb69201mouseAbcamab72663rabbitAcris Antibodies GmbHBM4033mouseAviva Systems BiologyOAAF05375rabbitSt John's LaboratorySTJ32097mouseGeneTexGTX86915rabbitGeneTexGTX86905rabbitOriGene TechnologiesTA311966rabbitOriGene TechnologiesTA310024ratAbbexaabx15590rabbitAbbexaabx15591rabbitAbbiotec252937mouseAbnova CorporationMAB10703mouseAbnova CorporationMAB5267mouseBioss Inc.bs-0567RrabbitCell Signaling Technology4705SrabbitAtlas AntibodiesHPA002642rabbitGenWay Biotech, Inc.GWB-MX190ArabbitGenWay Biotech, Inc.GWB-D9743Emouseantibodies-onlineABIN656138rabbitantibodies-onlineABIN219716rabbitNovus BiologicalsNBP1-71421-0.1mgrabbitNovus BiologicalsNBP1-71421-0.05mgrabbitBioLegend328009mouseBD Biosciences610634mouseBioLegend328002mouseBD Biosciences610634mouseAbcamab72665rabbitAbcamab55988rabbitBioworld TechnologyBS7053rabbitSanta Cruz Biotechnology, Inc.sc-166665mouseBioworld TechnologyBS7052rabbitR&D SystemsAF1864goatR&D SystemsFAB1864AmouseThermo Scientific PierceMA5-15568mouseAntibodiesThermo Scientific PierceMA1-81134mouseAntibodiesAbD Serotec (Bio-Rad)MCA1187mouseAbD Serotec (Bio-Rad)MCA1187TmouseLife Technologies132600mouseProteintech Group Inc10569-1-APrabbitRaybiotech, Inc.119-14178mouseCreative BiomartCAB-3671MHmouseMerck MilliporeCBL497mouseFitzgerald Industries International10R-1984mouseEMD MilliporeAB1921rabbitEMD MilliporeAB1949rabbit TABLE 6Non-limiting examples of commercially available ELISA Kits for β1(ITGB1, human)Company NameCatalogue numberRangeSensitivityantibodies-onlineABIN833710nanaMerck MilliporeECM470nanaDLdevelopDL-ITGb1-Hu1.56-100 ng/mLnaBiomatikE91042Hu1.56-100 ng/mL0.64 ng/mL TABLE 7Non-limiting examples of commercially available antibodies for β1ITGB1 (Human, Unconjugated)Company NameCatalogue numberHostAbgentAM2241bmouseBiorbytorb86390rabbitLifeSpan BioSciencesLS-C84969-100mouseNovus BiologicalsNB110-55545rabbitAbbexaabx12778rabbitBethyl Laboratories,A303-735ArabbitInc.Abcamab5189RabbitSt John's LaboratorySTJ60344RabbitCell Signaling4706SRabbitTechnologyBioss Inc.bs-0486RRabbitAntigenix AmericaMA290020Inc.GenWay Biotech, Inc.GWB-312F4DmouseFitzgerald Industries20R-2722rabbitInternationalGeneTexGTX50784rabbitThermo ScientificMA1-80764mousePierce AntibodiesR&D SystemsAF1778goatAbbiotec251162rabbitBioworld TechnologyBS1817rabbitAbgentAM2241bmouseEnzo Life Sciences,BML-IG6060-mouseInc.0100BIOCARECME 386 ArabbitMEDICALProSci, Inc48-392RabbiteBioscience14-0299-82mouse TABLE 8Non-limiting Examples of commercially availableantibodies for CD44 (human and unconjugated.CatalogueCompanyNumberHosteBioscience16-0441-81RatNovusNBP1-31121RabbitThermoPA5-32327RabbitGentexGTX50755RabbitCell Signaling5640SMouseAbcamab103552RabbitAbnovaH00000960-M03Mouseantibodies-onlineABIN871672RabbitR & D SystemsAF3660SheepBD Biosciences555476MouseAbbiotec252831MouseBethyl LaboratoriesA303-872ARabbitProteintech Group15675-1-APRabbitEnzo Life SciencesALX-801-089-MouseC100Cell Science852.603.020Merck Millipore217594-100ULRatLife Technologies336700MouseSanta Cruzsc-53503MouseProSci79-668RabbitMP Biomedicals08D526000MouseCedarlaneCLX47APMouse TABLE 9Non-limiting examples of commercially availableELISA Kits for CD44.CatalogueCompanyNumberCell Sciences850.570.192MyBioSource.comMBS335446Sino BiologicalSEK12211Biotrend ChemikalienBMA-27215Antibodies onlineABIN366268Enzo Life SciencesALX-850-053-KI01DRG InternationalEIA4876Kamiya BiomedicalKT-032CompanyabcamAB45912-2NovusNBP1-87599CUSABIOCSB-E11846H Antibodies directed against OPN, CD44 and integrins (α5, β1, β3, β5) are included within the scope of this invention as they can be produced by well established procedures known to those of skill in the art. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc), intravenous (iv) or intraperitoneal (ip) injections of the relevant antigen with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1 are different alkyl groups. Animals may be immunized against the antigen, immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with ⅕ to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled, and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response. Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Pat. Nos. 6,025,155 and 6,077,677 as well as U.S. Patent Application Publication Nos. 2002/0160970 and 2003/0083293. In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. As used herein, the term “purified” in the expression “purified antibody” is simply meant to distinguish man-made antibody from an antibody that may naturally be produced by an animal against its own antigens. Hence, raw serum and hybridoma culture medium containing anti-OPN antibody are “purified antibodies” within the meaning of the present invention. As used herein, the terminology “blood sample” is meant to refer to blood, plasma or serum. As used herein, the terminology “cell sample” is meant to refer to a sample containing cells expressing the desired GPCR(s) in sufficient amount to detect a cellular response in in order to classify the subject into one of functional groups FG1, FG2 and FG3. The cells in the cell sample may be any type of cells as long as they express the desired GPCR to be tested. The cells used herein naturally express one or more receptors coupled to Giproteins and were selected in part for their accessibility for collection from subjects. Hence, cells such as osteoblasts, osteoclasts, peripheral blood mononuclear cell (PBMC) (inherently including principally lymphocytes but also monocytes) and myoblasts are advantageously accessible and may conveniently be used in the methods of the present invention. Blood cells (e.g., PBMCs, platelets (thrombocytes), etc.) in particular are particularly accessible and provide for a more rapid testing. Any blood cell can be used for the methods of the present invention so long as it possesses at least one GPCR receptor coupled to a Gi protein. The cells can be fresh or frozen and may or may not have been cultured (expanded) prior to testing. The “sample” may be of any origin including blood, saliva, tears, sputum, urine, feces, biopsy (e.g., muscle biopsy), as long as it contains cells expressing the desired GPCR(s). The articles “a,” “an” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The term “including” and “comprising” are used herein to mean, and re used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”. The terms “such as” are used herein to mean, and is used interchangeably with, the phrase “such as but not limited to”. The present invention is illustrated in further details by the following non-limiting examples. EXAMPLE 1 AIS Endophenotype and Brace Treatment Outcome METHODS: A retrospective study was performed with 67 AIS patients having had a blood test (cell-based assay), seen between January 2007 and November 2012 and having completed treatment with TLSO braces respecting standard prescription criteria (23 h per day). AIS patients were stratified according to the method developed by Moreau et al. 2004 (Moreau et al., 2004; Akoume et al., 2010) based upon the measurement of a differential signaling impairment of receptors coupled to G inhibitory proteins (Gi) allowing their classification into three functional groups (i.e., biological endophenotypes FG1, FG2 or FG3). Cobb angles were measured by single blind observer in brace and at the end of treatment and compared to their initial values. Progression of the curvature was defined by 6° angle increase (Nachemson et al., 1995). Treatment was considered a success if final Cobb angle was 45° or no surgery was required (Richards et al., 2005). Association between group classification and treatment outcome was analysed with Chi2test in a contingency table. Logistic regression models were performed for odds ratio calculation. Group comparability at time of prescription was verified using ANOVA and Chi2test: groups were not different on mean Cobb angle for all curves, Risser sign (i.e., amount of calcification of human pelvis as a measure of maturity) nor age. Results: The patient distribution is reported in Table 10 (15 in FG1, 27 in FG2, and 25 in FG3). TABLE 10Statistical analysis of the patient distribution comparing3 success criteria (Cobb at the end of treatment ≤45°,Cobb angle progression ≤6° and no need for surgery)OddssuccessfailureratioFinal Cobb ≤45°FG16 (40%)9 (60%)1FG216 (59%)11 (41%)2.18p = 0.235FG321 (84%)4 (16%)7.88p = 0.007Total43 (64%)24 (36%)χ2= 8.4 (p = 0.015)Cobb angle progression ≤6°FG16 (40%)9 (60%)1FG213 (48%)14 (52%)1.39p = 0.612FG315 (60%)10 (40%)2.25p = 0.224Total33 (49%)34 (51%)χ2= 1.6 (p = 0.444)No need for surgeryFG18 (53%)7 (47%)1FG220 (74%)7 (26%)2.5p = 0.177FG322 (88%)3 (12%)6.4p = 0.02Total50 (74%)17 (25%)χ2= 5.96 (p = 0.05) Globally, in all patients who had brace success, the majority were from FG2 and FG3. There was a clear association between the functional group and success of the treatment regarding the progression of curvature ≤45° criteria. Group FG3 patients were more likely to have success with brace treatment than in group FG1. The association was in the same direction for group FG2. Regarding the ≥6° of progression criteria, an increased proportion of success was noted in FG3. Success in treatment in regard to preventing surgery was statistically different between the groups (Chi 2 (2, 67)=5.96, p=0.05). It is 6.4 times more likely to prevent surgery than to have one in group FG3 compared to FG1 (p=0.02). Again, a tendency towards increased chance of preventing surgery was found in group FG2 compared to FG1. In order to confirm the above results and determine whether the specific type of brace treatment used influenced outcome, a retrospective study was performed with 90 AIS patients previously stratified among three biological endophenotypes according to a cell-based assay, as described above, allowing their classification into three functional groups (FG1, FG2 or FG3). Patients completed the non-rigid/dynamic (SpineCor™) brace treatment following standard prescription criteria. Cobb angles were measured by a single blind observer in brace and at the end of treatment and compared to their initial values. Progression of the curvature was defined by a 6° Cobb increase and treatment was considered a success if final Cobb angle was ≤45° or no surgery was required. Association between group classification and treatment outcome was analysed with Chi2 test. Logistic regression models were performed for odds ratio calculation. Group comparability at time of prescription was verified using ANOVA and Chi2 test: no differences for mean Cobb angle, Risser sign, BMI nor age. Results. The patient distribution is reported in Table 11 (24 in FG1, 27 in FG2, and 39 in FG3). As for the first study with rigid brace treatment, globally, in all patients who had brace success, the majority were from FG3. There was a clear association between the functional group and the success of the treatment regarding the final Cobb angle ≤45° criteria (Chi2=6.7, p=0.034) and in regard to preventing progression of 6° (Chi2=15.7, p<0.001). Being classified as FG3 was 4 times (p=0.028) and 7.6 times (p=0.001) more likely to lead to treatment success than failure compared to FG1, respectively for the ≤45° final Cobb and ≤6° progression criteria. There was no significant difference in treatment outcomes between groups FG1 and FG2. TABLE 11Statistical analysis of the patient distribution treated withSpineCor ™ brace comparing 2 success criteria(Cobb at the end of treatment ≤45° and Cobbangle progression)OddssuccessfailureratioFinal Cobb ≤45°FG115 (63%)9 (37%)1FG217 (63%)10 (37%)1.02p = 0.973FG334 (87%)5 (13%)4.08p = 0.028Total66 (73%)24 (27%)χ2= 6.7 (p = 0.034)Cobb angle progression ≤6°FG15 (21%)19 (79%)1FG28 (30%)19 (70%)1.60p = 0.474FG326 (67%)13 (33%)7.60p = 0.001Total39 (43%)51 (57%)χ2= 15.7 (p < 0.001) Conclusion. Globally, in all patients who had brace success, the majority were from FG2 and FG3. Outcomes of bracing were most favorable for patients presenting the FG3 endophenotype, independently of the type of bracing. There was a clear association between the functional group and success of the treatment regarding the progression of curvature 45° criteria and the Cobb angle progression ≤6. Furthermore, results showed a tendency towards increased chance of preventing Cobb angle progression (≤6) and surgery in group FG2 compared to FG1. EXAMPLE 2 Circulating OPN Level Variations with Age in AIS and Control Subjects Data was obtained with AIS patients (N=884) in Phase 2 followed at Sainte-Justine Hospital, at the Shriners Hospital or Montreal Children's Hospital, in Montreal, Québec, Canada. Age matched control subjects (N=254) were recruited from primary and secondary schools in Montreal. The plasma was collected in tubes containing EDTA and circulating OPN levels were measured in blood samples from control and AIS subjects of age 9 to 18 by ELISA. As shown inFIGS.2A-2B, circulating OPN blood level generally increases until between the age of 11 and 12 years old and then begin to decrease with age. OPN levels are significantly higher in AIS than control subjects at all times and follow generally the same variation pattern with age. EXAMPLE 3 Circulating OPN Level Variations Upon Brace Treatment in Subjects Having High and Low Levels Of OPN The effect of brace treatment on the level of circulating OPN in AIS subjects was studied. Data was obtained with AIS patients in Phase 2 followed at Sainte-Justine Hospital, at the Shriners Hospital or Montreal Children's Hospital, in Montreal, Québec, Canada. The plasma was collected in tubes containing EDTA and OPN was measured with ELISA (IBL International, catalogue #JP27158). Circulating OPN levels were measured in blood samples from control and AIS subjects every 6 months during four years. Subjects were separated in two groups.FIG.3Apresents OPN levels for subjects which had initial (i.e., before the beginning of brace treatment) circulating OPN levels below 600 ng/mL, treated with a brace (N=94) and age-matched untreated control subjects (N=330).FIG.3Bshows OPN levels for subjects which had initial circulating OPN levels 600 ng/ml, treated (N=153) with a TLSO brace and age-matched untreated control subjects (N=310). As shown inFIG.3A, in subjects having initial low levels of circulating OPN (i.e., below about 600 ng/ml), brace treatment first increased OPN levels. OPN levels were significantly higher in subjects treated with a brace, 6 months after treatment and returned to the same level than subjects not treated with a brace after about 12 to 18 months of brace treatment. Brace treatment then induced a decrease in OPN levels which was maintained during the rest of the study, i.e., up to 48 months (FIG.3A). In subjects having high levels of OPN at the beginning of the study (i.e., about 600 ng/ml), brace treatment had the opposite effect. It produced an important decrease in circulating OPN level within the first 6 months. Then, OPN level increased slowly until it reached about 600 ng/ml (i.e., about the same level as untreated subjects) about 24 months after the beginning of treatment and decreased again after. Circulating OPN levels remained below that of AIS subjects not treated with a brace, except for a short period around 24 months of treatment, where OPN levels reached a peak and overlapped with OPN levels of untreated subjects (FIG.2B). Based on the results presented inFIGS.3A and3B, it appears that when the treatment begins with circulating OPN levels below about 600 ng/mL, brace treatment generally first causes an increase in OPN production, whereas when treatment begins with OPN concentrations at or above this value, brace treatment induces a reduction in circulating levels of OPN. These results show that long term brace treatment generally decreases the circulating level of OPN and suggest the presence of a retroinhibition mechanism which regulates circulating OPN levels when they reach around 600 ng/ml. EXAMPLE 4 Association Between OPN and SCD44 Levels and Curve Progression in AIS Subjects According to their Functional Group The relation between curve progression and OPN and sCD44 levels was followed in AIS subjects. An association between OPN levels and curve progression was observed. In FG1 subjects, low levels of OPN (≤than about 500 ng/ml) correlated with curve progression (see for examplesFIGS.6A-6B,8A-8B,9A-9B and10A-10B) while high levels (e.g., at or above 1000 ng/ml) were generally associated with absence of curve progression or smaller rate of progression (see for exampleFIGS.2A-2B and7A-7B). For FG2 and FG3 subjects, high levels of OPN were more often associated with curve progression (see for exampleFIG.17B).FIGS.4A-19Bshows examples of OPN and sCD44 levels variations observed with time and curve progression in AIS subjects for each functional group. No clear correlation was observed between curve progression and sCD44. EXAMPLE 5 OPN Enhances Gi-Mediated Cell Signalling in FG1 Subjects and Decreases Gi-Mediated Signalling in FG2 and FG3 Subjects The variation in Gi-mediated cell signaling in response to OPN in each functional group (FG1, FG2 and FG3) was studied.FIGS.20and21show the response to OPN (increasing doses) on osteoblasts isolated from patients classified into functional groups FG1, FG2 and FG3. OPN enhances Gi signaling in the FG1 functional group and aggravates the impairment in the FG2 (hypersensitive) and FG3 (sensitive) functional groups (FIG.20). Furthermore, MC3T3-E1 cells were used to check the effect of the knockdown of OPN and its receptors. MC3T3-E1 osteoblasts cells were transiently transfected in serum-free medium, using Lipofectamine™ RNAiMAX reagent (Invitrogen) according to the manufacturers instructions and functional experiments were performed 48 h post transfection. Knock down of OPN expression in osteoblasts by siRNAs (CCA CAG CCA CM GCA GUC CAG AUU A (SEQ ID NO: 14)) increases Gi-mediated transduction in FG2 and FG3 subgroups while it tends to decrease the response in FG1 (FIG.21). EXAMPLE 6 Differential Effect of HA, CD44 and Integrins on Gi-Mediated Cell Signalling in FG1, FG2 and FG3 Functional Groups MC3T3-E1 cells were also used to check the effect of the knockdown of OPN's receptors by RNAi. Experimental conditions were as described for Example 5. The sequence of RNA oligonucleotides used for the knockdowns are: integrin β1 (CCU MG UCA GCA GUA GGA ACA UUA U (SEQ ID NO: 15)), integrin β3 (CCU CCA GCU CAU UGU UGA UGC UUA U (SEQ ID NO: 16)); integrin β5 (AGAAUGUCUGCUAAUCCACCCAAAA, HSS-105572, Life technologies (SEQ ID NO: 17), CUGAGGGCAAACCUUGUCAAAAAUG, HSS-105573, Life technologies (SEQ ID NO: 18); and GAAAUGGCUUCAAAUCCAUUAUACA, HSS-179984, life technologies, (SEQ ID NO: 19)) and CD44 (GM CM GGA GUC GUC AGA MC UCC A (SEQ ID NO: 20)). The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole. | 85,806 |
11860170 | DETAILED DESCRIPTION I. Definitions The terms “a,” “an,” or “the” as used herein not only includes aspects with one member, but also includes aspects with more than one member. The term “about” as used herein to modify a numerical value indicates a defined range around that value. If “X” were the value, “about X” would indicate a value from 0.9X to 1.1X, and more preferably, a value from 0.95X to 1.05X. Any reference to “about X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.” When the modifier “about” is applied to describe the beginning of a numerical range, it applies to both ends of the range. Thus, “from about 500 to 850 nm” is equivalent to “from about 500 nm to about 850 nm.” When “about” is applied to describe the first value of a set of values, it applies to all values in that set. Thus, “about 580, 700, or 850 nm” is equivalent to “about 580 nm, about 700 nm, or about 850 nm.” “Activated acyl” as used herein includes a —C(O)-LG group. “Leaving group” or “LG” is a group that is susceptible to displacement by a nucleophilic acyl substitution (i.e., a nucleophilic addition to the carbonyl of —C(O)-LG, followed by elimination of the leaving group). Representative leaving groups include halo, cyano, azido, carboxylic acid derivatives such as t-butylcarboxy, and carbonate derivatives such as i-BuOC(O)O—. An activated acyl group may also be an activated ester as defined herein or a carboxylic acid activated by a carbodiimide to form an anhydride (preferentially cyclic) or mixed anhydride —OC(O)Raor —OC(NRa)NHRb(preferably cyclic), wherein Raand Rbare members independently selected from the group consisting of C1-C6alkyl, C1-C6perfluoroalkyl, C1-C6alkoxy, cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl. Preferred activated acyl groups include activated esters. “Activated ester” as used herein includes a derivative of a carboxyl group that is more susceptible to displacement by nucleophilic addition and elimination than an ethyl ester group (e.g., an NHS ester, a sulfo-NHS ester, a PAM ester, or a halophenyl ester). Representative carbonyl substituents of activated esters include succinimidyloxy (—OC4H4NO2), sulfosuccinimidyloxy (—OC4H3NO2SO3H), -1-oxybenzotriazolyl (—OC6H4N3); 4-sulfo-2,3,5,6-tetrafluorophenyl; or an aryloxy group that is optionally substituted one or more times by electron-withdrawing substituents such as nitro, fluoro, chloro, cyano, trifluoromethyl, or combinations thereof (e.g., pentafluorophenyloxy, or 2,3,5,6-tetrafluorophenyloxy). Preferred activated esters include succinimidyloxy, sulfosuccinimidyloxy, and 2,3,5,6-tetrafluorophenyloxy esters. “FRET partners” refers to a pair of fluorophores consisting of a donor fluorescent compound such as cryptate and an acceptor compound such as Alexa 647, when they are in proximity to one another and when they are excited at the excitation wavelength of the donor fluorescent compound, these compounds emit a FRET signal. It is known that, in order for two fluorescent compounds to be FRET partners, the emission spectrum of the donor fluorescent compound must partially overlap the excitation spectrum of the acceptor compound. The preferred FRET-partner pairs are those for which the value R0 (Förster distance, distance at which energy transfer is 50% efficient) is greater than or equal to 30 Å. “FRET signal” refers to any measurable signal representative of FRET between a donor fluorescent compound and an acceptor compound. A FRET signal can therefore be a variation in the intensity or in the lifetime of luminescence of the donor fluorescent compound or of the acceptor compound when the latter is fluorescent. VCAM-1 (vascular cell adhesion protein 1, CD106, INCAM-110) refers to a cell surface sialoglycoprotein expressed by cytokine activated endothelium. VCAM-1 has a number of functions including the regulation of leukocyte migration, leukocyte-endothelial cell adhesion and signal transduction and may play a role in a number of inflammatory diseases. VCAM-1 is distributed across non-leukocyte and leukocyte cells. VCAM-1 is a member of the Ig superfamily of adhesion molecules, is expressed at high levels on cytokine stimulated vascular endothelial cells, and at minimal levels on unstimulated endothelial cells. VCAM-1 is also present on follicular and inter-follicular dendritic cells of lymph nodes, myoblasts, and some macrophages. VCAM-1_HUMAN, accession P19320 is SEQ ID NO: 1. VCAM-1 has 739 amino acids and a mass of 81,276 Da. This isoform has been chosen as the ‘canonical’ sequence. A2M (alpha-2-macroglobulin) refers to a plasma protein found in the blood that mainly acts as an antiprotease and is able to inactivate a variety of proteinases. For example, it functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. It functions as an inhibitor of coagulation by inhibiting thrombin. Further, A2M sometimes acts as a carrier protein because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, TGF-β, insulin, and IL-113. A2M is mainly produced by the liver, and also locally synthesized by macrophages, fibroblasts, and adrenocortical cells. In humans it is encoded by the A2M gene. A2M HUMAN, accession P01023 is SEQ ID NO: 2. A2M has 1474 amino acids and a mass of approximately 720 kDa. II. Embodiments The present disclosure provides a homogenous solution phase time-resolved FRET assay (TR-FRET) to detect VCAM-1 and A2M presence or levels in a biological sample such as whole blood. In conjunction with other markers levels, VCAM-1 and A2M can be used as an aid in determination of fibrosis in liver diseases such as NASH, Hepatitis C and Hepatitis B. Forster resonance energy transfer or fluorescence resonance energy transfer (FRET) is a process in which a donor molecule in an excited state transfers its excitation energy through dipole-dipole coupling to an acceptor fluorophore, when the two molecules are brought into close proximity, typically less than 10 nm such as, <9 nm, <8 nm, <7 nm, <6 nm, <5 nm, <4 nm, <3 nm, <2 nm, or less than <1 nm. Upon excitation at a characteristic wavelength, the energy absorbed by the donor is transferred to the acceptor, which in turn emits the energy. The level of light emitted from the acceptor fluorophore is proportional to the degree of donor acceptor complex formation. Biological materials are typically prone to autofluorescence, which can be minimized by utilizing time-resolved fluorometry (TRF). TRF takes advantage of unique rare earth elements such as lanthanides, (e.g., europium and terbium), which have exceptionally long fluorescence emission half-lives. Time-resolved FRET (TR-FRET) unites the properties of TRF and FRET, which is especially advantageous when analyzing biological samples. If one anti-VCAM-1 antibody is labeled with a donor fluorophore and a second anti-VCAM-1 antibody is labeled with an acceptor fluorophore, and an anti-A2M antibody is labeled with a donor fluorophore (or an acceptor fluorophore) and an isolated A2M protein is labeled with an acceptor fluorophore (or a donor fluorophore), in which the two acceptor fluorophores are different, TR-FRET can occur in the presence of VCAM-1 in the sample and the presence of A2M in the sample would disrupt TR-FRET signal associated with the anti-A2M antibody binding to the isolated A2M protein (FIG.1). The use of the FRET phenomenon for studying biological processes implies that each member of the pair of FRET partners will be conjugated to compounds that will interact with one another, and thus bring the FRET partners into close proximity with one another. Upon exposure to light, the FRET partners will generate a FRET signal. In the methods according to the disclosure, an energy donor and an energy acceptor are each conjugated to a different anti-VCAM-1 antibody. An energy donor or an energy acceptor is conjugated to an anti-A2M antibody. Further, an energy donor or an energy acceptor is conjugated to an isolated A2M protein. For example, two anti-VCAM-1 antibodies that bind to two different epitopes in VCAM-1, and an anti-A2M antibody that bind to an epitope in A2M can be used. The energy transfer between the two FRET partners depends upon each binding to the analyte. Förster or fluorescence resonance energy transfer (FRET), is a physical phenomenon in which a donor fluorophore in its excited state non-radiatively transfers its excitation energy to a neighboring acceptor fluorophore, thereby causing the acceptor to emit its characteristic fluorescence. As such, in one aspect, the present disclosure provides an assay method for detecting the presence or amount of VCAM-1 and A2M in a sample, the method comprising: contacting the sample with a first anti-VCAM-1 antibody, which binds to a first epitope of VCAM-1, wherein the first anti-VCAM-1 antibody is labeled with a first donor fluorophore; contacting the sample with a second anti-VCAM-1 antibody, which binds to a second epitope of VCAM-1, wherein the second anti-VCAM-1 antibody is labeled with a first acceptor fluorophore; contacting the sample with an anti-A2M antibody, which binds to an epitope of A2M, wherein the anti-A2M antibody is labeled with a second donor fluorophore; contacting the sample with an isolated A2M protein, wherein the isolated A2M protein is labeled with a second acceptor fluorophore; incubating the sample for a time sufficient to obtain dual labeled VCAM-1 and labeled A2M; and exciting the sample having dual labeled VCAM-1 and labeled A2M using one or more light sources to detect at least one fluorescence emission signal associated with fluorescence resonance energy transfer (FRET), wherein the first and second acceptor fluorophores are different. In another aspect, the present disclosure provides an assay method for detecting the presence or amount of VCAM-1 and A2M in a sample, the method comprising: contacting the sample with a first anti-VCAM-1 antibody, which binds to a first epitope of VCAM-1, wherein the first anti-VCAM-1 antibody is labeled with a first donor fluorophore; contacting the sample with a second anti-VCAM-1 antibody, which binds to a second epitope of VCAM-1, wherein the second anti-VCAM-1 antibody is labeled with a first acceptor fluorophore; contacting the sample with an anti-A2M antibody, which binds to an epitope of A2M, wherein the anti-A2M antibody is labeled with a second acceptor fluorophore; contacting the sample with an isolated A2M protein, wherein the isolated A2M protein is labeled with a second donor fluorophore; incubating the sample for a time sufficient to obtain dual labeled VCAM-1 and labeled A2M; and exciting the sample having dual labeled VCAM-1 and labeled A2M using one or more light sources to detect at least one fluorescence emission signal associated with fluorescence resonance energy transfer (FRET), wherein the first and second acceptor fluorophores are different. In some embodiments, the first and second donor fluorophores are the same and the sample is excited using one light source. In other embodiments of this aspect of the disclosure, the first and second donor fluorophores are different and the sample is excited using two different light sources. In this aspect of the disclosure, two anti-VCAM-1 antibodies, one labeled with a donor fluorophore and one labeled with an acceptor fluorophore, are used. The two anti-VCAM-1 antibodies bind to two different epitopes on VCAM-1. An anti-A2M antibody labeled with a donor fluorophore (or an acceptor fluorophore) and an isolated A2M protein labeled with an acceptor fluorophore (or a donor fluorophore) are also used. The two anti-VCAM-1 antibodies binding to two different epitopes on VCAM-1 bring the first donor fluorophore and the first acceptor fluorophore in proximity to each other. The anti-A2M antibody and the isolated A2M protein to bring the second donor fluorophore and the second acceptor fluorophore in proximity to each other. The donor fluorophore in its excited state can transfer its excitation energy to the acceptor fluorophore to cause the acceptor fluorophore to emit its characteristic fluorescence. In some embodiments, the two acceptor fluorophores are different and emit fluorescence at different wavelengths. Thus, the appearance of the fluorescence emission signal is proportional to the presence or level of VCAM-1 in the sample and the disappearance of the fluorescence emission signal is proportional to the presence or level of A2M in the sample. In some embodiments of the two aspects of the disclosure described above, the methods described herein further comprise detecting the presence or amount of an additional biomarker. To detect the additional biomarker, the methods comprise: contacting the sample with an additional antibody, which binds to a first epitope of the additional biomarker, wherein the additional antibody is labeled with a third donor fluorophore; contacting the sample with a further antibody, which binds to a second epitope of the additional biomarker, wherein the further antibody is labeled with a third acceptor fluorophore; incubating the sample for a time sufficient to obtain dual labeled additional biomarker; and exciting the sample having dual labeled additional biomarker using a light source to detect two fluorescence emission signals associated with fluorescence resonance energy transfer (FRET), wherein the first, second, and third acceptor fluorophores are different. In some embodiments, the first acceptor fluorophore is Alexa Fluor 488, the second acceptor fluorophore is Alexa Fluor 546, and the third acceptor fluorophore is Alexa Fluor 647. In addition to VCAM-1 and A2M, the additional biomarker that can be detected using the methods described herein can be selected from the group consisting of M65 (CK18 full-length), ICAM-1, eSelectin, syndecan1 (CD138), adiponectin, chitinase-3-like-1 (YKL40), ACY1, osteopontin, GDF15, cathepsin D, M30 (CK18 fragment), ANGPTL4, FGF21, THBS2, MMP2, ANGPTL3, HA (hyaluronic acid), IP10, VAP1, MCP1, IL2Rα, EMMPRIN, FABP1, IL6, angiopoietin1, collagen IV-α1, FGF19, VEGF, galectin1, galectin3, MMP1, MMP9, haptoglobin, TIMP1, resistin, galectin3BP, e-cadherin, ApoA1, galectin 9, HBEGF, and MMP3. In some embodiments, the additional biomarker is ANGPTL4, FGF21, THBS2, MMP2, ANGPTL3, or HA (hyaluronic acid). In some embodiments, the additional biomarker is IP10, VAP1, MCP1, IL2Rα, EMMPRIN, or FABP1. In some embodiments, the additional biomarker is IL6, angiopoietin1, collagen IV-α1, FGF19, VEGF, or galectin1. In some embodiments, the additional biomarker is galectin3, MMP1, MMP9, haptoglobin, TIMP1, or resistin. In some embodiments, the additional biomarker is galectin3BP, e-cadherin, ApoA1, galectin 9, HBEGF, or MMP3. In certain aspects, the FRET assay is a time-resolved FRET assay. The fluorescence emission signal or measured FRET signal is directly correlated with the biological phenomenon studied. In fact, the level of energy transfer between the donor fluorescent compound and the acceptor fluorescent compound is proportional to the reciprocal of the distance between these compounds to the 6thpower. For the donor/acceptor pairs commonly used by those skilled in the art, the distance Ro (corresponding to a transfer efficiency of 50%) is in the order of 1, 5, 10, 20 or 30 nanometers. In certain aspects, the sample is a biological sample. Suitable biological samples include, but are not limited to, whole blood, urine, a fecal specimen, plasma or serum. In a preferred aspect, the biological sample is whole blood. In certain aspects, the FRET energy donor compound is a cryptate, such as a lanthanide cryptate. In certain aspects, the cryptate has an absorption wavelength between about 300 nm to about 400 nm such as about 325 nm to about 375 nm. In certain aspects, as shown inFIG.5, cyptate dyes (Lumi4-Tb inFIG.5) have four fluorescence emission peaks at about 490 nm, about 548 nm, about 587 nm, and 621 nm. Thus, as a donor, the cryptate is compatible with fluorescein-like (green zone) and Cy5 or DY-647-like (red zone) acceptor (e.g., green acceptor, NIR acceptor, or orange acceptor inFIG.5) to perform TR-FRET experiments. In certain aspects, the introduction of a time delay between a flash excitation and the measurement of the fluorescence at the acceptor emission wavelength allows to discriminate long lived from short-lived fluorescence and to increase signal-to-noise ratio. Cryptates as FRET Donors In certain aspects, the terbium cryptate molecule “Lumi4-Tb” from Lumiphore, marketed by Cisbio bioassays is used as the cryptate donor. The terbium cryptate “Lumi4-Tb” having the formula below, which can be coupled to an antibody by a reactive group, in this case, for example, an NHS ester: An activated ester (an NHS ester) can react with a primary amine on an antibody to make a stable amide bond. A maleimide on the cryptate and a thiol on the antibody can react together and make a thioether. Alkyl halides react with amines and thiols to make alkylamines and thioethers, respectively. Any derivative providing a reactive moiety that can be conjugated to a antibody can be utilized herein. For example, in some embodiments, when an anti-VCAM-1 antibody or an anti-A2M antibody is used, the maleimide on the cryptate can react with a thiol on the antibody. In certain other aspects, cryptates disclosed in WO2015157057, titled “Macrocycles” are suitable for use in the present disclosure. This publication contains cryptate molecules useful for labeling biomolecules. As disclosed therein, certain of the cryptates have the structure: In certain other aspects, a terbium cryptate useful in the present disclosure is shown below: In certain aspects, the cryptates that are useful in the present invention are disclosed in WO 2018/130988, published Jul. 19, 2018. As disclosed therein, the compounds of Formula I are useful as FRET donors in the present disclosure: wherein when the dotted line is present, R and IV are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkyl optionally substituted with one or more halogen atoms, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl or alkylcarbonylalkoxy or alternatively, R and R′ join to form an optionally substituted cyclopropyl group wherein the dotted bond is absent; R2and R3are each independently a member selected from the group consisting of hydrogen, halogen, SO3H, —SO2—X, wherein X is a halogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, or an activated group that can be linked to a biomolecule, wherein the activated group is a member selected from the group consisting of a halogen, an activated ester, an activated acyl, optionally substituted alkylsulfonate ester, optionally substituted arylsulfonate ester, amino, formyl, glycidyl, halo, haloacetamidyl, haloalkyl, hydrazinyl, imido ester, isocyanato, isothiocyanato, maleimidyl, mercapto, alkynyl, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl, cyclic anhydride, alkoxyalkyl, a water solubilizing group or L; R4are each independently a hydrogen, C1-C6alkyl, or alternatively, 3 of the R4groups are absent and the resulting oxides are chelated to a lanthanide cation; and Q1-Q4are each independently a member selected from the group of carbon or nitrogen. FRET Acceptors In order to detect a FRET signal, a FRET acceptor is required. The FRET acceptor has an excitation wavelength that overlaps with an emission wavelength of the FRET donor. In the present disclosure, two FRET signals of the acceptors are detected with one FRET signal proportional to the concentration level of VCAM-1 present in the sample (e.g., a patient's blood sample) and the other FRET signal inversely proportional to the concentration level of A2M present in the sample (e.g., a patient's blood sample). A known amount of calibrators, i.e., standard curves (FIGS.2A-2D), can be used to interpolate the concentration levels of VCAM-1 and A2M. As described above, when an anti-VCAM-1 or anti-A2M antibody is used, the cryptate donor (FIG.3) can be used to label the antibody. Lumi4-Tb has 3 spectrally distinct peaks, at 490, 550 and 620 nm, which can be used for energy transfer (FIG.5). Subsequently, a first acceptor can be used to label an anti-VCAM-1 antibody (which can bind to a different epitope on VCAM-1 from the epitope on VCAM-1 bound by the first anti-VCAM-1 antibody). A second acceptor can be used to label an anti-A2M antibody. In the present disclosure, the first acceptor and the second acceptor are different and emit two fluorescence emission signals at different emission wavelengths. The acceptor molecules that can be used include, but are not limited to, fluorescein-like (green zone), Cy5, DY-647, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647 (FIG.4), allophycocyanin (APC), and phycoerythrin (PE). Donor and acceptor fluorophores can be conjugated using a primary amine on an antibody. Other acceptors include, but are not limited to, cyanin derivatives, D2, CY5, fluorescein, coumarin, rhodamine, carbopyronine, oxazine and its analogs, Alexa Fluor fluorophores, Crystal violet, perylene bisimide fluorophores, squaraine fluorophores, boron dipyrromethene derivatives, NBD (nitrobenzoxadiazole) and its derivatives, DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid), allophycocyanin (APC), and phycoerythrin (PE). In one aspect, fluorescence can be characterized by wavelength, intensity, lifetime, and polarization. Antibodies In one aspect, a human anti-VCAM-1/CD106 antibody (e.g., Catalog #MAB809 (R&D systems), from monoclonal mouse IgG1Clone #HAE-2Z and shown to be specific for human VCAM-1/CD106) can be used to conjugate to a donor fluorophore (e.g., cryptate) and a different human anti-VCAM-1/CD106 antibody (e.g., Catalog #MA5-16429 (Thermo Fisher Scientific), from monoclonal mouse IgG1 Clone #1.G11B1) can be used to conjugate to an acceptor fluorophore, or vice versa. Other commercial anti-VCAM-1 antibodies are available in the art. A human anti-A2M antibody (e.g., Catalog #10C-CR2005M1 (Fitzgerald Industries International, Inc), from monoclonal mouse IgG1Clone #3127485 and shown to be specific for human A2M) can be used to conjugate to a donor fluorophore (e.g., cryptate) and an isolated A2M protein (e.g., Catalog #ab77935 (Abcam), from human plasma) can be used to conjugate to an acceptor fluorophore, or vice versa. Other commercial anti-A2M antibodies are available in the art. The methods herein for detecting the presence or levels of VCAM-1 and A2M can use a variety of samples, which include a tissue sample, blood, biopsy, serum, plasma, saliva, urine, or stool sample. Production of Antibodies The generation and selection of antibodies not already commercially available can be accomplished several ways. For example, one way is to express and/or purify a polypeptide of interest (i.e., antigen) using protein expression and purification methods known in the art, while another way is to synthesize the polypeptide of interest using solid phase peptide synthesis methods known in the art. See, e.g.,Guide to Protein Purification, Murray P. Deutcher, ed.,Meth. Enzymol., Vol. 182 (1990);Solid Phase Peptide Synthesis, Greg B. Fields, ed.,Meth. Enzymol., Vol. 289 (1997); Kiso et al.,Chem. Pharm. Bull.,38:1192-99 (1990); Mostafavi et al.,Biomed. Pept. Proteins Nucleic Acids,1:255-60, (1995); and Fujiwara et al.,Chem. Pharm. Bull.,44:1326-31 (1996). The purified or synthesized polypeptide can then be injected, for example, into mice or rabbits, to generate polyclonal or monoclonal antibodies. One skilled in the art will recognize that many procedures are available for the production of antibodies, for example, as described in Antibodies,A Laboratory Manual, Harlow and Lane, Eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988). One skilled in the art will also appreciate that binding fragments or Fab fragments which mimic antibodies can also be prepared from genetic information by various procedures (see, e.g.,Antibody Engineering: A Practical Approach, Borrebaeck, Ed., Oxford University Press, Oxford (1995); and Huse et al.,J. Immunol.,149:3914-3920 (1992)). In addition, numerous publications have reported the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected target antigen (see, e.g, Cwirla et al.,Proc. Natl. Acad. Sci. USA,87:6378-6382 (1990); Devlin et al.,Science,249:404-406 (1990); Scott et al.,Science,249:386-388 (1990); and Ladner et al., U.S. Pat. No. 5,571,698). A basic concept of phage display methods is the establishment of a physical association between a polypeptide encoded by the phage DNA and a target antigen. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target antigen bind to the target antigen and these phage are enriched by affinity screening to the target antigen. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods, a polypeptide identified as having a binding affinity for a desired target antigen can then be synthesized in bulk by conventional means (see, e.g., U.S. Pat. No. 6,057,098). The antibodies that are generated by these methods can then be selected by first screening for affinity and specificity with the purified polypeptide antigen of interest and, if required, comparing the results to the affinity and specificity of the antibodies with other polypeptide antigens that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptide antigens in separate wells of microtiter plates. The solution containing a potential antibody or group of antibodies is then placed into the respective microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed and a labeled secondary antibody (e.g., an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 minutes and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide antigen is present. The antibodies so identified can then be further analyzed for affinity and specificity. In the development of immunoassays for a target protein (VCAM-1 and/or A2M), the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ, e.g., certain antibody combinations may interfere with one another sterically, assay performance of an antibody may be a more important measure than absolute affinity and specificity of that antibody. Those skilled in the art will recognize that many approaches can be taken in producing antibodies or binding fragments and screening and selecting for affinity and specificity for the various polypeptides of interest, but these approaches do not change the scope of the present invention. A. Polyclonal Antibodies Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of interest and an adjuvant. It may be useful to conjugate the polypeptide of interest to a protein carrier that is immunogenic in the species to be immunized, such as, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent. Non-limiting examples of bifunctional or derivatizing agents include maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (conjugation through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, and R1N═C═NR, wherein R and R1are different alkyl groups. Animals are immunized against the polypeptide of interest or an immunogenic conjugate or derivative thereof by combining, e.g., 100 μg (for rabbits) or 5 μg (for mice) of the antigen or conjugate with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with about ⅕ to 1/10 the original amount of polypeptide or conjugate in Freund's incomplete adjuvant by subcutaneous injection at multiple sites. Seven to fourteen days later, the animals are bled and the serum is assayed for antibody titer. Animals are typically boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same polypeptide, but conjugation to a different immunogenic protein and/or through a different cross-linking reagent may be used. Conjugates can also be made in recombinant cell culture as fusion proteins. In certain instances, aggregating agents such as alum can be used to enhance the immune response. B. Monoclonal Antibodies Monoclonal antibodies are generally obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. For example, monoclonal antibodies can be made using the hybridoma method described by Kohler et al.,Nature,256:495 (1975) or by any recombinant DNA method known in the art (see, e.g., U.S. Pat. No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal (e.g., hamster) is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies which specifically bind to the polypeptide of interest used for immunization. Alternatively, lymphocytes are immunized in vitro. The immunized lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances which inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), the culture medium for the hybridoma cells will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and/or are sensitive to a medium such as HAT medium. Examples of such preferred myeloma cell lines for the production of human monoclonal antibodies include, but are not limited to, murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center; San Diego, Calif.), SP-2 or X63-Ag8-653 cells (available from the American Type Culture Collection; Rockville, Md.), and human myeloma or mouse-human heteromyeloma cell lines (see, e.g., Kozbor,J. Immunol.,133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)). The culture medium in which hybridoma cells are growing can be assayed for the production of monoclonal antibodies directed against the polypeptide of interest. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined using, e.g., the Scatchard analysis of Munson et al.,Anal. Biochem.,107:220 (1980). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal. The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such asE. colicells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody, to induce the synthesis of monoclonal antibodies in the recombinant host cells. See, e.g., Skerra et al.,Curr. Opin. Immunol.,5:256-262 (1993); and Pluckthun,Immunol Rev.,130:151-188 (1992). The DNA can also be modified, for example, by substituting the coding sequence for human heavy chain and light chain constant domains in place of the homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA,81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al.,Nature,348:552-554 (1990); Clackson et al.,Nature,352:624-628 (1991); and Marks et al.,J. Mol. Biol.,222:581-597 (1991). The production of high affinity (nM range) human monoclonal antibodies by chain shuffling is described in Marks et al.,BioTechnology,10:779-783 (1992). The use of combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries is described in Waterhouse et al.,Nuc. Acids Res.,21:2265-2266 (1993). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma methods for the generation of monoclonal antibodies. Human Antibodies As an alternative to humanization, human antibodies can be generated. In some embodiments, transgenic animals (e.g., mice) can be produced that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al.,Nature,362:255-258 (1993); Bruggermann et al.,Year in Immun.,7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369, and 5,545,807. Alternatively, phage display technology (see, e.g., McCafferty et al.,Nature,348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, using immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats as described in, e.g., Johnson et al.,Curr. Opin. Struct. Biol.,3:564-571 (1993). Several sources of V-gene segments can be used for phage display. See, e.g., Clackson et al.,Nature,352:624-628 (1991). A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described in Marks et al.,J. Mol. Biol.,222:581-597 (1991); Griffith et al.,EMBO J.,12:725-734 (1993); and U.S. Pat. Nos. 5,565,332 and 5,573,905. In certain instances, human antibodies can be generated by in vitro activated B cells as described in, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275. C. Antibody Fragments Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J. Biochem. Biophys. Meth.,24:107-117 (1992); and Brennan et al.,Science,229:81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered fromE. colicells and chemically coupled to form F(ab′)2fragments (see, e.g., Carter et al.,BioTechnology,10:163-167 (1992)). According to another approach, F(ab′)2fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See, e.g., PCT Publication No. WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. The antibody fragment may also be a linear antibody as described, e.g., in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific. D. Antibody Purification When using recombinant techniques, antibodies can be produced inside an isolated host cell, in the periplasmic space of a host cell, or directly secreted from a host cell into the medium. If the antibody is produced intracellularly, the particulate debris is first removed, for example, by centrifugation or ultrafiltration. Carter et al.,BioTech.,10:163-167 (1992) describes a procedure for isolating antibodies which are secreted into the periplasmic space ofE. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants. The antibody composition prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains (see, e.g., Lindmark et al.,J. Immunol. Meth.,62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (see, e.g., Guss et al.,EMBO J.,5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker; Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered. Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt). E. Bispecific Antibodies Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab′)2bispecific antibodies). Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e.g., Millstein et al.,Nature,305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually performed by affinity chromatography. Similar procedures are disclosed in PCT Publication No. WO 93/08829 and Traunecker et al.,EMBO J.,10:3655-3659 (1991). According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance. In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain with a second binding specificity in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. See, e.g., PCT Publication No. WO 94/04690 and Suresh et al.,Meth. Enzymol.,121:210 (1986). According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side-chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side-chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side-chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Heteroconjugate antibodies can be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well-known in the art, and are disclosed in, e.g., U.S. Pat. No. 4,676,980. Suitable techniques for generating bispecific antibodies from antibody fragments are also known in the art. For example, bispecific antibodies can be prepared using chemical linkage. In certain instances, bispecific antibodies can be generated by a procedure in which intact antibodies are proteolytically cleaved to generate F(ab′)2fragments (see, e.g., Brennan et al.,Science,229:81 (1985)). These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody. In some embodiments, Fab′-SH fragments can be directly recovered fromE. coliand chemically coupled to form bispecific antibodies. For example, a fully humanized bispecific antibody F(ab′)2molecule can be produced by the methods described in Shalaby et al.,J. Exp. Med.,175: 217-225 (1992). Each Fab′ fragment was separately secreted fromE. coliand subjected to directed chemical coupling in vitro to form the bispecific antibody. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. See, e.g., Kostelny et al.,J. Immunol.,148:1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al.,Proc. Natl. Acad. Sci. USA,90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers is described in Gruber et al.,J. Immunol.,152:5368 (1994). Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. See, e.g., Tutt et al.,J. Immunol.,147:60 (1991). III. Vascular Cell Adhesion Protein 1 (VCAM-1) VCAM-1 is a transmembrane cellular adhesion protein that mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. Upregulation of VCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription (e.g., in response to Tumor necrosis factor-alpha (TNFα) and Interleukin-1 (IL-1)). VCAM-1 is encoded by the vascular cell adhesion molecule 1 gene (VCAM-1; Entrez GeneID:7412) and is produced after differential splicing of the transcript (Genbank Accession No. NM_001078 (variant 1) or NM_080682 (variant 2)), and processing of the precursor polypeptide splice isoform (Genbank Accession No. NP_001069 (isoform a) or NP_542413 (isoform b)). The human VCAM-1 polypeptide sequence is set forth in, e.g., Genbank Accession No. NP_001069. The human VCAM-1 mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_001078. One skilled in the art will appreciate that VCAM-1 is also known as VCAM-1, V-CAM1, INCAM-100, CD antigen 106, cluster of differentiation 106, and CD106. In certain aspects, the methods described herein are used to measure and/or detect VCAM-1. In certain aspects, the concentration or level of VCAM-1 is measured. In certain aspects, the biological sample in which VCAM-1 is measured is whole blood. In certain aspects, the concentration of VCAM-1 is about 100 ng/mL to about 1500 ng/mL. In certain aspect, the concentration of VCAM-1 is about 100 ng/mL, 110 ng/mL, 120 ng/mL, 130 ng/mL, 140 ng/mL, 150 ng/mL, 160 ng/mL, 170 ng/mL, 180 ng/mL, 190 ng/mL, 200 ng/mL, 210 ng/mL, 220 ng/mL, 230 ng/mL, 240 ng/mL, 250 ng/mL, 260 ng/mL, 270 ng/mL, 280 ng/mL, 290 ng/mL, 300 ng/mL, 310 ng/mL, 320 ng/mL, 330 ng/mL, 340 ng/mL, 350 ng/mL, 360 ng/mL, 370 ng/mL, 380 ng/mL, 390 ng/mL, 400 ng/mL, 410 ng/mL, 420 ng/mL, 430 ng/mL, 440 ng/mL, 450 ng/mL, 460 ng/mL, 470 ng/mL, 480 ng/mL, 490 ng/mL, 500 ng/mL, 510 ng/mL, 520 ng/mL, 530 ng/mL, 540 ng/mL, 550 ng/mL, 560 ng/mL, 570 ng/mL, 580 ng/mL, 590 ng/mL, 600 ng/mL, 610 ng/mL, 620 ng/mL, 630 ng/mL, 640 ng/mL, 650 ng/mL, 660 ng/mL, 670 ng/mL, 680 ng/mL, 690 ng/mL, 700 ng/mL, 710 ng/mL, 720 ng/mL, 730 ng/mL, 740 ng/mL, 750 ng/mL, 760 ng/mL, 770 ng/mL, 780 ng/mL, 790 ng/mL, 800 ng/mL, 810 ng/mL, 820 ng/mL, 830 ng/mL, 840 ng/mL, 850 ng/mL, 860 ng/mL, 870 ng/mL, 880 ng/mL, 890 ng/mL, 900 ng/mL, 910 ng/mL, 920 ng/mL, 930 ng/mL, 940 ng/mL, 950 ng/mL, 960 ng/mL, 970 ng/mL, 980 ng/mL, 990 ng/mL, 1000 ng/mL, 1010 ng/mL, 1020 ng/mL, 1030 ng/mL, 1040 ng/mL, 1050 ng/mL, 1060 ng/mL, 1070 ng/mL, 1080 ng/mL, 1090 ng/mL, 1100 ng/mL, 1110 ng/mL, 1120 ng/mL, 1130 ng/mL, 1140 ng/mL, 1150 ng/mL, 1160 ng/mL, 1170 ng/mL, 1180 ng/mL, 1190 ng/mL, 1200 ng/mL, 1210 ng/mL, 1220 ng/mL, 1230 ng/mL, 1240 ng/mL, 1250 ng/mL, 1260 ng/mL, 1270 ng/mL, 1280 ng/mL, 1290 ng/mL, 1300 ng/mL, 1310 ng/mL, 1320 ng/mL, 1330 ng/mL, 1340 ng/mL, 1350 ng/mL, 1360 ng/mL, 1370 ng/mL, 1380 ng/mL, 1390 ng/mL, 1400 ng/mL, 1410 ng/mL, 1420 ng/mL, 1430 ng/mL, 1440 ng/mL, 1450 ng/mL, 1460 ng/mL, 1470 ng/mL, 1480 ng/mL, 1490 ng/mL, or 1500 ng/mL. In certain aspects, the normal control concentration of VCAM-1 or reference value is about 100 to about 500 ng/mL. In certain aspect, the amount of VCAM-1 is about 100 ng/mL, 110 ng/mL, 120 ng/mL, 130 ng/mL, 140 ng/mL, 150 ng/mL, 160 ng/mL, 170 ng/mL, 180 ng/mL, 190 ng/mL, 200 ng/mL, 210 ng/mL, 220 ng/mL, 230 ng/mL, 240 ng/mL, 250 ng/mL, 260 ng/mL, 270 ng/mL, 280 ng/mL, 290 ng/mL, 300 ng/mL, 310 ng/mL, 320 ng/mL, 330 ng/mL, 340 ng/mL, 350 ng/mL, 360 ng/mL, 370 ng/mL, 380 ng/mL, 390 ng/mL, 400 ng/mL, 410 ng/mL, 420 ng/mL, 430 ng/mL, 440 ng/mL, 450 ng/mL, 460 ng/mL, 470 ng/mL, 480 ng/mL, 490 ng/mL, or 500 ng/mL. In certain aspects, the concentration of VCAM-1 in the biological sample is deemed elevated when it is at least 10% to about 60% greater than the normal control concentration of VCAM-1. In certain aspects, the concentration of VCAM-1 in the biological sample is deemed elevated when it is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and/or 60% greater than the normal control concentration of VCAM-1. In certain aspects, the concentration of VCAM-1 in the biological sample is deemed elevated when it is at least 550 ng/mL (e.g., at least 560 ng/mL, 570 ng/mL, 580 ng/mL, 590 ng/mL, 600 ng/mL, 610 ng/mL, 620 ng/mL, 630 ng/mL, 640 ng/mL, 650 ng/mL, 660 ng/mL, 670 ng/mL, 680 ng/mL, 690 ng/mL, 700 ng/mL, 710 ng/mL, 720 ng/mL, 730 ng/mL, 740 ng/mL, 750 ng/mL, 760 ng/mL, 770 ng/mL, 780 ng/mL, 790 ng/mL, 800 ng/mL, 810 ng/mL, 820 ng/mL, 830 ng/mL, 840 ng/mL, 850 ng/mL, 860 ng/mL, 870 ng/mL, 880 ng/mL, 890 ng/mL, 900 ng/mL, 910 ng/mL, 920 ng/mL, 930 ng/mL, 940 ng/mL, 950 ng/mL, 960 ng/mL, 970 ng/mL, 980 ng/mL, 990 ng/mL, 1000 ng/mL, 1010 ng/mL, 1020 ng/mL, 1030 ng/mL, 1040 ng/mL, 1050 ng/mL, 1060 ng/mL, 1070 ng/mL, 1080 ng/mL, 1090 ng/mL, 1100 ng/mL, 1110 ng/mL, 1120 ng/mL, 1130 ng/mL, 1140 ng/mL, 1150 ng/mL, 1160 ng/mL, 1170 ng/mL, 1180 ng/mL, 1190 ng/mL, 1200 ng/mL, 1210 ng/mL, 1220 ng/mL, 1230 ng/mL, 1240 ng/mL, 1250 ng/mL, 1260 ng/mL, 1270 ng/mL, 1280 ng/mL, 1290 ng/mL, 1300 ng/mL, 1310 ng/mL, 1320 ng/mL, 1330 ng/mL, 1340 ng/mL, 1350 ng/mL, 1360 ng/mL, 1370 ng/mL, 1380 ng/mL, 1390 ng/mL, 1400 ng/mL, 1410 ng/mL, 1420 ng/mL, 1430 ng/mL, 1440 ng/mL, 1450 ng/mL, 1460 ng/mL, 1470 ng/mL, 1480 ng/mL, 1490 ng/mL, or 1500 ng/mL). In some embodiments, the concentration of VCAM-1 in the biological sample is deemed elevated when it is at least 650 ng/mL (e.g., at least 660 ng/mL, 670 ng/mL, 680 ng/mL, 690 ng/mL, 700 ng/mL, 710 ng/mL, 720 ng/mL, 730 ng/mL, 740 ng/mL, 750 ng/mL, 760 ng/mL, 770 ng/mL, 780 ng/mL, 790 ng/mL, 800 ng/mL, 810 ng/mL, 820 ng/mL, 830 ng/mL, 840 ng/mL, 850 ng/mL, 860 ng/mL, 870 ng/mL, 880 ng/mL, 890 ng/mL, 900 ng/mL, 910 ng/mL, 920 ng/mL, 930 ng/mL, 940 ng/mL, 950 ng/mL, 960 ng/mL, 970 ng/mL, 980 ng/mL, 990 ng/mL, 1000 ng/mL, 1010 ng/mL, 1020 ng/mL, 1030 ng/mL, 1040 ng/mL, 1050 ng/mL, 1060 ng/mL, 1070 ng/mL, 1080 ng/mL, 1090 ng/mL, 1100 ng/mL, 1110 ng/mL, 1120 ng/mL, 1130 ng/mL, 1140 ng/mL, 1150 ng/mL, 1160 ng/mL, 1170 ng/mL, 1180 ng/mL, 1190 ng/mL, 1200 ng/mL, 1210 ng/mL, 1220 ng/mL, 1230 ng/mL, 1240 ng/mL, 1250 ng/mL, 1260 ng/mL, 1270 ng/mL, 1280 ng/mL, 1290 ng/mL, 1300 ng/mL, 1310 ng/mL, 1320 ng/mL, 1330 ng/mL, 1340 ng/mL, 1350 ng/mL, 1360 ng/mL, 1370 ng/mL, 1380 ng/mL, 1390 ng/mL, 1400 ng/mL, 1410 ng/mL, 1420 ng/mL, 1430 ng/mL, 1440 ng/mL, 1450 ng/mL, 1460 ng/mL, 1470 ng/mL, 1480 ng/mL, 1490 ng/mL, or 1500 ng/mL). In some embodiments, the concentration of VCAM-1 in the biological sample is elevated when it is 650 ng/mL to 1500 ng/mL (e.g., 650 ng/mL to 1400 ng/mL, 650 ng/mL to 1300 ng/mL, 650 ng/mL to 1200 ng/mL, 650 ng/mL to 1100 ng/mL, 650 ng/mL to 1000 ng/mL, 650 ng/mL to 900 ng/mL, 650 ng/mL to 800 ng/mL, 650 ng/mL to 700 ng/mL, 700 ng/mL to 1500 ng/mL, 800 ng/mL to 1500 ng/mL, 900 ng/mL to 1500 ng/mL, 1000 ng/mL to 1500 ng/mL, 1100 ng/mL to 1500 ng/mL, 1200 ng/mL to 1500 ng/mL, 1300 ng/mL to 1500 ng/mL, or 1400 ng/mL to 1500 ng/mL). In certain aspects, the methods herein can be used to discriminate between nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH), by measuring a quantity of VCAM-1 contained in blood collected from a subject; and determining that the subject is affected with or possibly affected with NASH in a case that the quantity of VCAM-1 is elevated or larger than a reference value. In certain aspects, the methods herein can be used to determine the presence of fibrosis such as hepatic fibrosis by measuring a quantity of VCAM-1 contained in blood collected from a subject; and determining that the subject has or possibly has a symptom of hepatic fibrosis in a case that the quantity of VCAM-1 is elevated or larger than a reference value. In certain aspects, the method herein can be used to determine a degree of progression of a symptom of nonalcoholic fatty liver disease (NAFLD), by measuring a quantity of VCAM-1 contained in blood collected from a subject as the quantity of VCAM-1 is larger than a reference value. In certain aspects, the methods herein can be used to determine the degree of progression of a symptom of NAFLD, NAFL or NASH by monitoring the level of VCAM-1. IV. Alpha-2-Macroglobulin (A2M) A2M is a plasma protein found in the blood that mainly acts as an antiprotease and is able to inactivate a variety of proteinases. For example, it functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. It functions as an inhibitor of coagulation by inhibiting thrombin. Further, A2M sometimes acts as a carrier protein because it also binds to numerous growth factors and cytokines, such as platelet-derived growth factor, basic fibroblast growth factor, TGF-β, insulin, and IL-1β. A2M is mainly produced by the liver, and also locally synthesized by macrophages, fibroblasts, and adrenocortical cells. In humans it is encoded by the A2M gene. A2M HUMAN, accession P01023, is SEQ ID NO: 2. A2M has 1474 amino acids and a mass of approximately 720 kDa. The human A2M mRNA (coding) sequence is set forth in, e.g., Genbank Accession No. NM_000014.5. One skilled in the art will appreciate that A2M is also known as A2MD, CPAMD5, FWP007, S863-7, or transcuprein. In certain aspects, the methods described herein are used to measure and/or detect A2M. In certain aspects, the concentration or level of A2M is measured. In certain aspects, the biological sample in which A2M is measured is whole blood. In certain aspects, the concentration of A2M is about 0.1 mg/mL to about 10 mg/mL. In some embodiments, the concentration of A2M is about 0.09 mg/mL, 0.1 mg/mL, 0.11 mg/mL, 0.12 mg/mL, 0.14 mg/mL, 0.16 mg/mL, 0.18 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL, 1.2 mg/mL, 1.4 mg/mL, 1.6 mg/mL, 1.8 mg/mL, 2 mg/mL, 2.2 mg/mL, 2.4 mg/mL, 2.6 mg/mL, 2.8 mg/mL, 3 mg/mL, 3.2 mg/mL, 3.4 mg/mL, 3.6 mg/mL, 3.8 mg/mL, 4 mg/mL, 4.2 mg/mL, 4.4 mg/mL, 4.6 mg/mL, 4.8 mg/mL, 5 mg/mL, 5.2 mg/mL, 5.4 mg/mL, 5.6 mg/mL, 5.8 mg/mL, 6 mg/mL, 6.2 mg/mL, 6.4 mg/mL, 6.6 mg/mL, 6.8 mg/mL, 7 mg/mL, 7.2 mg/mL, 7.4 mg/mL, 7.6 mg/mL, 7.8 mg/mL, 8 mg/mL, 8.2 mg/mL, 8.4 mg/mL, 8.6 mg/mL, 8.8 mg/mL, 9 mg/mL, 9.2 mg/mL, 9.4 mg/mL, 9.6 mg/mL, 9.8 mg/mL, or 10 mg/mL. In certain aspects, the normal control concentration of A2M or reference value is about 1 mg/mL to about 5 mg/mL. In some embodiments, the normal control concentration of A2M is about 0.9 mg/mL, 1 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.4 mg/mL, 1.6 mg/mL, 1.8 mg/mL, 2 mg/mL, 2.2 mg/mL, 2.4 mg/mL, 2.6 mg/mL, 2.8 mg/mL, 3 mg/mL, 3.2 mg/mL, 3.4 mg/mL, 3.6 mg/mL, 3.8 mg/mL, 4 mg/mL, 4.2 mg/mL, 4.4 mg/mL, 4.6 mg/mL, 4.8 mg/mL, or 5 mg/mL. In certain aspects, the concentration of A2M in the biological sample is deemed elevated when it is at least 10% to about 60% greater than the normal control concentration of A2M. In certain aspects, the concentration of A2M in the biological sample is deemed elevated when it is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and/or 60% greater than the normal control concentration of A2M. In certain aspects, the concentration of A2M in the biological sample is deemed elevated when it is at least 5.5 mg/mL (e.g., a least 5.6 mg/mL, 5.7 mg/mL, 5.8 mg/mL, 5.9 mg/mL, 6 mg/mL, 6.5 mg/mL, 7 mg/mL, 7.5 mg/mL, 8 mg/mL, 8.5 mg/mL, 9 mg/mL, 9.5 mg/mL, or 10 mg/mL). In some embodiments, the concentration of A2M in the biological sample is deemed elevated when it is at least 6.5 mg/mL (e.g., a least 6.6 mg/mL, 6.7 mg/mL, 6.8 mg/mL, 6.9 mg/mL, 7 mg/mL, 7.5 mg/mL, 8 mg/mL, 8.5 mg/mL, 9 mg/mL, 9.5 mg/mL, or 10 mg/mL). In certain aspects, the methods herein can be used to discriminate between nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH), by measuring a quantity of A2M contained in blood collected from a subject; and determining that the subject is affected with or possibly affected with NASH in a case that the quantity of A2M is elevated or larger than a reference value. In certain aspects, the methods herein can be used to determine the presence of fibrosis such as hepatic fibrosis by measuring a quantity of A2M contained in blood collected from a subject; and determining that the subject has or possibly has a symptom of hepatic fibrosis in a case that the quantity of A2M is elevated or larger than a reference value. In certain aspects, the method herein can be used to determine a degree of progression of a symptom of nonalcoholic fatty liver disease (NAFLD), by measuring a quantity of A2M contained in blood collected from a subject as the quantity of A2M is larger than a reference value. In certain aspects, the methods herein can be used to determine the degree of progression of a symptom of NAFLD, NAFL or NASH by monitoring the level of A2M. V. Device Various instruments and devices are suitable for use in the present disclosure. Many spectrophotometers have the capability to measure fluorescence. Fluorescence is the molecular absorption of light energy at one wavelength and its nearly instantaneous re-emission at another, longer wavelength. Some molecules fluoresce naturally, and others must be modified to fluoresce. A fluorescence spectrophotometer or fluorometer, fluorospectrometer, or fluorescence spectrometer measures the fluorescent light emitted from a sample at different wavelengths, after illumination with light source such as a xenon flash lamp. Fluorometers can have different channels for measuring differently-colored fluorescent signals (that differ in their wavelengths), such as green and blue, or ultraviolet and blue, channels. In one aspect, a suitable device includes an ability to perform a time-resolved fluorescence resonance energy transfer (FRET) experiment. Suitable fluorometers can hold samples in different ways, including cuvettes, capillaries, Petri dishes, and microplates. The assays described herein can be performed on any of these types of instruments. In certain aspects, the device has an optional microplate reader, allowing emission scans in up to 384-well plates. Others models suitable for use hold the sample in place using surface tension. Time-resolved fluorescence (TRF) measurement is similar to fluorescence intensity measurement. One difference, however, is the timing of the excitation/measurement process. When measuring fluorescence intensity, the excitation and emission processes are simultaneous: the light emitted by the sample is measured while excitation is taking place. Even though emission systems are very efficient at removing excitation light before it reaches the detector, the amount of excitation light compared to emission light is such that fluorescent intensity measurements exhibit elevated background signals. The present disclosure offers a solution to this issue. Time resolve FRET relies on the use of specific fluorescent molecules that have the property of emitting over long periods of time (measured in milliseconds) after excitation, when most standard fluorescent dyes (e.g., fluorescein) emit within a few nanoseconds of being excited. As a result, it is possible to excite cryptate lanthanides using a pulsed light source (e.g., Xenon flash lamp or pulsed laser), and measure after the excitation pulse. As the donor and acceptor fluorescent compounds attached to the antibodies move closer together, an energy transfer is caused from the donor compound to the acceptor compound, resulting in a decrease in the fluorescence signal emitted by the donor compound and an increase in the signal emitted by the acceptor compound, and vice-versa. The majority of biological phenomena involving interactions between different partners will therefore be able to be studied by measuring the change in FRET between two fluorescent compounds coupled with compounds which will be at a greater or lesser distance, depending on the biological phenomenon in question. The FRET signal can be measured in different ways: measurement of the fluorescence emitted by the donor alone, by the acceptor alone or by the donor and the acceptor, or measurement of the variation in the polarization of the light emitted in the medium by the acceptor as a result of FRET. One can also include measurement of FRET by observing the variation in the lifetime of the donor, which is facilitated by using a donor with a long fluorescence lifetime, such as rare earth complexes (especially on simple equipment like plate readers). Furthermore, the FRET signal can be measured at a precise instant or at regular intervals, making it possible to study its change over time and thereby to investigate the kinetics of the biological process studied. In certain aspects, the device disclosed in PCT/IB2019/051213, filed Feb. 14, 2019 is used, which is hereby incorporated by reference. That disclosure in that application generally relates to analyzers that can be used in point-of-care settings to measure the absorbance and fluorescence of a sample with minimal or no user handling or interaction. The disclosed analyzers provide advantageous features of more rapid and reliable analyses of samples having properties that can be detected with each of these two approaches. For example, it can be beneficial to quantify both the fluorescence and absorbance of a blood sample being subjected to a diagnostic assay. In some analytical workflows, the hematocrit of a blood sample can be quantified with an absorbance assay, while the signal intensities measured in a FRET assay can provide information regarding other components of the blood sample. One apparatus disclosed in PCT/IB2019/051213 is useful for detecting an emission light from a sample, and absorbance of a transillumination light by the sample, which comprises a first light source configured to emit an excitation light having an excitation wavelength. The apparatus further comprises a second light source configured to transilluminate the sample with the transillumination light. The apparatus further comprises a first light detector configured to detect the excitation light, and a second light detector configured to detect the emission light and the transillumination light. The apparatus further comprises a dichroic mirror configured to (1) epi-illuminate the sample by reflecting at least a portion of the excitation light, (2) transmit at least a portion of the excitation light to the first light detector, (3) transmit at least a portion of the emission light to the second light detector, and (4) transmit at least a portion of the transillumination light to the second light detector. One suitable cuvette for use in the present disclosure is disclosed in PCT/IB2019/051215, filed Feb. 14, 2019. One of the provided cuvettes comprises a hollow body enclosing an inner chamber having an open chamber top. The cuvette further comprises a lower lid having an inner wall, an outer wall, an open lid top, and an open lid bottom. At least a portion of the lower lid is configured to fit inside the inner chamber proximate to the open chamber top. The lower lid comprises one or more (e.g., two or more) containers connected to the inner wall, wherein each of the containers has an open container top. In certain aspects, the lower lid comprises two or more such containers. The lower lid further comprises a securing means connected to the hollow body. The cuvette further comprises an upper lid wherein at least a portion of the upper lid is configured to fit inside the lower lid proximate to the open lid top. VI. Example This example illustrates a method of this disclosure for detecting the presence and amounts of VCAM-1 and A2M in a TR-FRET assay. As shown inFIG.1, VCAM-1 binds to an anti-VCAM-1 antibody (MAB-1) labeled with a donor fluorophore and a second anti-VCAM-1 antibody (MAB-2) labeled with an acceptor fluorophore (NIR acceptor). An isolated A2M protein labeled with an acceptor fluorophore (Green acceptor) binds to an anti-A2M antibody (MAB) labeled with a donor fluorophore. The VCAM-1 analyte is in a sample from a patient (i.e., whole blood sample) and it binds to both anti-VCAM-1 antibodies simultaneously resulting in a FRET signal. The A2M analyte in a sample from a patient (i.e., whole blood sample) competes with the isolated A2M protein for binding to the anti-A2M antibody, thus, disrupting the FRET signal from the anti-A2M antibody labeled with the donor fluorophore and the isolated A2M protein labeled with the acceptor fluorophore. If one anti-VCAM-1 antibody is labeled with a donor fluorophore and a second anti-VCAM-1 antibody is labeled with an acceptor fluorophore, and an anti-A2M antibody is labeled with a donor fluorophore and an isolated A2M protein is labeled with an acceptor fluorophore, in which the two acceptor fluorophores are different, TR-FRET can occur in the presence of the VCAM-1 and the FRET signal would disappear in the presence of the A2M in the sample (FIG.1). The increase in FRET signal of the acceptor fluorophore on the anti-VCAM-1 antibody is proportional to the level of VCAM-1 present in the patient's blood as interpolated from a known amount of VCAM-1 calibrator (FIGS.2A-2D). Table 1 below shows the corresponding numerical data forFIG.2Cand Table 2 below shows the corresponding numerical data forFIG.2D. The FRET signal of the acceptor fluorophore on the anti-A2M antibody or the isolated A2M protein is inversely proportional to the level of A2M present in the patient's blood as interpolated from a known amount of A2M calibrator (FIGS.2E-2G). Table 3 below shows the corresponding average numerical data forFIG.2G, which includes 4 replicates. TABLE 1STDS [ng/mL]Delta F %20004271500351100023750011920049801440800 TABLE 2STDS [ng/mL]Delta F %200050115003831000266500129200568022401100 TABLE 3Conc.(ug/mL)F/B MeanF/B Std% CV40000.3930.0092.17%20000.5320.0071.36%10000.6660.0101.47%5000.7950.0070.93%2000.9050.0131.39%01.0000.0000.00% A multiplex assay was also performed to generate standard curves for VCAM-1 and A2M simultaneously in whole blood (FIGS.2H and2I). Three separate calibration curves were prepared: 1) VCAM-1 only 2) A2M only 3) VCAM-1 and A2M combined. The concentration of both VCAM-1 and A2M were held constant between the individual calibration curves and the combined curve. All three calibration curves were tested at 5 and 30 minutes. Table 4 below shows the corresponding numerical data forFIG.2Hand Table 5 below shows the corresponding numerical data forFIG.2I. TABLE 4MultiplexSingleplexMultiplexSingleplexVCAM-1Cals atCals atCals atCals at(ng/mL)5 mins5 mins30 mins30 mins2000308%291%276%300%1000164%145%160%147%50095%81%81%80%25051%41%47%42%10018%19%17%19%00%0%0%0% TABLE 5MultiplexSingleplexMultiplexSingleplexA2MCals atCals atCals atCals at(μg/mL)5 mins5 mins30 mins30 mins40000.340.330.330.3320000.450.430.450.4310000.580.580.560.595000.710.720.740.712000.830.880.870.8801.001.001.001.00 FIGS.2J and2Killustrate that when A2M and VCAM-1 are multiplexed, the results are comparable to the single plex assay.FIG.2Jshows that the multiplexed VCAM-1 results overlay with the single plex VCAM-1 results while A2M results do not show a dose response. Similarly,FIG.2Kshows that the multiplexed A2M results overlay with the single plex A2M results while VCAM-1 results do not show a dose response. Table 6 below shows the corresponding numerical data forFIGS.2J and2K. TABLE 6TargetedBackCalc.TargetedBackCalc.VCAM1VCAM1A2MVCAM1ID(ng/mL)(ng/mL)Recovery(ug/mL)(ng/mL)RecoveryVCAM120002074.4104%0N/AN/ACal 1VCAM11000948.995%0NAN/ACal 2VCAM1500489.398%02.57N/ACal 3VCAM1250246.999%0N/AN/ACal 4VCAM110095.295%0N/AN/ACal 5VCAM10N/AN/A0N/AN/ACal 6A2M01.3N/A40004076.5102%Cal 1A2M0N/AN/A20001947.297%Cal 2A2M0N/AN/A1000987.999%Cal 3A2M02.9N/A500536.8107%Cal 4A2M03.8N/A250245.998%Cal 5A2M05.2N/A0N/AN/ACal 6 Donor fluorophore, Lumi4-Tb (also called Tb-H22TRENIAM-5LIO-NHS,FIG.3), can be used to label an anti-VCAM-1 antibody, an anti-A2M antibody, or an isolated A2M protein. Lumi4-Tb has 3 spectrally distinct peaks, at 490, 550, and 620 nm, which can be used for energy transfer (FIG.5). The acceptor fluorophores that can be used include but are not limited to: AlexaFluor 488, AlexaFluor 546, and AlexaFluor 647 (FIG.5). Donor and acceptor fluorophores can be conjugated to antibodies using primary amines on antibodies. The sequence of human VCAM-1 (accession P19320) is shown below: SEQ ID NO: 1:MPGKMVVILGASNILWIMFAASQAFKIETTPESRYLAQIGDSVSLTCSTTGCESPFFSWRTQIDSPLNGKVTNEGTTSTLTMNPVSFGNEHSYLCTATCESRKLEKGIQVEIYSFPKDPEIHLSGPLEAGKPITVKCSVADVYPFDRLEIDLLKGDHLMKSQEFLEDADRKSLETKSLEVTFTPVIEDIGKVLVCRAKLHIDEMDSVPTVRQAVKELQVYISPKNTVISVNPSTKLQEGGSVTMTCSSEGLPAPEIFWSKKLDNGNLQHLSGNATLTLIAMRMEDSGIYVCEGVNLIGKNRKEVELIVQEKPFTVEISPGPRIAAQIGDSVMLTCSVMGCESPSFSWRTQIDSPLSGKVRSEGTNSTLTLSPVSFENEHSYLCTVTCGHKKLEKGIQVELYSFPRDPEIEMSGGLVNGSSVTVSCKVPSVYPLDRLEIELLKGETILENIEFLEDTDMKSLENKSLEMTFIPTIEDTGKALVCQAKLHIDDMEFEPKQRQSTQTLYVNVAPRDTTVLVSPSSILEEGSSVNMTCLSQGFPAPKILWSRQLPNGELQPLSENATLTLISTKMEDSGVYLCEGINQAGRSRKEVELIIQVTPKDIKLTAFPSESVKEGDTVIISCTCGNVPETWIILKKKAETGDTVLKSIDGAYTIRKAQLKDAGVYECESKNKVGSQLRSLTLDVQGRENNKDYFSPELLVLYFASSLIIPAIGMIIYFARKANMKGSYSLVEAQKSKV The sequence of human A2M (accession P01023) is shown below: SEQ ID NO: 2:MGKNKLLHPSLVLLLLVLLPTDASVSGKPQYMVLVPSLLHTETTEKGCVLLSYLNETVTVSASLESVRGNRSLFTDLEAENDVLHCVAFAVPKSSSNEEVMFLTVQVKGPTQEFKKRTTVMVKNEDSLVFVQTDKSIYKPGQTVKFRVVSMDENFHPLNELIPLVYIQDPKGNRIAQWQSFQLEGGLKQFSFPLSSEPFQGSYKVVVQKKSGGRTEHPFTVEEFVLPKFEVQVTVPKIITILEEEMNVSVCGLYTYGKPVPGHVTVSICRKYSDASDCHGEDSQAFCEKFSGQLNSHGCFYQQVKTKVFQLKRKEYEMKLHTEAQIQEEGTVVELTGRQSSEITRTITKLSFVKVDSHFRQGIPFFGQVRLVDGKGVPIPNKVIFIRGNEANYYSNATTDEHGLVQFSINTTNVMGTSLTVRVNYKDRSPCYGYQWVSEEHEEAHHTAYLVFSPSKSFVHLEPMSHELPCGHTQTVQAHYILNGGTLLGLKKLSFYYLIMAKGGIVRTGTHGLLVKQEDMKGHFSISIPVKSDIAPVARLLIYAVLPTGDVIGDSAKYDVENCLANKVDLSFSPSQSLPASHAHLRVTAAPQSVCALRAVDQSVLLMKPDAELSASSVYNLLPEKDLTGFPGPLNDQDNEDCINRHNVYINGITYTPVSSTNEKDMYSFLEDMGLKAFTNSKIRKPKMCPQLQQYEMHGPEGLRVGFYESDVMGRGHARLVHVEEPHTETVRKYFPETWIWDLVVVNSAGVAEVGVTVPDTITEWKAGAFCLSEDAGLGISSTASLRAFQPFFVELTMPYSVIRGEAFTLKATVLNYLPKCIRVSVQLEASPAFLAVPVEKEQAPHCICANGRQTVSWAVTPKSLGNVNFTVSAEALESQELCGTEVPSVPEHGRKDTVIKPLLVEPEGLEKETTFNSLLCPSGGEVSEELSLKLPPNVVEESARASVSVLGDILGSAMQNTQNLLQMPYGCGEQNMVLFAPNIYVLDYLNETQQLTPEIKSKAIGYLNTGYQRQLNYKHYDGSYSTFGERYGRNQGNTWLTAFVLKTFAQARAYIFIDEAHITQALIWLSQRQKDNGCFRSSGSLLNNAIKGGVEDEVTLSAYITIALLEIPLTVTHPVVRNALFCLESAWKTAQEGDHGSHVYTKALLAYAFALAGNQDKRKEVLKSLNEEAVKKDNSVHWERPQKPKAPVGHFYEPQAPSAEVEMTSYVLLAYLTAQPAPTSEDLTSATNIVKWITKQQNAQGGFSSTQDTVVALHALSKYGAATFTRTGKAAQVTIQSSGTFSSKFQVDNNNRLLLQQVSLPELPGEYSMKVTGEGCVYLQTSLKYNILPEKEEFPFALGVQTLPQTCDEPKAHTSFQISLSVSYTGSRSASNMAIVDVKMVSGFIPLKPTVKMLERSNHVSRTEVSSNHVLIYLDKVSNQTLSLFFTVLQDVPVRDLKPAIVKVYDYYETDEFAIAEYNAPCSKDLGNA | 75,631 |
11860171 | DETAILED DESCRIPTION OF THE INVENTION To describe the present invention more specifically, the technical solutions of the present invention will be described in detail with reference to the accompanying drawings and detailed embodiments. The description merely indicates how the present invention is achieved, but is not construed as limiting the specific scope of the present invention. The scope of the present invention is defined by the claims. Example 1: Accurate Detection of Each Marker I. Solution Preparation Preparation of the mixed working solution of a calibration product and a quality control product is shown in Table 1: TABLE 1Preparation of the mixed working solution of a calibrationproduct and a quality control productVolumeVolumeConcentrationof theConcentrationof theConcentrationof theaqueousof theaqueousof theprimarysolutionsecondarysolutionmixedstockof 50%stockof 50%workingsolutionVolumemethanolsolutionVolumemethanolsolutionAnalytemg/mlμlμlmg/mlμlμlμg/mlAngiotensin I1100900100506755Angiotensin II11099010250.25Aldosterone11099010250.25Cortisol1——1000252518-hydro-0.110090010200.2corticosterone Preparation and Concentration Gradient of the Calibration Product are Shown in Table 2: TABLE 2Preparation of the calibration productVolumeName ofof theVolume18OH-theworkingofCorti-workingsolutionmatrixAng IAng IIAldosteroneCottisolcosteronepg/mlsolutionμLμL(ng/mL)(pg/mL)(pg/ml)(ng/mL)(pg/ml)LLMICalibration159850.315151.512productS5CalibrationCalibration159850.315151.512productproductS1S5CalibrationCalibration37.5962.50.7537.537.53.7530productproductS2S5CalibrationCalibration159851.575757.560productproductS3S7CalibrationCalibration50950525025025200productproductS4S7CalibrationCalibration2008002010001000100800productproductS5S7CalibrationMixed1099050250025002502000productworkingS6solutionCalibrationMixed20980100500050005004000productworkingS7solutionNote:the matrix for the preparation of the calibration product is a PBS (1X) buffer solution containing 4% BSA, stored at 2-8° C. Preparation of the quality control product is shown in Table 3: TABLE 3Preparation of the quality control productVolume of theName of theworkingName ofVolume ofworking solutionsolution μLmatrixmatrix μlPreparationQuality controlCalibration20Plasma980productproduct Lproduct S5of theQuality controlQuality control100Quality1100qualityproduct Mproduct Hcontrolcontrolproduct LQuality controlMixed working6.6Quality993.4product Hsolutioncontrolproduct LNote:the matrix of the quality control product is blood plasma which is subpackaged and frozen immediately after being prepared. Preparation of the internal standard working solution is shown in Table 4: TABLE 4Preparation of the internal standard working solutionConcentrationVolumeVolume ofof theConcentrationof theConcentrationthemixedof theaqueousof theaqueousinternalprimarysolutionsecondarysolutionstandardstockwith 50%stockwith 50%workingsolutionVolumemethanolsolutionVolumemethanolsolutionAnalytemg/mlμlμlmg/mlμlμlng/mlAngiotensin0.110090010501000050I-ISAngiotensin0.1109901252.5II-ISAldosterone-d70.1109901252.5Cortisol-d411009001002020018-hydrocorti-0.11009001252.5costerone-d4 Preparation of Other Solution Preparation of phenylmethylsulfonyl fluoride (PMSF): 0.174 g PMSF was taken and added to 10 mL methanol and prepared into a 100 mM PMSF methanol solution; storage condition was 2-8° C. Buffer formation solution: 12.11 g TRIS and 7.4 g ethylene diamine tetraacetic acid (EDTA) were added to a 100 mL volumetric flask, and added with deionized water to 90 mL, and then subjected to ultrasonic treatment for 30 min to be evenly dissolved. Deionized water was added to the scale line and mixed well. The solution was transferred to a reservoir vessel made of polypropylene. The PH value of the solution was adjusted within 5.45-5.60, and then the solution was stored at −20° C. Buffer formation solution A: the solution was prepared at the same day of the detection analysis; 100 μL of 100 mM PMSF (angiotensin converting enzyme inhibitor) solution was added to 10 mL of the buffer formation solution, thus preparing the buffer formation solution A (pH value ranges from 5.4 to 5.6). Stop buffer containing internal standard: 1 mL of the internal standard working solution was mixed with 9 mL water and 250 μL formic acid into the stop buffer containing internal standard. II. Sample Pretreatment 1. Sample thawing: a plasma sample to be tested and a quality control sample to be tested were placed into ice water (0° C.) for thawing till melted. 2. Sampling: 50 μL of the buffer formation solution A was added to a 96-well plate, and 400 μL of the calibration product, quality control product and quality control sample to be tested were taken and added to two plates prepared in the step (1), and then the remaining sample was immediately frozen. 3. Sample incubation: the sample in the step 2 was sealed with a silicone pad and subjected to vortex treatment for a short period of time, and then put to a 37° C. water bath for 3 h, 3 h later, 400 μL of the stop buffer containing internal standard was added, and the mixed solution was centrifuged for 1 min at 4° C. and 4000 rpm; 800 μL supernatant was taken and added to an automatic magnetic bead extractor, ready for sample extraction. 4. Magnetic bead extraction: additives of each column in the adaptive 96-well plate of the magnetic bead extractor are shown in Table 5: TABLE 5Additives of each column in the adaptive 96-well plateof the magnetic bead extractorFirstSecondSecondFourthFifthSixthcolumncolumncolumncolumncolumncolumn(7)(8)(9)(10)(11)(12)ActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solution The magnetic bead is the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (Biosepur, Art.No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A). The activating agent is a solution of 50% ethanol including the magnetic bead; the balanced solution is an aqueous solution containing 1% formic acid; the washing liquid 1 is an aqueous solution containing 10% methanol; the washing liquid 2 is isooctane, and the eluent solution is an aqueous solution containing 50% methanol. 5. The sample pretreatment step of the magnetic bead extractor is shown in Table 6; the pretreatment time of each batch of samples is about 10 min. TABLE 6Sample pretreatment step of the magnetic bead extractorMixingSolventMagneticColumns of thetimeamountabsorption timeNo.Instruction96-well plate(S)(μl)(S)1Activating1 (7)60300302Activating2 (8)60300303Sampling3 (9)90800304Drip washing4 (10)60300305Drip washing5 (11)60300306Eluting6 (12)60100307Waste1 (7)101000discharge 6. After the extraction was completed by the magnetic bead extractor, the solution to be tested in the columns 6 and 12 of the 96-well plate was transferred to the 96-well loading plate for detection on the machine. The existing magnetic bead extractor may accommodate two 96-well plates for parallel operation for one time. Therefore, the pretreatment flux is 32 samples/batch; a 96-channel magnetic bead extractor may be also available; and the extraction steps are kept same. III. Sample Detection In the process of the liquid chromatography tandem-mass spectrometry, gradient elution was applied in the liquid chromatography; separation conditions of the object to be detected for the reversed phase chromatography were established as follows: chromatographic column was a Phenomenex C8 chromatographic column; flow rate was 0.4 mL/min; column temperature was 40° C.; where the mobile phase A was an aqueous solution containing 1 mM ammonium fluoride, and the mobile phase B was a methanol solution containing 1 mM ammonium fluoride (with 5% isopropanol); the volume ratio of the mobile phase A to the mobile phase B was 90-5%:10-95%. The gradient elution program is shown in Table 7. TABLE 7Gradient elution programTimeFlow rateMobile phase AMobile phase B(min)(mL/min)(%)(%)00.480200.30.480200.60.450503.30.450503.40.45954.20.45954.30.480204.80.48020 During the mass spectrometric detection, quantitative detection was performed by a triple quadrupole mass spectrometer with a model of CalQuant-S independently developed and researched by CALIBAR. The mass spectrometry was performed by a positive/negative ion mode (ESI+) of an ESI source and a multi-reaction monitoring MRM mode. The corresponding mass spectrometry is shown in Table 8, and the mass spectrometry conditions are shown in Table 9: TABLE 8Mass spectrometryQ1Q3DWELLIDDPCECXP433.1619.230Angiotensin I-1139308433.1647.45Angiotensin I-2145257437.3660.530Angiotensin I-IS-21202416437.3631.15Angiotensin I-IS-21303010523.9263.430Angiotensin II-1140317523.9784.35Angiotensin II-2140288527.3263.330Angiotensin II-IS-1140317527.3791.45Angiotensin II-IS-2140287363.3121.11018-hydrocorticosterone-11404010363.3269.21018-hydrocorticosterone-21403810367.2121.11018-hydrocorticosterone-IS-11404010367.2273.21018-hydrocorticosterone-IS-21403810359.2188.925Aldosterone-NEG-1−125−26−8359.2331.310Aldosterone-NEG-2−125−23−8367.2194.225Aldosterone-NEG-IS-1−125−26−8367.2339.410Aldosterone-NEG-IS-2−125−23−8363.2309.230Cortisol-1175258363.2121.215Cortisol-2175328367.2313.330Cortisol-IS-1175258367.2121.215Cortisol-IS-2175328 TABLE 9Mass spectrometry conditionsMass spectrometry conditionsValueCurtain gas CUR25 psiAtomized gas GS155 psiAuxiliary heating gas GS255 psiIon source heating temperature500° C.Collision gas CAD10 psiSpray voltage5500 V/−4500 V A standard curve was established by the internal standard method. Records of validation on the linear relation is shown in Table 10 with a measuring unit of ng/mL. TABLE 10Results of validation on the standard curveCoefficient ofClinical linearCompoundRegression equationWeightassociation rrangeAngiotensin IY = 0.00957X + 0.0008361/X20.9970.3-100 ng/mlAngiotensin IIY = 0.0000762593X + 0.003031/X20.99615-5000 pg/mlAldosteroneY = 0.00250X − 0.030291/X20.99715-5000 pg/mlCortisolY = 0.04826X + 0.139741/X20.9981.5-500 ng/ml18-hydrocorticosteroneY = 0.000956X + 0.078011/X20.99912-4000 pg/ml The standard curve was formulated by the PBS buffer solution matrix containing 4% BSA and subjected to synchronous treatment with the sample to be tested for detection. The test chromatogram is shown inFIG.1, representing angiotensin I (Ang I), angiotensin II (Ang II), 18-hydrocorticosterone (18-OH CORT), aldosterone (Aldo) and Cortisol from top to bottom in order. As can be seen fromFIG.1, the method provided by the example may accurately detect the 5 markers simultaneously. By the validation on accuracy and precision (Table 11), the detection linear relation is good within the scope of concentration. TABLE 11Accuracy and precision validated by the methodSampleTheoreticalMeasuredIm-CompoundsizeconcentrationvalueAccuracyprecisionAngiotensin61.8 ng/ml1.79 ng/ml99.44%2.50%I4.8 ng/ml4.81 ng/ml100.2%2.08%34.8 ng/ml34.5 ng/ml99.13%1.58%Angiotensin650 pg/ml49.3 pg/ml98.60%1.42%II200 pg/ml198 pg/ml99.00%3.08%1700 pg/ml1702 pg/ml100.1%2.12%Aldosterone650 pg/ml50.3 pg/ml100.6%3.00%200 pg/ml198 pg/ml99.20%3.47%1700 pg/ml1693 pg/ml90.58%1.13%Cortisol650 ng/ml50.7 ng/ml101.40%2.46%65 ng/ml65.6 ng/ml100.92%1.12%215 ng/ml214 ng/ml99.53%3.46%18-6160 pg/ml159 ng/ml99.38%2.38%hydrocorti-280 pg/ml289 pg/ml104%2.01%costerone1480 pg/ml1489 pg/ml100.6%3.20% Example 2: Comparison of the Extraction Effect Between the Magnetic Bead Bonded with a Balanced Hydrophilic-Lipophilic Polymer and the Solid Phase Extraction Column The preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. The extraction was performed respectively by the different three methods: 1, extraction by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80A); 2, extraction by PEP 96 Well Microplates of the SPE plate filled with a balanced hydrophilic-lipophilic polymer; 3, extraction by PEP 96 Waters Oasis HLB of the SPE plate filled with a balanced hydrophilic-lipophilic polymer; after elution, the sample was subjected to liquid chromatography tandem-mass spectrometry; the test result of the treated sample was surveyed to measure the peak areas of the 5 markers in the sample S1, as shown in Table 12. TABLE 12Effects of different treatment methods on theextraction effects of the markersHLBPEP 96Waters OasismagneticWellHLBMarker/Treatment methodbeadMicroplates96 WellplatesAngiotensin IPeak area646058765031(theoreticalconcentration:0.3 ng/ml)Angiotensin IIPeak area896774627270(theoreticalconcentration:15 pg/ml)AldosteronePeak area456336873852(theoreticalconcentration:15 pg/ml)CortisolPeak area1636531457314135(theoreticalconcentration:1.5 ng/ml)18-Hydro-Peak area1019821820corticosterone(theoreticalconcentration:12 pg/ml) As can be seen from Table 12, different sample treatment methods will affect the extraction results of the 5 markers in the sample; compared with the SPE plate embedded the balanced hydrophilic-lipophilic polymer, the magnetic bead has more significant extraction effect on the 5 markers. Meanwhile, the two SPE plates of balanced hydrophilic-lipophilic polymer are compared, and there is a difference in effects to some extent; PEP 96 Well Microplates of the SPE plate are superior to the Waters Oasis HLB 96 Wellplates; compared with the two SPE plates, the HLB magnetic bead has significantly improved extraction effect on the 5 markers. Moreover, the solid phase extraction is featured by easy blocking, complex operation and too stronger matrix effect, and the like. Therefore, the method of magnetic bead is applied. The magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof is used to adsorb the 5 markers, and then the magnetic bead extractor may extract the magnetic bead which absorbs the markers from the biological sample matrix, thus effectively removing the interference elements in the biological sample. Therefore, after extraction and enrichment of the magnetic bead, compared with the solid phase extraction column, the eluent solution extracted by the magnetic bead has more purified components; the markers have smaller disturbing influences in the detection process; the mass spectrometry ionization efficiency is higher, and better detection sensitivity may be achieved. Example 3: Effects of the Magnetic Bead Bonded Different Polymer Materials on the Extraction Effects of the Markers In this example, the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. Based on the search result of the literature on the single and separate detection of the 5 different markers: the mixed anion exchange polymer SPE may be used in the sample pretreatment of the angiotensin detection. Therefore, comparisons were further made on the solid phase extraction column bonded a mixed anion exchange polymer (AgelaCleanert PAX 96 Wellplates) filler, the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A), the magnetic bead bonded a mixed anion exchange polymer (MAX magnetic bead, Biosepur, Art.No.: BNMA1430-SY; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A), and the magnetic bead bonded a mixed cation exchange polymer (MCX magnetic bead, Biosepur, Art.No.: BNMA8300001-0-P; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A) in this example. The 5 markers were adsorbed and extracted in the 4 ways, and after elution, the sample was subjected to liquid chromatography tandem-mass spectrometry; the test result of the treated sample was surveyed to measure the peak areas of the 5 markers in the sample S1, as shown in Table 13. TABLE 13Effects of the magnetic bead bonded different materials on theextraction effects of the markersCleanertHLBMAXMCXMarker/Magnetic beadPAX 96magneticmagneticmagneticbonding materialWellplatesbeadbeadbeadAngiotensin IPeak4961646052614884(theoreticalareaconcentration:0.3 ng/ml)Angiotensin IIPeak7230896772417067(theoreticalareaconcentration:15 pg/ml)AldosteronePeak2134456341343242(theoreticalareaconcentration:15 pg/ml)CortisolPeak145467163653145349148580(theoreticalareaconcentration:1.5 ng/ml)18-Hydro-Peak3821019682666corticosteronearea(theoreticalconcentration: 12pg/ml) As can be seen from Table 13, in the same way of being bonded the anionic polymer, the extraction effect of the MAX magnetic bead is obviously superior to that of the AgelaCleanert PAX 96 Wellplates solid phase extraction column; meanwhile, different materials are bonded on the surface of the magnetic bead, which has a large impact on the extraction effects of the 5 markers in the blood sample. Comparisons are made on the magnetic beads bonded the three fillers of the hydrophilic-lipophilic polymer, the mixed anion exchange polymer and the mixed cation exchange polymer; among them, the magnetic bead bonded the hydrophilic-lipophilic polymer may significantly improve the extraction effect on a portion of low-content indexes (aldosterone and 18-hydrocorticosterone) and may achieve better balance on the extraction and enrichment effects of the 5 markers with greater differences in physical and chemical properties. The reason is probably as follows: the balanced hydrophilic-lipophilic polymer may not only achieve the adsorption on high polar compounds, but also may effectively adsorb the low polar compounds; but anionic and cationic polymers have stronger adsorptive selectivity and thus, only selectively achieve the adsorption on high polar anions or cations, and hardly achieve the adsorption on low polar compounds simultaneously, leading to a low extraction efficiency of small molecules such as, aldosterone and 18-hydrocorticosterone, incapable of achieving the sensitivity requirements of clinical test. Example 4: Comparison of the Extraction Process Between the Magnetic Bead and the Solid Phase Extraction Column When a biological sample was pretreated by the conventional solid phase extraction column, the 96-well SPE plate (Agela PEP 96 Well Microplates) was firstly extracted by the solid phase extraction column for activation and balance, and then the biological sample solution to be tested was loaded on the SPE plates; the target compound was extracted and adsorbed by a PEP filler selectively; a portion of inorganic salt and other impurities were washed from the SPE column via drip washing, and then the target component was eluted from the extraction column by an eluting agent with stronger binding capacity to the solid phase, afterwards, the eluent solution was blown-dried with nitrogen gas, redissolved and loaded for sample detection. The operation process has more manual steps, is complex and time-consuming; it takes about 2 h to treat a batch of samples. Moreover, the inter-well extraction efficiency of the sample greatly varies from the difference of the biological sample matrix; all the indexes need to be calibrated via isotope internal standards. Further, due to the specificity of the biological sample matrix, partial samples (in especial hemolysis, lipemia and other samples) may cause the blocking of the SPE column, leading to reduced pretreatment efficiency of the total batch of the samples and retest of the blocked sample. Additives of each column in the adaptive 96-well plate of the magnetic bead extractor are shown in Table 14 (the same as Table 5 above): TABLE 14Additives of each column in the adaptive 96-well plateof the magnetic bead extractorFirstSecondSecondFourthFifthSixthcolumncolumncolumncolumncolumncolumn(7)(8)(9)(10)(11)(12)ActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solutionActivatingBalancedSampleWashingWashingEluentagentsolutionliquid 1liquid 2solution The magnetic bead is the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A). The activating agent is a solution of 50% ethanol including the magnetic bead; the balanced solution is an aqueous solution containing 1% formic acid; the washing liquid 1 is aqueous solution containing 10% methanol; the washing liquid 2 is isooctane, and the eluent solution is an aqueous solution containing 50% methanol. The sample pretreatment step of the magnetic bead extractor is shown in Table 15 (the same as Table 6); the pretreatment time of each batch of samples is about 10 min. TABLE 15Sample pretreatment step of the magnetic bead extractorMixingSolventMagneticColumns of thetimeamountabsorptionNo.Instruction96-well plate(S)(μl)time (S)1Activating1 (7)60300302Activating2 (8)60300303Sampling3 (9)90800304Drip4 (10)6030030washing5Drip5 (11)6030030washing6Eluting6 (12)60100307Waste1 (7)101000discharge After the extraction was completed by the magnetic bead extractor, the solution to be tested in the columns 6 and 12 of the 96-well plate may be transferred to the 96-well loading plate for detection on the machine. The existing magnetic bead extractor may accommodate two 96-well plates for parallel operation for one time. Therefore, the pretreatment flux is 32 samples/batch; a 96-channel magnetic bead extractor may be also available; and the extraction efficiency is kept same. The pretreatment of the sample 96 may be achieved in 10 min. The pretreatment efficiency is greatly improved to achieve the high degree of automation of clinical sample pretreatment. Example 5: Effects of Different pH Values of the Buffer Formation Solution on the Extraction Effect of Markers During the incubation, angiotensinogen in the plasma sample will be converted into angiotensin I under the catalysis of renin activity, and due to the addition of the angiotensin converting enzyme, the content of the angiotensin I increases. Therefore, the incubation process will directly affect the content of the angiotensin I in the sample. In this example, the preparation, extraction and detection of the low, moderate and high-quality control samples were performed by the method provided in Example 1. The pH values of the buffer formation solution added to the pre-incubation samples were respectively 4.0, 5.0, 5.5, 6.0 and 7.4; the post-incubation samples were absorbed and extracted by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer on the surface thereof (HLB magnetic bead, Biosepur, Art.No.: BNMA7300001-0; granularity: 30-50 μm; specific surface area: 600 m2/g and pore diameter: 80 A); after elution, the samples were subjected to liquid chromatography tandem-mass spectrometry to survey the test results of the post-incubation samples at different pH values, thus measuring the peak areas of the angiotensin I in the low, moderate and high quality control samples after incubation, as shown in Table 16. TABLE 16Effects of different pH values on the marker (angiotensin I) during the incubationMarker/pH4.05.05.56.07.4Angiotensin IPeak area639209814412461010307296902in the lowMeasured3.214.676.035.254.47quality controlconcentrationsample after(ng/ml)incubationAngiotensin IPeak area122006142264179340157004109414in the moderateMeasured5.977.288.817.545.55quality controlconcentrationsample after(ng/ml)incubationAngiotensin IPeak area206258247818291260248104206134in the highMeasured10.2812.3414.9912.3110.07quality controlconcentrationsample after(ng/ml)incubation As can be seen from Table 16, the pH value of the buffer formation solution must be controlled within a suitable scope during the incubation, or, the catalytic activity of renin will be affected seriously, leading to a larger deviation in the angiotensin I generated after the incubation. The measured renin activity is low under nonideal pH conditions, and when ARR is calculated by aldosterone/renin activity, it is easy to cause a higher value of ARR, leading to a false-positive result. Example 6: Effects of the Different Eluting agent Additives on the Eluting Effects of the Markers In this example, the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. Different additives were added to the eluting agent to prepare different eluting agents, thus eluting the magnetic bead sample; after being eluted, the sample was subjected to liquid chromatography tandem-mass spectrometry to survey the test results of the sample after being eluted by different eluting agents, thus measuring the peak areas of the 5 markers in the sample S1, as shown in Table 17. TABLE 17Effects of different eluting agents on the extraction effects of the markersAqueous solutionwith 0.1%AqueousAqueousAqueousformic acid andsolutionsolutionsolutionaqueous solutionMarker/Eluentwith 75%with 50%with 25%with 50%solutionMethanolmethanolmethanolmethanolmethanolAngiotensin IPeak45435784646057996620(theoreticalareaconcentration:0.3 ng/ml)Angiotensin IIPeak59326161896772466876(theoreticalareaconcentration:15 pg/ml)AldosteronePeak49953875456340912674(theoreticalareaconcentration:15 pg/ml)CortisolPeak169965167501163651121041161041(theoreticalareaconcentration:1.5 ng/ml)18-Hydro-Peak7077801019613732corticosteronearea(theoreticalconcentration:12 pg/ml) As can be seen from Table 17, when methanol is directly used as the eluting agent, each index has poor shape of detection peak and there are serious solvent effects; the situation is improved to some extent when the aqueous solution with 75% methanol is used for elution; and the aqueous solution with 50% methanol may effectively improve the peak shape; the elution efficiency is inadequate when 25% methanol is used; after an acid is added to the eluting agent, the ionization efficiency of aldosterone is obviously inhibited and the peak area decreases, thus affecting the detection sensitivity. Example 7: Effects of Different Additives on the Test Result of Liquid Chromatography-Tandem Mass Spectrometry In this example, the preparation, extraction and detection of the sample at the minimum concentration point (S1) of the standard curve were performed by the method provided in Example 1. Mobile phase A was an aqueous solution and mobile phase B was a methanol solution (with 5% isopropanol) as basic mobile phases. Different additives were added to the mobile phase A and mobile phase B to prepare different mobile phases for liquid chromatography tandem-mass spectrometry; the test results of the sample detected under different mobile phases were surveyed, thus measuring the peak areas of the 5 markers in the sample S1, as shown in Table 18. TABLE 18Effects of different additives on the extraction effects of the markers1 mM ammonium0.03%Marker/Mobile phaseAdditive-freefluorideformic acidAngiotensin IPeak area436864607967(theoreticalconcentration:0.3 ng/ml)Angiotensin IIPeak area806989678355(theoreticalconcentration:15 pg/ml)AldosteronePeak area266845631656(theoreticalconcentration:15 pg/ml)CortisolPeak area139964163651147501(theoreticalconcentration:1.5 ng/ml)18-Hydro-Peak area7061019739corticosterone(theoreticalconcentration:12 pg/ml) As can be seen from Table 18, compared with the condition free of an additive, the addition of 1 mM ammonium fluoride both in the mobile phase A and the mobile phase B may effectively improve the detection sensitivity; but the test result of the sample greatly varies from the type of the additives added; compared with the addition of 0.03% formic acid, the addition of 1 mM ammonium fluoride will cause decreased Ang I response to some extent, but may further improve the detection sensitivity of the other 4 indexes, especially for the low-content aldosterone. Therefore, the detection sensitivity of the 5 markers may satisfy the clinical demand more. Example 8: Effects of the Selection of Anionic and Cationic Modes on the Detection of Aldosterone In this example, research shows that during the mass spectrometric detection of the 5 markers, the cationic mode should be chosen to detect angiotensin I, angiotensin II, cortisol and 18-hydrocorticosterone, while the anionic mode needs to be chosen to detect aldosterone; this is because when the cationic mode is chosen, there exists a peak diagram of cortisone, an isomer of aldosterone, nearby the detection peak of aldosterone to cause larger interference, and CV % is greater than 15%; but when the anionic mode is applied for detection, the test result is more stable and accurate, and CV% is less than 8.33%. Example 9: Screening, Confirmed and Typing Diagnosis System for Primary Aldosteronism In this example, a screening, confirmed and typing diagnosis system for primary aldosteronism is used for the screening, confirmed and typing diagnosis of primary aldosteronism; the specific method is as follows: (1) A marker test module is used to obtain test values of the 5 markers for one time by the method provided by Example 1. (2) ARR is calculated by a data analysis module and according to the test values of aldosterone and angiotensin I; the calculation formula of the ARR is as follows: ARR=concentration of aldosterone/production rate of angiotensin I; the production rate of angiotensin I is calculated by detecting the concentration of angiotensin I in the pre-incubation sample and in the post-incubation sample and according to the following formula: production rate of angiotensin I=(concentration of angiotensin I after incubation—concentration of angiotensin I before incubation)/incubation time, where the concentration of angiotensin I before incubation is very low and thus, may be basically ignored. (3) A positive or negative result is judged in combination with a cut-off value 20.4 of the ARR and a concentration of a hypertension therapeutic affecting the ARR (see details in the patent invention 2021106374412 of the prior application). When the test result of ARR is less than 20.4, and if the patient is simultaneously detected to contain the drug capable of reducing ARR in vivo, a false-positive result may be judged, and a confirmed experiment or drug withdrawal needs to be performed for examination. When the test result of ARR is less than 20.4, and if the patient is simultaneously detected to contain the drug capable of increasing ARR in vivo, a positive result may be judged. When the test result of ARR is greater than 20.4, and if the patient is simultaneously detected to contain the drug capable of increasing ARR in vivo, a false-positive result may be judged, and a confirmed experiment or drug withdrawal needs to be performed for examination. When the ARR is greater than 20.4, and if the patient is simultaneously detected to contain the drug capable of reducing ARR in vivo, a positive result may be judged. (4) When a positive result is judged, the PA typing is performed according to the test values of aldosterone, angiotensin II, cortisol and 18-hydrocorticosterone; the specific typing diagnosis method is as follows: a, adrenal venous sampling (AVS): SI (a ratio of cortisol in adrenal veins to arterio-venous cortisol) ≥2:1, indicating successful intubation; LI (a ratio of the aldosterone-cortisol ratio at the dominant side to the aldosterone-cortisol ratio at the non-dominant side) ≥2:1, indicating secretion from the dominant side; CI (a ratio of the aldosterone-cortisol ratio at non-dominant side to the arterio-venous aldosterone-cortisol ratio) <1:1, the contralateral is inhibited; b, 18-hydrocorticosterone (18-OHB): the level of 18-OHB in the plasma of aldosteronoma patients at 8:00 a.m. in a lying position is usually >100 ng/dl, while for the patients with idiopathic aldosteronism, the level of 18-OHB is usually <100 ng/dl; c, primary and secondary hypertension is subjected to typing diagnosis in combination with the test value of angiotensin II. Example 10: Selection for the ARR Cut-Off Value 61 cases of patients receiving secondary hypertension screening were chosen in this example, of which 20 cases were diagnosed with PA. 1. Inclusion Criteria The patients whose standing position PAC was greater than 15 ng/dL, standing position ARR was greater than 30 and aldosterone inhibition ratio after CCT was less than 30% were brought into the group PA. Before screening, the patients were requested to withdraw diuretics or aldosterone receptor antagonists at least for 4 weeks, and other antihypertensive drugs such as, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor inhibitors (ARB), calcium ion antagonists (CCB) and beta receptor blockers at least for 2 weeks. Before blood sampling, serum potassium should be corrected to the normal range as much as possible. The patients who were grouped into the PA group kept in a standing position at 05:00 a.m. in the following day, and 2 h later, the blood sample was collected in the standing position at 07:00 a.m. 2. AVS Judgment Criteria Bilateral synchronous blood sampling stimulated by non-adrenocorticotrophic hormone was applied. The content of the 5 markers in the blood sample was detected by the method provided by Example 1. A ratio of cortisol in adrenal veins to arterio-venous cortisol is defined as a selectivity index (SI); SI >2 indicates successful blood sampling. A ratio of the aldosterone-cortisol ratio at the dominant side (a standardized aldosterone value) to the contralateral standardized aldosterone value is defined as a lateralized index (LI); when LI is greater than 2, it is believed that there is unilateral dominant secretion, and when LI is less than 2, it is believed that there is no obvious unilateral dominant secretion. 3. Sample collection: for all the sample collection, the subject was requested to receive the treatment of overnight fasting for 8 h above; sample transfer, centrifugation and separation should be ensured to be completed within 1 h, thus avoiding possible pre-analysis factors. All the samples were kept at −80° C for test before being analyzed. 4. ARR calculation: the content of the 5 markers in the blood sample was detected by the method provided by Example 1. Based on the calculation formula of the ARR: ARR=concentration of aldosterone/renin activity (production rate of angiotensin I); the renin activity is calculated by detecting the concentration of angiotensin I in the pre-incubation sample and in the post-incubation sample and according to the following formula: production rate of angiotensin I=(concentration of angiotensin I after incubation—concentration of angiotensin I before incubation sample)/incubation time, where the concentration of angiotensin I before incubation is very low and thus, may be basically ignored. The screening, confirmed and typing diagnosis system for primary aldosteronism provided in Example 8 was used for the screening, confirmed and typing diagnosis of primary aldosteronism. 5. Analysis by a Statistical Method The data was processed by R language software. Based on the analysis of the correlation between the ARR value and the presence of PA patients or not, the analysis result is shown in Table 19. TABLE 19Comparison of the test result between the ARR value andthe presence of PA patients or notYoudenCriteriaindexSensitivity95% CISpecificity95% CI+LR−LR>13.80.662394.4472.7-99.971.7955.1-85.03.350.077>20.40.816294.4472.7-99.987.1872.6-95.77.370.064>290.756483.3358.6-96.492.3179.1-98.410.830.18>37.20.670972.2246.5-90.394.8782.7-99.414.080.29>39.10.559861.1135.7-82.794.8782.7-99.411.920.41 As can be seen from Table 19, when the ARR cut-off value is 20.4, the detection sensitivity, degree of sensitivity and 95% CI are in higher levels and have obvious advantages relative to other cut-off values. The effect of sensitivity is the most crucial index to judge whether ARR is negative or positive by the screening, confirmed and typing diagnosis system for primary aldosteronism provided by the present invention. Missing detection may be prevented effectively only by high sensitivity. Even though it is false-positive, the false-positive result may be further confirmed by PA typing according to the test values of the subsequent aldosterone, angiotensin II, cortisol and 18-hydrocorticosterone. However, if there is lack of sensitivity, originally positive patients are erroneously judged as negative and thus, are not subjected to the subsequent typing diagnosis, which is very easy to cause missing detection. Therefore, 20.4 is applied as the ARR cut-off value, which may effectively prevent missing detection. Meanwhile, when LC-MS/MS of the ARR is greater than 20.4, the Youden index is also up to the maximum value (YI=0.82), which indicates that the screening effect of primary aldosteronism is optimal and closest to the real value. The ARR cut-off value of 20.4 is applied for analysis; and results are shown in Table 20: TABLE 20Analysis result of ARR > 20.4ROC area under the curve (AUC)0.95695% confidence interval b0.866-0.993Significance level P (area = 0.5)<0.0001Youden index J0.8162Relative standard>20.4Sensitivity94.44Specificity87.18 As can be seen from Tables 19 and 20, when LC-MS/MS of the ARR is greater than 20.4, the Youden index is also up to the maximum value (YI=0.82); sensitivity and specificity are respectively 94.4% (95% CI:72.7-99.9) and 87.2% (95% CI: 72.6-95.7); area under the curve (AUC) is up to 0.956 (FIG.2). The cut-off value of ARR is commonly believed as 30 in the prior art. The cut-off value of ARR needs to be adjusted 20.4 when the screening, confirmed and typing diagnosis system for primary aldosteronism provided in the present invention is applied. This is mainly because the ARR value is directly correlated to the detection sensitivity of aldosterone and renin activity. Aldosterone is universally detected by chemiluminesent immunoassay previously, but the test value is higher such that the calculated result of the ARR value is greater than the actual value. Therefore, it needs to set a cut-off value of 30 to judge whether PA is positive or negative accurately more. When the sample pretreatment (extraction by the magnetic bead bonded with a balanced hydrophilic-lipophilic polymer silica gel) and detection method (HPLC-tandem mass spectrometry) provided by the present invention are applied, the test value may reflect the content of the aldosterone reagent more accurately. Therefore, the previous cut-off value of 30 of ARR has not conformed to the PA screening and typing system provided by the present invention; the cut-off value of ARR needs to be adjusted 20.4 such that the screening and typing result has a higher sensitivity, and the diagnostic result is more accurate. Even though the present invention is disclosed above, the present invention is not limited thereto. Any person skilled in art can make various alterations and modifications within the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subjected to the scope defined by the claims. | 40,484 |
11860172 | DETAILED DESCRIPTION The present invention provides new methodologies that allow mass spectrometry analysis of a lipid containing sample (e.g., DESI-MS imaging) to be performed in a non-destructive matter, so that other analyses of the same sample can be performed after the mass spectrometry analysis is performed. A particular embodiment of the invention relates to mass spectral tissue imaging using DESI. DESI-MS imaging has been increasingly applied in the biomedical field. Methods of the invention, which use a variety of solvent systems for imaging, allows DESI-MS imaging of chemical compounds to be performed on lipid containing samples (e.g., tissue sections), while the morphology of the tissue remains unmodified. After DESI-MS imaging, the tissue can be used for H&E staining, immunohistochemistry, and any other tissue analysis technique to obtain more information on the distribution of its chemical constituents. A frozen mouse brain from a male mouse was purchased from Rockland Immunochemicals, Inc. (Gilbertsville, PA, USA) and stored at −80° C. until it was sliced into coronary sections of varying thickness (2 μm, 3 μm, 5 μm and 15 μm) using a Shandon SME Cryotome cryostat (GMI, Inc., Ramsey, MN, USA) and thaw mounted onto glass slides. The glass slides were stored in a closed container at −80° C. until analysis, when they were allowed to come to room temperature and dried in a dessicator for approximately 15 minutes. All human tissue samples were handled in accordance with approved institutional review board (IRB) protocols at Indiana University School of Medicine. Six human bladder cancer and paired normal samples, four human prostate cancer and paired normal samples and one human kidney cancer and paired normal sample were obtained from the Indiana University Medical School Tissue Bank. All tissue samples were flash frozen in liquid nitrogen at the time of collection and subsequently stored at −80° C. until sliced into 5 or 10 μm thick sections. The 5 and 15 μm thick sections were used for DESI-MS imaging experiments followed by either p63 immunohistochemistry or H&E stain, respectively. Tissue sections not analyzed by DESI-MS were used in control experiments. The thin tissue sections were thaw mounted to glass slides; each slide containing one section of tumor tissue and one section of adjacent normal tissue from the same patient. The glass slides were stored in closed containers at −80° C. Prior to analysis, they were allowed to come to room temperature and then dried in a dessicator for approximately 15 minutes. The DESI ion source was a lab-built prototype, similar to a commercial source from Prosolia Inc. (Indianapolis, IN USA), configured as described elsewhere (Watrous J D, Alexandrov T, & Dorrestein P C (2011), Journal of Mass Spectrometry 46(2):209-222). It consists of an inner capillary (fused silica, 50 μm i.d., 150 μm o.d.) (Polymicro Technologies, AZ, USA) for delivering the spray solvent and an outer capillary (250 μm i.d., 350 μm o.d.) for delivering nitrogen nebulizing gas. The DESI spray was positioned 2.5 mm from the tissue sample at an incident angle of 54°. A low collection angle of 10° was chosen to ensure the most efficient collection of the material being desorbed. The distance between the spray and the inlet was 6.0 mm. Multiple spray solvent systems were tested in the experiments, including ACN, H2O, MeOH, ethanol (EtOH), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), chloroform (CHCl3), acetone and many of their binary mixtures in a ratio of (1:1). The only tertiary mixture investigated was of ACN:H2O:DMF at different v/v proportions, such as (8:3:1 and 1:1:1). DESI-MS experiments were carried out in the negative ion mode, using a 5 kV spray voltage and a flow rate of 0.5-1.5 μL/min depending on the solvent system of choice. The nebulizing gas (N2) pressure was set for all experiments at 175-180 psi. The mass spectrometer used was a LTQ linear ion trap mass spectrometer controlled by XCalibur 2.0 software (Thermo Fisher Scientific, San Jose, CA, USA). Analysis were performed using an imaging approach. The tissues were scanned using a 2D moving stage in horizontal rows separated by a 150 μm vertical step for the mouse brain imaging assay (FIG.3), and 250 μm vertical step for the human tissue imaging assays. For the DESI-MS assay shown inFIG.2, the same mouse brain section was imaged 10 times with DMF:EtOH (1:1) and each analysis was performed in 10 lines of 250 μm. After one analysis was concluded, the moving stage was set to coordinate 0,0 (x,y) for the new analysis to be performed. The surface moving stage included an XYZ integrated linear stage (Newport, Richmond, CA, USA) and a rotary stage (Parker Automation, Irwin, PA, USA). A software program allowed the conversion of the XCalibur 2.0 mass spectra files (.raw) into a format compatible with the Biomap software. Spatially accurate images were assembled using the BioMap software. The color scale is normalized to the most intense (100% relative intensity) peak in the mass spectra. Tissue sections were subjected to H&E staining after DESI-MS imaging analysis or after being dried in a dessicator (control sections). All chemicals used for the H&E staining were purchased from Sigma-Alrich (St. Louis, MO, USA). The H&E staining was performed at room temperature: dip in MeOH for 2 minutes, rinse in water (10 dips), stain in Harris modified hematoxylin solution for 1.5 minutes, rinse in water (10 dips), 1 quick dip in 0.1% ammonia (blueing agent), rinse in water (10 dips), counterstain in Eosin Y (8 seconds), rinse in 100% EtOH (10 dips), rinse again in 100% EtOH (10 dips), rinse in Xylene (6 dips) and rinse again in Xylene (6 dips). Sections were allowed to dry and covered with a glass cover slide. Immunohistochemistry assays were performed in the Veterinary Department at Purdue University by Dr. Carol Bain, in accordance to their standard protocol. The primary antibody p63 (4A4):sc-8431 was purchased from Santa Cruz Biotechnology, INC (Santa Cruz, CA, USA). Pathological evaluation of the human tissue sections that were either H&E stained or subjected to p63 immunohistochemistry was performed by Dr. Liang Cheng, at IU School of Medicine in a blind fashion. Optical images of tissue sections were obtained using a SM-LUX Binocular Brightfield Microscope (Leitz, Wetzlar, Germany) under 16, 25 and 40× magnification. The solvent system used in DESI tissue imaging is taught to be an important technical parameter for optimization (Badu-Tawiah A, Bland C, Campbell D I, & Cooks R G (2010), Journal of the American Society for Mass Spectrometry 21(4):572-579; and Green F M, Salter T L, Gilmore I S, Stokes P, & O'Connor G (2010), Analyst 135(4):731-737). Many studies have shown that the chemical and physical properties of the solvent system used affect the molecular information obtained during DESI-MS tissue imaging (Ellis S R, et al. (2010), J. Am. Soc. Mass Spectrom. 21(12):2095-2104). Optimization of the spray composition allows targeted classes of compounds to be enhanced depending on the overall goal. Besides the chemical information, the effect of the solvent system on the morphology of the tissue being analyzed is a factor in DESI-MS imaging. Commonly used DESI-MS imaging solvent systems, such as mixtures of water with methanol or acetonitrile (Wiseman J M, Ifa D R, Venter A, & Cooks R G (2008), Nature Protocols 3(3):517-524), with or without an acidic modifier, yield extensive chemical information but are known to cause depletion and destruction of the tissue sections, precluding any consecutive analysis to be performed. To overcome these problems, different solvents such as ACN, H2O, MeOH, ethanol (EtOH), tetrahydrofuran (THF), N,N-dimethylformamide (DMF), chloroform (CHCl3), acetone and mixtures of these were investigated in the analysis of 15 μm thick serial coronary mouse brain tissue sections. A binary mixture of MEOH:H2O (1:1, v/v) or ACN:H2O (1:1, v/v) has been commonly used in DESI imaging of brain tissue, yielding high signal intensity for polar lipids and free fatty acids (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010), Angewandte Chemie-International Edition 49(5):873-876; and Wiseman J M, Ifa D R, Song Q Y, & Cooks R G (2006), Angewandte Chemie-International Edition 45(43):7188-7192). The majority of the ions observed in the mass spectra obtained from the solvent systems tested here correspond to commonly observed lipid species in brain tissue when using standard MeOH:H2O (1:1), such as deprotonated free fatty acids, phosphatidylserines (PS), phosphatidylinositols (PI) and sulfatides (ST) (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010), Angewandte Chemie-International Edition 49(5):873-876). Variations in the relative abundance of the lipid species and in the total ion signal obtained were observed depending on the solvent composition. For instance, spectra obtained when using pure methanol as the solvent system showed higher relative abundance of fatty acid dimers in the m/z 500-700 region of the mass spectrum. In particular, it was observed that pure DMF yielded spectra with high total abundance and with chemical information which is very similar to that which is obtained using MeOH:H2O. Interestingly, the DMF spray was observed to not cause tissue destruction. The effect of DMF in the tissue was further explored by combining this solvent with other solvents in binary (1:1 v/v) and tertiary mixtures. The combination of DMF with either ACN, EtOH, THF or CHCl3yielded very high ion signal and chemical information similar to what is seen using MeOH:H2O. Combinations of DMF with either H2O or MeOH greatly enhanced the signal of low molecular weight compounds, such as small metabolites, FAs and FA dimers (Eberlin L S, Ferreira C R, Dill A L, Ifa D R, & Cooks R G (2011) Desorption Electrospray Ionization Mass Spectrometry for Lipid Characterization and Biological Tissue Imaging. Biochimica Et Biophysica Acta-Molecular And Cell Biology Of Lipids accepted). In terms of spray stability and total ion abundance, the combinations of DMF with either EtOH or ACN are great solvent systems for tissue imaging experiments. The change in chemical information obtained by DESI-MS using different solvent combinations can be compared to the use of different matrices in MALDI imaging, but in DESI-MS imaging experiments, the “matrix” is delivered in real-time, spot-by-spot, without the need for sample preparation or without causing spatial delocalization of molecules. Importantly, none of these solvent combinations were observed to cause visual damage to the 15 μm thick tissue sections that were analyzed.FIG.1shows the physical and chemical effect of two of the new solvent systems developed, DMF:EtOH (1:1) and DMF:H2O (1:1) in comparison to the standard MEOH:H2O solvent system. DESI-MS conditions were kept identical in all analyses performed. It is striking to observe that while extensive chemical information was obtained from the tissue sections when using DMF:H2O and DMF:EtOH, tissue integrity was preserved. As observed in the optical images shown of the DESI-MS experiment, damage to the tissue was insignificant when using DMF solvent systems. To confirm preservation of tissue integrity, H&E staining was performed on the tissue sections previously analyzed by DESI-MS. H&E staining is a commonly used histochemical protocol to evaluate cellular structure and tissue morphology by light microscopy. Careful microscopic examination of the H&E tissue sections revealed no damage or change in the cellular morphology of the sample after DESI analysis using DMF:EtOH and DMF:H2O solvent systems, while the tissue analyzed using MeOH:H2O was found to be altered and damaged, as was macroscopically observed. DESI-MS analysis of sequential mouse brain tissue sections of 2, 3 and 5 μm thicknesses was also performed, and sequential H&E staining of the tissue sections also revealed that no morphological damage occurred following DESI-MS analysis using DMF:EtOH or DMF:ACN as the solvent system. The physical and chemical effect of the DMF:EtOH solvent system was further investigated by performing several DESI-MS analyses of the same mouse brain tissue section. The same tissue region of a 5 μm and a 15 μm thick tissue section were analyzed 10 times using the DESI-MS moving stage system. Mass spectra were recorded for 10 rows (250 μm step size) of each mouse brain section and after 10 analyses had been performed, each tissue section was H&E stained and observed under brightfield microscopy under 16-40× magnification.FIGS.2Aand B show the mass spectra obtained from the gray matter region of the 5 μm thick mouse brain tissue section from the 5throw scanned using DMF:EtOH in the 1stand 10thDESI-MS analysis, respectively. The ion count is approximately 60 times greater in the 1stanalysis of the mouse brain compared to the ion count obtained in the 10thanalysis of the same region. The ion count of the main ion observed in the gray matter region of the 5throw scanned, m/z 834.4 (PS 18:0/22:6), was plotted as a function of the DESI-MS analysis number for both the 5 μm and a 15 μm thick tissue sections, shown inFIG.2C. Interestingly, the signal of the typical ion of m/z 834.4 obtained in the 3rdor even 4thDESI-MS analysis is still observed at high intensities. Furthermore, the decay profile of the ion count is consistent with the extraction mechanism proposed for DESI-MS (Costa A B & Cooks R G (2008), Chem. Phys. Lett. 464(1-3):1-8). While a MeOH:H2O spray extracts the chemical compounds from the tissue cells resulting in tissue damage, the DMF based solvent system is able to extract the chemical compounds from the tissue section without disturbing the tissue morphology. H&E staining of both a 5 μm and a 15 μm thick tissue section after ten DESI-MS imaging analyses revealed no damage to the tissue, indicating that the repetitive removal of the phospholipids by the DESI solvent spray does not affect the morphology of the cells. In fact, the extraction process that occurs in DESI-MS is comparable to the fixative procedures commonly used in histology for lipid removal (DiDonato D & Brasaemle D L (2003), Journal of Histochemistry & Cytochemistry 51(6):773-780), such as the alcohol wash used in the initial step of the H&E staining data. This alcohol wash step extracts the majority of cellular phospholipids while the cellular cytoskeletal elements are kept intact. Since hematoxylin stains nucleoproteins and eosin stains intracellular and extracellular proteins, the removal of the lipid content with conservation of the tissue integrity by DESI-MS should not interfere with this standard histochemistry protocol. Importantly, the use of DMF based solvent systems or even other solvent systems with similar morphologically-friendly properties allows pathological evaluation to be performed on the same tissue section previously analyzed by DESI-MS but with acquisition of complementary results. All combinations of DMF with other solvents used in the DESI-MS assays on mouse brain tissue sections were found to not destroy the native morphology of the tissue. Other pure solvents, such as ACN, DMF, THF, ethanol and others did not cause damage to the tissue integrity as observed in the H&E stains. A few other combinations that did not contain DMF, such as ACN:EtOH (1:1), MeOH:CHCl3(1:1) and ACN:CHCl3(1:1), did not destroy the native morphology of the tissue. The morphological effect that the DESI spray has on tissue appear to be related to the physical and chemical properties of the solvent systems itself. While solubility of the proteic cellular and extracellular components of the tissue section in the DESI spray solvent system plays a role in the conservation of the tissue morphology integrity, the physical properties of the solvent system such as surface tension and its effects on the dynamics of the DESI spray primary droplets also impact the damage caused to the tissue. When solubilization of cellular and extracellular components that keep cellular morphological integrity intact occurs, the tissue becomes more susceptible to the mechanical action of the DESI spray droplets. Therefore, tissue damage should be related to both solubilization of tissue components and mechanical action of the DESI spray system. The fact that the morphologically-friendly solvent systems described here do not disturb tissue integrity appear to be related to the physical properties of the DESI spray primary droplets, but also on the solubility of tissue components on the solvent system. Chemical information and image quality are important factors in DESI-MS imaging applications. The geometric parameters of the DESI spray as well as the choice of solvent system, gas pressure and solvent flow are important when optimizing imaging conditions. When the solvent system is modified, it is important to observe that the spray spot is stable and that the ion signal intensity is maximized for obtaining good quality 2D chemical images. FIG.3shows ion images of a mouse brain tissue section obtained using DMF:EtOH as the solvent system. The spray geometry and gas pressure conditions used in this imaging experiment are standard for DESI-MS imaging applications (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010), Angewandte Chemie-International Edition 49(5):873-876; and Ifa D R, Wiseman J M, Song Q Y, & Cooks R G (2007), International Journal of Mass Spectrometry 259(1-3):8-15). In terms of solvent flow, it was observed that at a regular DESI-MS imaging flow rate of 1.5 μL/min a larger spot size is obtained with DMF solvent combinations as compared to standard mixtures of water with MEOH or ACN at the same flow. The high stability and larger diameter of the spray spot can be associated with the higher boiling point of DMF (153° C.), when compared to the boiling point of solvents as MeOH and ACN. Smaller diameter spray spots can be achieved by using either a lower solvent flow rate or by mixing DMF with a higher ratio of a solvent with higher volatility, such as a mixture of DMF:EtOH (1:2). A solvent flow of 0.5 μL/min was used for the mouse brain imaging experiments using binary mixtures of DMF so that a spot size of approximately 180 μm was obtained. Lower solvent consumption as a result of using a lower flow rate is advantageous in DESI-MS imaging applications. In the images shown inFIG.3, two distinctive MS peak patterns associated with the lipid compositions representative of the gray and white matter of the brain were observed in the negative-ion mode for the brain section analyzed (Eberlin L S, Ifa D R, Wu C, & Cooks R G (2010), Angewandte Chemie-International Edition 49(5):873-876). The ion images of m/z 834.3, PS 18:0/22:6 (FIG.3A), m/z 885.6, PI 18:0/20:4 (FIG.3D) and m/z 303.3, FA 20:4 (FIG.3E) show a homogeneous distribution in the brain gray matter, which are complementary and distinct from the ion images of m/z 888.8 (ST 24:1,FIG.3B) and m/z 890.7 (ST 24:0,FIG.3C), which are homogeneously distributed in the mouse brain white matter.FIG.3Fshows the optical image of the same tissue section which was H&E stained after DESI-MS imaging was performed. This order of analysis in which ambient MS imaging is performed followed by histochemical analysis of the same tissue section is comparable to the “post-acquisition staining” methodology used in MALDI-MS imaging (Schwamborn K, et al. (2007), International Journal of Molecular Medicine 20(2):155-159). The high-quality 2D DESI-MS ion images can be directly compared and even overlaid with the H&E stained tissue section, allowing a better correlation between the spatial distribution of the lipid species detected and the substructures of the mouse brain. The capability to perform DESI-MS imaging and histochemical analysis of the same tissue section is important in the investigation of diseased tissue. The comparison of histological features from stained sections with corresponding molecular images obtained by ambient imaging MS is important for accurate correlations between molecular signatures and tissue disease state. This is especially true in the analysis of cancerous tissue sections which are very often highly heterogeneous, with regions of containing various tumor cell concentrations (Agar NYR, et al. (2011), Neurosurgery 68(2):280-290), infiltrative normal tissue (Dill A L, et al. (2011), Chemistry—a European Journal 17(10):2897-2902), precancerous lesions (Eberlin L S, et al. (2010), Analytical Chemistry 82(9):3430-3434), etc. Integration of DESI-MS imaging into a traditional histopathology workflow required that the mass spectrometric analysis not interfere with the morphology of the tissue section. Provided this is the case, the combination of the two different types of data (as represented by the case of superimposed images) greatly increases discrimination between different tissue types including that between diseased and healthy tissue. To investigate this capability, human bladder, kidney and prostate cancer tissues along with adjacent normal samples were analyzed by DESI-MS imaging in the negative ion mode using one of our histology compatible solvent system and sequentially H&E stained. The lipid species present in the tissue sections were identified based on collision-induced dissociation (CID) tandem MS experiments and comparison of the generated product ion spectra with literature data (Hsu F F & Turk J (2000), Journal of the American Society for Mass Spectrometry 11(11):986-999).FIG.4shows a series of negative ion mode DESI-MS ion images of species commonly observed in human bladder transitional cell carcinoma and adjacent normal tissue from sample UH0210-13.FIGS.4A, B, C, D and E show the ion images obtained for the ions at m/z 788.4 (PS(18:0/18:1)), m/z 885.6 (PI(18:0/20:4)), m/z 835.6 (PI(16:0/18:1)), m/z 281.6 (FA 18:1) and m/z 537.2 (FA dimer). As previously reported for DESI-MS imaging of human bladder cancer in combination with statistical analysis using a standard ACN:H2O (1:1) solvent system, the ions that most significantly contribute to the discrimination between cancerous and normal bladder tissue are the free fatty acid and the fatty acid dimers, which consistently appear at increased intensities in the ion images of cancerous tissue when compared to normal tissue using the morphology friendly solvent system, DMF:EtOH (1:1) (FIGS.4Cand D; Dill A L, et al. (2011), Chemistry—a European Journal 17(10):2897-2902; and Dill A L, et al. (2009), Analytical Chemistry 81(21):8758-8764). Representative mass spectra obtained for the cancerous and normal tissue sections are shown inFIGS.4Gand H. Many other individual ions observed in the mass spectra were found at different intensities in the normal and cancerous tissues as observed in extracted DESI-MS ion images. The optical image of the same tissue sections stained with H&E after DESI-MS imaging analysis is shown inFIG.4Fand were used to obtain a histopathological diagnosis. Detailed pathological examination of the H&E stained sections confirmed that there was no morphological damage to the tissue sections as a result of DESI-MS imaging analysis, allowing a straightforward diagnosis of the sections as cancerous and normal. No difference in cell morphology or tissue integrity was observed at the microscopic scale when the H&E stained tissue section of the DESI-MS imaged tissue was compared to a control tissue section. The non-destructive nature of the DMF based solvent system enables ion images to be overlaid with the H&E stain of the same tissue section for unambiguous diagnosis and correlation. For example, a small region of tissue within the cancerous section detected by DESI-MS as negative for bladder cancer based on the distribution of the FA dimer m/z 537.2 was confirmed as normal tissue by pathological evaluation of the overlaid DESI-MS ion image and H&E stain of the same tissue section. This unambiguous correlation is made possible through the use of the morphologically friendly solvent systems so that the histological data can be considered in combination with the DESI-MS imaging data. H&E stained serial sections of the same sample imaged using standard ACN:H2O (1:1) revealed that the tissue integrity was completely destroyed and were inadequate for pathological evaluation. The same histological observation that DESI imaging is histology compatible was obtained in the analysis of the H&E stained sections of five other human bladder cancer and paired normal samples, four human prostate cancer and paired normal samples and one kidney cancer and paired normal sample initially imaged by DESI-MS with a morphologically friendly solvent system. Previously reported molecular information that allowed a diagnosis to be obtained for these types of cancer was consistent using the new solvent system. The capability of DESI-MS imaging to be histology compatible was further investigated by performing immunohistochemical (IHC) analysis with p63 antibody on bladder and prostate cancer tissue sections, which was performed after DESI-MS imaging. The gene p63 is one of the most commonly used basal cell-specific markers in the diagnosis of prostate cancer, whose expression is known to be down-regulated in adenocarcinoma of the prostate when compared to normal prostate tissue (Signoretti S, et al. (2000), American Journal of Pathology 157(6):1769-1775). Negative IHC staining of tumor protein p63 is commonly used as a clinical tool for identifying prostate cancerous tissue. The role of p63 in bladder carcinogenesis is not as clear as in prostate cancer (Comperat E, et al. (2006), Virchows Archiv 448(3):319-324), and positive staining of p63 is typically associated with both benign and malignant bladder epithelial cells. Two bladder cancer samples and two prostate cancer samples were subjected to p63 IHC after DESI-MS imaging on the same tissue section. Detailed pathological evaluation of the tissue sections that were subjected to IHC after DESI-MS imaging confirmed that the DESI-MS analysis of the tissue lipid content did not interfere with the p63 IHC protocol, as the tissue remained intact after the imaging experiment. p63 IHC of the bladder sample was found to be positive for both cancerous and normal tissue sections. For the prostate cancer sample, UH0002-20, it was subjected to p63 IHC after DESI-MS imaging and again no damage to the morphology of the tissue was observed (FIG.5), allowing a diagnosis of cancerous and adjacent normal tissue to be achieved, which correlated with the cholesterol sulfate signal previously reported as a possible prostate cancer biomarker using DESI-MS imaging (Eberlin L S, et al. (2010), Analytical Chemistry 82(9):3430-3434.). Also, these findings confirmed that protein position in the tissue samples remained unchanged, which is probably due to the insolubility of these proteins in the solvent combinations used. The results reported here introduce a novel capability of histologically compatible ambient molecular imaging by DESI-MS. The feasibility of DESI-MS imaging to be performed while tissue integrity and cell morphology is conserved allows ambient mass spectrometric analysis of tissue to be combined with traditional histopathology with the goal of providing better disease diagnostics. As DESI-MS imaging using histologically friendly solvent systems does not interfere with pathological analysis, the technique could be included as the initial step in the clinical tissue analysis workflow. Methods reported herein will allow DESI-MS to be more broadly applied in the biomedical field, such as in intraoperative applications. In additional to biomedical applications, the morphologically compatible solvent system allows DESI-MS imaging to be combined to other analytical techniques for chemical analysis of the same tissue section. INCORPORATION BY REFERENCE References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. EQUIVALENTS The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. | 28,510 |
11860173 | DETAILED DESCRIPTION Introduction Embodiments of the disclosure relate to systems and techniques for detection of hazardous environmental contaminants, such as but not limited to antineoplastic drugs used in the treatment of cancer, while minimizing exposure of the test operator to the contaminants. A kit for such testing can include a collection device and a testing device. Throughout this disclosure, example systems, kits, and methods will be described with reference to collection, testing, and detection of antineoplastic agents, but it will be understood that the present technology can be used to collect, test, and detect any particle, molecule, or analyte of interest. A collection device can include an integrated swab and testing apparatus such as a lateral flow assay test strip. Beneficially, the collection device provides a fluid-tight fluid path between the swab and the test strip, such that the user is protected from collected liquid as it is transferred from the swab to the test strip. The collection device can also include a cap or other container for sealing the swab after collection of the antineoplastic agent. Optionally, a reservoir for containing fluid such as a buffer solution can be disposed in the cap for flushing the swab after sample collection. The swab can be constructed from a special material having desired pickup efficiency and shedding efficiency for detecting trace amounts of antineoplastic agents, and is provided on a handle having sufficient length so that the user can swab a surface without physically contacting the surface or the swab. The handle can perform a double function by serving as the cartridge enclosing the test strip, and interior features of the handle can form the fluid path between the swab and the test strip. A liquid, for example a buffer solution, can be provided on the swab material so that the user removes a pre-wetted swab to swipe the surface in one implementation. In another implementation, the user sprays the surface with a liquid and collects this liquid with the swab material. Tris buffer and ChemoGlo solution are two suitable buffer solutions that can be implemented in contamination collection devices described herein. The collection kit can further include a template, guide, or instructions to delineate a specific dimensional area for testing. In order to obtain an accurate test result for contaminants that are hazardous even in trace amounts, a precise method of marking (demarcation) and then performing the sampling procedure (for example, to sample all of the demarcated area and only the demarked area) can be a very important step to ensure an accurate result. There are several factors that can be key to obtaining an accurate drug concentration measurement given in the following formula: C=∝*A*ηp*ηeVb where C is the concentration, a is the contamination surface density (ng/ft{circumflex over ( )}2), A is the surface area swabbed and tested, ηpis the pick-up efficiency, ηeis the extraction from the swab density, and Vbis the fluid volume of the buffer solution used to help extract and carry the contamination to the test strip. A goal of the described testing can be to have a high concentration signal with low variability. Excessive “noise” or variation in the variables may cause the test to either give false positive or false negative results. Test kits described herein can include mechanisms and/or instructions to users to assist in reducing the variation of each term in the above concentration equation. After swabbing the surface, the user places the cap over the swab to form a liquid-tight, sealed compartment that encapsulates the swab material and any absorbed liquid. The cap can additionally lock to the handle. A reservoir in the handle and/or cap can contain a buffer or diluent solution used as an agent to help remove the particles of interest embedded on the swab material into the fluid of the container. The collection device advantageously prevents liquid from spilling and contaminating surfaces or users, but provides for controlled delivery of fluid to a detection device such as a test strip. Fluid can be wicked directly from the swab material to the test strip in some embodiments, and in other embodiments fluid can be released from the swab material along a fluid path to a receiving zone of an assay test strip. It will be understood that signals generated by embodiments of lateral flow assay devices described herein can be detected using any suitable measurement system, including but not limited to visual inspection of the device and optical detection using an optical reader. In one aspect, the testing device can be an immunoassay reader, for example a lateral flow assay and reader device, with an interface that alerts the user to the presence and/or degrees of contamination. After sample collection with the integrated swab material, the assay test strip can be inserted into a reader to image the indicators on the strip, analyze the image(s), determine a level of contamination, and report the determined level of contamination to the user. The reader can have more than one method of entering data regarding the sample and can have various ways of saving, storing, displaying, uploading and alerting the appropriate personnel when unacceptable levels of contamination are detected. In one example, after detecting contamination in an initial test there can be several possible next steps. A first option can be to use another more sensitive test strip to determine an advanced level of detection. A second option can be to use another similar test strip in an area near the initial test area to determine the spread of the contamination. A third option can be to initiate any specified decontamination protocol in the area of the test surface (and potentially surrounding areas). It will be understood that some or all of these non-limiting options can initiated simultaneously or sequentially. The described swabs, buffer solutions, and test devices can be configured to pick up and detect trace amounts of antineoplastic agents and/or chemotherapeutic drugs in some embodiments. It will be appreciated that the described systems can be adapted to collect and detect quantities of other biohazardous chemicals, drugs, pathogens, or substances in other embodiments. Further, the disclosed systems can be used in forensic, industrial, and other settings. Although the disclosed detection devices are typically described herein with reference to test strips and lateral flow assay reader devices, it will be appreciated that the described hazardous contaminant detection aspects described herein can be implemented in any suitable detection system. For example, features described herein can be implemented in reader devices that analyze other types of assays, such as but not limited to molecular assays, and provide a test result. Further, the fluid including any collected contaminants can be transferred from the collection device to a centrifuge, spectrometer, chemical assay, or other suitable test device to determine the presence and/or concentration of one or more hazardous substances in the sample. Drugs successfully treat many types of illnesses and injuries, but virtually all drugs have side effects associated with their use. Not all adverse side effects classify as hazardous, however. In the present disclosure, the term “hazardous drugs” is used according to the meaning adopted by the American Society of Health-System Pharmacists (ASHP), which refers to a drug as hazardous if studies in animals or humans have indicated that exposures to them have any one of four characteristics: genotoxicity; carcinogenicity; teratogenicity or fertility impairment; and serious organ damage or other toxic manifestation at low doses in experimental animals or treated patients. Although described in the example context of ascertaining the concentration of hazardous drugs such as antineoplastic agents, it will be appreciated that the disclosed test strips and reading techniques can be used to detect the presence and/or concentration of any analyte of interest. An analyte can include, for example, drugs (both hazardous and non-hazardous), antibodies, proteins, haptens, nucleic acids and amplicons. Various embodiments will be described below in conjunction with the drawings for purposes of illustration. It should be appreciated that many other implementations of the disclosed concepts are possible, and various advantages can be achieved with the disclosed implementations. Overview of Example Contaminant Collection FIG.1illustrates example steps of a testing method100using a system contaminant collection device according to the present disclosure, such as but not limited to those shown inFIGS.2A-2C and4A-6E. One, some, or all of the depicted blocks ofFIG.1can be printed as graphical instructions on the packaging or instruction materials of an assay and/or collection kit, or can be presented on a graphical user interface of a display screen of an assay reader device, a test area terminal, or a personal computing device of the user. At block105, the user can identify a sample location and gather testing supplies including a collection kit and protective wear. The collection kit can include a collection device including a swab material on an elongate handle, as described herein, for example in a sealed package. In some examples, the swab is pre-wetted with buffer solution and packaged in a sealed pouch. In some implementations, a removable cap can seal the swab material prior to use. The assay cartridge can form some or all of the handle. The assay cartridge may include an assay device housed inside a cartridge having a window or port aligned with a reaction zone of the assay device. In one implementation, the assay device is a test strip, for example but not limited to a lateral flow assay test strip. Also at block105the user can put on clean gloves prior to each sample collection and/or opening of the collection kit, both to protect the user from potential contamination on the surface and to protect the collection device from any contamination on the user's hands. The collection kit can also include a template for demarcating the area to be tested on the test surface, though in some examples the template may be provided separately. The collection kit may also include additional buffer fluid for wetting the test surface, though in some examples this can be provided on a pre-moistened swab material. At block110, the user can establish a test area on the test surface. For example, the user can place a template over the intended location to clearly demarcate the area that will be swabbed. The template can be a physical template as shown, or may be provided via augmented reality (e.g., smart glasses, a heads up display, a projection onto the test surface). Also at block110the user can open the collection kit packaging, including opening the integrated swab and assay cartridge. The test area may be one square foot in some embodiments, for example demarcated as a 12 inches by 12 inches (144 square inches) region. Other examples can use greater or smaller areas for collection including 10 inches by 10 inches, 8 inches by 8 inches, 6 inches by 6 inches and 4 inches by 4 inches, non-square rectangular regions (e.g., a 9 inches by 16 inches rectangle), and non-rectangular regions (e.g. circles). Different-sized templates may be specified for use with different test surfaces. The particular template used can be indicated to a reader device, for example via a manual user input or via a barcode or other identifying pattern on the template scanned by the reader device. For example, a template providing a swab area of a 12 inches by 12 inches region can be indicated for use in sampling a countertop, while a smaller template demarcating a smaller swab area can be indicated for swabbing an IV pole. The reader device can adjust its test result calculations to account for the actual area tested, as indicated by the particular template used for the sampling procedure. At block115, the user can swab the entire test area with the pre-moistened swab. The user can swab the test area using slow and firm strokes. As shown, the user can methodically pass the swab in straight lines along the height of the test area all the way across the width of the test area. In embodiments using augmented reality templates, the user can be provided with a visual indication of one or more of the already-swabbed portions of the test region, to-be-swabbed portions of the test region, and swab pattern. As the user swabs the surface, the swab material of the collection device can pick up contaminant particles and/or any buffer liquid provided on the test surface. After swabbing is complete, the user can seal the exposed swab material of the collection device, for example by applying a cap that engages with the assay cartridge and/or handle and seals the swab material. Optionally, the cap can include an additional quantity of buffer solution and a mechanism for releasing this buffer solution onto the swab material to flush collected contaminants downstream to the assay device. At block120, the user can use a timer to allow the sample to develop for a period of time. For example, the sample can develop for about one minute, about two minutes, about three minutes, about four minutes, about five minutes, about six minutes, or some other amount of time. Other development times are possible. In some embodiments the timer can be built in to the programming of the reader device that reads the assay. The development time can vary depending on the particular test that is being performed and the particular operating parameters of the assay device. In some embodiments, at least some liquid may be transferred from the swab material to the assay device during swabbing, and the development time can include some or all of the time taken at block115to swab the surface. In some embodiments, a valve or frangible seal can isolate the assay device from the collected liquid during swabbing at block115, and after completion of the swabbing the user can cause the breaking or opening of this seal to transfer the liquid from the swab material to the assay device. In such embodiments, development time may not include any of the swabbing time. At block125, the user can insert the assay cartridge into an assay reader device. The assay cartridge can be inserted into the reader device prior to or after the sample is developed, depending upon the operational mode of the device. In some embodiments, the user may sequentially insert multiple cartridges for testing different aspects of the test surface or for ensuring repeatability of test results. Although the cartridge shown in block125is not depicted with swab material, it will be appreciated that in some embodiments the integrated swab material may remain affixed to the cartridge as it is inserted (via the end opposite the integrated swab material) into the reader device. At block130, the assay reader device reads portions of the inserted cartridge (including, for example, detecting optical signals from exposed areas of a capture zone of a test strip housed in the cartridge), analyzes the signals to determine optical changes to test zone location(s) and optionally control zone location(s), determines a result based on the optical changes, and displays the result to the user. The device can optionally store the result or transmit the result over a network to a centralized data repository. As illustrated, the device displays a negative result for the presence of Doxorubicin in the sample. In other embodiments the device can display a specific detected concentration level in the sample and/or determined for the test area, and optionally can display confidence values in the determined result. After testing the user can dispose of the collection device and assay (for example in compliance with hazardous waste regulations). Optionally, the user can connect the reader device to its power supply, execute any needed decontamination procedures, re-test a decontaminated surface, and perform required reporting of the result. Though not illustrated inFIG.1, further steps can include operating the reader device to perform analysis of the test strip. An example of the cartridge inserted into a reader device is shown inFIG.3A, and an example of the reader device displaying test results is shown inFIG.3B. FIGS.2A-2Cshow example steps of using a collection device with an integrated swab and test strip during various portions of the process100. Specifically,FIG.2Ashows a user holding a collection device200according to one implementation of the present disclosure. The collection device includes an integrated swab handle/assay cartridge225.FIG.2Aalso shows the user removing a cap205from a distal end215of the integrated swab handle/assay cartridge225to expose a swab material210. This can happen before block115of the testing method100.FIG.2Aalso shows a window220in the integrated swab handle/assay cartridge225for viewing a reaction zone of a test strip contained within the integrated swab handle/assay cartridge225.FIG.2Bshows the user holding the integrated swab handle/assay cartridge225with the swab material210applied to a test surface230, for example during block115of the testing method100.FIG.2Cshows the user holding the collection device200after completion of the swabbing with the cap205reapplied, for example after block115of the testing method100. Further details of collection devices with an integrated swab and test strip are described below. FIGS.3A and3Billustrate an example reader device300that can be included in or used with hazardous contamination detection kits described herein.FIG.3Aillustrates the reader device300with an assay cartridge330inserted into a cartridge receiving aperture305, andFIG.3Billustrates the reader device300without an inserted cartridge. Examples of the assay cartridge330include but are not limited to the integrated handle and cartridge shown inFIGS.2A-2C and4A-6E. The reader device300can be an assay reader device having an aperture305for receiving an assay test strip and cartridge330. The aperture305can also be configured to position the test strip so that analyte binding regions are positioned in an optical path of imaging components located inside of the device300. The device can also use these or additional imaging components to image a bar code on the cartridge, for example to identify which imaging techniques and analysis to perform. Some embodiments of the device300can be configured to perform an initial scan, for example using a bar code scanner to image one or more bar codes. A bar code can identify the type of test to be performed, the person conducting the test, the location of the test, and/or the location in the facility of the test surface (for example pharmacy, nursing area, cabinet #, bed #, chair #, pump #, etc.). After reading the bar code identifier the cartridge is then inserted into the reader as shown inFIG.3A. The device300can have a button310that readies the device for use and provides an input mechanism for a user to operate the device. The device300can also include a display315for displaying instructions and/or test results to the user. After insertion of the test strip, the device300can read a bar code on the assay test strip to identify the name and/or concentration range of the drug. The device300can image the inserted test strip, and analyze the signals representing the imaged test strip to calculate results, display the results to the user, and optionally transmit and/or locally store the results. The results can be calculated and displayed as contamination with an indication of positive or negative (for example, +/−; yes/no; etc.), and/or the actual contamination per area (for example, Drug Concentration=0.1 ng/cm2) and/or per volume (for example, Drug Concentration=3 ng/ml) Some embodiments of the device300may simply display the result(s) to the user. Some embodiments of the device300may also store the result(s) in an internal memory that can be recalled, for example, by USB connection, network connection (wired or wireless), cell phone connection, near field communication, Bluetooth connection, and the like. The result(s) can also automatically be logged into the facility records and tracking system. The device300can also be programmed to automatically alert any additional personnel as required, without further input or instruction by the user. For example, if the device300reads contamination levels that are above the threshold of human uptake and considered hazardous to for human contact, a head pharmacist, nurse, manager, or safety officer can be automatically notified with the results and concentration of contamination to facilitate a rapid response. The notification can include location information, such as but not limited to a geographic position (latitude/longitude) or description of location (Hospital A, Patient Room B, etc.). That response may include a detailed decontamination routine by trained personnel or using a decontamination kit provided together or separately from the hazardous contamination detection kit. In some embodiments, device300can be a special-purpose assay reader device configured with computer-executable instructions for identifying trace concentrations of contaminants in the samples applied to test strips. Further components of the device300are discussed below with respect to the diagram ofFIG.7. Overview of Example Collection Devices with Integrated Swab and Test Strip As described herein, the contaminant collection devices according to the present disclosure can be “closed systems,” referring to the transfer of fluid from the swab material to the assay test strip via a liquid-tight transfer mechanism. For example, the swab material and detection device (such as a test strip) can be fluidically coupled together within a housing to provide a fluid tight seal between the swab material and the test strip (and any intervening fluid path of the collection device). Beneficially, harmful fluids, drugs, or vapors can be completely contained within such a collection device and not vented into the atmosphere or spilled during transfer between the collector and test device, which would possibly cause harm to the user. Fluid-tight can refer to being liquid impermeable, gas or vapor impermeable, or both, depending upon the properties of the contaminant that the collection kit is designed to detect. Beneficially, this can provide protection to a user of the kit from the potential contaminants in the fluid of the collection device. The various collection devices disclosed herein are described at times using relative position terms. As used herein, the “upper” surface of a collection device refers to the surface through which the reaction zone of the test device is visible. The “lower” surface opposes this upper surface. The “distal” end refers to the end of the collection device from which swabbing material extends or protrudes. The “proximal” end opposes the distal end, and is typically the end that would be positioned closest to the user during swabbing. In some cases, the proximal end includes the leading edge or surface during insertion into a reader device (e.g., the edge or surface that enters the reader device before other edges or surface of the collection device). An “elongate” body of the collection device as described herein refers to the length of the body (extending between the proximal and distal ends) being greater than a width of the body (extending perpendicularly to the length along the upper or lower surface). For example, the length of an elongate body may be two times, three times, four times, or five times greater than the width (or another multiple of the width, where the multiple is greater than one). It will be understood, however, that implementations of the present disclosure are not limited to the specific shapes, sizes, and configurations of the example implementations described with reference toFIGS.2A-2C and4A-6E, and the present disclosure can be implemented in devices having other suitable shapes, sizes, and configurations. A reaction zone of a test device, such as an assay test strip, can be visible through the housing of a collection device as described herein. Such a reaction zone can be configured to produce an optically-detectable change in appearance in the presence of a hazardous contaminant. This change can include one or more optically-detectable lines that develop if the hazardous contaminant is (or is not) present in the applied sample, as described below. A test strip can also include a sample receiving zone, for example positioned where the fluid path within the collection device is configured to provide a liquid sample from the absorbent swab material. The sample receiving zone can evenly distribute the sample and direct it to a downstream region of the test strip. The sample receiving zone can optionally include compounds (e.g., buffer salts, surfactants, proteins, etc.) that facilitate interaction between the liquid sample and molecules in other zones. The liquid sample can flow, for example, via capillary action downstream along a substrate of the test strip towards the reaction zone. A conjugate release zone can be disposed along this fluid path, for example containing diffusibly bound molecules that are conjugated to colored or fluorescent label particles. The term “diffusibly bound” refers to reversible attachment or adsorption of the labeled conjugate to the conjugate release zone such that the material moves with the lateral flow when contacted with the liquid sample. The conjugate release zone is configured to release the labeled conjugate upon contact with the moving liquid sample. The liquid sample and labeled conjugate can be carried downstream along the lateral flow path from the conjugate release zone to the reaction zone, where one or more detection zones (formed as lines in some examples, also referred to herein as a reaction zone) have non-diffusibly bound capture reagents immobilized within the zone. The term “non-diffusibly bound” refers to attachment of the capture reagents to the material of the detection zone such that the capture reagent is immobilized and therefore does not move with the lateral flow when contacted with the liquid sample. In competitive assay implementations, the labeled conjugate can compete with the target contaminant molecule for binding with the capture reagents, such that a greater intensity of a detection line indicates a smaller quantity of target contaminant. Competitive assay implementations may be suitable for small molecules, such as some antineoplastic agents. In sandwich assay implementations, the labeled conjugate can bind with a first site of target contaminant and a second site of the target contaminant can bind with the capture reagent immobilized in the detection zone, such that a greater intensity of a detection line indicates a greater quantity of the target contaminant. FIGS.4A-4Ddepict an embodiment of a collection device400with an integrated swab material435and test device430according to the present disclosure. Specifically,FIG.4Ashows a front (distal end), top (upper surface), and side perspective view of the collection device400, andFIG.4Bshows a front, bottom (lower surface), and side perspective view of the collection device400.FIG.4Cshows a top, side perspective view of the collection device400without its cap, andFIG.4Dshows the top, side perspective view of the collection device400without its upper cartridge portion to reveal the inner components. The model of the collection device400depicted inFIGS.4A-4Dcan correspond to the collection device200depicted inFIGS.2A-2C.FIGS.4A-4Dare described together below, except where a specific one ofFIGS.4A-4Dis noted. The collection device400includes an elongate body405that forms an integrated handle and cartridge. The elongate body405can be formed from an upper shell and a lower shell coupled together. This elongate body405serves to enclose the test strip430and fluid path495as well as provide an elongate handle for a user to grasp while swabbing a test surface. The elongate body405includes an aperture420on its upper surface exposing a detection region of the test strip430. Signals generated at the detection region of the test strip430can be detected through a transparent or translucent material forming a window425in the aperture420. The window425also maintains a sealed compartment for the test strip430, which may become saturated with a liquid containing hazardous contaminants. The window425may be flat, or may follow the contours of the aperture420. On its lower surface, the integrated handle and cartridge405can include a track recess490that can engage a correspondingly shaped protrusion or rail of a reader device when the elongate body405is inserted into the reader device. The track recess490can be formed in the lower surface and the proximal end498of the collection device400. In other embodiments the track recess490can be replaced with a rail or track, with the reader device having a corresponding track recess. Other suitable alignment features can be used in other embodiments. The lower surface can include additional mechanical features (e.g., grooves, detents, protrusions) that mate with corresponding features of the reader device. FIGS.4A and4Bshow a cap410secured to the distal end499of the elongate body405. The cap405includes a grip tab415to facilitate its removal by a user. The cap405also includes a reattachment clip411. When the user snaps off the cap for sample collection, the cap can be reattached after use by this small clip. The cap410covers the swab material435, which is visible inFIGS.4C and4Dand extends beyond the distal end499of the elongate body405. Some embodiments can include a semi-rigid sheet of material within or along a surface the swab material435, which can assist in sample collection by acting as a squeegee and/or backer that supports the swab material435. For example, a ledge440of the upper surface of the elongate body405can counter pressure placed on the swab material435by a test surface during swabbing and keep the swab material435firmly engaged with the test surface. In some embodiments, the cap410can be broken away from the elongate body405, folded at a hinge region to be flush with the bottom of the elongate body405, and secured there with mechanical mating features. The cap410can then be folded back to its original position to re-seal the swab material435after swabbing a test surface. The user can moisten the test surface using a solution, hold the elongate body405, and pass the swab material435along the test surface, for example as described with respect toFIG.1. The solution can be provided separate from the collection device400. In this implementation, the user may not have to perform steps of extracting the collected sample from the swab material435and homogenizing the sample. Instead, the test strip430has been extended along a longitudinal axis of the device in a direction away from the reaction zone and leading to a portion that is widened into the swab material435, allowing the test strip430to function as a collection device in addition to functioning as a detection device. As such, fluid picked up by the swab material435is drawn directly into the test strip430(which can be a lateral flow assay test strip), for example via capillary action along the fluid path495. In the embodiment ofFIGS.4A-4D, the fluid path495is formed by the continuous material extending from the swab material435region to the test strip430region. Absorbed fluid can begin to saturate the test strip430as enough fluid is absorbed by the swab material435. As such, processing (e.g., binding of any collected contaminants to compounds in or on the test strip430) can begin to take place even during swabbing of the test surface and development (e.g., the appearance of one or more optically-detectable signals (such as lines) at the reaction zone) can occur (including one or more optically-detectable signals at a test region if the contents of the absorbed liquid include an analyte of interest and/or one or more optically-detectable signals at a control region confirming that the test strip430functions as intended). Signals generated on the test strip430can continue to develop until required processing time for result verification is complete. In some embodiments the test strip430and swab material435can be a unitary piece of the same material. In other embodiments the swab material435can be a different piece of material, potentially a different type of material, affixed to the test strip430. For example, the swab material435can be any suitable material having desired pickup and/or shedding efficiency for a target contaminant, including but not limited to materials described herein. The test strip430may be any suitable material, including but not limited to materials having structures (e.g., fibers and/or channels) to wick fluid via capillary action in the direction depicted for the fluid path495. FIGS.5A-5Gdepict another embodiment of a collection device500with an integrated swab material535and a test device530according to the present disclosure. Specifically,FIG.5Ashows a back (proximal end), top (upper surface), and side perspective view of the collection device500with a cap510in a first orientation515A, andFIG.5Bshows a front (distal end), top, and side perspective view of the collection device500with the cap510in a second orientation515B.FIGS.5A and5Bare described together below, except where a specific one ofFIGS.5A and5Bis noted. The collection device500includes an elongate body505that forms an integrated handle and cartridge. The elongate body505can be formed from an upper shell506A and a lower shell506B coupled together. This elongate body505serves to enclose the test strip530and fluid path595as well as provide an elongate handle for a user to grasp while swabbing a test surface. The elongate body505includes an aperture520on its upper surface exposing a detection region of the test strip530. Signals generated at the detection region of the test strip530can be detected through a transparent or translucent material forming a window525in the aperture520. The window525also maintains a sealed compartment for the test strip530, which may become saturated with a liquid containing hazardous contaminants. The window525may be flat, or may follow the contours of the aperture520. On its lower surface, the integrated handle and cartridge505can include a track recess590that can engage a correspondingly shaped protrusion or rail of a reader device when the elongate body505is inserted into the reader device. The track recess590can be formed in the lower surface and the proximal end598of the collection device500. In other embodiments the track recess590can be replaced with a rail or track, with the reader device having a corresponding track recess. Other suitable alignment features can be used in other embodiments. The lower surface can include additional mechanical features (e.g., grooves, detents, protrusions) that mate with corresponding features of the reader device. FIGS.5A and5Bshow a cap510secured to the distal end599of the elongate body505. The cap510covers the swab material535when applied and extends beyond the distal end599of the elongate body505. The cap510includes a swab receiving member540. InFIG.5A, the cap510is in a first orientation515A, while inFIG.5B, the cap510is in a second orientation515B in which it has been flipped over (e.g., rotated 180 degrees around its longitudinal axis) compared to the first orientation515A. As will be described in further detail below, this change in orientation provides certain benefits. In the first orientation515A, the swab material535(visible inFIG.5Athrough the cap510) abuts a surface of the swab receiving member540, thereby preventing the distal end599of the elongate body505from extending fully into the cap510. This protects the swab material535from contamination during shipment and storage. The user may receive the device with the cap in the first orientation515A, remove the cap510, perform swabbing as described herein, and then reapply the cap in the second orientation515B in which the cap is rotated 180 degrees from the position it was in when it was first attached to the device. In the second orientation515B, the swab material535(not visible inFIG.5B) is fully received by the swab receiving member540, and the distal end599of the elongate body505extends fully (e.g., to its maximum possible distance) into the cap510. This flushes the swab material535with a solution stored in the cap as the swab material breaks the seal containing the solution. It will be understood that the cap510may not include a swab receiving member540in some cases, or may include a swab receiving member540that is shaped and sized differently than in this example implementation. In some implementations, the cap510includes a tab555that engages a detent550on the upper surface of the elongate body505. Further details of the structure of the cap510and its orientations are described with respect toFIGS.5E-5F. FIG.5Cshows a top view of the collection device500without its upper cartridge portion506A (with only the lower cartridge portion506B) to reveal the inner components.FIG.5Calso depicts the collection device500without the cap510, illustrating how the swab material535extends from the distal end599of the elongate body505in a direction away from a reaction zone of the test strip530, and depicting how the swab material535can have a tapered distal end536. Some embodiments can include a semi-rigid sheet of material within or along a surface the swab material535, which can assist in sample collection by acting as a squeegee and/or backer that supports the swab material535.FIG.5Dshows the swab material535, a retaining member560configured to retain the swab material535, and the test strip530.FIGS.5C and5Dare described together below, except where a specific one ofFIGS.5C and5Dis noted. As shown inFIG.5C, the test strip530is housed in an enclosure570of the elongate body505. The enclosure570can be considered as the interior cavity of the elongate body505, formed by interior surfaces of the upper cartridge portion506A, lower cartridge portion506B, and retaining member560. The enclosure570can be substantially sealed, for example in a fluid-tight manner, within the elongate body505. “Substantially sealed” refers to how the enclosure570is designed to prevent egress of potentially contaminated fluid from its interior, but still includes the fluid path595that allows passage of fluid from the swab material535into the enclosure570to contact the test strip530. For example, the enclosure570can have a fluid-tight seal along a seam or junction between the upper cartridge portion506A and lower cartridge portion506B, and can have a fluid-tight seal along a seam or junction at a distal aperture of the elongate body505between the upper cartridge portion506A, the lower cartridge portion506B, and the retaining member560. The channel565can enable fluid in the swab material535to flow to the test strip530. The cap510(not pictured inFIGS.5C and5D) can complete the seal around the enclosure570by preventing egress of liquid from the swab material535into the environment of the collection device500. The retaining member560couples the swab material to the elongate body505and also provides a fluid path595between the swab material535and the test strip530. Specifically, the retaining member560includes a distal collar563forming a recess564that holds a proximal portion of the swab material535. The retaining member can include a fluid transfer member561that extends away from the distal collar563and forms a channel565leading from the recess564to an aperture562disposed adjacent to the test strip530. It will be understood that the retaining member560is not limited to the specific configuration described with respect to this example, and can be implemented using different structures that provide a fluid path595between the swab material535and the test strip530. As described in more detail below with respect toFIG.5G, when the cap510is pressed onto the elongate body505in the second orientation515B, this can cause compression of the swab material535(and can release additional fluid into the swab material535), pushing fluid through the channel565in the direction depicted for the fluid path595. The distal collar563can include a contoured surface566that engages with (and in some examples forms a seal with) internal features of the cap510. Thus, the retaining member560secures the swab material535, provides a fluid path595along which fluid can travel from the swab material535to the test strip530, and also forms a seal to enclose the fluid path595leading into the enclosure570. The user may receive the collection device500packaged with the cap510applied in the first orientation515A. As shown inFIG.5A, in this first orientation515A, the cap510may not engage the detent550on the upper surface of the elongate body505. The swab material535can be pre-moistened, for example with a liquid designed to optimize pickup efficiency of the target contaminant from a test surface. In the first orientation515A, the cap510can form a seal with the upper and lower cartridges506A,506B to maintain the swab material535in its pre-moistened condition. The user can remove the cap510and pass the moistened swab material along the test surface, for example as described above with respect toFIG.1. After the collection procedure is complete, the user can put the cap510back onto the elongate body505in the second orientation515B. In this second orientation515B, as described in further detail below, additional fluid stored within the cap is released onto the swab material535, causing it to over-saturate and release fluid along the fluid path595through the channel565to the test strip530. FIGS.5E-5Fillustrate how the cap510is configured to both compress the swab material535and also flush additional solution through the swab material535after the swab material535has been used to collect a sample.FIG.5Eshows a perspective view of the cap510and the swab receiving member540positioned within the interior of the cap510. The swab receiving member540has a lip543that engages with an interior wall of the cap510. The lip543can include a contoured surface548shaped to match the shape of contoured surface566of the swab retaining member560. A gasket544is disposed along the contoured surface566to seal with the contoured surface566. In the depicted example, the gasket544and contoured surface548are arch-shaped, however the particular design can vary in other implementations. A wall546, formed in this example as a rectangular tube, extends away from the lip543to form a slot542sized and positioned to receive the swab material535when the cap510is applied to the elongate body505in the section orientation515B. The wall546may assume other geometric configurations in other designs such that the shape of the slot542generally corresponds to the exterior shape of the swab material535. In some implementations, the slot542may taper along its length to encourage liquid to move backwards through the swab material535(e.g., in a direction from the distal end599of the collection device500toward the proximal end598of the collection device500) as the swab material535is inserted into the slot542. An aperture545is formed at a distal end of the slot542. InFIG.5E, the aperture545is covered by a frangible seal541. The frangible seal541can be formed from a liquid-tight material that may be pierced, broken, or detached from the wall546upon application of a certain amount of force from the swab material535(as described with more detail with respect toFIG.5G). When the seal541is opened, fluid can flow from the reservoir513into the slot542(and any swab material535positioned in the slot542). The swab receiving member540may be a separately manufactured component from the body of the cap510, and can be inserted into the cap510through its proximal aperture514. The cap510can have a ledge511that retains the swab receiving member540in its proper position within the cap510(e.g., positioned so that the swab material535will be received in the slot542). For example, the reservoir513of the cap510can be filled with liquid, for example a buffer solution designed to flush collected contaminant off of the swab material535, and then the swab receiving member540with the frangible seal541sealing the aperture545can be inserted into the cap510. This can seal the liquid in the reservoir513of the cap until the frangible seal541is broken. Once in place, the swab receiving member540can be affixed in place with suitable means including but not limited to pressure, adhesive, ultrasonic welding, and mechanical fasteners. FIG.5Ealso depicts the structure of the tab555in greater detail. The tab555includes a protrusion558on its surface that faces the upper cartridge portion506A. The protrusion558has a sloped or curved surface556that faces proximally as the cap510is inserted onto the elongate body505and a flat surface557that faces distally as the cap510is inserted onto the elongate body505. This shape can correspond to the shape of the detent550on the elongate body505in order to snap the cap510into place and/or lock the cap510to the elongate body505when in the second orientation515B. It will be understood that other configurations of the tab555can be suitably implemented in embodiments of the present disclosure. FIG.5Fshows a cross-sectional view of the distal end599of the collection device500with the swab material535omitted for ease of viewing other components. In this view, the cap510is fully applied to the elongate body505in the second orientation515B. This cross-sectional view illustrates how the recess564of the retaining member560tapers (e.g., has an increasingly smaller cross section) towards the aperture545. This cross-sectional view also illustrates how the recess564of the retaining member560aligns with the slot542of the swab receiving member540when the cap510is applied to the elongate body505in the second orientation515B. The reservoir513has a height H along a dimension between the upper surface and the lower surface of the elongate handle505. This height H can correspond to the height of the elongate handle505along the same dimension. As illustrated inFIG.5F, the slot542and recess564are vertically offset (e.g., not centered) along the height H. This results in a mismatch between the swab material extending from the recess564and the slot542when the cap is in the first orientation515A, such that the swab material535abuts the lip543rather than entering the slot542. This can prevent the swab material535from breaking the frangible seal541when the cap is applied in the first orientation515A. The vertical offset of the recess564can be the same as the vertical offset of the slot542along the height H so that they align with one another when the cap510is applied to the elongate body505in the second orientation515B. In this example, the cap510has a rectangular cross section, and the second orientation515B represents reapplication of the cap after a 180 degree rotation (about the cap's longitudinal axis around its length dimension) of the cap510relative to the first orientation515A. Other implementations can structure the cap510and the distal end599of the elongate body505so that other rotations from the first orientation515A yield the second orientation515B. For example, in another implementation the cap510may have a square, circular, or other rotationally symmetric cross section, and can be rotated any number of degrees less than 360 degrees to switch to the second orientation515B from the first orientation515A.FIG.5Falso illustrates how the gasket544can seal the negative space formed by the joining of the slot542and the recess564. Beneficially, this can isolate any contaminants collected by the swab material535within the sealed enclosure570, protecting the environment and/or user of the collection device500from exposure to potentially hazardous compounds. FIG.5Gshows a cross-sectional view of the distal end599of the collection device500with the swab material535, and the cap510fully applied to the elongate body505in the second orientation515B.FIG.5Galso includes a legend580depicting dimensions corresponding to the height (H), length (L), and width (W) of the collection device500as shown inFIGS.5F-5G. This cross-sectional view illustrates the positioning of the swab material535relative to the receiving member540.FIG.5Gdepicts the swab material535in its original, uncompressed shape, and as such it appears to spatially overlap with the receiving member540due to the taper of the slot542. It will be appreciated that in use the swab material535would be compressed to correspond to the taper of the slot542. In addition, the length of the swab material535is greater than the combined length of the slot542and the recess564, such that the tapered distal end536extends distally beyond the position of the frangible seal541. This can cause the frangible seal541to break or otherwise open, releasing the fluid of the reservoir513into the swab material535. The tapered distal end536can also facilitate insertion of the swab material535into the tapered slot542. As such, the application of the cap510in the second orientation515B, as shown inFIG.5G, beneficially provides both flushing of the swab material535(by breaking the seal541) and compression of the swab material535to encourage flow of fluid (and any collected contaminants) through the channel565and onto the test strip530. As described above, the test strip530can be a lateral flow assay test strip, which may have a predetermined development time for viewing of results of the test. In some embodiments, this development time can begin upon application of the cap510in the second orientation515B, which causes liquid to flush the swab material535and carry any collected sample to the test strip530. After the development time, a user and/or reader device can view the detection region of the test strip530to determine the presence and/or concentration of the target contaminant at the test surface, for example based on the number and/or intensity of lines that develop in the detection region. FIGS.6A-6Edepict another embodiment of a collection device600with an integrated swab material635and test device630according to the present disclosure. Specifically,FIG.6Ashows a front (distal end), top (upper surface), and side perspective view of the collection device600with a cap610applied, andFIG.6Bshows the front, top, and side perspective view of the collection device600with the cap610removed to expose the swab material635.FIGS.6A and6Bare described together below, except where a specific one ofFIGS.6A and6Bis noted. The collection device600includes an elongate body605that forms an integrated handle and cartridge. The elongate body605can be formed from an upper shell606A and a lower shell606B coupled together. This elongate body605serves to enclose the test strip630and fluid path as well as provide an elongate handle for a user to grasp while swabbing a test surface. The elongate body605includes an aperture620on its upper surface exposing a detection region of the test strip630. Signals generated at the detection region of the test strip630can be detected through a transparent or translucent material forming a window625in the aperture620. The window525also maintains a sealed compartment for the test strip630, which may become saturated with a liquid containing hazardous contaminants. The window625may be flat, or may follow the contours of the aperture620. On its lower surface, the integrated handle and cartridge605can include a mechanical feature (not shown) that can engage a correspondingly shaped mechanical feature of a reader device when the elongate body605is inserted into the reader device. The swab material635is secured by a retaining member660. A collar650secures the retaining member660to the elongate body605. As described with respect toFIGS.6D and6E, the collar650can be slidably engaged with the elongate body605such that a user can move the collar650(and the attached retaining member660and optionally the cap610) toward the proximal end698of the collection device. In some embodiments, a user can press a button651to release the collar650, retaining member660, and swab material635(optionally with the cap610attached) from the elongate body605. The user can separate the collection device600into a collection portion (including the collar650, retaining member660, swab material635, and optionally the cap610) and a test portion (including the elongate body605and the test strip630). Beneficially, this can allow the user to connect the collar650to a different elongate body605A containing another test strip630A (not illustrated), for example to apply the same collected sample to multiple test strips. As described in more detail with respect toFIGS.6D and6E, one or both of the collection portion and the test portion can have sealing features such that their interior enclosures are sealed during the decoupling of the collection portion from the test portion. Beneficially, this can prevent potentially hazardous contamination from spilling during the decoupling and as the separated portions are handled, and can maintain any additional collected sample within the collection portion. FIG.6Cshows a top view of the collection device600without its upper cartridge portion606A (with only the lower cartridge portion606B) to reveal the inner components.FIG.6Calso depicts the collection device600with the cap610removed, illustrating how the swab material635extends from the distal end699of the elongate body605. Some embodiments can include a semi-rigid sheet of material within or along a surface the swab material635, which can assist in sample collection by acting as a squeegee and/or backer that supports the swab material635. The retaining member660couples the swab material635to the elongate body605. As shown inFIG.6C, the test strip630is housed in an enclosure670of the elongate body605. The enclosure670can be considered as the interior cavity of the elongate body605, formed by interior surfaces of the upper cartridge portion606A, lower cartridge portion606B, and retaining member660. The enclosure670can be substantially sealed, for example in a fluid-tight manner, within the elongate body606. “Substantially sealed” refers to how the enclosure670is designed to prevent egress of potentially contaminated fluid from its interior, but still includes the fluid path (a second portion695B which is depicted inFIGS.6C and6E) that allows passage of fluid from the swab material635into the enclosure670to contact the test strip630. For example, the enclosure670can have a fluid-tight seal along a seam or junction between the upper cartridge portion606A and lower cartridge portion606B, and can have a fluid-tight seal along a seam or junction at a distal aperture of the elongate body605between the upper cartridge portion606A, the lower cartridge portion606B, and the retaining member660. A dosing mechanism680, described in further detail with respect toFIGS.6D and6E, selectively passes fluid eluted from the swab material635along the second portion695B of the fluid path to the test strip630. The dosing mechanism680can be open to receive fluid from the swab material635and can seal the second portion695B of the fluid path in the enclosure670when in a “fluid entry” configuration. The dosing mechanism680can also seal off (e.g., fluidically isolate) the swab material635from the enclosure670and open the fluid path into the enclosure670when in a “fluid delivery” configuration. FIG.6Calso depicts the swab receiving member640of the cap610. The swab receiving member640is sized and positioned to receive the swab material635when the cap610is applied to the retaining member660. A reservoir613formed in the cap610can hold a liquid, for example a buffer solution designed to promote shedding of collected contaminants from the swab material635. When a user actuates a pump button611, this can break a frangible seal641(visible inFIGS.6D and6E) of the swab receiving member640, thereby saturating the swab material635. It will be understood that embodiments of the present disclosure can be implemented with a cap that does not include a swab receiving member and/or reservoir in the cap. The user may receive the collection device600packaged with the cap610applied. The swab material635can be pre-moistened, for example with a liquid designed to optimize pickup efficiency of the target contaminant from a test surface. The cap610can include gasket(s) or other sealing mechanisms to maintain the pre-moistened condition of the swab material635prior to use. The user can remove the cap610and pass the moistened swab material635along the test surface, for example as described above with respect toFIG.1. After the collection procedure is complete, the user can put the cap610back onto the retaining member660and activate the pump button611to flush any collected contaminants from the swab material635. FIG.6Ddepicts a cross-sectional view of the distal end699of the collection device600showing the collar650in a first position and the dosing mechanism680in the fluid entry configuration, andFIG.6Edepicts a cross-sectional view of the distal end699of the collection device600showing the dosing mechanism680in the fluid delivery configuration. FIGS.6D and6Edepict additional details of the cap610, including a seal612in the pump button assembly. The cap610also includes the frangible seal641positioned between the reservoir613and the swab material635. The pump button611can be a deformable material that allows it to be compressed into a pump chamber614of the push button assembly, for example by a finger of a user. The user can depress the pump button611a number of times to increase the pressure within the reservoir613, causing the seal641to break. The pump button611can include vents (e.g., small apertures) to allow air to be drawn into the pump chamber614when the pump button611is released. The filter612can be a gas-permeable, liquid impermeable material to prevent any liquid within the reservoir613from being released into the environment of the collection device600. In some embodiments, the filter612can be hydrophobic. The user may depress the pump button611an additional number of times to flush fluid through the swab material. FIGS.6D and6Ealso depict additional details of the retaining member660. The swab material635is retained in a collar664of the retaining member660. The retaining member660forms sample receiving bladder663on the proximal side of the collar664for containing any fluid eluted from the swab material635along the first portion695A of the fluid path between the swab material635and the test strip630. The first portion695A of the fluid path extends between the proximal side of the swab material635and the distal end of the dosing mechanism680. The collar664includes apertures661that allow fluid to pass from the swab material635into the sample receiving bladder663. A number of fins662are disposed on the proximal surface of the collar664along support structures665that extend between (and form) the apertures661. Beneficially, the fins662can promote turbulence in fluid eluted from the swab material635into the sample receiving bladder663, mixing the fluid into a homogenous solution. After depressing the pump button611the desired number of times to flush the swab material635, the sample receiving bladder663contains a substantially homogenous solution including any collected contaminants. This solution can be provided to the test strip630via the dosing mechanism680. For example, the user can slide the collar650proximally along the elongate body605in the direction of arrow697. This can also cause movement of the retaining member660that is coupled to the collar650relative to the elongate body605, where the motion of the retaining member660is toward the proximal end698of the elongate body605. A tubular end652of the retaining member660can slide into a corresponding recess654surrounding the dosing mechanism680, until the collar650has transitioned from the first position shown inFIG.6Dto the second position shown inFIG.6E. The dosing mechanism680can include a tubular body681forming an interior lumen686. A piston682can be disposed within the interior lumen686for selectively sealing and opening the dosing mechanism. The piston682can have an enlarged distal end683having a diameter that substantially corresponds to the interior diameter of the interior lumen686, such that the enlarged distal end683can both prevent fluid from entering the distal aperture of the interior lumen686and push fluid through the proximal aperture of the interior lumen686. The piston682can have an elongate length extending between the enlarged distal end683and a proximal seal684. The proximal seal684can be formed as a dish-shaped member that seals the proximal aperture of the interior lumen686when in the configuration shown inFIG.6D. The piston682can be biased in a position that causes the proximal seal684to seal the proximal aperture, for example by a spring685or other biasing element (e.g., shape memory alloy, magnets). The enlarged distal end683can be spring-loaded and intended to push through a pre-slit valve. The pre-slit valve can be connected to the retaining member660assembly as a seal, while the enlarged distal end683is part of the cartridge assembly. This action can dose the correct volume (e.g. 75 microliters plus or minus 50-150 microliters, in some implementations) through and onto the strip. The collar650can be biased toward the retaining member660so that releasing its locking feature and sliding it away from the retaining member660would release the assembly of the retaining member660from the cartridge. The collar650can have connections to the enlarged distal end683closer to the proximal seal684which can control its movement. Overview of Example Assay Reader Devices and Operations FIG.7illustrates a schematic block diagram of one possible embodiment of components of an example assay reader device700. The components can include a processor710linked to and in electronic communication with a memory715, working memory755, cartridge reader735, connectivity module interface745, and display750. Connectivity module745can include electronic components for wired and/or wireless communications with other devices. For example, connectivity module745can include a wireless connection such as a cellular modem, satellite connection, or Wi-Fi, or via a wired connection. Thus, with connectivity module745the assay reader device can be capable of sending or uploading data to a remote repository via a network and/or receiving data from the remote repository. As such, the test data of such assay reader devices can be stored and analyzed, alone or in the aggregate, by remote devices or personnel. A module having a cellular or satellite modem provides a built-in mechanism for accessing publicly available networks, such as telephone or cellular networks, to enable direct communication by the assay reader device with network elements or other testing devices to enable electronic test result transmission, storage, analysis and/or dissemination without requiring separate intervention or action by the user of the device. In some embodiments connectivity module745can provide connection to a cloud database, for example a server-based data store. The cloud based connectivity module can enable ubiquitous connectivity of assay reader devices without the need for a localized network infrastructure. The cartridge reader735can include one or more photodetectors740for reading an assay held in an inserted cartridge and optionally any information on the inserted cartridge, for example a barcode printed on the cartridge, and one or more light emitting devices742for illuminating the inserted cartridge at one or more wavelengths of light. The cartridge reader735can send image data from the one or more photodetectors to the processor710for analysis of the image data representing the imaged assay to determine a test result of the assay. The cartridge reader735can further send image data from the one or more photodetectors representing the imaged cartridge for use in determining which one of a number of automated operating processes to implement for imaging the assay and/or analyzing the image data of the assay. The photodetector(s)740can be any device suitable for generating electric signals representing incident light, for example a PIN diode or array of PIN diodes, a charge-coupled device (CCD), or a complementary metal oxide semiconductor (CMOS) sensor, to name a few examples. The cartridge reader735can also include a component for detecting cartridge insertion, for example a mechanical button, electromagnetic sensor, or other cartridge sensing device. An indication from this component can instruct the processor710to begin an automated assay reading process without any further input or instructions from the user of the device700. Processor710can be configured to perform various processing operations on image data received from the cartridge reader735and/or connectivity module interface745in order to determine and store test result data, as will be described in more detail below. Processor710may be a general purpose processing unit implementing assay analysis functions or a processor specially designed for assay imaging and analysis applications. The processor710can be a microcontroller, a microprocessor, or ASIC, to name a few examples, and may comprise a plurality of processors in some embodiments. As shown, the processor710is connected to a memory715and a working memory755. In the illustrated embodiment, the memory715stores test result determination component725, data communication component730, and test data repository705. These modules include instructions that configure the processor710of device700to perform various module interfacing, image processing, and device management tasks. Working memory755may be used by processor710to store a working set of processor instructions contained in the modules of memory715. Alternatively, working memory755may also be used by processor710to store dynamic data created during the operation of device700. As mentioned above, the processor710may be configured by several modules stored in the memory715. The test result determination component725can include instructions that call subroutines to configure the processor710to analyze assay image data received from the photodetector(s)740to determine a result of the assay. For example, the processor can compare image data to a number of templates or pre-identified patterns to determine the test result. In some implementations, test result determination component725can configure the processor710to implement adaptive read processes on image data from the photodetector(s)740to improve specificity of test results and to reduce false-positive results by compensating for background and non-specific binding. The data communication component730can determine whether a network connection is available and can manage transmission of test result data to determined personnel and/or remote databases. If the device700is not presently part of a network, the data communication component730can cause local storage of test results and associated information in the test data repository705. In some case, the device700can be instructed to or automatically transmit the stored test results upon connection to a network. If a local wired or wireless connection is established between the device700and another computing device, for example a hospital, clinician, or patient computer, the data communication component730can prompt a user of the device700to provide a password in order to access the data in the repository705. The processor710can be configured to control the display750to display captured image data, imaged barcodes, test results, and user instructions, for example. The display750may include a panel display, for example, a LCD screen, LED screen, or other display technologies, and may implement touch sensitive technologies. Processor710may write data to data repository705, for example data representing captured images of assays, instructions or information associated with imaged assays, and determined test results. While data repository705is represented graphically as a traditional disk device, those with skill in the art would understand that the data repository705may be configured as any storage media device. For example, data repository705may include a disk drive, such as a hard disk drive, optical disk drive or magneto-optical disk drive, or a solid state memory such as a FLASH memory, RAM, ROM, and/or EEPROM. The data repository705can also include multiple memory units, and any one of the memory units may be configured to be within the assay reader device700, or may be external to the device700. For example, the data repository705may include a ROM memory containing system program instructions stored within the assay reader device700. The data repository705may also include memory cards or high speed memories configured to store captured images which may be removable from the device700. AlthoughFIG.7depicts a device having separate components to include a processor, cartridge reader, connectivity module, and memory, one skilled in the art would recognize that these separate components may be combined in a variety of ways to achieve particular design objectives. For example, in an alternative embodiment, the memory components may be combined with processor components to save cost and improve performance. Additionally, althoughFIG.7illustrates a number of memory components, including memory715comprising several modules and a separate memory755comprising a working memory, one of skill in the art would recognize several embodiments utilizing different memory architectures. For example, a design may utilize ROM or static RAM memory, internal memory of the device, and/or an external memory (e.g., a USB drive) for the storage of processor instructions implementing the modules contained in memory715. The processor instructions may be loaded into RAM to facilitate execution by the processor710. For example, working memory755may comprise RAM memory, with instructions loaded into working memory755before execution by the processor710. Overview of Example Networked Testing Environment Aspects of the present disclosure relate to a contamination test data management system. There are drug preparation systems, surface contamination tests, and healthcare worker safety procedures in the hospital and other healthcare delivery environments. These three areas are connected only to the extent that they have a common goal: to reduce or eliminate healthcare worker exposure to hazardous drugs, and to ensure patients are provided correct drug doses. The described hazardous contamination detection kits, systems and techniques improve upon existing approaches by linking these three areas, sensing patterns and trends, and targeting worker feedback and training. By creating and analyzing associations between data regarding dose preparation, personnel activities, and contamination test results, the disclosed systems can provide information to healthcare workers and management targeted at risk identification, feedback, and training. A beneficial outcome can include behavioral and/or workflow changes to reduce exposure risk in the test areas. There are several existing solutions for assisting with pharmacy (or other clinical setting) drug preparation workflow. Each of these systems is designed to enhance patient safety through automated preparation or verification steps in compounding drugs. These systems are often used with hazardous drugs, such as chemotherapy agents, because there is little room for error with these drugs due to the health risks of exposure to even trace amounts. One such system performs automated dose calculation, weight-based (gravimetric) preparation and verification, integrated drug and consumable barcode verification, real-time automated documentation of the compounding process, and step-by-step compounding guidance. Other examples can employ a camera that captures images of products used in dose preparation and optionally an integrated weighing scale design with step-by-step guidance and automatic documentation. While these systems help automate several aspects of drug preparation, they do not address pre- and post-preparation issues in the pharmacy, such as managing data associated with surface contamination testing (for example, floors, walls, hoods, etc.). They also do not manage data associated with air testing, nor data from testing individuals via fingertip, urine, blood or any other personal exposure monitoring. Surface wipe tests are available from companies such as ChemoGLO™ which provide quantitative analysis of the antineoplastic agents 5-fluorouracil, ifosfamide, cyclophosphamide, docetaxel and paclitaxel. An example existing kit contains enough materials to conduct six surface wipes. The wipes and samples are sent to an outside laboratory, and reports are provided back to the test location within three to four weeks. Such tests and delayed reports are disconnected processes from day to day activities in the pharmacy. Hazardous drugs, particularly chemotherapy drugs, are known to contaminate surfaces and air in pharmacies and other patient care settings, which presents a significant health risk to pharmacy and other healthcare workers. Further, the United States Pharmacopeia (Cpater 797, 28th Rev) recommends sampling of surfaces for contamination with hazardous drugs at least every six months. With improved testing technology, better feedback and improved outcomes, the frequency of testing is expected to become a more routine activity. FIG.8depicts a high level schematic block diagram of an example networked test system environment800. Hazardous contamination detection kits described herein can be used in the networked test system environment800to improve contamination detectin, risk identification, feedback, and training. The networked environment800includes a user interface805, dose preparation system820, surface contamination test825, and reporting system815in network communication with a central server810(and/or one another) via a network. The network can be any suitable data transfer network or combination of networks including wired networks and/or wireless networks such as a cellular or other publicly accessible network, WiFi, and the like. The user interface805supports system interaction by the test operator and can be located in the work area, for example in or near the testing environment. This facilitates interaction without the test operator having to remove and reapply personal safety equipment in order to use the system. The dose preparation system820can be hardware associated with a gravimetric dose preparation system, a scale, robotics, or devices that are designed to assist in the preparation of safe drug doses for the patient. The surface contamination test825can include a local test processing system which is in network communication with at least the central server810. For example, the local test processing system can be the assay reader device800ofFIG.8. Central server810can implement the algorithms, decisions, rules, and heuristics involved with management of contaminant testing data, and can store data (individual and aggregate), handle data input and/or output, generate reports, provide the user interface, and the like. Though referred to as a central server, these functions could be carried out in a distributed fashion, virtually, in any location. The reporting user interface815can provide raw and processed data to the user or safety manager regarding the relationship between activities in the pharmacy and test results. In some implementations, the above descriptions apply to tests that are performed immediately in a pharmacy, hospital, or other clinical setting. However, the described testing is not limited to architectures where instant, immediate, or real-time connectivity is available. For example, if a local wipe test processing system is not available, data from a remote system can be transmitted to the central server using any number of methods. Results from tests may be fed in to an interface manually, electronically encoded, or in machine readable format. Data networks (e.g., internet, wireless, virtual private, cloud-based) can be used to input data from a remote lab (outside the pharmacy, hospital, or clinic) that performs testing either immediately or at a later time. The main difference between immediate local contamination detection versus remote testing is a potential time delay. As described above, current contaminant detection occurs in a two-step process with the steps performed at different locations. First, collection happens at site of possible contamination. Collection occurs a time A. Second, detection of the contamination occurs in a laboratory facility geographically separate from the contamination. Detection occurs at a time B, which is weeks or even months after collection occurred. The present disclosure provides a system including collection device and detection device in one kit. Using the disclosed kit, collection and detection occur at the site of possible contamination, and detection occurs within minutes of collection. For example, collected fluid can be provided onto an assay immediately (for example, within seconds such as but not limited to within 1, 2, 3, 4, 5, 10, or 15 seconds) after agitation of the fluid within a container as described herein. The collected fluid can be provided to the assay for up to 3 hours (360 minutes) after agitation in some embodiments. In some embodiments, instructions for use include a recommendation to the user not to apply the collected fluid to the assay more than 3 hours after collection because accuracy may decrease after 3 hours. After the fluid is added to the assay it can take around five minutes to fully develop in some non-limiting examples. In one advantageous implementation, the assay is read by a detection system around the time of its complete development. As such, the disclosed kits can provide test results indicating the presence, absence, and/or degree of contamination between 2-365 minutes after completion of sample collection, in some embodiments. Laboratory testing of embodiments of test kits described herein has demonstrated that reliable results can be obtained within about 5 minutes of completion of sample collection, and in some cases in as little as 2 minutes of completion of sample collection. This represents a dramatic improvement in the time to obtain a test result indicating the presence, absence, and/or degree of contamination of a hazardous drug over prior systems. Embodiments of the system800described herein directly link activities performed in the test environment to test results. For example, the system800can directly link contaminant test results to when activities (for example, during antineoplastic drug preparation, dosing, and the like) were performed, who performed these activities (for example, through authentication), where the activities occurred (which hood, nearby floor, air test), and other events (such as spills, wasting of materials, or improper waste disposal) which can be manually or automatically recorded. In some embodiments, the central server810can perform analysis of the related information to identify trends in hazardous contamination levels, and can output recommendations for preventing or mitigating hazardous contamination levels in certain areas. FIG.9depicts a flow chart of an example process900for test data generation, analysis, and reporting that can be implemented in some embodiments of the system800ofFIG.8. The dose preparation system820, whether volumetric, gravimetric, photographic, or bar code scanning, can be capable of keeping a record of every dose that was prepared in a particular pharmacy hood or other work area or clinical care area, when the dose was prepared and/or administered, and who prepared and/or administered the dose (for example, the identity of the pharmacy technician). As described above, this information can be correlated with the results of the contamination test. The correlation algorithm can, in some embodiments, match detected contamination with specific personnel who might have created or contributed to the contamination. For example, if three technicians worked in a hood, and only one worked with compound x, and compound x was identified in a contamination test, then the technician who worked with compound x might be targeted for training or follow up testing. The correlation algorithm can, in some embodiments, provide contamination test guidance by limiting tests to compounds that were actually used over a period of time, or used since the last contamination test. In a scenario where more than one test is required to screen for multiple possible contaminants, the cost may increase for a number of reasons. For example, it may take a longer period of time to perform testing due to more samples being needed. The time it takes to run a test may be longer. Sample preparation may be more complex. Each test may have an incremental cost, so tailoring tests may lower the overall cost. Advantageously, the dose preparation system could direct the user, or an automated system, to perform only contamination tests for drugs that were prepared in a specific location or hood. The correlation algorithm can, in some embodiments, improve the specificity of contamination tests by utilizing a priori knowledge of drugs that were prepared in the hood. For example, if a contamination test shows a positive result, but is not capable of indicating which of a family of possible contaminants actually has been identified, the database of drugs prepared in the hood could be queried for all of those possible drugs, and the test result narrowed to the ones actually prepared. In some implementations, further testing can be performed for those specific drugs. The correlation algorithm can, in some embodiments, determine systematic issues with devices used in preparing drugs. Drug preparation systems can have the capability to store information representing the products and devices used in drug preparation. For example, information on syringe types (manufacturer, volume etc.), closed system transfer devices, connectors, spikes, filters, needles, vials, and IV bags, to name a few examples, can be stored along with the drug and diluent data in the preparation systems database. Failures can be linked to specific devices and directly help with risk mitigation. The correlation algorithm can, in some embodiments, identify drug manufacturers, dose and containers that systematically fail, resulting in detected contamination. The correlation algorithm can identify procedures that commonly cause contamination, such as reconstitution steps. The system800can provide some or all of these analytics, alone or in combination, in various embodiments. The system800can be designed to implement workflows that are initiated based on a set of conditions. For example, one condition that can trigger a workflow is the detection of contamination. Examples of workflows are described below. A decontamination workflow can include the following procedures. The system800can instruct a user how to contain and decontaminate a specific area, depending on what area the test was performed in. Instructions can include audio, text, video, and the like. After decontamination, the workflow can continue to instructions on performing repeat contamination tests to ensure the area was properly decontaminated. If testing fails again, the decontamination procedure can be repeated. The system800can be configured to provide instructions through the user interface805and/or dose preparation system820(or any other means of communication, including printed instructions, other displays, voice output and input, direct messages to designated users, etc.). These instructions can be configured to be specific for certain sources of contaminants. Another example workflow is repeat testing of the area of contamination, prior to decontamination. This may be a useful workflow if the specificity of a particular test is not high. The objective could be to re-test with the same test, or perform further tests to identify more specifically, what the source and/or level of contamination is. A follow-on step could be specific decontamination instructions, already described above. In various workflows, system800can be configured to receive, prompt, and/or wait for input during the workflow to acknowledge completion of each step. The system800can be configured to capture decontamination procedure evidence, such as photographic, video, audio, proximity information for future review, training, documentation, and the like. System800can be configured to identify risks from preparation issues. For example, the system800can analyze data already captured by a drug preparation system, or provide means to capture data regarding drug preparation issues, problems or errors. For example, when material is wasted, the user involved can be questioned about whether there was a spill or any surface contamination that caused the wasting. System800can link wasting with positive contamination tests, if wasting is commonly caused by spills. System800can be adapted for use in non-pharmacy healthcare environments including, but not limited to, hospitals, clinics, hospice environments, and veterinary treatment centers. The system800can be adapted to other areas of patient care, such as the patient floor, nursing, drug delivery (e.g., infusion, injection), patient room, bathroom, etc. Contamination tests can be performed in any of these settings, and this data can be fed back to the system800. As described above, detected contamination can be correlated with personnel, protocols followed, specific drugs, devices, locations, and any other parameter of interest. Any parameter around the delivery of drugs that can be encoded can be correlated with the presence of contamination to provide feedback to risk managers, clinical and pharmacy personnel. Further, dose preparation and dispensing can occur in many locations outside the pharmacy, and similar workflows can be employed in those areas, including remote contamination test preparation and execution. The physical location of specific functions performed by the system800are not restricted to the pharmacy or hospital data center. Any structure or function of the system800, including the database, correlation and analysis, data entry, data display, reporting, etc., can be carried out in any system in any location. A system model may be to have a central web-based service, for example. Another model may be to have remote reporting and notification capability through remote devices like smart phones, pagers, computers, displays, applications etc. Supply of devices can be automated through any of the previously described systems. For example, pharmacies may be provided resupply of test kits by system800, and such resupply can be automated in some embodiments by managing an inventory of kits and initiating a resupply when stock falls below a certain level. Implementing Systems and Terminology Implementations disclosed herein provide systems, methods and apparatus for detection of the presence and/or quantity of antineoplastic agents or other environmental contaminants. One skilled in the art will recognize that these embodiments may be implemented in hardware or a combination of hardware and software and/or firmware. The assay reader device may include one or more image sensors, one or more image signal processors, and a memory including instructions or modules for carrying out the processes discussed above. The device may also have data, a processor loading instructions and/or data from memory, one or more communication interfaces, one or more input devices, one or more output devices such as a display device and a power source/interface. The device may additionally include a transmitter and a receiver. The transmitter and receiver may be jointly referred to as a transceiver. The transceiver may be coupled to one or more antennas for transmitting and/or receiving wireless signals. The functions described herein may be stored as one or more instructions on a processor-readable or computer-readable medium. The term “computer-readable medium” refers to any available medium that can be accessed by a computer or processor. By way of example, and not limitation, such a medium may comprise RAM, ROM, EEPROM, flash memory, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium that can be used to store desired program code in the form of instructions or data structures and that can be accessed by a computer. It should be noted that a computer-readable medium may be tangible and non-transitory. The term “computer-program product” refers to a computing device or processor in combination with code or instructions (e.g., a “program”) that may be executed, processed or computed by the computing device or processor. As used herein, the term “code” may refer to software, instructions, code or data that is/are executable by a computing device or processor. The various illustrative logical blocks and modules described in connection with the embodiments disclosed herein can be implemented or performed by a machine, such as a general purpose processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor can be a microprocessor, but in the alternative, the processor can be a controller, microcontroller, or state machine, combinations of the same, or the like. A processor can also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. Although described herein primarily with respect to digital technology, a processor may also include primarily analog components. For example, any of the signal processing algorithms described herein may be implemented in analog circuitry. A computing environment can include any type of computer system, including, but not limited to, a computer system based on a microprocessor, a mainframe computer, a digital signal processor, a portable computing device, a personal organizer, a device controller, and a computational engine within an appliance, to name a few. The methods disclosed herein comprise one or more steps or actions for achieving the described method. The method steps and/or actions may be interchanged with one another without departing from the scope of the claims. In other words, unless a specific order of steps or actions is required for proper operation of the method that is being described, the order and/or use of specific steps and/or actions may be modified without departing from the scope of the claims. It should be noted that the terms “couple,” “coupling,” “coupled” or other variations of the word couple as used herein may indicate either an indirect connection or a direct connection. For example, if a first component is “coupled” to a second component, the first component may be either indirectly connected to the second component or directly connected to the second component. As used herein, the term “plurality” denotes two or more. For example, a plurality of components indicates two or more components. The term “determining” encompasses a wide variety of actions and, therefore, “determining” can include calculating, computing, processing, deriving, investigating, looking up (e.g., looking up in a table, a database or another data structure), ascertaining and the like. Also, “determining” can include receiving (e.g., receiving information), accessing (e.g., accessing data in a memory) and the like. Also, “determining” can include resolving, selecting, choosing, establishing and the like. The phrase “based on” does not mean “based only on,” unless expressly specified otherwise. In other words, the phrase “based on” describes both “based only on” and “based at least on.” The previous description of the disclosed implementations is provided to enable any person skilled in the art to make or use implementations of the present disclosure. Various modifications to these implementations will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other implementations without departing from the scope of the present disclosure. Thus, the present disclosure is not intended to be limited to the implementations shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. | 93,823 |
11860174 | DETAILED DESCRIPTION OF THE INVENTION A first aspect of the present disclosure is directed to an isolated antibody or antigen binding fragment thereof, wherein said antibody or antigen binding fragment there of binds to cannabidiol (IUPAC name 2-[(1R,6R)-3-methyl-6-prop-1-en-2-ylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol) having the structure of Formula I The term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired binding activity, i.e., binding cannabidiol. An “isolated” antibody as used herein refers to an antibody which has been identified, separated and/or recovered from a component of its natural environment. For example, a composition comprising an antibody as described herein will be isolated and purified from a cell culture or other synthetic environment to greater than 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% by weight of the antibody. In one embodiment, the antibody of the disclosure is an immunoglobulin (Ig) molecule and comprises four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains linked by disulfide bonds. Five types of mammalian Ig heavy chains are known: α, δ, ε, γ, and μ, wherein the type of heavy chain defines the class (isotype) of the antibody. Antibodies of the disclosure can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA), and subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The heavy chain may contain two regions, the constant region (CH) and the variable region (VH). The constant region shares high homology in all naturally occurring antibodies of the same isotype within the same species. Like the heavy chain, a light chain may also consist of one constant domain (CL) and one variable domain (VL). In mammals there are two types of immunoglobulin light chain, i.e., lambda (λ) and kappa (κ). The unique binding property or antigen binding specificity of a given antibody is determined by the variable (V) regions. In particular, three variable loops in each of the light (VL) and the heavy (VH) chain variable regions, known as complementarity determining regions (CDRs), are responsible for the antigen binding specificity. These regions of the antibody of the present invention are described in more detail infra. An antibody fragment of the disclosure is a molecule containing an antigen binding region or antigen binding domain of a full antibody but is not the full antibody, e.g., the VHregion, the VLregion, or a combination of both regions. In one embodiment, the antibody fragment comprises a single-chain polypeptide containing one, two, or three of the CDRs of the light-chain variable domain. In another embodiment, the antibody fragment comprises a single-chain polypeptide containing one, two, or three of the CDRs of the heavy chain variable region. In another embodiment, the antibody fragment of the disclosure is a single domain antibody (also referred to as a nanobody), e.g., a peptide chain of about 110 amino acids long comprising one heavy chain variable region domain or one light chain variable region domain of a full antibody. In another embodiment, the antibody fragment is a fragment antigen-binding (F(ab)) fragment or a F(ab′)2fragment. Antibodies and antibody fragments of the present disclosure also encompass mutants, variants, or derivatives of the disclosed antibodies or fragments thereof which retain the essential epitope binding features of an Ig molecule. For example, the single domain antibodies can be derived from camelid (VHH domains) or cartilaginous fish (V-NAR) variable domains, alone or fused to an Fc domain. In another embodiment, the antibody fragment comprises the heavy chain and light chain variable regions fused together to form a single-chain variable domain antibody (scFv) or a single-chain variable domain with an Fc portion (i.e., a scFv-Fc, e.g., a minibody.). In another embodiment, the antibody fragment is a divalent or bivalent single-chain variable fragment, engineered by linking two scFvs together either in tandem (i.e., tandem scFv), or such that they dimerize to form diabodies. In yet another embodiment, the antibody is a trivalent single chain variable fragment, engineered by linking three scFvs together, either in tandem or in a trimer formation to form triabodies. In another embodiment, the antibody is a tetrabody single chain variable fragment. In another embodiment, the antibody is a “linear antibody” which is an antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen binding regions (see Zapata et al.Protein Eng.8(10):1057-1062 (1995), which is hereby incorporated by reference in its entirety). Antibody and antibody fragments disclosed herein can be mono-valent, bi-valent, or tri-valent with regard to binding domains, and the binding domains may be mono-specific, bi-specific, or tri-specific in binding specificity by design. As noted above, the VHand VLregions of an antibody are subdivided into regions of hypervariability, termed complementarity determining regions (CDR). The CDRs are interspersed with regions that are more conserved in each family of V genes, termed framework regions (FR). These FR regions are specific to place in the proper spatial configuration the contact amino acid residues of the CDRs that are responsible for most of the binding capacity of the antibody. Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs in each of the variable regions of the heavy chain and the light chain are designated CDR1, CDR2 and CDR3 for each of the variable regions (i.e., L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region, and H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region). The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991), which is hereby incorporated by reference in its entirety) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. In one embodiment, the antibody or binding fragment thereof described herein is a chimeric antibody. A chimeric antibody is an antibody where one portion of the amino acid sequence of each of the heavy and light chains is homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular class, while the remaining segment of each chain is homologous to corresponding sequences in another species or class. Typically the variable region of both light and heavy chains mimics the variable region of antibodies derived from one species of mammal, while the constant portions are homologous to sequences of antibodies derived from another. For example, the variable region can be derived from presently known sources using readily available B-cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. Methods of making chimeric antibodies are well known in the art, see e.g., U.S. Pat. No. 4,816,567; and Morrison et al., “Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains”Proc. Natl. Acad. Sci. USA81:6851-6855 (1984), which are hereby incorporated by reference in their entirety. In another embodiment, the antibody or binding fragment thereof is a CDR-grafted antibody. A “CDR-grafted antibody” is an antibody which comprises heavy and light chain variable region sequences of one species, where one or more of the CDR regions are replaced with CDR regions of another species. For example, in one embodiment the CDR grafted antibody comprises human or humanized heavy and light chain variable regions, where one or more of the CDRs within these regions are replaced with one or more CDRs from another species, e.g., murine CDRs. In another embodiment, the antibody or binding fragment thereof is a humanized antibody. A humanized antibody is an antibody or a variant, derivative, analog or portion thereof which comprises a framework region having substantially the amino acid sequence of a human antibody and a complementary determining region having substantially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a non-human antibody CDR. Likewise, the term “substantially” in the context of a FR refers to a FR having an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a human FR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab′, F(ab′)2, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., the donor antibody) and all or substantially all of the framework regions are those of a human or humanized immunoglobulin framework sequence (i.e., the acceptor antibody). Methods of humanizing antibodies are well known in the art, see e.g., Almagro and Fransson, “Humanization of Antibodies,”Frontiers in Bioscience13:1619-1633 (2008), U.S. Pat. No. 6,054,297 to Carter et al., U.S. Pat. No. 8,343,489, and U.S. Patent Application Publication No. US20100261620 to Almagro et al., which are hereby incorporated by reference in their entirety. The human or humanized framework sequences can be chosen based on known structure, i.e., a fixed framework sequence, sequence homology to the framework sequences of the donor antibody (e.g., the antibody from which the CDRs are derived), i.e., a best-fit framework sequence, or a combination of both approaches. Regardless of the method chosen to select the human framework sequence, the sequences can be selected from mature framework sequences, germline gene sequences, or consensus framework sequences. Compatible human framework sequences are those that are similar in both length and sequence to the framework sequence of the donor antibody sequence (i.e., the antibody from which the CDRs are derived) to ensure proper folding of the antibody and binding domain formation. In one embodiment, the humanized framework sequence of a humanized antibody of the disclosure comprises a consensus framework sequence. A consensus framework sequence is derived from a consensus immunoglobulin sequence, which is the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see e.g., WINNAKER, “From Genes to Clones: Introduction to Gene Technology” (1987); Carter et al.,Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al.,J. Immunol.,151:2623 (1993), which are hereby incorporated by reference in their entirety). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid residue occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. In another embodiment, a humanized antibody or binding fragment thereof as disclosed herein comprises a fixed framework region. Human heavy chain and light chain FR sequences known in the art can be used as heavy chain and light chain “acceptor” framework sequences (or simply, “acceptor” sequences) to humanize a non-human antibody using techniques known in the art (see e.g., Sims et al.,J. Immunol.,151:2296 (1993); Chothia et al.,J. Mol. Biol.,196:901 (1987), which are hereby incorporated by reference in their entirety). In one embodiment, human heavy chain and light chain acceptor sequences are selected from the framework sequences listed in publically available databases such as V-base or in the international ImMunoGeneTics® (IMGT®) information system. Humanized antibodies or binding fragments thereof as described herein may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In one embodiment, the humanized antibody disclosed herein comprises the light chain as well as at least the variable domain of a heavy chain. The humanized antibody may further comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In another embodiment, the humanized antibody comprises only a humanized light chain. In another embodiment, the humanized antibody comprises only a humanized heavy chain. In another embodiment, the humanized antibody comprises only a humanized variable domain of a light chain and/or a humanized variable domain of a heavy chain. In one embodiment, the antibodies and binding fragments thereof as described herein are human antibodies. Methods of producing human antibodies that are known in the art are suitable for use in accordance with the present disclosure. For example, one can produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al.,Proc. Natl Acad. Sci. USA90:2551 (1993); Jakobovits et al.,Nature362:255-258 (1993); U.S. Pat. No. 5,545,806 to Lonberg et al, U.S. Pat. No. 5,569,825 to Lonberg et al, and U.S. Pat. No. 5,545,807 to Surani et al, which are hereby incorporated by reference in their entirety. The antibodies and binding fragments thereof described herein can be human antibodies or humanized antibodies (fully or partially humanized) as described supra. Alternatively, the antibodies and binding fragments thereof can be animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, or a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.). Methods of antibody production, in particular, monoclonal antibody production, may be carried out using the methods described herein and those well-known in the art (MONOCLONAL ANTIBODIES-PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporated by reference in its entirety). Generally, the process involves obtaining immune cells (lymphocytes) from the spleen of an animal which has been previously immunized with the antigen of interest, i.e., 2-(6-isopropenyl-3-methyl-2-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol, either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is achieved by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation of Specific Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion,”Eur J Immunol6:511 (1976), which is hereby incorporated by reference in its entirety). The immortal cell line, which may be murine or derived from cells of other mammalian species, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and have good fusion capability. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. In accordance with the present invention, a hybridoma producing an anti-CBD antibody as described herein is referred to as the NAYAM BJ9003.236 hybridoma. Antibodies produced by this hybridoma comprise a variable heavy chain region having an amino acid sequence of SEQ ID NO: 7 as described in more detail herein, and a variable light chain region having an amino acid sequence of SEQ ID NO: 8, as described in more detail herein. In another embodiment, monoclonal antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., “Phage Antibodies: Filamentous Phage Displaying Antibody Variable Domains,”Nature348:552-554 (1990), which is hereby incorporated by reference in its entirety. Clackson et al., “Making Antibody Fragments using Phage Display Libraries,”Nature352:624-628 (1991); and Marks et al., “By-Passing Immunization. Human Antibodies from V-Gene Libraries Displayed on Phage,”J. Mol. Biol.222:581-597 (1991), which are hereby incorporated by reference in their entirety, describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al.,BioTechnology10:779-783 (1992), which is hereby incorporated by reference in its entirety), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al.,Nuc. Acids. Res.21:2265-2266 (1993), which is hereby incorporated by reference in its entirety). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. Alternatively, monoclonal antibodies can be made using recombinant DNA methods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, which is hereby incorporated by reference in its entirety. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such asE. colicells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, generate monoclonal antibodies. The polynucleotide(s) encoding a monoclonal antibody can further be modified using recombinant DNA technology to generate alternative antibodies. For example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted for those regions of a human antibody to generate a chimeric antibody. Alternatively, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be substituted for a non-immunoglobulin polypeptide to generate a fusion antibody. In other embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Furthermore, site-directed or high-density mutagenesis of the variable region can be used to optimize specificity and affinity of a monoclonal antibody. In one embodiment, the anti-cannabidiol antibody or binding fragment thereof as described herein comprises a heavy chain variable region. The heavy chain variable region comprises a complementarity-determining region 1 (H-CDR1) comprising an amino acid sequence of SEQ ID NO: 1 (Asn-Phe-Tyr-Glu-Met-Trp), or a modified amino acid sequence of SEQ ID NO: 1, said modified sequence containing 1 or 2 amino acid residue modifications as compared to SEQ ID NO: 1. The heavy chain variable region further comprises a complementarity-determining region 2 (H-CDR2) comprising an amino acid sequence of SEQ ID NO: 2 (Ser-Arg-Asn-Lys-Ala-Glu-Asp-Tyr-Thr-Thr-Glu-Tyr-Ser-Ala-Ser), or a modified amino acid sequence of SEQ ID NO: 2, said modified sequence containing 1, 2, 3, or 4 amino acid residue modifications as compared to SEQ ID NO: 2. The heavy chain variable region further comprises a complementarity-determining region 3 (H-CDR3) comprising an amino acid sequence of SEQ ID NO: 3 (Ile-Tyr-Tyr-Cys-Ala-Arg-Asp-Lys), or a modified amino acid sequence of SEQ ID NO: 3, said modified sequence containing 1, 2, or 3 amino acid residue modifications as compared to SEQ ID NO: 3. The antibody or binding fragment thereof as described herein may alternatively or further comprise a light chain variable region. The light chain variable region comprises a complementarity-determining region 1 (L-CDR1) having an amino acid sequence of SEQ ID NO: 4 (Asp-Leu-Ser-Gln-Tyr-Leu-Phe), or a modified amino acid sequence of SEQ ID NO: 4, said modified sequence containing 1 or 2 amino acid residue modifications as compared to SEQ ID NO: 4. The light chain variable region further comprises a complementarity-determining region 2 (L-CDR2) having an amino acid sequence of SEQ ID NO: 5 (Arg-Val-Ser-Arg-Leu-Thr-His), or a modified amino acid sequence of SEQ ID NO: 5, said modified sequence containing 1 or 2 amino acid residue modifications as compared to SEQ ID NO: 5. The light chain variable region further comprises a complementarity-determining region 3 (L-CDR3) having an amino acid sequence of SEQ ID NO: 6 (Gln-Gln-Ser-Arg-Leu-Ile-Pro-Asn-Thr), or a modified amino acid sequence of SEQ ID NO: 6, said modified sequence containing 1, 2, or 3 amino acid residue modifications as compared to SEQ ID NO: 6. Suitable amino acid modifications to the heavy chain CDR sequences and/or the light chain CDR sequences of the anti-CBD antibody disclosed herein include, for example, conservative substitutions or functionally equivalent amino acid residue substitutions that result in variant CDR sequences having similar or enhanced binding characteristics to those of the CDR sequences disclosed herein. Conservative substitutions are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. Alternatively, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally grouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry, 2nd ed, WH Freeman and Co., 1981, which is hereby incorporated by reference in its entirety). Non-conservative substitutions can also be made to the heavy chain CDR sequences and the light chain CDR sequences as disclosed herein. Non-conservative substitutions involve substituting one or more amino acid residues of the CDR with one or more amino acid residues from a different class of amino acids to improve or enhance the binding properties of CDR. The amino acid sequences of the heavy chain variable region CDRs and/or the light chain variable region CDRs of the anti-CBD antibody described herein may further comprise one or more internal neutral amino acid insertions or deletions that do not alter CBD binding. In one embodiment, the H-CDR3 having an amino acid sequence of SEQ ID NO: 3, further contains one or more internal neutral amino acid insertions or deletions that do not alter CBD binding. In another embodiment, the L-CDR1, having an amino acid sequence of SEQ ID NO: 4 further contains one or more internal neutral amino acid insertions or deletions that do not alter CBD binding. In one embodiment of the present disclosure, the anti-CBD antibody or binding fragment thereof comprises a heavy chain variable region with a H-CDR1 having the amino acid sequence of SEQ ID NO: 1; a H-CDR2 having the amino acid sequence of SEQ ID NO: 2; and a H-CDR3 having the amino acid sequence of SEQ ID NO: 3. An exemplary heavy chain variable region comprising the aforementioned CDR regions has the amino acid sequence of SEQ ID NO: 7 as shown below. The CDR regions of the variable heavy chain of SEQ ID NO: 7 are underlined and the flanking framework regions (i.e., FR1-FR4) are shown in bold. (SEQ ID NO: 7)Glu-Val-Lys-Leu-Val-Glu-Ser-Gly-Gly-Gly10-Leu-Val-Gln-Pro-Gly-Gly-Ser Leu-Arg-Leu20-Ser-Cys-Ala-Thr-Ser-Gly-Phe-Thr-Phe-Ser30-Asn-Phe-Tyr-Glu-Met-Trp-Val-Arg-Gln-Ser40-Pro-Gly-Lys Arg-Leu-Glu-Trp-Ile Ala-Ala50Ser Arg-Asn-Lys-Ala-Glu-Asp-Tyr-Thr-Thr60- Glu-Tyr-Ser-Ala-Ser-Val-Lys-Gly- Arg-Phe70-Ile-Val-Ser-Arg-Asp-Thr-Ser-Gln-Ser-Ile80-Leu-Tyr-Leu-Gln-Met-Asp -Ala-Leu-Arg-Ala90-Glu-Asp-Thr-Ala-Ile-Tyr-Tyr-Cys-Ala-Arg100-Asp-Lys-Asp-Tyr-Gly-Ser-Ser-Tyr-Trp-Tyr110-Phe-Asp-Val-Trp-Gly-Ala-Gly-Thr-Thr-Val120-Thr-Val-Ser In one embodiment of the present disclosure, the anti-CBD antibody or binding fragment thereof comprises a light chain variable region with a L-CDR1 having the amino acid sequence of SEQ ID NO: 4; a L-CDR2 having the amino acid sequence of SEQ ID NO: 5; and a L-CDR3 having the amino acid sequence of SEQ ID NO: 6. An exemplary light chain variable region comprising the aforementioned CDR regions has the amino acid sequence of SEQ ID NO: 8 as shown below. The CDR regions of the variable light chain of SEQ ID NO: 8 are underlined and the framework regions (i.e., FR1-FR4) are shown in bold. (SEQ ID NO: 8)Asp-Ile-Gln-Asn-Thr-Gln-Thr-Pro-Ser-Ser10-Leu-Ser-Ala-Ser-Leu-Gly-Asp-Arg-Val-Ser20-Ile-Ser-Cys-Arg-Ala-Ser-Gln-Asp-Leu-Ser30-Gln-Tyr-Leu-Phe-Trp-Tyr-Gln-Gln-Lys-Pro40-Gly-Gln-Pro-Pro-Lys-Leu-Leu-Ile-Tyr-Arg50-Val-Ser-Arg-Leu-Thr-His-Gly-Val-Pro-Asp60-Arg-Phe-Ser-Gly-Ser-Gly-Ser-Gly-Thr-Asp70-Phe-Thr-Leu-Thr-Ile-Asp-Pro-Asn-Glu-Glu80-Asp-Asp-Thr-Ala-Thr-Tyr-Phe-Cys-Gln-Gln90-Ser-Arg-Leu-Ile-Pro-Asn-Thr-Phe-Gly-Gly100-Gly-Thr-Lys-Leu-Glu-Ile-Lys-Arg- In one embodiment, the anti-CBD antibody or binding fragment thereof as described herein comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 7 and/or a light chain variable region having an amino acid sequence of SEQ ID NO:8. In another embodiment, the antibody or binding fragment thereof comprises a heavy chain variable region having an amino acid sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 7, and/or a light chain variable region having an amino acid sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 8. In another embodiment, the antibody or binding fragment thereof comprises a heavy chain having an amino acid sequence of SEQ ID NO: 9 as shown below. The CDRs of SEQ ID NOs: 1, 2, and 3 are underlined. (SEQ ID NO: 9)Glu Val Lys Leu Val Glu Ser Gly Gly Gly10Leu ValGln Pro Gly Gly-Ser Leu-Arg Leu20-Ser-Cys-Ala Thr-Ser Gly-Phe-Thr Phe Ser30-Asn-Phe-Tyr-Glu-Met-Trp-Val-Arg-Gln-Ser40Pro-Gly-Lys Arg-Leu-Glu-Trp-IleAla-Ala50Ser Arg-Asn-Lys-Ala Glu-Asp-Tyr-Thr-Thr60-Glu-Tyr-Ser-Ala-Ser-Val-Lys-Gly-Arg-Phe70-Ile-Val-Ser-Arg-Asp Thr-Ser Gln-Ser-Ile80-Leu-Tyr-Leu-Gln-Met-Asn-Ala-Leu-Arg-Ala90-Glu-Asp-Thr-AlaIle-Tyr-Tyr-Cys-Ala-Arg100-Asp-Lys-Asp-Tyr-Gly-Ser-Ser-Tyr-Trp-Tyr110-Phe-Asp-Val-Trp-Gly-Ala-Gly-Thr-Thr-Val120-Thr-Val-Ser-Ser-Glu-Ser-Ala-Arg-Asn-Pro130-Thr-Ile-Tyr-Pro-Leu-Thr-Leu-Pro-Pro-Ala140-Leu-Cys-Ser-Asp-Pro-Val-Ile-Ile Gly-Cys150-Leu-Ile-His-Asn-Tyr-Phe-Pro-Ser-Gly-Thr160-Met-Asn-Val-Thr-Trp-Gly-Lys-Ser-Gly-Lys170-Asp-Ile-Thr-Thr-Val Asn-Phe-Pro-Pro-Ala180-Leu-Ala-Ser-Gly-Gly-Arg-Tyr-Thr-Met-Ser190-Ser-Gln-Leu-Thr-Leu-Pro-Ala-Val-Glu-Cys200-Pro-Glu-Gly-Glu-Ser-Val-Lys-Cys-Ser-Val210-Gln-His-Asp-Ser-Asn-Pro-Val-Gln-Glu-Cys220-Asp-Val-Asn-Cys-Ser-Gly-Pro-Thr-Pro-Pro230-Pro-Pro-Ile-Thr-Ile-Gly-Ser-Cys-Gln-Pro240-Ser-Leu-Ser-Leu-Gln-Arg-Pro-Ala-Leu-G1u250-Asp-Leu-Leu Leu-Gly-Ser-Asp-Ala-Gln-Ile260-Thr-Cys-Thr-Leu-Asp-Gly-Leu-Arg-Asn-Pro270-Glu-Gly-Ala-Val-Phe-Thr-Trp-Glu-Pro-Ser280-Thr-Gly-Lys-Asp-Ala-Val-Gln-Lys-Lys-Ala290-Val-Gln-Asn-Ser-Cys-Gly-Cys-Tyr-Ser-Val300-Ser-Ser-Val-Leu-Pro-Gly-Cys-Ala-Glu-Arg310-Trp-Asn-Ser-Gly-Ala-Ser-Phe-Lys-Cys-Thr320-Val-Thr-His-Pro-Glu-Ser-Gly-Thr-Leu-Thr330Gly-Thr-Ile-Ala-Lys-Val-Thr-Val-Asn-Thr340-Phe-Pro-Pro-Gln-Val-His-Leu-Leu-Pro-Pro350-Pro-Ser-Glu-Glu-Leu-Ala-Leu-Asn-Gly-Leu360-Leu-Ser-Leu-Thr-Cys-Leu-Val-Arg-Ala-Phe370-Asn-Pro-Lys-Glu-Val-Leu-Val-Arg-Val-Ser380-Ala-Glu-Asp-Trp-Lys-Gln-Gly-Asp-Gly-Tyr390-Ser-Cys-Met-Val-Gly-His-Glu-Ala-Leu-Pro400-Met-Asn-Phe-Thr-Gln-Lys-Thr-Ile-Asp-Arg410-Leu-Ser-Gly-Lys-Pro-Thr-Gln-Val-Asn-Val420-Ser,Val-Ile-Met-Ser-Glu-Gly-Asp-Gly-Ile430-Tyr-Cys. In another embodiment, the antibody or binding fragment thereof comprises a light chain having an amino acid sequence of SEQ ID NO: 10 as shown below. The light chain CDRs of SEQ ID NOs: 4, 5, and 6 are underlined. (SEQ ID NO: 10)Asp-Ile-Gln-Asn-Thr-Gln-Thr-Pro-Ser-Ser10-Leu-Ser-Ala-Ser-Leu-Gly-Asp-Arg-Val-Ser20-Ile- Ser-Cys-Arg-Ala-Ser-Gln-Asp-Leu-Ser30Gln-Tyr-Leu-Phe-Trp-Tyr-Gln-Gln-Lys-Pro40-Gly-Gln-Pro Pro LysLeu Leu Ile TyrArg50Val Ser Arg Leu Thr HisGlyVal Pro Asp60Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp70Phe Thr Leu Thr Ile Asp Pro Asn Glu Glu80Asp Asp Thr Ala Thr Tyr Phe CysGln Gln90Ser ArgLeu Ile Pro Asn ThrPhe Gly Gly100Gly Thr LysLeu Glu Ile Lys Arg Cys-Pro110-Glu-Gly-Glu-Ser-Val-Lys-Cys-Ser-Val-Gln120-His-Asp-Ser-Asn-Pro-Val-Gln-Glu-Leu-Asp130-Val-Asn-Cys-Ser-Gly-Pro-Thr-Pro-Pro-Pro140-Pro-Ile-Thr-Ile-Gly-Ser-Cys-Gln-Pro-Ser150-Leu-Ser-Leu-Gln-Arg-Pro-Ala-Leu-Glu-Asp160-Leu-Leu Leu-Gly-Ser-Asp-Ala-Gln-Ile-Thr170-Cys-Thr-Leu-Asp-Gly-Leu-Arg-Asn-Pro-Glu180-Gly-Ala-Val-Phe-Thr-Trp-Glu-Pro-Ser-Thr190-Gly-Lys-Asp-Ala-Val-Gln-Lys-Lys-Ala-Val200-Gln-Asn-Ser-Cys In one embodiment, the anti-CBD antibody or binding fragment thereof as described herein comprises a heavy chain having an amino acid sequence of SEQ ID NO: 9 and/or a light chain having an amino acid sequence of SEQ ID NO: 10. In another embodiment, the antibody or binding fragment thereof comprises a heavy chain having an amino acid sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 9, and/or a light chain having an amino acid sequence that shares at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 10. Antibody “specificity” refers to selective recognition of the antibody or binding portion thereof as described herein for a particular epitope or epitopes of CBD. The term “epitope” includes any determinant capable of specific binding to an immunoglobulin or T-cell receptor or otherwise interacting with a molecule. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally have specific three dimensional structural characteristics, as well as specific charge characteristics.FIG.3shows the epitopic determinants of CBD and identifies the amino acid residues within the CDR regions of the anti-CBD antibody that are critical for binding these epitopes of CBD. In particular, amino acid residue 34 (SEQ ID NO: 7) of the heavy chain CDR1, amino acid residues 52 and 56 (SEQ ID NO: 7) of the H-CDR2, amino acid residue 102 (SEQ ID NO: 7) of the H-CDR3, and amino acid residue 96 (SEQ ID NO: 8) of the light chain CDR3 are critical for antibody binding to CBD. The antibody or antigen binding fragment thereof as described herein binds specifically to CBD. The antibody or binding fragment thereof as described herein binds with the highest affinity to the naturally occurring 2-(6-isopropenyl-3-methyl-2-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol isoform of CBD. The antibody or binding fragment thereof as described herein also binds to other isoforms of CBD, albeit at lower affinity relative to its binding to the 42-isomer of CBD. “Binds specifically” as used herein refers to an antibody binding to its antigen and is not intended to exclude low-level, non-specific binding that may occur between random proteins. “Binds specifically” as used herein is not intended and does not imply that the antibody will not bind to any protein or molecule other than the molecule(s) as disclosed herein since antibodies can cross-react with any molecule or protein that includes the relevant epitope. Another aspect of the present disclosure relates to an antibody or binding portion thereof that competes for binding to CBD with the anti-CBD antibody described herein, i.e., the antibody comprising a heavy chain variable region comprising an H-CDR1 of SEQ ID NO: 1, an H-CDR2 of SEQ ID NO: 2, and an H-CDR3 of SEQ ID NO: 3, and a light chain variable region comprising L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6. In accordance with this aspect of the disclosure, the antibody or binding portion thereof competes for binding to the Δ-2 isomer of CBD. In accordance with this aspect of the disclosure, a competitive binding assay, such as Bio-Layer Interferometry (BLI) can be utilized to identify an antibody or binding portion thereof that competes for binding to CBD with the anti-CBD antibody described in detail herein. Other competitive binding assays known in the art can also be utilized to identify a competitive binding antibody in accordance with this aspect of the disclosure. Another aspect of the present disclosure is directed to one or more isolated polynucleotides encoding the antibody or binding fragment thereof as described herein. In one embodiment, the isolated polynucleotide encodes all or at least a portion of the heavy chain variable region having the amino acid sequence of SEQ ID NO: 7. In one embodiment, the isolated polynucleotide encodes all or at least a portion of the heavy chain having the amino acid sequence of SEQ ID NO: 9. In another embodiment, the isolated polynucleotide encodes all or at least a portion of the light chain variable region having the amino acid sequence of SEQ ID NO: 8. In another embodiment, the isolated polynucleotide encodes all or at least a portion of the light chain having the amino acid sequence of SEQ ID NO: 10. In another embodiment, the isolated polynucleotide encodes a combination of all or portions of SEQ ID NOs: 7, 8, 9, and 10. The nucleic acid molecules described herein include isolated polynucleotides, portions of expression vectors or portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation, and vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion, and/or display of the antibodies or binding fragments thereof described herein. The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the invention may be produced by other techniques such a PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art. The polynucleotides of the invention may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pIII. Another embodiment of the disclosure is directed to a vector comprising at least one polynucleotide as described herein. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides described herein into a given organism or genetic background by any means. Another embodiment of the disclosure is directed to one or more expression vectors comprising the polynucleotides encoding the antibody or binding fragment thereof as described herein. The polynucleotide sequences encoding the heavy and light chain variable domains, Fab fragments, or full-length chains of the antibodies disclosed herein are combined with sequences of promoter, translation initiation, 3′ untranslated region, polyadenylation, and transcription termination to form one or more expression vector constructs. In accordance with this embodiment, the expression vector construct encoding the anti-CBD antibody or binding portion thereof can include the nucleic acid encoding the heavy chain polypeptide, a fragment thereof, a variant thereof, or combinations thereof. The heavy chain polypeptide can include a variable heavy chain (VH) region and/or at least one constant heavy chain (CH) region. The at least one constant heavy chain region can include a constant heavy chain region 1 (CH1), a constant heavy chain region 2 (CH2), and a constant heavy chain region 3 (CH3), and/or a hinge region. In some embodiments, the heavy chain polypeptide can include a VH region and a CH1 region. In other embodiments, the heavy chain polypeptide can include a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region. The expression construct can also include a nucleic acid sequence encoding the light chain polypeptide, a fragment thereof, a variant thereof, or combinations thereof. The light chain polypeptide can include a variable light chain (VL) region and/or a constant light chain (CL) region. The expression construct also typically comprises a promoter sequence suitable for driving expression of the antibody or binding fragment thereof. Suitable promoter sequences include, without limitation, the elongation factor 1-alpha promoter (EF1a) promoter, a phosphoglycerate kinase-1 promoter (PGK) promoter, a cytomegalovirus immediate early gene promoter (CMV), a chimeric liver-specific promoter (LSP) a cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a tetracycline responsive promoter (TRE), a transthyretin promoter (TTR), a simian virus 40 promoter (SV40) and a CK6 promoter. Other promoters suitable for driving gene expression in mammalian cells that are known in the art are also suitable for incorporation into the expression constructs disclosed herein. The expression construct can further encode a linker sequence. The linker sequence can encode an amino acid sequence that spatially separates and/or links the one or more components of the expression construct (heavy chain and light chain components of the encoded antibody). Another embodiment of the invention is a host cell comprising the polynucleotides and/or vectors described herein. The antibodies and binding fragments thereof described herein can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art (see e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), which are hereby incorporated by reference in their entirety). The host cell chosen for expression may be of mammalian, e.g., COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, or lymphoma cell. The host cell can alternatively be a yeast cell, an insect cell, a plant cell, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g., a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any of the natural or engineeredE. colispp.,Klebsiellaspp., orPseudomonasspp. strains. The antibodies described herein can be prepared by any of a variety of techniques using the isolated polynucleotides, vectors, and host cells described supra. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies via conventional techniques, or via transfection of antibody genes, heavy chains and/or light chains into suitable bacterial or mammalian cell hosts, in order to allow for the production of antibodies, wherein the antibodies may be recombinant. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Transfecting the host cell can be carried out using a variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., by electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is sometimes preferable, and sometimes preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. As noted above, exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin,Proc. Natl. Acad. Sci. USA,77: 4216-4220 (1980), which is hereby incorporated by reference in its entirety). Other suitable mammalian host cells include, without limitation, NS0 myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody described herein. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies described herein. The antibodies and antibody binding fragments are recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification. Protease inhibitors may be used to inhibit proteolytic degradation during purification. One skilled in the art will appreciate that purification methods suitable for the antibody of interest may require modification to account for changes in the character of the antibody upon expression in cell culture. Another aspect of the present invention is directed to method of detecting cannabidiol in a sample. This method involves contacting the sample with the antibody or antigen binding fragment that binds cannabidiol as described herein, and detecting the antibody or antigen binding fragment thereof bound to cannabidiol, i.e., the anti-CBD antibody-CBD complex, if present in the sample. Methods described herein involving the detection of CBD in a sample involve the use of a detectably labeled anti-CBD antibody as described herein. Accordingly, in one aspect the anti-CBD antibody as described herein may be coupled to a detectable label. Suitable detectable labels are well known in the art and include detectable tags (e.g., a poly-histidine (His6-) tag, a glutathione-S-transferase (GST-) tag, or a maltose-binding protein (MBP-) tag); radioactive labels (e.g., carbon (14C) or phosphorous (32P)); fluorescent labels (e.g., hydroxycoumarin, succinimidyl ester, aminocoumarin, methoxycoumarin, Cascade Blue™, Hydrazide, Pacific Blue™ Maleimide, Pacific Orange®, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5™ conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7™ conjugate, Red 613, PE-Texas Red®, PerCP, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5™ conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY™-FL, TRITC, X-Rhodamine (XRITC), Lissamine™ Rhodamine B, Texas Red®, Allophycocyanin (APC), an APC-Cy7™ conjugate, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, Cy2™, Cy3™, Cy3B™, Cy3.5™, Cy5™, Cy5.5™ or Cy7™); luminescent labels (e.g., luminol); bioluminescent labels (e.g., luciferase, luciferin, and aequorin); or enzymatic labels (e.g., luciferin, 2,3-dihydrophthalazinedi ones, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase), alkaline phosphatase, b-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase). Alternatively, the CBD-antibody can be bound by a detectable label, for example, bound by a secondary antibody that contains a detectable label. Detection assays for detecting the anti-CBD antibody bound to a cannabidiol in a sample are well known in the art and include, for example, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescent activated cell sorting (FACS). Another aspect of the present invention is directed to a method of determining the bioactivity of cannabidiol. This method involves providing a sample containing a known amount of cannabidiol, and contacting the sample with the antibody or antigen binding fragment that binds cannabidiol as described herein. The method further involves detecting the antibody or antigen binding fragment thereof bound to said cannabidiol in the sample; measuring the amount of detected antibody or antigen binding fragment thereof bound to said cannabidiol; and determining the bioactivity of the cannabidiol in the sample based on said measuring. As discussed supra, CBD binds the CB2 receptor, and the bioactivity of CBD in any CBD containing sample (e.g., plant extract) is measured by the binding affinity of CBD for its receptor. The anti-CBD antibody of the present invention simulates the CB2 receptor, acting as a surrogate agent by which the binding affinity of CBD to its receptor can be measured in vitro. The antibody binds most strongly to the native, natural form of CBD (i.e., 2-(6-isopropenyl-3-methyl-2-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol)). Solvent extraction, heating, high speed shear of the CBD molecule, the type of plant and the part of the plant from which the CBD is extracted, etc., all affect antibody-CBD binding. Thus, the bioactivity of CBD in any composition containing CBD can readily be determined by measuring the binding affinity between the anti-CBD antibody described herein and CBD in the composition, and comparing it to the binding affinity between the anti-CBD antibody and the natural isoform of CBD. Bioactivity of CBD in a sample is expressed as a percentage of maximal activity, i.e., the percentage of activity relative to the natural isoform of CBD. Assays and methods for detecting anti-CBD antibody and CBD complexes, along with suitable detectable labels are described supra. Additional assays suitable for quantitatively assessing binding affinity include, without limitation, radioactive binding assays, non-radioactive binding assays (e.g., fluorescence resonance energy transfer assays or surface plasmon resonance), solid phase ligand binding assays, and liquid phase ligand binding assays (e.g., immunoprecipitation). Another aspect of the present invention is directed to a kit that comprises the anti-cannabidiol antibody as described herein. In one embodiment, the kit is an assemblage of materials or components, including at least one of the inventive antibodies or antigen binding fragments as described herein that is useful for measuring the bioactivity of CBD in a sample. The exact nature of the components configured in the inventive kit depends on its intended purpose. Instructions for use may be included in the kit. “Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to test for bioactive CBD or to quantify the amount of CBD. Optionally, the kit also contains other useful components, such as, substrates, syringes, applicators, pipetting or measuring tools, or other useful reagents as will be readily recognized by those of skill in the art. The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in assays. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of a purified antibody that binds specifically to CBD as described herein. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components. Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s). EXAMPLES Examples are provided below to illustrate the present invention. These examples are not meant to constrain the present invention to any particular application or theory of operation Example 1 Production of the Anti-Cannabidiol Antibody Cannabidiol (2-[(1R,6R)-6-isopropenyl-3-methylcyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol) was obtained from 1) Hebrew University of Jerusalem, Rehovot, Israel, and 2) ImmunAG, LP, Goa, India. Step 1—5% Cannabidiol was dissolved in Caproic acid (C5H11COOH) and this solution was injected into BALB/Lac mice. 0.2 ml were intravenously injected into the lateral tail vein with a 27-28 mm gage needle. The injections were repeated every other day for 14 days, for a total of 7 injections. Step 2—Each injection was followed by an in vivo electroporation, which significantly enhanced the immune response. This was done by sending 80 pulses of 100 microseconds at 0.3 Hz with an electrical field magnitude of 2500 V/cm. Step 3—Spleen cells of the mice immunized with Cannabidiol were isolated. Monoclonal antibodies were made by cell culture that involved fusing myeloma cells with the spleen cells immunized with Cannabidiol-creating a fused “Splenocyte”. The fusion of the B cells with myeloma cells was done by electrofusion. The BTX ECM 2001 Electrofusion generator cell fusion applications manufactured by BTX Harvard Apparatus, Holliston Mass. USA were used for the fusion. The two cells were fused by dielectrophoresis which used a high frequency alternating current. Once the cells were brought together, a pulsed voltage was applied. The pulsed voltage caused the cell membrane to permeate and subsequently the membranes of the two cells to combine and fuse. After this, an alternative voltage was applied again for a brief period of time to stabilize the process. The end result was that the mixed cytoplasm and the cell membranes were completely fused. The two nuclei fused later within the cell making the resulting “heterokaryon” cell. Step 4—The fused cells were incubated in HAT (hypoxanthine-aminopterin-thymidine) medium for roughly 10 to 14 days. The aminopterin blocked the pathway for nucleotide synthesis and killed any unfused myeloma cells. Only the B cell-myeloma hybrids survive. These cells produced antibodies (a property of B cells) and are immortal (a property of the myeloma cells). The incubated medium was then diluted into multi-well plates to such an extent that each well only contained one cell. Step 5—The next stage was a rapid primary screening technique, ELISA, to identify and select only those hybridomas that produce antibodies of appropriate specificity. Solid-phase enzyme immunoassay (EIA) was used to detect the presence of Cannabidiol in liquid samples. The Cannabidiol from the sample were attached to the surface. Then the antibody produced in the incubated medium was applied over the surface so it could bind to the Cannabidiol. This antibody was linked to a Cytochrome P450 enzyme (CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 all worked) and the final step was the addition of a substance containing Cytochrome P450 substrate (such as Pentalenolactone). The subsequent reaction produced a detectable color change in the substrate. Step 6—The B cells that produced the desired antibodies were cloned to produce numerous identical daughter clones. Supplemental media cultures containing interleukin-6 were used to establish a hybridoma colony. They grew well in culture medium RPMI-1640 (with antibiotics and fetal bovine serum) and produced numerous antibodies. The smaller multi-well plates used to grow the hybridomas, were changed to larger tissue culture flasks. The culture supernatant yielded 46 μg/ml to 72 μg/ml of Cannabidiol monoclonal antibody, which was maintained at −20° C. or lower until used. Example 2 Analyzing the Bioactivity of CBD by Immunoelectrophoresis Immunoelectrophoresis was used test the Cannabidiol-Antibody-Complex (“CAC”). There are several variants to this procedure, all of which require CAC. Agarose as 1% gel slabs of about 1 mm thickness, buffered at high pH (around 8.6) is a preferred medium. An electrophoresis equipment with a horizontal cooling plate appear to works best. FIG.1is a separation and identification of CAC using Counter-immunoelectrophoresis. This is similar to immunodiffusion, but with the addition of an electrical field across the agarose medium. There is more rapid migration of the Cannabidiol and antigen out of their respective wells towards one another to form a line of precipitation, indicating binding Example 3 Analyzing the Bioactivity of CBD by Immunofluorescence For this experiment, the anti-CBD antibody as described herein was labeled with the fluorescent molecule, fluorescein.FIG.2shows the fluorescein labeled anti-CBD antibody bound to CBD in various CBD containing samples. The bioactivity of CBD is quantified by measuring the intensity of the light emitted by the fluorescein label of the bound anti-CBD antibody and comparing it to the intensity of light emitted by fluorescein labeled anti-CBD antibody bound to the same amount of the Δ2-isomer of CBD. Materials and Methods for Examples 4-7 CHO cells and membrane preparation. These were stably transfected with cDNA encoding human CB2 receptors. The CB2-transfected cells were used in binding assays with [3H]-CP55940, [3H]-WIN55212-2 or [35S]-GTPγS (Bmax=72.5 pmol mg−1 protein). The clones used in the assays were the same as those used in the sPAP reporter assay described by Green et al. (1998). Cells were maintained at 37° C. and 5% CO2in DMEM (f-12 HAM) with 2 mm Glutamine, Geneticin (600 μg ml−1) and Hygromycin (300 μg ml−1). Because receptor over expression may lead to the activation of effector mechanisms to which receptors in natural membranes are not normally coupled (Kenakin, “Agonist-receptor Efficacy II: Agonist Trafficking of Receptor Signals,”Trends in Pharmacological Sciences16(7):232-238 (1995), which is hereby incorporated by reference in its entirety)), the assays were performed with cells expressing fewer CB2receptors than the cells used in the binding assays. CHO cells were suspended in 50 mm Tris buffer (pH 7.4) and 0.32 m sucrose and homogenized with an Ultra-Turrex homogenizer. The homogenate was diluted with 50 mm Tris buffer (pH 7.4) and centrifuged at 50,000×g for 1 hour to isolate the membranes. CHO—CB2binding. A filtration procedure was used to measure [3H]-CP55940 and [3H]-WIN55212-2 binding. This is a modification of the method described by Compton et al., “Cannabinoid Structure-activity Relationships: Correlation of Receptor Binding and In Vivo Activities,”Journal of Pharmacology and Experimental Therapeutics265(1):218-226 (1993), which is hereby incorporated by reference in its entirety. Binding assays were performed with [3H]-CP55940 or [3]-WIN55212-2, 1 mm MgCl2, 1 mm EDTA, 2 mg ml−1 bovine serum albumin (BSA) and 50 mm Tris buffer, total assay volume 500 μl. Binding was initiated by the addition of cell membranes (20-30 μg protein). Assays were carried out at 30° C. for 90 minutes before termination by addition of ice-cold wash buffer (50 mm Tris buffer, 1 mg ml−1 BSA) and vacuum filtration using a 12-well sampling manifold (Brandel Cell Harvester) and Whatman GF/B glass-fibre filters that had been soaked in wash buffer at 4° C. for 24 hours. Each reaction tube was washed three times with a 4 ml aliquot of buffer. The filters were oven-dried for 60 minutes and then placed in 5 ml of scintillation fluid (Ultima Gold XR, Packard). Radioactivity was quantified by liquid scintillation spectrometry. Specific binding was defined as the difference between the binding that occurred in the presence and absence of 1 μm reference cannabidiol. Protein assays were performed using a Bio-Rad Dc kit. Unlabeled and radio labelled cannabidiol were each added in a volume of 50 μl following dilution in assay buffer (50 mm Tris buffer containing 10 mg ml−1 BSA). The concentration of [3H]-CP55940 or [3H]-WIN55212-2 used in displacement assays was 0.5 nm. The concentrations of cannabidiol that produced a 50% displacement of radio ligand from specific binding sites (IC50 values) were calculated using GraphPad Prism (GraphPad Software, San Diego, U.S.A.). Competitive binding curves were fitted with minimum values for displacement of radio ligand from specific binding sites constrained to zero. Dissociation constant (Ki) values were calculated using the equation of Yung-Chi et al., “Relationship Between the Inhibition Constant (Ki) and the Concentration of Inhibitor Which Causes 50 Percent Inhibition (ISO) of an Enzyme Reaction,”Biochemical Pharmacology22(23): 3099-3108 (1973), which is hereby incorporated by reference in its entirety, and dissociation constant values of [3H]-CP55940 and [3]-WIN55212-2 shown in Table 1. TABLE 1Kivalues were calculated by the Cheng & Prusoff equation(n = 3 or 4) using KD values of 0.8 nm for [3H]-CP55940 inmembranes of CB2 cells and a Kd value of 2.1 nm for[3H]-WIN55212-2 in membranes of CB2 cells.Labelled cannabinoidUnlabeled cannabinoidCB2 Ki(nM)[3H]-CP55940CP559401.8 ± 0.2L7596336.4 ± 2.2L75965611.8 ± 2.5AM63031.2 ± 12.4SR1445285.6 ± 1.1[3H]-W1N55212-2AM63037.5 ± 15.4SR1445284.1 ± 1.3 Generation and binding of the anti-CBD antibody. Reference CBD was extracted from the inflorescence of the Avidekel plant. 5% reference CBD was dissolved in caproic acid (C5H11COOH). 0.2 ml of this solution was injected with a 27-28 mm gauge needle into the lateral tail vein of BALB/Lac mice. The injections were repeated every other day for 14 days for a total of 7 injections. Each injection was followed by an in vivo electroporation of 80 pulses of 100 microseconds at 0.3 Hz with an electrical field magnitude of 2500 V/cm. Following cannabidiol immunization, mouse splenocytes were extracted and isolated. They were fused with myeloma cells by dielectrophoresis using a BTX ECM 2001 Electrofusion Generator, manufactured by BTX Harvard Apparatus, Holliston, Mass. USA. The fused cells were incubated in a hypoxanthine-aminopterin-thymidine medium (with respective concentrations 0.1 mM, 0.4 μM, and 0.016 mM) for between ten and fourteen days, resulting in the survival of only the B cell-myeloma hybrids. Following limiting dilution to one cell per plate, ELISA was used to select hybridomas that produced antibodies with higher binding to the pure CBD molecule. The antibody was linked to a Cytochrome P450 enzyme. Pentalenolactone was used as the Cytochrome P450 substrate. The hybridoma producing the antibody with the highest binding affinity, as measured by a molar weight increase in the Cannabidiol Antibody Complex (CAC), was cloned using supplemental media cultures containing interleukin-6. Cloned hybridomas grew in culture medium RPMI-1640 with antibiotics and fetal bovine serum. A/G purification was used to extract monoclonal antibodies from hybridomas. The culture supernatant contained 46 micrograms/milliliter to 72 micrograms/milliliter of Cannabidiol monoclonal antibody (MCA). This antibody was maintained at −20° C. or lower until used. Fluorescence labelled ELISA was used to measure binding for each sample. The molar weight of the CAC was divided by the molar weight of the gold standard reference CAC to derive binding affinity values. Ultracentrifugal CBD extraction. Plant tissue (from the inflorescence) was ultrasonically fractioned. The pulp and plasma were separated by centrifugation. The plasma fraction was further fractionated and studied by analytical ultra centrifuge to obtain the sedimentation coefficient of CBD. Isopycnic density gradient preparative ultracentrifugation (up to 130,000 RPM), using sodium bromide and cesium chloride, was then done to collect the purified CBD samples. This is not a commercially viable process but it provided enough mg of CBD to conduct the bioactivity test. Solvent CBD extraction. For the solvent procedure, dried plant material was extracted at around 20° C. with ethanol, followed by methylene chloride, and separated uncarboxylated cannabinoids from carboxylated cannabinoids. CBD isolation and analysis. Each fraction was identified by using the following methods: Silica gel eluting with CHCl3; silica gel eluting with C6H6-MeOH—AcOH (88%: I 0%: 2%) (Mechoulam et al., “A New Tetrahydrocannabinolic Acid,”Tetrahedron Letters10(28):2339-2341 (1969); Korte et al., “Chemical Classification of Plants. XXVI. Hashish Constituents by Thin-Layer Chromatography,”Journal of Chromatography13(1):90-98 (1964), which are hereby incorporated by reference in their entirety); Cannabinoid reference standards. Following CBD isolation and identification, Fast Blue B Salt colors were used for qualitative analysis. The cannabinoids were then analyzed after trimethyl-sililation, by GLC using OV225 (50′ SCOT column) or OV17 (2% on Chromosorb W, 5′ column). Acid cannabinoids were estimated after decarboxylation by heating in pyridine. Fluorescence labelled ELISA was used to measure the bioactivity of sample. Example 4 A Novel, Valid, Scalable CBD Bioactivity Test Highly pure, naturally occurring CBD molecules were extracted from the Avidekel plant, obtained in 2014 from Tikun Olam, Israel, via sonic fractionation and ultra centrifugal separation. [3H]-CP55940 displacement assays were performed for this reference sample using membrane fractions of CHO cells expressing recombinant human CB2. Additionally, binding of an MCA, described herein, was tested for this reference sample. The resulting displacement and binding values were used as a reference standard against which 26 CBD samples (acquired from Natural Hemp Solutions, Atlanta Ga.) were compared. A correlation between the CHO CB2binding and the MCA binding across 26 samples was investigated. If the MCA binding correlated to the CHO—CB2, it could be used instead for quicker, more efficient testing. Pearson correlation analysis was performed using R on the CB2and CAC binding values of the 26 CBD samples. The binding affinities to both the recombinant human CB2and the highest-affinity MCA are listed in Table 2 as a proportion relative to the binding affinity shown by a highly pure CBD molecule. Using the Pearson correlation analysis, it was found that these were highly correlated (Pearson coefficient=0.97). Thus, the bioactivity of CBD can be predicted using the MCA with high accuracy. TABLE 2Binding affinities for the MCA versus the CB2complex in 26 CBD-producing plant samples.They were highly correlated (r = .97).MCACB2SampleAffinityAffinity10.780.8120.340.4230.250.2940.320.350.340.3660.310.3370.240.2580.290.3490.320.3100.290.31110.250.25120.310.32130.350.33140.310.31150.250.26160.30.31170.270.24180.250.24190.320.29200.210.24210.260.24220.440.41230.330.41240.810.8250.30.22260.190.22270.780.81280.340.42 A successful and scalable bioactivity test for CBD has been validated. Bioactivity values are expressed as a proportion between 0 and 1 as compared to the CHO—CB2binding of the purest CBD molecule able to be isolated. The lower the number, the lower the bioactivity. If a CBD molecule has a bioactivity below 0.5, one could expect to observe CBD—CB2binding at half the strength of a molecule with a bioactivity of 1. If a molecule had an observed bioactivity of 0.2, one could expect the binding affinity to be at ⅕ the strength of a molecule with a bioactivity of 1. This test will illuminate the distribution of bioactive molecules throughout various parts of the plant. Example 5 Cannabis CBD Bioactivity by Plant Organ It is well known, among growers, that the yield of CBD is variable across different organs in the cannabis plant, with the inflorescence producing the highest output (The tip of secreting hairs located mainly on female-plant contain resin glands that have a considerable amount of cannabinoids. These glands are fewer in number in the leaves (Zuardi, “Cannabidiol: From an Inactive Cannabinoid to a Drug with Wide Spectrum of Action,”Revista Brasileira de Psiquiatria30(3):271-280 (2008), which is hereby incorporated by reference in its entirety)). However, the bioactivity of CBD extracted from different organs has never been studied before. Using the bioactivity test, validated in experiment 1, 4 regions from 48 different cultivars of cannabis obtained from the USA, India, China, and the Czech Republic were examined. Inflorescence, petioles, apical buds/leaves, and stalks were tested separately. A combination of sonic fractionation and ultra centrifugal separation was used on the inflorescence to obtain purified samples. Cold solvent extraction was also used to obtain CBD from the inflorescence, petioles, apical buds/leaves, and stalks. A 1×5 ANOVA and appropriate post-hoc comparisons were conducted on bioactivity with plant organ as the only factor. Bioactivity means and standard errors were plotted. Levene's test indicated heteroscedastic variances between the organs F(4,235)=6.05,p<0.001. As such, a robust ANOVA as described by Wilcox, Introduction to Robust Estimation & Hypothesis Testing. 3rdedition. Elsevier, Amsterdam, The Netherlands. (2012), which is hereby incorporated by reference in its entirety, was conducted. It found a significant difference between the bioactivities of centrifuge-extracted inflorescence CBD (M=0.96, SD=0.02, solvent-extracted CBD from the inflorescence (M=0.86, SD=0.04), solvent-extracted CBD from the petioles (M=0.54, SD=0.03), solvent-extracted CBD from the apical buds/leaves (M=0.4, SD=0.04), and solvent-extracted CBD from the stalks (M=0.19, SD=0.02), F(4, 70.38)=9885.21, p<0.001. (20% trimmed means are presented above.) Robust post-hoc comparisons (Mair et al., “Robust Statistical Methods in R Using the WRS2 Package,” Technical Report, Harvard University (2016), which is hereby incorporated by reference in its entirety), revealed significant differences between each of the CBD source categories (See Table 3 for the psihat values of each comparison, and their associated confidence intervals). TABLE 3Psihat and corresponding confidence interval values (in brackets) for robust one-way ANOVApost-hoc comparisons of bioactivity. Psihat values for each post-hoc comparions wereobtained using 20% trimmed means. Corresponding 95% confidence interval valuesare presented in brackets. All associated p-values were <.001.ApicalInfluorescencePetiolesbuds/LeavesStalksInfluorescence−.205−.769−.669−.344Centrifuge[−.222 to −.187][−.781 to −.756][−.685 to −.654][−.360 to −.329]Influorescence−.563−.464−.140[−.583 to −.545][−.486 to −.443][−.161 to −.119]Petioles0.99.424[.082 to .117][.407 to .441]Apical.325buds/LeaveS[.306 to .344] In summary, the highest bioactivity CBD was found in the pods of each plant, with decreasing bioactivity in the petioles, apical buds/leaves, and stalks respectively (SeeFIG.4). Individual bioactivity scores obtained per plant are provided in Table 4. TABLE 448 cultivars of cannabis, and their associated bioactivitylevels by plant organ.Ultra-centrifugedInflo-ApicalCultivarInflorescencerescencePetioleBud/LeafStalkUniko B0.9560.9020.5170.360.169Kompolti0.980.8810.5380.3830.192Fedora 170.9760.8650.520.430.191Fedora 170.9190.8920.5090.4680.194Fedora 170.950.9070.5690.4090.201Ferimon 120.920.8660.5170.3920.19Santhica 270.9850.850.5570.4530.162Epsilon 680.9910.8590.5750.370.171Futura 750.9580.8230.5570.410.189Futura 750.9630.850.530.4430.176Felina 320.9740.880.570.3780.189Felina 340.9460.8170.5880.3920.183Juso 140.9560.8320.5050.4280.199Bialobrzeskie0.9730.8360.5610.3910.216Beniko0.9840.8440.5160.4340.175Chamaeleon0.9690.850.5250.3840.17Chamaeleon0.9720.9070.5130.3970.226Carmagnola0.9610.8940.5330.2830.224Carmagnola0.9730.8620.5650.4310.21Carmagnola0.9320.8770.5610.3870.185selezionataTiborszallasi0.940.8820.5310.3590.192Fibranova0.9740.8930.510.4080.23Delta-llosa0.9490.8520.540.3970.228Delta-4050.9820.8580.5610.3780.179Novgorod-0.9470.8880.5690.3930.204Seversky, cvBernburgskaya0.970.8050.5520.3850.193Odnodomnaya,bmSzegedi 90.9360.880.5210.3730.166Fibrimulta 1510.9710.8760.5310.3580.189Glukhovskaya 100.9890.8070.5540.3780.192Zheltostebel′nayaKrasnodarsky 100.9650.8760.5760.430.191FBAlpine Rocket0.9510.8410.620.4270.198Alpine Rocket0.9470.790.5170.4360.206Hindu Kush0.9350.8870.5290.4250.183Nortern Light0.9930.8710.5460.360.221Snow White0.9310.8170.5060.3490.159Top 440.9730.8390.5150.380.189Top 440.9340.8610.5530.3250.188F1 Fraise0.9660.8630.5140.4310.197B520.9430.9140.5430.4290.226Peace Maker0.9460.8480.5340.3460.197Big Bud0.9510.90.5360.3790.2Big Skunk0.9670.8670.5090.3690.184F Fraise0.9310.8750.5170.4050.213Hawaii Maui0.9850.840.4850.3820.194WauiHaze0.9930.8840.5960.4570.158Swaziland0.9810.8340.5790.3970.192Mexican Sativa0.9630.8250.5280.4510.207Ruderalis Indica0.9420.7890.4990.3980.186 These results showed a difference between extraction methodologies. The combination of sonic fractionation and ultra centrifugal separation produced CBD with the highest bioactivity. Sonic fractionation and ultra centrifugal extraction are labor- and equipment-intensive laboratory procedures, not fit for large-scale manufacture. By contrast, ethanol solvent extraction causes a small amount of degradation in bioactivity, but is scalable and relatively inexpensive to carry out. Ergo, it is a far more common procedure for commercial CBD production. Among plant organs subjected to ethanol extraction, the results indicate a canonical pattern of CBD bioactivity. The inflorescence produced the highest bioactivity CBD molecules, with levels five times higher than CBD extracted from the stalk. Inflorescence should be used exclusively for the production of high bioactivity CBD. If commercial CBD suppliers have mixed in biomass from the stem and bark of the plant before extraction, it has lily led to low bioactivity in their products. Example 6 Testing the Bioactivity of a Novel, Non-Cannabis, Plant Source of CBD Using a plant from the Humulus family that produces CBD, a new plant was developed called Humulus Kriya. It does not produce THC, is from a family of plants considered GRAS (FDA Title 21, Volume 3, Sec 182.2-CAS 8060-28-4) and has been certified by FSSAI (Food Safety and Standards Authority of India) as a “Food Ingredient”. It should not fall under the Scheduled List classification. The bioactivity profile of the various parts of H. Kriya was tested using the same methods as in Example 5. The samples were made of six H. Kriya plants, provided by ImmunAG, LLP, India, and thirty one samples of ImmunAG oil extract. Welch's t was used to compare CBD bioactivity of H. Kriya from all five groups to the cannabis samples. Individual bioactivity scores obtained per plant are provided in Table 5. TABLE 56 samples of ImmunAG, and their associated bioactivitylevels by plant organ.Ultra-centrifugedInflo-ApicalCultivarInflorescencerescencePetioleBud/LeafStalkH. Kriya #30.9640.8290.5360.4650.212H. Kriya #50.9470.7980.5320.4140.195H. Kriya #60.9560.8830.5490.3990.215H. Kriya #110.9610.960.5510.4450.188H. Kriya #140.9410.8350.5190.4020.21H. Kriya #170.9320.8510.5360.3550.182 The centrifuged pod CBD from H. Kriya (M=0.95, SD=0.01) showed no difference in bioactivity compared to cannabis samples, t(8.6594)=1.74,p=0.12. The solvent extracted pod CBD (M=0.86, SD=0.06) showed no difference, t(5.3803)=0.007,p=0.99. The solvent-extracted petiole CBD (M=0.54, SD=0.01) showed no difference, t(14.123)=0.373,p=0.715. The solvent-extracted leaf CBD (M=0.41, SD=0.04) showed no difference, t(6.164)=−1.0212, p=0.346. The solvent-extracted stem CBD (M=0.20, SD=0.01) showed no difference, t(7.322)=−1.143, p=0.289. It appears as though H. Kriya has an identical CBD bioactivity profile to the cannabis strains tested. Comparisons are shown inFIG.5. Identical CBD bioactivity was found between H. Kriya and Cannabis for CBD extracted from various parts of the plant. H. Kriya appears to be a viable cannabis alternative for CBD research. CBD from H. Kriya has no risk of THC contamination. It has been certified as a food ingredient by the Food Safety and Standards Authority of India. Example 7 Examining the Bioactivity of Commercially Available CBD Products The bioactivity of commercial CBD samples has never been examined. The results of commercial, cannabis-based, products were analyzed over the past 2 years. These samples were sent directly by vendors (Natural Hemp Solutions, Centuria Foods, BSPG, Isodiol, Hammer Enterprises, etc.) or sent by 3rd parties. The bioactivity results for individual vendors have not been published the bioactivity results for all of the vendors together has been anonymously presented. There are many cannabimimetic molecules other than CBD. The two announced sources of CBD from non-hemp/cannabis sources are yeast and humulus. Samples of CBD could not be extracted from yeast. The bioactivity of CBD extracted from H. Kriya (ImmunAG) was tested and compared to commercial cannabis products. The minimum bioactivity in commercial samples was 0.11 and the maximum was 0.41. The minimum bioactivity in ImmunAG was 0.72, and the maximum was 0.98. Bioactivity scores for both classes of product are shown inFIG.6. When comparing the CBD bioactivity in ImmunAG (M=0.88, SD=0.06) to products on the market (M=0.23, SD=0.07), Welch's t found a significant difference in bioactivity, t(41.288)=53.41,p<1.001. Commercial CBD bioactivity were low, having values consistent with the lower bioactive organs—stalks, stems, barks and leaves. It is possible that suppliers have been using biomass rich in stalk, stem and leaves to comply with regulations and increase mass. The caution is that low bioactive CBD may not produce desirably intense immunologic cell signals. Commercial CBD bioactivity was also quite variable, with a minimum of 0.11 and a maximum of 0.41. The highest commercial sample had almost four times the potency of the lowest sample. Left unchecked, low bioactivity CBD are likely to confound medical use or research and produce spurious results. ImmunAG samples ranged from 0.72 to 0.98, with the lowest ImmunAG bioactivity higher than the highest commercial cannabis-based CBD bioactivity. This is not surprising because ImmunAG is only made from the inflorescence of H. Kriya. An audit revealed that carefully regulated processing conditions also enabled ImmunAG to maintain significantly high bioactivity. Discussion of Examples 4-7 It was found that use of mono clonal antibody testing of CBD bioactivity was viable. It was found that CBD extracted from different plant organs had different bioactivity, with inflorescence having the highest bioactivity, and stalks/stems having the lowest. A non cannabis CBD-producing plant, H. Kriya, that has a bioactivity profile similar to cannabis, was evaluated. It was found that hemp/cannabis based CBD products sold commercially have low bioactivity. It was found that commercial CBD products made from it H. Kriya had the highest bioactivity. CBD-CB2 interactions are responsible for a wide range of immunologic effects. The samples studied had widely varying levels of bioactivity. It is likely that bioactivity levels have been silently confounding historical research results. Scientific studies utilizing CBD for medical research should strive to use products with the highest bioactivity levels. Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. | 81,659 |
11860175 | DETAILED DESCRIPTION It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described exemplary embodiments. Thus, the following more detailed description of the exemplary embodiments, as represented in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely representative of exemplary embodiments. Reference throughout this specification to “one embodiment” or “an embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” or the like in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of the embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation. As used herein and in the appended claims, the singular forms “a,” “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a transport path” includes a plurality of such transport paths and equivalents thereof known to those skilled in the art, and so forth, and reference to “the transport path” is a reference to one or more such transport paths and equivalents thereof known to those skilled in the art, and so forth. As, for example, illustrated schematically inFIG.1, in a number of embodiments, the devices, systems and/or methods hereof are operable to test transport properties of a gas detection or other system10via application of a driving force other than an analyte gas or a simulant gas (that is, a gas simulating the analyte gas by evoking a response from an analytical electrode of the system) from a container to one or more inlets22or an inlet system of an enclosing housing20of system10. In a number of embodiments, the driving force may, for example, be the application of exhaled breath to inlet(s)22. Housing20may, for example, include a mass transport path into an interior thereof (for example, a diffusion path) in fluid connection with inlet22. System10, may, for example, include, one or more filters24in fluid connection with inlet22either external or internal to housing20. The path may, for example, include or be in fluid connection with a mass transport or diffusion member or barrier30(for example, a membrane through which gas is mobile (for example, via diffusion) but through which a liquid has limited or no mobility). Housing20encloses a sensor40which is sensitive to the presence of exhaled breath. For example, sensor40may be sensitive to an environmental gas (the concentration of which is changed by the presence of exhaled breath), to a gas within exhaled breath, to a change in humidity, to a change in temperature, to a change in pressure, to a change in flow etc. A response of sensor40to exhaled breath provides a measurement of the transport properties and/or functionality of one or more transport paths of system10. Filter24may, for example, be used to filter out interferent gasses (that is, gasses other than the analyte gas to which the sensor is responsive) or to filter out inhibitors or poisons. In a number of representative embodiments discussed herein, devices, systems and/or methods hereof decrease or eliminate the necessity to bump check a gas detection instrument with stored calibration (for example, an analyte or a simulant) gas. Such representative embodiments of systems, devices and/or methods may, for example, combine an internal, electronic check or interrogation of sensor functionality, connection, and/or correction without the application of an analyte gas or a simulant therefor (as, for example, described in U.S. Pat. No. 7,413,645) with a transport path test using, for example, a “secondary” sensor sensitive responsive to a driving force other than the presence of an analyte gas or a simulant gas (for example, a driving force/variable change arising from the presence of exhaled human breath as described above). Many gas detection devices, instruments or systems (for example, portable gas detection instruments) include amperometric electrochemical gas sensors. These sensors are often referred to as “fuel cell” type sensors, which refers to a primary principle of operation. Such electrochemical gas sensors are typically combined or integrated into a device, system or instrument with a battery or other power supply, appropriate electronic driving circuitry (for example, including a potentiostat), a display, and one or more alarms (or other means of communicating to the user the presence of a dangerous level of harmful or toxic gas or a condition of dangerous oxygen depletion or enrichment). The sensor, circuitry and displays are typically contained in a rugged, sealed housing. As used in connection with such an instrument, the term “sealed” refers to protection of the sensor, circuitry, and displays from harmful environmental hazards (for example, dusts, condensing vapors, such as paints or coatings, and water and/or other liquids). However, the sealed housing must continually provide for the efficient transfer of the target or analyte gas(es) from outside the instrument housing into a housing of the sensor itself. Often, this result is accomplished with one or more porous diffusion membranes that keep dusts, vapors, and liquids out of the instrument housing, but allow one or more analyte gases of interest to be transported into the sensor itself. This transport is typically accomplished by gaseous diffusion or by pumping an analyte gas stream into or across the face of the sensor. As described above, the need to bump check a gas detection system/device with a calibration or simulant gas from a container is decreased or eliminated by providing a sensor (for example, a secondary sensor) that is sensitive to or responds to a driving force or variable change in the vicinity of the inlet of the system, such as, for example, the presence of exhaled breath. In a number of embodiments, components which make a sensor responsive to oxygen are provided in an amperometric electrochemical sensor (which is functional to detect an analyte other than oxygen). Exhaled human breath typically includes 4 to 5 volume-percent (vol-%) of carbon dioxide (CO2) and 15.8 to 16.8 vol-% oxygen (O2). In contrast, ambient air includes approximately 20.8 vol-% O2and 0.035 vol-% CO2. Thus, when a user exhales in the vicinity of one or more inlets into the housing of the detection system or instrument, the exhaled breath displaces the volume of gas (ambient air) within a diffusion volume in a sensor therein with the exhaled breath. A response to the decreased concentration of oxygen in exhaled breath as compared to ambient air may be used to test the transport properties of whatever gas transport path or mechanism may be used in the gas detection device (for example, including one or more gas diffusion membranes). The same result may, for example, be accomplished by incorporating, within or along with, for example, a toxic gas, a combustible or other sensor channel, a sensing element (which may be the same as or different from the sensing element for the analyte) that responds to any or all components of exhaled breath. For example, a similar result may be obtained by including a sensor or sensing functionality that responds to the increased concentration of CO2in exhaled breath as compared to ambient air. In that regard, exhaled breath contains approximately 5 vol % CO2, as compared to ambient air, which contains approximately 600 ppm CO2(0.06 vol-%). A sensor or sensing system to measure CO2concentration may, for example, include an electrochemical sensor and/or a non-dispersive infrared sensor. Amperometric or fuel cell-type gas sensors typically include at least two electrocatalytic electrodes (an anode and a cathode), at least one of which is a gas diffusion electrode or working electrode. The working electrode can be either the anode or the cathode in any given sensor. The gas diffusion electrode typically includes fine particles of an electrocatalytic material adhered to one side of a porous or gas-permeable membrane. The electrocatalytic side of the working electrode is in ionic contact with the second electrode (the counter electrode, whether the anode or the cathode) via an electrolyte (for example, a liquid electrolyte, a solid electrolyte, a quasi-solid state electrolyte or an ionic liquid). A liquid electrolyte is typically a solution of a strong electrolyte salt dissolved in a suitable solvent, such as water. An organic solvent may also be used. Quasi-solid state electrolytes can, for example, include a liquid electrolyte immobilized by a high-surface-area, high-pore-volume solid. The working electrode and the counter electrode are also in electrical contact via an external circuit used to measure the current that flows through the sensor. Additionally, although by no means necessary, a third or reference electrode, is often included. The reference electrode is constructed in a way that its potential is relatively invariant over commonly occurring environmental conditions. The reference electrode serves as a fixed point in potential space against which the operating potential of the working electrode may be fixed. In this way, electrochemical reactions that would not normally be accessible may be used to detect the analyte gas of interest. This result may be accomplished via control and driving circuitry which may, for example, include a potentiostat. FIGS.2A through2Cillustrate a schematic diagram of an instrument or system100including at least one electrochemical sensor or sensor system110. System100includes a system housing102including an inlet or inlet system104which places an interior of system housing102in fluid connection with the ambient environment. In the illustrated embodiment, electrochemical sensor system110includes at least one primary sensor responsive to at least one analyte gas. System100further includes at least one secondary sensor which is responsive to a driving force or variable change outside of system housing102in the vicinity of inlet104other than a change in concentration of the analyte gas or a simulant gas (that is, a gas other than the analyte gas to which the primary sensor is responsive) applied to system100from a container. A system50for creating such a driving force or variable change is illustrated schematically inFIG.2A. System50may, for example, change the concentration of a gas, change humidity, change temperature, change pressure, change flow or diffusion etc. in the vicinity of system inlet104. The secondary sensor is responsive to the driving force created by system50. The response of the secondary sensor to the driving force is indicative of the state of the path or transport path between inlet104and the secondary sensor. In general, the transport path is the path via which a gas is transported from outside housing102(via inlet104) to the secondary sensor (whether by, for example, diffusion or pumping). The transport path between inlet104and the secondary sensor and the transport path between inlet104and the primary sensor may, for example, be the same or similar and are exposed to generally the same conditions over the life of system100. The secondary sensor may, for example, be positioned in close proximity to the primary sensor. The response of the secondary sensor to the driving forces provides an indication of the state of the transport between system inlet104and the primary sensor. In a number of representative embodiments described herein, system50represents a person who exhales in the vicinity of inlet104. In the case of exhaled breath, the driving force may be any one of (or more than one of), for example, a change in the concentration of a gas (for example, oxygen or carbon dioxide), a change in humidity, a change in temperature, a change in pressure, or a change in flow. The secondary sensor may thus include a gas sensor, a humidity sensor, a temperature sensor, a pressure sensor and/or a flow sensor. In the case that, for example, the secondary sensor is a humidity sensor, a temperature sensor, a pressure sensor or a flow sensor, system50need not be a person who exhales in the vicinity of system inlet104. System50may, for example, be any system or device suitable to create a change in humidity, a change in temperature, a change in pressure, or a change in flow. The degree of change in the variable of interest may, for example, be controlled to monitor for a corresponding response of the secondary sensor. In the case of a change in temperature, system50may, for example, including a heating element. In the case of a change in pressure or a change in flow, system50may, for example, include a small, manually operated air pump such as a bellows. In a number of representative embodiments hereof, the secondary sensor includes a gas sensor responsive to the concentration of a gas which is changed by exhalation in the vicinity of system inlet104. In several such embodiments, sensor110includes a housing120having a gas inlet130(formed in a lid122of sensor housing120) for entry of analyte gas and human breath into sensor110. In the illustrated embodiment, inlet130is in fluid connection with a gas diffusion volume or space118. Electrolyte saturated wick materials140a,140band140cseparate a first working electrode150a(responsive to the presence of analyte gas) and a second working electrode150b(responsive to the presence of human breath) from reference electrode(s)170and counter electrode(s)180within sensor110and provide ionic conduction therebetween via the electrolyte absorbed therein. First working electrode150a, reference electrode170and counter electrode180, in cooperation with electrolyte saturated wick materials140a,140band140cform a portion of the primary sensor. Second working electrode150b, reference electrode170and counter electrode180, in cooperation with electrolyte saturated wick materials140a,140band140cform a portion of the secondary sensor. Electronic circuitry190as known in the art is provided, for example, to maintain a desired potential between working electrodes150aand150band reference electrode(s)170, to process an output signal from sensor110and to connect/communicate with other components of system100(including, for example, one or more displays, communication systems, power supplies etc.). In the illustrated embodiment, first working electrode150aand second working electrode150bare located to be generally coplanar within sensor housing120. In the illustrated embodiment, first working electrode150ais formed by depositing a first layer of catalyst154aon a diffusion membrane152(using, for example, catalyst deposition technique known in the sensor arts). Second working electrode150bis also formed by depositing a second layer of catalyst154bon diffusion membrane152(using, for example, catalyst deposition techniques known in the sensor arts). Methods of fabricating electrodes on diffusion membranes are, for example, described in U.S. Patent Application Publication No. 2011/0100813. Catalyst layers154aand154bmay or may not be formed using the same electrocatalytic material. It is immaterial whether second gas diffusion or working electrode150bis operated as an anode or cathode with respect to the operation of first gas diffusion or working electrode150a. FIGS.3A through3Dillustrate an embodiment of a sensor210that is similar in design and operation to sensor110. Like elements of sensor210are numbered similarly to corresponding elements of sensor110with the addition of 100 to the reference numbers of the elements of sensor210. As illustrated inFIG.3A, reference electrode270, counter electrode280and electrolyte absorbent wicks240a,240band240care supported within housing220via a support member284. A printed circuit board292is connected to housing220and may form a part of the electronic circuitry of sensor210. As, for example, illustrated inFIGS.3A and3C, a housing lid222includes a first gas inlet230aand a second gas inlet230b. First gas inlet230aand a second gas inlet230bmay, for example, be in fluid connection with an inlet system204(including, for example, one or more inlets) formed in a housing202of an instrument or system200(seeFIG.3B). First inlet230acan, for example, be designed for use in connection with a first working electrode250afor an analyte gas such as hydrogen sulfide. A first catalyst layer254aof first working electrode250a, which is deposited upon a first diffusion membrane252a, may, for example, include iridium in the case that the analyte gas is hydrogen sulfide (H2S). Second inlet230bis designed for use in connection with the application of exhaled breath to second working electrode250b. Second working electrode250bis formed by deposition of a second catalyst layer254bupon a second diffusion membrane252b. Separate gas inlets230aand230bmay, for example, be designed or optimized for passage of two different gases. In that regard, first gas inlet230amay be optimized (for example, in dimension and/or shape) for the analyte gas of interest, while second gas inlet230bmay be optimized for a component of exhaled breath. In the case of an aqueous electrolyte, the material(s) (which can be the same or different) of the gas diffusion membranes can be generally hydrophobic in nature to minimize or eliminate any flow of the aqueous electrolyte therethrough. In the case of a non-aqueous (for example, organic) electrolyte, the material of the gas diffusion membranes can be generally oleophobic in nature to minimize or eliminate any flow of the non-aqueous electrolyte therethrough. The material(s) can also be hydrophobic and oleophobic. Such materials are referred to as “multiphobic”. The materials can also be chemically or otherwise treated to minimize or eliminate liquid electrolyte flow or leakage therethrough. In general, the term “hydrophobic” as used herein refers to materials that are substantially or completely resistant to wetting by water at pressures experienced within electrochemical sensors (and thus limit flow of aqueous electrolyte therethrough). In general, the term “oleophobic” as used herein refers to materials that are substantially or completely resistant to wetting by low-surface tension liquids such as non-aqueous electrolyte systems at pressures experienced within electrochemical sensors (and thus limit flow of non-aqueous electrolyte therethrough). As used herein, the phrase “low-surface tension liquids” refers generally to liquids having a surface tension less than that of water. Hydrophobic, oleophobic, and multiphobic materials for use in electrodes are, for example, discussed in U.S. Pat. No. 5,944,969. Gas diffusion membranes for use herein can, for example, be formed from polymeric materials such as, but not limited to, polytetrafluoroethylene (for example, GORETEX®), polyethylene or polyvinylidene fluoride (PVDF). Such polymeric materials can, for example, include a pore structure therein that provides for gas diffusion therethrough. In sensors110and210, first working electrodes150aand250ashare a common electrolyte, a common counter electrode (180and280) and a common reference electrode (170and270) with second working electrodes150band250b, respectively. In certain situations, depending, for example, upon the analyte gas to be detected and the associated electrochemistry, it may not be desirable or possible to have a common electrolyte, counter electrode and/or reference electrode.FIG.3Eillustrates another embodiment of a sensor210′, which is similar in operation and construction to sensors110and210. Unlike sensors110and210, in the embodiment of210′, first working electrode150a′ and second working electrode150b′ are positioned in separate cells within housing220′ which are not in fluid connection. In this manner, a different electrolyte can be used in connection with electrolyte saturated wick materials140a′,140b′ and140c′ than the electrolyte used in connection with electrolyte saturated wick materials140a″,140b″ and140c″. Likewise, reference electrode170a′ may be formed differently from reference electrode170b′, and/or counter electrode180a′ may be formed differently from counter electrode180b′. In the illustrated embodiment, separate inlets230a′ and230b′ are formed in a common lid or cap222′ to be in fluid connection with first working electrode150a′ and second working electrode150b′, respectively. FIGS.3F and3Gillustrate another embodiment of a sensor310, which is similar in operation and construction to sensors110and210. Sensor310includes a housing320having a gas inlet330(formed in a lid322of sensor housing320) for entry of analyte gas and human breath into sensor310. In the illustrated embodiment, inlet330is formed as an extending slot in lid322and is in fluid connection with a gas diffusion member318. Gas diffusion member318is, for example, formed from a porous polymeric material and provides for relatively quick lateral diffusion of gas to a first working electrode350a(responsive to the presence of analyte gas) and a second working electrode350b(responsive, for example, to the presence of human breath) to reduce response times of sensor310. First working electrode350a, second working electrode350b, and remainder of the components of sensor330, may, for example, be formed in the same manner as described above for working electrode150a, second working electrode150band the remainder of the components of sensor110. Gas diffusion member318may, for example, be stiffer in construction than diffusion membrane352aof first working electrode350aand diffusion membrane352bof second working electrode350b(upon which, catalyst layers354aand354b, respectively, are deposited). In addition to providing relatively quick lateral diffusion, gas diffusion member318may also protect diffusion membranes352aand352bfrom “pinching” as a result of mechanical compression. Although the transport paths for first working electrodes250a,250a′ and350aand for second working electrodes250b,250b′ and350bof sensor210,210′ and310are slightly different, all transport paths in a particular instrument experience generally the same environments and environmental conditions. Therefore, a challenge with a driving force such as, for example, exhaled breath and the measured response of second working electrodes250b,250b′ and350bthereto provides an indication of the functionality of all transport paths in the system or instrument. In a number of embodiments described above, amperometric oxygen (or other) sensors operated in a diffusion mode are responsive to a driving force created in the vicinity of the inlet system (for example, exhaled breath) to test one or more transport paths. Such sensors may also be used in an instrument with a plenum or manifold which supplies a test gas (via pumping) to one or more sensors or sensing elements in fluid connection with the plenum. In this way, a single sensor responsive to a driving force such as exhaled breath provides information on the flow state of all transport paths (including, for example, membranes and membrane-protected or equipped sensors or sensing elements) in fluid contact with the plenum. This is especially true if the sensor responsive to the driving force such as exhaled breath is placed upstream of all the other sensors. FIG.3Hillustrates an embodiment of an instrument or system400including a plurality of individual sensors410,420,430and440within a common housing. At least one of sensors410-440may, for example, be a non-analytical oxygen sensor as described above which is responsive to, for example, oxygen concentration change resulting, for example, from exhaled breath. In a number of embodiments, sensor410, which is the first sensor in the flow path (that is, forced flow path), in system400is, for example, a non-analytical oxygen sensor. In such an embodiment, sensors420,430and440may, for example, independently be a sensor for the detection of H2S, CO2, CO, NO2, NO, SO2, HCN, HCl, NH3, H2, CH4, C2H4, Cl2, EtOH or other analyte gases of interest. In a number of embodiments, at least one of sensors420,430and440is an analytical oxygen sensor. Working electrodes414,424,434, and444, reference electrodes416,426,436, and446, counter electrodes418,428,438, and448, as well as the remaining components of sensors410,420,430, and440, respectively, may, for example, be formed in the manner described above. As is clear to one skilled in the art, system400may, for example, include fewer than or greater than four sensors. As used herein, “analytical”, “analytical electrode” and like terms refer to a working or sensing electrode with sufficient characteristics to provide an accurate or analytical indication of the concentration of the gas being sensed. Such characteristics include, for example, sufficient response range to provide accurate indications of test gas content over the desired range of concentration, long-term baseline stability, resistance to changes resulting from changes in environmental conditions, etc. “Non-analytical”, “pseudo-analytical” and like terms refer to a working or sensing electrode with sufficient range and accuracy to be useful to accomplish an exhaled breath test or other flow path test as described herein. Stability and accuracy are not as important in this aspect as the exhaled breath test or other flow path test hereof occurs over a short time frame, and the response is entirely contained within that time frame. That is, there is no need to refer to an earlier established calibration event. Referring again toFIG.3H, each of sensor410,420,430and440is in fluid connection with a plenum402. Test gas from the ambient environment is forced through plenum402(in the direction of the arrows ofFIG.3H—that is, entering plenum402via an inlet402aand exiting plenum402via an exit402b) via pump406including a pump motor406a. Pump406is in fluid connection with the ambient atmosphere and with plenum402. Sensors410,420,430and440as well as pump406may, for example, be in communicative connection with a control system which may, for example, include a processor system404(including, for example, one or more microprocessors) and/or circuitry for control thereof and data collection/processing. Processor system404is, for example, in communicative connection with a memory system405. System400further includes at least one power source408(for example, one or more batteries). System400may also include at least one user interface system409in communicative connection with processor system404and memory system405to provide information to a user. User interface system409may, for example, include a display for visual signals. Information may also be provided via user interface system409via audible, tactile and/or olfactory signals. As described above, in a number of embodiments, sensor410is a non-analytical oxygen sensor and one of sensors420,430and440may be an analytical oxygen sensor. The output of the analytical oxygen sensor in ambient air (20.8 vol-% oxygen) provides an independent check of the health or state of function of system400. Such an analytical oxygen sensor may, for example, be used in any embodiment of systems hereof. As illustrated inFIG.3I, system400may also be operated in a diffusion mode when pump406ais not powered. In other embodiments, a sensor housing with multiple separate sensors in fluid connection with a common gas inlet may be provided in which no pump is present. Once again, separate and distinct electrochemical cells within a common housing including, for example, at least one sensor responsive to oxygen provides a flow check or transport path functionality check as described herein, wherein individual sensors may be formed without the design restriction of common components (as, for example, illustrated in connection withFIG.2A). As described above, sensor410may include oxygen sensitive chemistry (and components) described herein and other sensors420,430,440etc. may include entirely different sensing chemistry (and components) such as those described in U.S. Pat. Nos. 5,944,969, 5,667,653, and elsewhere. FIG.3Jillustrates another embodiment of instrument or system400(which may operate in a forced flow or pumped mode and/or in a diffusion mode). In the embodiment ofFIG.3J, system400includes one or more electrochemical sensors410,420and430and one or more combustible gas sensors represented by combustible gas sensor440c. Catalytic or combustible (flammable) gas sensors have been in use for many years to, for example, prevent accidents caused by the explosion of combustible or flammable gases. In general, combustible gas sensors operate by catalytic oxidation of combustible gases. As illustrated inFIG.3J, combustible gas sensor440cincludes a sensing element442c, which includes a heating element such as a platinum heating element wire or coil442c(i) encased in a refractory (for example, alumina) bead442c(ii). Bead442c(ii) is impregnated with a catalyst (for example, palladium or platinum) to form active or sensing element442c, which is sometimes referred to as a pelement, pellistor, or detector. A detailed discussion of pelements and catalytic combustible gas sensors which include such pelements is found, for example, in Mosely, P. T. and Tofield, B. C., ed.,Solid State Gas Sensors, Adams Hilger Press, Bristol, England (1987). Combustible gas sensors are also discussed generally in Firth, J. G. et al.,Combustion and Flame21, 303 (1973) and in Cullis, C. F., and Firth, J. G., Eds.,Detection and Measurement of Hazardous Gases, Heinemann, Exeter, 29 (1981). Sensing element442cmay react to phenomena other than catalytic oxidation that can change its output (i.e., anything that changes the energy balance on the bead) and thereby create errors in the measurement of combustible gas concentration. Among these phenomena are changes in flow, ambient temperature, humidity, and pressure. To minimize the impact of secondary effects on sensor output, the rate of oxidation of the combustible gas may be measured in terms of the variation in resistance of sensing element or pelement442crelative to a reference resistance embodied in an inactive, compensating element or pelement444c. The two resistances are typically part of a measurement circuit such as a Wheatstone bridge. The output or the voltage developed across the bridge circuit when a combustible gas is present provides a measure of the concentration of the combustible gas. The characteristics of compensating pelement444care typically matched as closely as possible with active or sensing pelement442c. Compensating pelement444c, however, typically either carries no catalyst or carries an inactivated/poisoned catalyst. Active or sensing pelement442cand compensating pelement446ccan, for example, be deployed within wells446c(i) and446c(ii) of an explosion-proof housing section448cand can be separated from the surrounding environment by a flashback arrestor, for example, a porous metal frit449c. Porous metal frit449callows ambient gases to pass into housing section448cbut prevents ignition of flammable gas in the surrounding environment by the hot elements. Such catalytic gas sensors may be mounted in instruments such as instrument400which, in some cases, must be portable and, therefore, carry their own power supply408. It may, therefore, be desirable to minimize the power consumption of a catalytic gas sensor. Combustible gas sensor440cmay provide an additional (or an alternative) sensor which is responsive to a flow path test as described herein. As described above, combustible gas sensors are sensitive to changes in flow, ambient temperature, humidity, and pressure. Moreover, combustible gas sensors are also sensitive to the concentration of oxygen in the environment surrounding the sensing element. Multiple sensors (of the same or different types) which are responsive to one or more driving forces of a flow path test hereof may, for example, be positioned at various positions along one or more flow paths of a system hereof to provide improved data specificity during a flow path test. In several studies of sensors hereof, sensors fabricated in the manner of sensor210hereof were studied wherein first gas diffusion or working electrode250awas used to detect hydrogen sulfide (H2S), while second gas diffusion or working electrode250bwas used to detect the oxygen component of exhaled breath. Sensors fabricated in the manner of, for example, sensor110, sensor210′, sensor310or sensor410would operate in the same or similar manner. In the specifically studied embodiments, first electrocatalyst layer254aincluded iridium (Ir) metal. Second electrocatalyst layer254bincluded platinum (Pt) metal, Other electrocatalysts suitable for reduction of oxygen may be used in second electrocatalyst layer254b. FIG.4illustrates the behavior sensor210when challenged with exhaled breath, followed by a mixture of 15 vol-% oxygen and 20 ppm hydrogen sulfide, followed by nitrogen. The H2S channel trace is the response of first working electrode250a(designed to detect hydrogen sulfide), and the O2channel trace is the response of second working electrode250b(designed to detect the oxygen component of exhaled breath). As illustrated, second working electrode250bresponds to the decreased oxygen content of exhaled breath which occurs at approximately the 50 second mark in the graph. A mixture of 15 vol-% oxygen and 20 ppm hydrogen sulfide was applied at approximately 100 seconds. Each of first working electrode250aand second working electrode250bresponded appropriately to this challenge gas. Finally, nitrogen was applied at 250 seconds. Upon application of nitrogen, second working electrode250b(designed for the detection of oxygen) responded appropriately to the challenge gas. The response of second working electrode250bto exhaled breath as shown inFIG.4may, for example, be used to determine that the transport paths (including gas diffusion members and/or membranes) of a portable gas detection instrument are, for example, not compromised by dust, vapors, and/or liquid. That is, based on the response of second working electrode250bto the decreased oxygen concentration of exhaled breath, it can be determined that there is appropriate flow through all gas diffusion members (for example, gas diffusion membranes252aand252b), whether they are part of sensor210itself or part of the overall instrument. This gas response, when combined with, for example, an internal sensor electronic interrogation signal (such as that described in U.S. Pat. No. 7,413,645), may be used to provide a check of both the internal conductive condition of an amperometric electrochemical sensor (or other sensor) and any gas transport path(s) (including, for example, associated gas diffusion membranes), whether part of the sensor cell itself or part of the overall instrument. In this manner, a test similar in overall result to a bump test is accomplished without the use of expensive and potentially hazardous calibration gas and equipment associated therewith. In a number of embodiments hereof for use in connection with an exhaled breath test or bump check, an amperometric oxygen (or other gas) sensing element is disposed within, for example, an amperometric toxic (or other) gas sensor for detecting an analyte of interest. In a number of the embodiments described above, both an analyte gas sensing working electrode and the oxygen sensing electrode are conventionally fabricated as gas diffusion electrodes. In many cases, such gas diffusion electrodes include a high surface area electrocatalyst dispersed on a porous support membrane. In embodiments in which an amperometric gas sensor is used in systems hereof as a secondary sensor to test one or more transport paths, because the secondary sensor (for example, an oxygen sensor) is not used to present an analytical signal (that is, it may be a non-analytical sensor), there may be no need to use either a gas diffusion electrode or a high surface area electrocatalyst. For example, a conductor such as a contact ribbon or another conductive member, which are often used to carry an electrical signal from a gas diffusion electrode, may have sufficient surface area and electrocatalytic activity to be used as an oxygen, CO2or other gas sensitive electrode. For example,FIG.5Aillustrates a ribbon450aand a wire450a′ which may be used to form a non-analytical sensor element in the systems hereof. Such ribbons or wires may, for example, be fabricated from an electrocatalytic material such as Platinum (Pt), Iridium (Ir), Gold (Au) or carbon (C). As illustrated inFIG.5Bsensor elements550aand550a′ hereof may, for example, be a conductive ribbon552aor a conductive wire552a′, respectively, upon which an electrocatalytic material554aand554a′ (for example, Pt, Ir, Au, C etc.), respectively, is coated or immobilized. The material of ribbon552aand wire552a′ may be the same or different from electrocatalytic material554aand554a′ immobilized thereon. The sensor elements or electrodes hereof for testing transport paths may take a wide variety of two-dimensional or three-dimensional shapes. For example,FIG.5Cillustrates a sensor element650ahereof including an extending ribbon652ahaving a rectangular end member653awhich is wider than extending ribbon652ato, for example, provide increased surface area per unit length as compared to a ribbon of the same length. Similarly,FIG.5Dillustrates a sensor element650a′ hereof including an extending ribbon652a′ having a rectangular end member653a′. In the embodiment ofFIG.5D, an electrocatalytic material654a′ is immobilized on end member653a′.FIG.5Eillustrates a sensor element750ahereof including an extending wire752ahaving a spiraled section753aon an end thereof, which may, for example, provide increased surface area per unit length as compared to an extending wire of the same length. Similarly,FIG.5Fillustrates a sensor element750a′ hereof including an extending wire752a′ having a spiraled section753a′ on an end thereof. In the embodiment ofFIG.5F, an electrocatalytic material754a′ is immobilized on spiraled section753a′. In the embodiments ofFIGS.5D and5F, electrocatalytic materials654a′ and754a′ may be the same or different as the material upon which the electrocatalytic material is immobilized. In the embodiments discussed above, a first electrode is used for sensing an analyte and a second electrode, formed separately from the first electrode, is used to, for example, detect oxygen concentration. In the representative example of a toxic gas sensor for detecting the analyte H2S, for example, the toxic gas channel (H2S, in that case) is fabricated to include the electrocatalyst iridium (Ir) and the oxygen-sensing electrode is fabricated to include the electrocatalyst platinum (Pt). Those electrocatalysts may, for example, be independently dispersed onto the same porous substrate, but in two distinct and different areas. The same or similar functionality may, for example, be achieved if mixtures of Pt and Ir are used. For example, such mixtures may be physical mixtures of high surface area catalytic powders or such mixtures may be alloys. In a number of embodiments, one electrocatalytic substance or material may, for example, be fabricated on top of another electrocatalytic substance or material in a two-step process. Moreover, the two electrocatalytic materials may, for example, be fabricated into an interdigitated electrode system.FIG.6illustrates an embodiment of an interdigitated electrode system850wherein a first branch850aof electrode system850includes a first electrocatalytic material and a second branch850bincludes a second electrocatalytic material. The first and second electrocatalytic materials of the two branches or “fingers”850aand850bof electrode system850may, for example, be fabricated to include the same electrocatalytic substance (or mixture of substances) or to include different electrocatalytic substances. In another embodiment of an electrode system950hereof illustrated inFIG.7, a first electrode950aand a second electrode950bare supported upon a gas porous disk960, which is formed as an annulus in the illustrated embodiment. Disk960may, for example, be fabricated from gas porous or permeable (that is, adapted to transport gas therethrough) polymer or another material that is inert in the electrolyte used in the sensor system. As described above, disk960serves as an electrode support onto which first working electrode950aand secondary working electrodes950bare fabricated, but on opposite sides of disk960as illustrated inFIG.7. First or upper electrode950a(in the orientation ofFIG.7) is formed as an annulus. Second or bottom electrode950bis formed as a disk centered on the annulus of disk960. Electrode system950further includes a first or upper electrolyte wick970aand a second or lower electrolyte wick970b. Electrode system also includes a first electrode current collector980aand a second electrode current collector980b. The configuration ofFIG.7may, for example, be vertically flipped or rotated 180° from its illustrated orientation and still function as intended. Many other shapes and configuration of electrodes are possible for use herein. Moreover, electrodes hereof may, for example, be stacked in multiple layers or other arrangements to produce sensors with a sensitivity for a multiplicity of target gases. In a number of embodiments hereof, a single working or sensing electrode, operated at a single bias potential, can be used that responds to both the analytical gas of interest (analyte) and to a another driving force (for example, a component of exhaled breath) to enable testing of one or more transport paths to the electrode(s) of the system. For example, in the representative sensor system described inFIG.2, the H2S working electrode also responds to exhaled breath. The response of the working electrode to exhaled breath can be used to test the function of the transport path.FIG.8Aillustrates the response of a representative embodiment of a single channel amperometric sensor having a single electrode, operated at a single bias potential, fabricated to include an electrocatalytic material that is responsive to an analytical gas of interest or analyte (H2S in the representative example), to exhaled breath and to nitrogen. The electrode may be fabricated from a single electrocatalytic material, a physical mixture of electrocatalytic materials or an alloy of electrocatalytic materials. The data shown inFIG.8Awas collected by operating a hydrogen sulfide (H2S) sensor at a constant bias potential of zero (0) mV versus an internal reference electrode. At this potential the working electrode (iridium (Ir), in this case) is sufficiently anodic to cause the Faradaic conversion of hydrogen sulfide to sulfur dioxide (SO2), as is widely reported in the electrochemical literature. This can be seen in the graph ofFIG.8A, beginning around the 100 second mark, and represents the analytical signal of the sensor. Prior to the application of hydrogen sulfide, a driving force was applied to the sensor in the form of exhaled breath. The associated sensor response can be seen at about the 50 second mark in the graph. There is a small, positive excursion of the trace, upon application of exhaled breath, which was probably a result of the changes in the local humidity of the atmosphere in fluid contact with the sensor, caused by the high humidity (near 98% RH) in exhaled breath. Finally, a second driving force was applied to the sensor by the application of nitrogen (N2) to the sensor at about 250 seconds. Again there is an excursion in the sensor signal, both for the application and removal of N2. In this case, the signal originates from non-Faradaic rearrangement of ions near the electrode surface as a result of the sudden change in oxygen concentration. In both cases the application of a driving force to the face of the sensor, either by exhaled breath or by nitrogen, causes a sufficient, transitory change in the sensor signal to be used to assess the condition of the flow elements and flow path into the sensor. As described above, these effects are observed on a single sensor, with one working electrode, operated at a single, constant bias potential. FIG.8Billustrates a portion of another embodiment of sensor410athat is similar in design and operation to sensor110. The remaining portion of sensor410amay, for example, be substantially identical in design to sensor110. Like elements of sensor410aare numbered similarly to corresponding elements of sensor110with the numerical addition of 300 and the addition of the designation “a” to the reference numbers of corresponding elements of sensor110. As illustrated inFIG.8B, a housing lid422aincludes a gas inlet430awhich may, for example, be designed for use in connection with a working electrode450afor an analyte gas such as hydrogen sulfide. A catalyst layer454aof working electrode450a, which is deposited upon a first diffusion membrane452a, may, for example, include iridium in the case that the analyte gas is hydrogen sulfide (H2S). In a number of the embodiments discussed above, one channel, for example, a toxic gas channel for the measurement of H2S is fabricated to have a working electrode including an iridium catalyst, while a second channel includes an oxygen sensing electrode including a platinum catalyst. As described above, those catalysts may, for example, be independently dispersed on the same porous substrate in two distinct areas. In the embodiment ofFIG.8Bworking electrode450ais operated at two bias potentials. At a first bias potential, working electrode450ais active for oxidizing or reducing a target gas or analyte that sensor410ais intended to detect (for example, H2S). At a second bias potential, which is different from the first bias potential, working electrode450ais active for oxidizing or reducing a component of exhaled breath utilized in an exhaled breath check as described above. The bias switching described above is controlled by the driving circuitry (for example, included upon a printed circuit board such as printed circuit board292) and logic of sensor410aand/or an instrument in which sensor410ais included. Gas inlet430amay, for example, be optimized (for example, in dimension and/or shape) for the analyte gas of interest and for a component of exhaled breath. One of the more important operational aspects of using bias switching in a sensor with interrogation features as described above is that of the phenomenon colloquially known as “cookdown” to those skilled in the art of amperometric electrochemical gas sensors. Cookdown refers to the decay of large extraneous (that is, extraneous to the application of gas sensing) currents that flow between the working and counter electrodes of an amperometric gas sensor when the bias applied to the working electrode is suddenly changed (with respect to the either an internal reference electrode, in a three electrode cell, or with respect to a combination counter/reference electrode in a two electrode cell). In the electrochemical arts, “Faradic current” usually refers to currents that flow in an electrochemical device when one substance is electrochemically converted to another, such as, for example, in an oxidation-reduction reaction, such as the reduction of oxygen (O2) to water in an acidic electrolyte: O2+4H++4e−2H2O 1.1 Conversely, non-Faradaic currents are those currents that flow in an electrochemical cell when no substance is converted and are a result of only the rearrangement of ions very close to the electrode surface. These phenomena may become important in considering the behavior of a sensor such as sensor410athat uses single working electrode450a, operated at two different bias potentials, to access the electrochemical reaction important for sensing the gas of interest and to access the potential region where, for example, oxygen (a component of exhaled breath) is reduced according to equation 1.1, above, to enable an exhaled breath test or flow check. In the example of a sensor with interrogation functionality described herein in which the intended target gas to be sensed is hydrogen sulfide (H2S), one would typically use a high surface area iridium (Ir) electrocatalyst (Ir black) as the working electrode surface. At an applied potential of zero (0) mV versus an iridium/air (Ir|air) or platinum/air (Pt|air) pseudo-reference electrode (as is commonly employed in sensors to sense H2S including an Ir working electrode), H2S is oxidized to sulfur dioxide (SO2) according to: H2S+2H2OSO2+6H++6e−1.2 The above reaction is a Faradaic reaction, and occurs only where there is H2S in the atmosphere supplied to the sensor (for example, sensor410a). In the absence of H2S, very small (near zero) non-Faradaic currents flow as a result of the continual rearrangement of ions very near the electrode surfaces. Such ionic rearrangements are a result of thermally induced Brownian motion. The phenomenon of cookdown becomes important when the potential or bias of the working electrode is suddenly changed. As described above, the same high surface area Ir electrode (for example, working electrode450ain the embodiment ofFIG.8B) may also be used to sense oxygen in the atmosphere supplied to the sensor (according to equation 1.1) if the potential of the working electrode is changed to approximately −600 mV (versus the internal reference electrode described above). This sudden change results in large negative currents (following the convention that reduction currents are presented as negative) that decay slowly over time to a steady state current that is indicative of the amount of oxygen present in the atmosphere sensed by the device. The decay over time is referred to as “cookdown.” In the case described above, there are two sources of the cookdown current. The first source of current is the electrochemically induced rearrangement of ions very near the electrode surface as a result of the newly applied potential, which is a non-Faradaic current. The second source of current is the electrochemical reduction of oxygen. The oxygen that is electrochemically reduced includes oxygen that is dissolved in the electrolyte of the sensor. In that regard, until the step change in potential to −600 mV, the electrode was operated in a region where the conversion depicted in equation 1.1 did not occur. Therefore, over time, the electrolyte becomes saturated with dissolved oxygen. The electrochemically reduced oxygen also includes the oxygen being supplied to the working electrode from the atmosphere applied to the sensor. The resultant current is Faradaic current, resulting from the conversion of oxygen to water, regardless of the source of oxygen. The current resulting from reduction of oxygen dissolved in the electrolyte may be accounted for during an exhaled breath test. Operated at a potential of −600 mV, a sensor with an interrogation functionality as described herein is able to undergo or perform some type of breath or flow check that involves the perturbation of delivery of oxygen to the sensor. This may, for example, be associated with the application of exhaled breath. During operation, a sensor such as sensor410awould be operated at −600 mV only during an exhaled breath or flow test/check. Its nominal operation would be at an applied potential of zero mV for the sensing of H2S. However, upon the completion of the exhaled breath or flow test/check, the external operational circuitry of sensor410awould suddenly return the applied bias of working electrode450ato 0 mV. This bias potential change would induce large, transitory positive currents, until sensor410areturned to its normal, near zero current in the absence of H2S. The large, positive, transitory current would be the non-Faradaic cookdown of sensor410ato its normal operating state. Such cookdown currents will occur every time the bias is switched to and from the region where sensor410awould reduce oxygen, as is necessary for the exhaled breath or flow test/check. FIGS.8C and8Dprovide data from examples of electrochemical sensors such as electrochemical sensor410ain which a single working electrode is operated at two different bias potentials. As described above, the sensor is operated at a first bias potential for the analytical sensing of the analyte gas of interest, and at a second, different bias potential, for operation as a pseudo-electrode for oxygen. In this context, the term “pseudo-electrode” refers to a working electrode-bias potential combination that gives a sufficient response to indicate a change in the oxygen content of the atmosphere being sensed, but may not have the analytical sensitivity or range to be relied upon to provide an analytical or accurate indication of the oxygen content of the atmosphere. Thus, when operated as a pseudo-electrode, the working electrode may be operating as a non-analytical electrode as described above. FIG.8Cprovides an example of operation of a sensor for the detection of the analyte hydrogen sulfide at two different biasing potentials. As described above, the tested sensor utilized an iridium (Ir) working electrode, operated a two different potentials to accomplish both the analytical detection of hydrogen sulfide and to act as a pseudo-electrode for detecting a change in the oxygen content of the atmosphere being detected (such as, for example, during an exhaled breath check). InFIG.8C, the upper, solid line represents the results of the application of 20 ppm H2S to the sensor with the Ir working electrode biased at 0 mV verses the internal Pt|air reference electrode, plotted against the left-hand, y-axis. The lower, broken-line trace represents the response of the same electrode, operated at −600 mV (versus the same internal reference electrode), plotted against the right-hand, y-axis to five consecutive 2.5 second applications of nitrogen. Referring to the broken line inFIG.4B, operation of the Ir working electrode at −600 mV, in air (20.8 vol-% oxygen) resulted in a current, from the electrochemical reduction of oxygen in air, of approximately −27 μA. The 2.5 sec pulses of nitrogen (simulating oxygen reduction associates with, for example, an exhaled breath test as described herein) resulted in positive deflections of the oxygen baseline, sufficient in magnitude to act as an indication of the condition of the flow system flow path elements of the sensor. FIG.8Dprovides data from an experiment similar to that ofFIG.8C, but for a carbon monoxide sensor. In the sensor of the experiments ofFIG.8D, the working electrode was platinum (Pt). The upper, solid line sets forth the results of the application of 60 ppm CO to the Pt working electrode, biased at 0 mV against the internal Pt|air reference electrode, plotted against the left-hand, y-axis. The lower, broken line sets forth the results of the same working electrode, operated at −600 mV against the internal reference electrode, plotted against the right-hand, y-axis. InFIG.8D, the current observed as a result of the reduction of oxygen from the atmosphere results in a steady state or baseline oxygen reduction current of approximately −5300 μA. The positive pulses inFIG.8Dwere the result of five consecutive 2.5 second long pulses of nitrogen. The positive deflections observed inFIG.4Dwere sufficient to assess the condition of the flow paths of the sensor. Many other types of sensor may include a working electrode operated at two potentials as described above. For example, similar behavior is observed for a chlorine (Cl2) or a chlorine dioxide (ClO2) sensor utilizing a gold (Au) working electrode. Further, a sulfur dioxide (SO2) sensor with either platinum or gold working electrodes could be operated in the same manner. In a number of other embodiments of sensor systems hereof, two sensing or working electrodes are provided which include the same electrocatalytic material immobilized thereon. The electrodes can, for example, be fabricated in an identical manner. In such embodiments, the analyte gas and, for example, a gas of interest in exhaled breath are each electroactive on the electrocatalytic material. In a number of embodiments, the function of the two electrodes is alternated (for example, each time the user activates a breath check as described above). Referring to, for example,FIG.6, the first and second electrocatalytic materials of the two branches or electrodes850aand850bof electrode system850would include the same electrocatalytic material. In a first instance of activation of the instrument including electrodes850aand850b, electrode850awould be used as the working electrode for the target analyte gas and electrode850bwould be used to, for example, detect a component of exhaled breath (for example, oxygen). The next time the user activates the internal breath check (a second instance), the functions of electrodes850aand850bwould be switched by the external circuitry and logic of the system or instrument including sensors850aand850b. That is, in the second instance, electrode850bwould be used as the working electrode for the target analyte gas and electrode850awould be used to detect the component of exhaled breath. In this manner, alternatively, each electrode area would be calibrated against the target gas of interest and the electronic life and health checks described below would be periodically applied to each electrode. Such a system and methodology provides a greater amount of surveillance and surety to the test methodology. A detection or sensing element switching scheme which may be adapted for user herein is described in U.S. Patent Application Publication No. 2011/0100090, the disclosure of which is incorporated herein by reference. The application of human breath to cause a perturbation in, for example, oxygen concentration as described herein is applicable, in most instances, to portable instrument applications, wherein a human user is available to provide a sample of exhaled breath to exercise the interrogation or test system of the sensor (as described above), thereby testing flow through the instrument and/or sensor inlet holes and membranes (that is, testing flow paths of the system). Analysis of oxygen concentration perturbation may also be extended to, for example, permanent sensing applications (in which a sensor is fixedly positioned for extended periods of time—typically until replacement), wherein there is no human user available to exhale breath into the instrument/sensor membranes. The instrument may, for example, be placed in a position which is not easily accessible by a human attendant. FIGS.9A and9Billustrate schematically an embodiment hereof in which a gas such as oxygen in the ambient atmosphere is used to test or interrogate a system via dynamic coulometric measurement. In the illustrated embodiment, a sensor such as sensor110described above is provided. Along with sensor110and other components of a permanent sensing application (as known to those skilled in the art, and which are similar to those describe above for portable sensor systems), a small volume or space (sometimes referred to herein as a diffusion volume) and an associated restrictor system or mechanism (that is, a system or mechanism which restricts of or limits (including eliminating) flow of molecules into the volume) are situated immediately adjacent to sensor110. The diffusion volume may, for example, incorporate all flow/diffusion paths into the sensor, including dust covers, filters, etc. The diffusion volume is relatively small and in no way interferes with the normal operation of sensor110, including normal sensing and calibration. However, the diffusion volume is provided with a restrictor mechanism that, when applied, may, for example, create a small, sealed volume immediately adjacent to the inlet diffusion means of sensor110, thereby disrupting flow/diffusion of oxygen from the ambient atmosphere into the volume. In the embodiment ofFIG.9A, sensor housing120is at least partially encompassed within a secondary housing or cap120′ to create a volume124′ adjacent inlet130formed in lid122of sensor housing120. FIG.9Aillustrates sensor110and diffusion volume124′ in normal (fully open) operation wherein volume124′ and sensor inlet130are in fluid connection with the ambient atmosphere in which the concentration of an analyte is to be tested.FIG.9Billustrates sensor110and volume124′ in a restricted, closed or interrogation/testing position via a controllable restrictor mechanism, closure or lid122′, which may be controllably altered between a fully open state as illustrated inFIG.9Aand a flow/diffusion restricted or closed state as illustrated inFIG.9B. In permanent sensing applications, restrictor mechanism122′ may, for example, be actuated remotely, either by user input, or automatically, by the sensing device itself via a local and/or remote control system150′ illustrated schematically inFIG.9B. Like other Figures hereof,FIGS.9A and9Bare not necessarily drawn to scale.FIGS.9C and9Dillustrate a cross-sectional view of sensor110incorporated within instrument or system100wherein closure122′ (seeFIG.9D) is an open state and a flow/diffusion restricted or closed state, respectively (FIGS.9C and9Dare not necessarily drawn to scale). As illustrated inFIG.9D, alternatively, a restrictor mechanism104a(illustrated in dashed lines) may be provided to restrict or close inlet104of system100so that the created diffusion volume incorporates all diffusion paths into sensor110, including dust covers, filters, etc. With closure122′(or restrictor mechanism104a) in an open state, and the absence of an alarm condition, with a nominal signal present on the oxygen sensitive channel of sensor110, and with a nominal response to the electronic sensor interrogation system described below, it is highly likely that there is present in (diffusion) volume124′ (adjacent to sensor130), ambient air with an oxygen concentration of approximately 20.8 vol-%. Upon actuation of restrictor mechanism122′ (or restrictor mechanism104a) to place it in, for example, a closed state as illustrated inFIG.9D, there is created, immediately adjacent to sensor inlet130, a small, closed and fixed volume124′ with a trapped volume of gas containing a fixed amount of oxygen. That amount of oxygen will be consumed by the oxygen sensitive channel of sensor110(described above), resulting in an asymptotically decreasing signal on the oxygen sensitive channel. In a number of embodiments hereof volume124′ is maintained relatively small to ensure a relatively quick depletion of the oxygen therein. For example, in a number of embodiment, volume124′ is in the range of 0.25 to 1.5 ml. In a number of embodiments, volume124′ is approximately 0.5 ml. It is not necessary to completely close the diffusion volume124′ adjacent to sensor110, but it is only necessary to sufficiently disrupt or restrict the diffusion of oxygen to the oxygen sensitive channel of sensor110to cause a change in signal that can be analyzed according to the principals of analytical coulometry, as described below. Altering or cycling restrictor mechanism112′ (or restrictor mechanism104a) between an open, a closed, or a restricted state provides differential data, all of which can be deconvoluted to assess the condition of the flow path and flow elements into sensor110. Coulometry, as described above, is an analytical electrochemical technique fundamentally involving the measurement of the passage of charge, in coulombs, involving a Faradaic conversion of substance, that is, electrolysis. The measurement of charge is a fundamental (as opposed to derived) measurement, and therefore, can be used to make absolute quantitative analytical measurements. Coulometry, or coulometric measurement, is typically performed using a coulometer, either electronic or electrochemical. Typically, coulometry is performed at constant potential and is often referred to as “bulk electrolysis.” Given a well behaved electrochemical reaction, presented in the general form: Ox+ne−→Red 1.3 a system can be easily set to reduce the oxidized species (Ox) at a constant potential until it is completely converted to the reduced species (Red). This is signaled by a drop in observed current to zero. The amount of electricity (the number of coulombs) necessary to cause this conversion is a direct measurement of the amount of oxidized species originally present in the system. There are a number of ways in which a system can be modulated or dynamically changed to perform a coulometric measurement in a shorter time than by completing bulk electrolysis. For the particular systems described herein, a volume of gas in communication with an oxygen sensor is suddenly closed off from the ambient atmosphere (wherein diffusion of the analytically important species or analyte is modulated). The oxygen in the trapped sample is electrochemically consumed by the sensor (via working electrode150bin the representative example) according to: O2+4H++4e−2H2O 1.4 If the volume of the sample is known, the absolute concentration of oxygen in the trapped sample can be calculated based on the charge necessary to completely consume it. Other techniques might be used to estimate the oxygen concentration including, for example, the rate of decay of the reduction current, or time to reach a predetermined fraction of the original, un-modulated current. Many other schemes might be imagined. The theory behind dynamic measurements is discussed, for example, in Stetter, J. R. and Zaromb, S.,J. Electroanal. Chem.,148, (1983), 271, the disclosure of which is incorporated herein by reference. At least three system conditions for the systems described herein can be related to the response of the oxygen sensitive channel of the sensor (for example, via a processing system192including appropriate circuitry and/or one or more processors such as a microprocessor). Each of those conditions and the corresponding oxygen channel response/output is illustrated inFIG.9E. For normal operation, including an initial 20.8 vol-% oxygen (see discussion above) and nominally open membranes, a signal decay curve similar to that labeled “Normal Response” is obtained. In this situation, the velocity of the signal decay is dependent only on the speed with which the trapped amount of oxygen diffuses to the oxygen sensitive element of the sensor (for example, working electrode150bof sensor110) and is consumed by the electrochemical reaction there present. In the situation wherein the diffusion membrane(s) of the sensor inlet are blocked by dust, or other foreign matter, the rate of diffusion of oxygen to the sensor is decreased and a signal response similar to that labeled “Membrane Blocked” is obtained. Alternatively, in the case of permanent sensing applications, it is possible that bulk matter may become deposited in the diffusion volume (for example, volume124′), however small it may be. This may, for example, occur when an insect nest or the like occludes the face of the sensor. This situation is depicted in the signal response labeled “Diffusion Space Blocked.” In this case, the gas volume trapped in the diffusion volume is reduced from the normal case by the bulk matter present in the closed diffusion space and the response is observed to drop more quickly than the normal response. In the case that oxygen variation (for example, as a result of a breath test or a flow/diffusion restriction test) is measured, sensing elements other than amperometric oxygen sensing element may, for example, be used. In that regard, any alternative oxygen sensing system may be used in place of an amperometric oxygen sensing. Representative examples of suitable oxygen sensing systems include, but are not limited to, a metal oxide semiconductor or MOS (also colloquially referred to as a “Figaro” sensor) oxygen sensing element, a high temperature potentiometric oxygen sensor (zirconia sensor), a combustible gas sensor, or a paramagnetic oxygen sensor. A particular oxygen sensing technology may, for example, be more suitable as a complement to a given toxic gas or other sensing technology for a particular use. For example, an MOS or zirconia-based oxygen sensing element may be well suited for use with an MOS toxic sensor or with a heated catalytic bead combustible gas sensor. FIG.10illustrates a decision tree diagram that depicts a representative embodiment of an operating mode or method for use in connection with sensors for an analyte in any of the systems hereof. The method illustrated inFIG.10assumes a successful complete calibration of the instrument (with a calibration gas) at some point in time, either at final assembly and testing or in the field. In daily use, when the instrument is turned on, as is typical, the instrument will perform its necessary self-diagnosis checks. Part of this self-diagnosis may, for example, include the application of an electronic interrogation of a sensor such as, for example, a life and health check similar to that described in U.S. Pat. No. 7,413,645. As described in U.S. Pat. No. 7,413,645, and as illustrated inFIG.11, a sensor generally can be described as a combination of resistances and capacitance in series. The resistance RR resulting from the reference electrode ofFIG.11is not part of the current path of the analytical signal of the sensor. The resistive portion of this circuit is primarily a result of the solution (ionic) resistance of the electrolyte interspersed between the working electrode (Rw) and the counter electrode (Rc). The capacitive portion (Cw) of the equivalent circuit is primarily a result of the micro solution environment found very close to the surfaces of the metallic particles that comprise the working electrode. As a result of electrostatic forces, the volume of solution very close to the electrode surface is a very highly ordered structure. This structure is important to understanding electrode processes. The volume of solution very close to the electrode surface is variously referred to as the diffusion layer, diffuse layer, and or the Helmholtz layer or plane. The magnitudes of the resistance and capacitance present in an electrochemical cell are a result of the nature and identities of the materials used in its fabrication. The resistance of the electrolyte is a result of the number and types of ions dissolved in the solvent. The capacitance of the electrode is primarily a function of the effective surface area of the electrocatalyst. In an ideal world, these quantities are invariant. However, the solution resistance present in an amperometric gas sensor that utilizes an aqueous (water-based) electrolyte may change, for example, as a result of exposure to different ambient relative humidity levels. As water transpires from the sensor, the chemical concentration of the ionic electrolyte increases. This concentration change can lead to increases or decreases in the resistivity of the electrolyte, depending on the actual electrolyte used. The response curves of sensors have the shape expected for the charging curve of a capacitor, that is a typical “RC” curve. In a number of embodiments, the analytical signal used to determine the “health” of a sensor is the algebraic difference in the observed potential just prior to the application of a current pulse and at the end of the current pulse. The magnitude of the potential difference observed as a function of the application of the current pulse is an indicator of the presence and the health of any sensor of the system hereof and provides an independent check of sensor system operability. Although limitations on the magnitude and duration of the current pulse have mostly to do with experimental convenience, the magnitude of the current pulse may, for example, be chosen to correspond to application of a reasonably expected amount of target gas. Sensor presence and health may be determined by the shape of the sensor's RC charging curve, being measured by observing the difference in sensor output at the beginning and the end of the current pulse. If the sensor is absent, the observed potential is equal to that which would be expected based on the magnitudes of the current pulse and the sensor load resistor. FIG.12illustrates a block diagram of one embodiment of an electronic interrogation circuit as described in U.S. Pat. No. 7,413,645 and as used in several embodiments of the systems described herein. InFIG.12, the voltage follower and the current follower sections function as known to one skilled in the art. See, for example, A. J. Bard and L. R. Faulkner, Electrochemical Methods: Fundamentals and Applications, John Wiley & Sons: New York (1980), the disclosure of which is incorporated herein by reference. The voltage follower maintains a constant potential between the reference electrode (R) and the working electrode (W). The current follower buffers and amplifies currents which flow in the electrochemical sensor between the counter electrode (C) and the working electrode (W). In an number of embodiments, the current pump applies electronic interrogation to the sensor by forcing a known current to flow between the counter electrode (C) and the working electrode (W). On or more additional electronic interrogation tests may, for example, be performed on one or more combustible gas sensors in an instrument. For example, U.S. patent application Ser. No. 13/795,452, filed Mar. 12, 2013, and entitled DIAGNOSTICS FOR CATALYTIC STRUCTURES AND COMBUSTIBLE GAS SENSORS INCLUDING CATALYTIC STRUCTURES, the disclosure of which is incorporated herein by reference, discloses an electronic interrogation test for a sensing element of a combustible gas sensor in which a variable related to reactance of the sensing element is measured, and the measured variable is related to an operational state or functionality of the sensing element. In a number of embodiments hereof wherein an electronic interrogation as described above or another electronic interrogation is used, redundant analytical sensors (that is, redundant analytical sensors for the same analyte) may facilitate continuous sensing of the analyte. For example, a two channel amperometric electrochemical sensor with redundant, identical analytical channels may be used. The electronic interrogation may, for example, be applied independently to each channel, in turn. In this embodiment, the benefits of electronic interrogation are obtained. However, because of the redundant, identical analytical channels, at no time would the sensing capability of the sensor for the analyte sensed by the redundant sensor be affected. Such embodiments might be particularly useful for permanent sensor system installations, or for any sensor installation wherein the analytical signal of the sensor system for a particular analyte cannot be interrupted, even for the short times necessary for the electronic interrogation described herein or another electronic interrogation. In a representative embodiment, a redundant carbon monoxide sensor system may, for example, include two independent platinum (Pt) working electrodes, a first working electrode and second working electrode, which may, for example, be dispersed on the same porous electrode support. Each working electrode is operated independently of the other, providing redundant indication of the absence or the presence and concentration of carbon monoxide applied to the sensor. At some predetermined time, either manually, remotely, or automatically, the first working electrode would undergo the electronic interrogation check, and the information necessary for the real-time correction of the analytical signal and/or maintenance of channel1would be collected. The second working electrode/channel2would be completely unaffected by this operation on channel1. Sometime after the completion of the electronic interrogation of the first working electrode/channel1, after the effects of the interrogation have passed and a correct baseline is re-established, the second working electrode/channel2would undergo the same electronic interrogation and signal collection, and the same data would be obtained for channel2. In this way, at no time is the analytical signal for carbon monoxide from the sensor interrupted. This redundant working electrode/channel configuration may, for example, be utilized in connection with electronic or other interrogations procedures other than the electronic interrogation described in connection withFIGS.10through12. Moreover, the configuration is applicable to sensors other than electrochemical sensors (for example, combustible gas sensors). A further embodiment is illustrated inFIGS.13A and13B. In this embodiment, a sensor410eincludes a first working electrode450e(i) and second working electrode450e(ii), which are identical analytical electrodes as described above. Sensor410efurther includes a third working electrode450e(iii), which is a pseudo-analytical or non-analytical electrode that is responsive to a driving force applied in the vicinity of an inlet430eformed in an upper portion422eof the sensor housing to effect a flow path test hereof. As described above, third working electrode450e(iii) may, for example, be responsive to some component of exhaled breath (oxygen, for example). In a number of embodiments, all three working electrodes450e(i),450e(ii) and450e(iii) are dispersed on the same porous support or diffusion membrane452e. Working electrodes450e(i),450e(ii) and450e(iii) are operated independently of each other, however. Working electrodes450e(i),450e(ii) and450e(iii) are in simultaneous fluid connection with the atmosphere being sensed via V-shaped gas inlet430e. Alternatively, each channel may have a separate gas inlet, or the various channels may be in fluid contact with the atmosphere being sensed in a multitude of combinations. Working electrode450e(iii) may, for example, be used the perform a flow path test as described herein by, for example, applying a driving force to inlet430eof sensor410e(for example, by applying exhaled breath). Working electrode450e(iii) would respond as previously described and would provide an indication of the operative state or functionality of the flow path into sensor410e. Following an electronic interrogation test as described above as an independent check of sensor health, the user may, for example, be prompted to perform a flow path test such as an exhaled breath test or a “bump check” hereof (without calibration gas) by exhaling closely into the instrument face. Embedded instrument software observes the resulting signal on, for example, second working electrode250b(designed to respond to some driving force/variable change associated with exhaled breath such as a change in oxygen concentration). In the embodiment of sensor210, the observed response is a result of the decreased oxygen content in exhaled human breath. The embedded instrument control software compares the result of the electronic interrogation test and the result of the exhaled breath test to established parameters. If the responses of either the electronic interrogation test or the flow path/exhaled breath test fail to meet these pre-established criteria, the instrument may prompt the user to perform a full calibration or some other maintenance. If the results of both the electronic interrogation test and the flow path/exhaled breath test meet or exceed the pre-established criteria, the instrument may indicate to the user that it is functioning properly and is ready for daily use. FIGS.14A through14Cillustrate further embodiments of operating methodologies or schemes for systems hereof.FIGS.14A and14Billustrate operating methodologies in which both a life and health test (that is, a test including an electronic interrogation or stimulation of a sensor) and a flow path test or interrogation hereof are performed. In the operating methodology ofFIG.14A, the electronic interrogation or sensor life and health test is performed first and the flow path test is performed thereafter. In the operating methodology ofFIG.14B, the flow path test or interrogation is preformed first and the electronic interrogation or sensor life and health test is performed thereafter. In the operating methodology ofFIG.14C, only a life and health or electronic interrogation test is performed. In an initial, set-up phase, a user may be provided with the ability to adjust certain limits and set points prior to using either the sensor electronic interrogation feature or the flow path test feature. Examples of such adjustments include, but are not limited to, changes between calculated and calibrated sensitivity and time since last calibration. Once the initial set-up is complete, the user may, for example, begin using the sensor interrogation features. A user may, for example, begin by initiating one of the interrogation methods. Alternatively, initiation of one of the interrogation methods may be set up to automatically occur after a certain length of time, at a certain date, at a certain time of day, etc. User initiation may, for example, be carried out in many different manners including, for example, actuating a button, transmitting a wireless command etc. Upon initiation, the system or instrument begins the test or interrogation process. The instrument then analyzes the data collected during the test process. The system or instrument (via, for example, a control system which may include a processor and/or other control circuitry) applies a predetermined algorithm or formula to the data and then compares the results from the algorithm or formula to the set points or thresholds earlier established (for example, during set up). If the data from, for example, an electronic interrogation or life and health test of a sensor is “non-conforming” or outside of one or more determined set points or thresholds, one or more of the following representative tasks may be performed either individually or in any combination: a) perform an automatic or automated (that is, without user intervention) gas calibration of the sensor, b) change the reporting parameters of the sensor/instrument, c) switch to a second sensing element in the sensor or a new sensor for a particular analyte, d) signal the user to perform a “gas calibration” or perform other maintenance, e) perform automated maintenance internal or external to the sensor system, and/or f) signal to the user an “end of life” error message. For options “a”, “b”, “f” the user may, for example, be provided a code providing information of what changes have occurred or another indication of any changes. Such information may be simply communicated to the user or the system may require a user's acknowledgement or approval of the changes. For options “c” and “d”, the system may, for example, require the user or instrument to repeat the “interrogation method” or signal the user or system to perform a “gas calibration”. In the case of an automatic or automated gas calibration, the user need not supply the gas or otherwise intervene. In that regard, a compressed gas container may be present in the vicinity of a permanent instrument. Alternatively, the test or calibration gas may be a generated in situ or otherwise released in a manner to enter the inlet of the instrument. In situ gas generation is well know to those skilled in the art. For example, hydrogen gas (H2) can easily be generated from an electrochemical gas generator, which then can be used to calibrate both hydrogen and carbon monoxide electrochemical sensors. Other gases of interest such as chlorine (Cl2) and chlorine dioxide (ClO2) can be electro-generated as well. Also, there are methods of storing a gas of interest in a solid matrix from which it can then be thermally released. After such an automated calibration, the user may be provided with an indication of any system parameter changes, error codes and/or the readiness of the instrument for further use. In the case of changing the parameters of the sensor/instrument, parameters such as gain, range or resolution, cross-sensitivity parameters, set points (for example, alarm set point), alarm signals (for example, the type of signal) and/or other parameters may be adjusted for one or more sensors of an instrument on the basis of the results of an interrogation method hereof. For example, based on those results, the resolution of, for example, an H2S sensor or other sensor of the instrument may be changed from 0.01 ppm to 0.1 ppm. Other parameters that can be changed based on the results of interrogation methods would include, but would not be limited to, changing the linear range of the sensor so that gas values above or below certain level would not be displayed or reported or be displayed or reported on a different format. Additionally, any corrections to the linearity of the sensor signal that are normally applied may be altered or adjusted based on the results of interrogation events. The electronic gain or amplification applied to the signal of a sensor may also be adjusted in the same manner. A set point for an alarm threshold may be changed. Likewise, the alarm type to be provided to a user may also or alternatively be changed. The user may be informed of such a change as described above. Such a parameter change or other parameter change may, for example, be made until the next interrogation or until the next gas calibration. Once again, the user may be informed of the change and may be requested to acknowledge the change. In the case of a multi-sensor system such as system400ofFIGS.3H and3I, redundancy of sensors may be provided. In that regard, if non-conforming results are obtained in an electronic interrogation of a sensor, the system may switch to a second sensing element in the sensor. Alternatively, a user may be alerted to remove the non-conforming analytical sensor and replace that sensor with a new sensor for a particular analyte. After switching to the second sensing element, the system may repeat the interrogation and/or a gas calibration may be performed (either an automated calibration or a user assisted calibration). Moreover, if a sensor is determined to no longer be suitable for detection of a particular analyte, it may be suitable for detection of another, different analyte at the same or at a different biasing potential. The system may, for example, switch the sensor to detection of a different analyte in an automated procedure. The biasing potential of the sensor may, for example, be changed to facilitate the sensing of the different analyte. As with other changes, the user may, for example, be notified and may be required to acknowledge or approve the change. In the case that the instrument signals the user to perform a gas calibration, the user will supply a test gas (for example, a gas including a known concentration of the analyte or a simulant therefor) to the instrument inlet. User initiated maintenance might include, for example, changing filters or dust covers. Many electrochemical sensors are equipped with external chemical filters to remove interfering gases (see, for example,FIG.1). The data from an electronic interrogation of a sensor and/or a flow path test may, for example, be used to signal the user that such a filter needs to be replaced. In the same way, many instruments, especially portable instruments, come equipped with filters or dust covers that protect the sensor and the internals of the instrument from intrusion of water, dust and other foreign materials. These dust covers and filters normally include at least part of the flow path into the sensor. The data from an electronic interrogation of a sensor and/or a flow path test may, for example, be used to signal the user that such filters or dust covers need to be replaced. Upon a certain result or combination of results from electronic interrogation, the instrument or system may initiate an automated maintenance procedure. For example, the bias potential of an electrochemical sensor may be altered via the instrument/system control system or controller. The bias potential of the working or sensing electrode of the electrochemical sensor may, for example, be altered 1) to increase its sensitivity to the target analyte, 2) to enhance the working electrode's ability to discriminate against an interfering gas (that is, a gas to which the working electrode is responsive other than the analyte of interest). Moreover, a regeneration procedure may be initiated. The biasing potential of the working or sensing electrode may, for example, be changed to remove (for example, via oxidation, reduction, or desorption), an interfering or inhibiting substance that may have formed on or near (or otherwise contaminated) the sensing electrode surface as a function of normal usage or as a result to exposure to an inhibiting agent or poison For example, the biasing potential of a sensing electrode may be changed for a period of time and then brought back to a potential at which the sensing electrode is sensitive to the target analyte. For example, a CO sensor which is typically operated at a bias potential of zero (0) mV may have its biasing potential increase to +500 mV for a period of time (for example, one hour). Subsequently, the sensing electrode is returned to its operating biasing potential of zero (0) mV. This procedure may, for example, improve cross-sensitivity to hydrogen (H2). In the case of a combustible gas sensor, the temperature of the sensing element may be increased for a period of time to “burn off” an inhibitor (for example, a sulfur-containing compound). Increasing the temperature of a sensing element in a combustible gas sensor to, for example, burn off an inhibitor in response to an electronic interrogation of the sensing element of the sensor is disclosed in U.S. patent application Ser. No. 13/795,452, filed Mar. 12, 2013. Instead of automating the above-identified maintenance procedures, a user may alternatively be provided an indication of the need to perform any of the procedures. The user may also be notified of an impending “end of life” of a sensor. For example, a user may be notified that the sensor should be replaced in “X” days or another time period. Likewise, the user may be notified of scheduled maintenance tasks required. For example, the user may be notified that a gas calibration is required in “X” days or another time period. Pre-planned or scheduled maintenance may, for example, be altered on the basis of the results of one or more interrogations. The life and health test or electronic interrogation test may be run on multiple sensors within the instrument. Such an electronic interrogation may also be run upon a sensor (whether analytical or non-analytical) which is responsive to the driving force associated with the flow path test (for example, a non-analytical oxygen sensor) to test the operational status or functionality of that sensor. The results of electronic interrogations of multiple sensors can be combined in an analytical algorithm to determine actions (as, for example, described above) based upon that data. As described above, it is common for portable gas detection instruments to contain several sensors with a plenum through which gas is pumped by and external or internal gas pump. The sensors typically included in such an instrument would be a combustible gas sensor, an analytical oxygen sensor (which may or may not include a non-analytical oxygen sensing element or electrode) and several toxic gas sensors such as carbon monoxide and hydrogen sulfide sensors. At least one of the toxic gas sensors may, for example, include a non-analytical oxygen sensor channel for performing a flow path test hereof. As described below, it is possible to monitor the current of the pump to determine the condition of flow through the plenum. In addition, under normal operating conditions, the output of the analytical oxygen sensor should correspond to that expected for value of 20.8 vol-% (atmospheric) oxygen. Finally, the results of electronic interrogation of any or all of the electrochemical sensors, along with the results of applying a driving force to those sensors with non-analytical channels intended to respond to such a driving force (that is a flow path test) may be combined together with, for example, pump current (and/or other pump interrogation) measurements and the output signal of the analytical oxygen sensor to give a high degree of reliability that all sensors in the plenum are experiencing correct flow and are operating as intended. If, however, the results of these tests, either singly or taken together indicate a non-conforming condition, the combination of signals provide a means of differentially informing the user of the nature of the non conforming condition. For example, if the pump current is correct, but the result of the flow path test (for example, applying a driving force to which the non-analytical channel is sensitive) for a particular sensor is non-conforming, then that particular sensor requires maintenance. If however, the pump current is non-conforming, but the output of the analytical oxygen sensor is as expected, this would indicate a potential problem with the pump itself, or with is associated driving circuitry. Once “conforming” results are obtained in the embodiment ofFIG.14A, the users (or the system) may move onto an inlet/flow path blockage testing as described herein. The system will enable the flow path test process and collect the associated data. The system then applies any associated algorithm/analysis and may, for example, compare the results to stored limits or thresholds. If the results are “non-Conforming, an error code or other indication may be provided to inform the user of any repair or replacement options (for example, replacement of filters etc.). If the results are “conforming”, any and all associated sensor output corrections are applied. The user may also be informed that the instrument is ready for use. In addition to sensor output corrections associated with the electronic interrogation of the sensor, the system may also apply one or more corrections to sensor output determined as a result of the flow path test. In that regard, sensors may, for example, be thought of as “molecule counters”. Analytical sensors are thus calibrated in a manner that a certain amount of analyte molecules react at the analytical working or sensing electrode(s) as they diffuse through the instrument and measured values are converted to, for example, a part per million (ppm) or percentage based equivalent readings based upon previous calibration. When the inlet is open and unobstructed, rates of diffusion are very repeatable under the same conditions. As any instrument inlet becomes blocked or flow paths are otherwise obstructed, the rate at which the molecules can diffuse from outside the instrument housing to the sensor can slow, thus lowering the rate at which molecules will encounter the active portion of the sensor (for example, the working electrode of an electrochemical sensor), and thereby lowering the output. By measuring partial blockages as a result of one or more flow path tests hereof, one can adjust the sensitivity of the sensor to maintain accurate readings regardless of such partial blockages. In a number of embodiments hereof, once a flow path test such as an exhaled breath test is complete, the system calculates a derivative of the sensor response, based on the function: Rate of change=dxdt=x(t+1)-xt(t+1)-t The equation shown above indicates a generalized derivative function. As is known to one skilled in the art, there are many arithmetic formulas which can be used to calculate a derivate from periodic data. FIG.15Aillustrates sensor response or output for a typical flow path test hereof in the form of an exhaled breath test as a function of time.FIG.15Billustrates a plot of the rate of change of the sensor response ofFIG.15A. A peak rate of change as percentage of the baseline is then calculated as follows: Peak rate of change=peakmaxbaseline Referring toFIG.15B, the peakmaxcorresponds to the maximum value of the derivative function immediately subsequent to the application of the driving force, resulting in the positive deflection shown inFIG.15A, and the baseline refers to a mean value of the derivative function prior to the application of the driving force. The peak rate of change values may be correlated with a correction factor as illustrated in the plot ofFIG.15C. From the plot ofFIG.15C, an associated lookup table or an associated algorithm/formula, the system may determine a correction factor for sensor sensitivity based upon the calculated peak rate of change. An embodiment of a control procedure and fault detection procedure for a gas detection system or instrument that may be operated in a forced flow mode (that is, using a pneumatic pump to draw environmental gasses to the one or more sensors of the instrument as described in connection withFIG.3I) is illustrated inFIGS.16A and16B. The control procedure and fault protection procedure may be used in connection with a system such as system400which may be operated in a forced flow mode as well as in a diffusion mode (that is, relying on diffusion to bring environmental gasses to the one or more sensors of the instrument as illustrated inFIG.3J). The illustrated procedure is discussed in further detail in U.S. Pat. No. 6,092,992, the disclosure of which is incorporated herein by reference, and provides another independent check of instrument or system operational state. Computer code for the procedure may be stored in memory system405and is discussed in connection with the pseudocode set forth in the appendix to the specification of U.S. Pat. No. 6,092,992. Under the illustrated procedure, when the power switch of the gas detection instrument or system such as system410is turned on, a pump initialization procedure begins. A control system, which may, for example, include a processor system404(for example, including a microprocessor) in communicative connection with memory system405) and/or control circuitry, preferably first checks to see if motor406of pump406ais connected within the instrument or system410by measuring if a motor signal (for example, back emf) is being generated. If no motor signal is detected, the pump initialization procedure is exited and the gas detection instrument may be readily operated in a diffusion mode. If motor406is detected, the duty cycle is set to 100% (percent on) for approximately 0.5 seconds. Microcontroller/processor404measures the power available from a power source such as a battery408, and then sets the duty cycle to a maximum duty cycle previously established for the measured battery voltage. A maximum duty cycle and a minimum duty cycle for given battery voltage ranges may, for example, be established experimentally for a given pump and motor combination to provide an acceptable flow rate. For example, for the motor and pump combination controlled via the pseudocode set forth in U.S. Pat. No. 6,092,992, a maximum duty cycle of 80% and a minimum duty cycle of 5% were experimentally established to provide an acceptable flow rate for a battery voltage of greater than approximately 3.6 volts. For a battery voltage equal to or between approximately 3.6 and 3.3 volts, the maximum and minimum duty cycles were experimentally determined to be 90% and 5%, respectively. For a battery voltage less than approximately 3.3 volts, the maximum and minimum duty cycles were experimentally determined to be 100% and 5%, respectively. In a number of embodiments, a PUMP CHECK procedure (seeFIG.16B) is initiated after the duty cycle is set to the maximum duty cycle for the measured battery voltage. The PUMP CHECK procedure first determines if a pump has been added to the gas detection instrument since the instrument has been turned on. If the pump is newly added, a fault is preferably indicated and the user is required to actuate a reset button to begin initialization of the newly added pump. Likewise, in a number of embodiments, removal of a pump results in a fault indication requiring the user to actuate the reset button to continue to operate the instrument in the diffusion mode. The PUMP CHECK procedure is exited if a fault condition has been detected and a fault indication has been given. Upon initialization after turning on the instrument, however, fault indications are preferably delayed for up to 15 seconds for centering. If no fault condition has been detected, the PUMP CHECK procedure determines if a PULSE CHECK procedure is in progress. During initialization, however, the PULSE CHECK procedure is disabled for a period of, for example, 30 seconds in a number of embodiments. If no PULSE CHECK procedure is in progress, processor404may, for example, attempt to adjust the duty cycle in a manner to achieve a motor signal (average back emf voltage) centered between a maximum acceptable average voltage and a minimum acceptable average voltage experimentally determined to efficiently provide an acceptable flow rate. For example, for the pump and motor combination in the pseudocode of U.S. Pat. No. 6,092,992, the maximum and minimum motor signals were established to be approximately 1.95 and 1.85 volts, respectively. Processor404thus attempts to adjust the duty cycle to achieve a motor signal of approximately 1.90 volts. A motor signal in the range of approximately 1.85 to 1.95 volts may, for example, be considered to be centered, however. If pump motor10is not centered within, for example, 15 seconds, a pump fault is indicated by an electronic alarm system90such as an alarm light and/or an alarm sound. If motor10is centered, the PUMP CHECK procedure checks whether it is time for a PULSE CHECK procedure. If yes, the PULSE CHECK procedure as described above is initiated. If no, processor404checks for faults. As discussed above, during operation of gas detection instrument or system400the average back emf or motor signal may, for example, be centered between 1.95 and 1.85 volts to maintain a suitable flow rate. Fault indications are enabled only when the motor signal is maintained in this range. If the duty cycle has been set to the minimum duty or the maximum duty for a defined period of time such as one second or more in controlling motor406, a fault is indicated. Moreover, if the motor signal is less than approximately 1.4 volts for a defined period of time such as one second or more, a fault is indicated. Further, if the rate of change of the duty cycle is greater than 5% during, for example, a five second interval, a fault is indicated. Like the maximum and minimum duty cycles and the target motor signal range, the 1.4 volt minimum motor signal and 5%/5 second rate of change thresholds or fault conditions are readily determined experimentally for the pump and motor combination in use. If no fault condition is identified, the PUMP CHECK procedure is exited. After initialization, the PUMP CHECK procedure or function may, for example, be called or executed periodically (for example, 10 times per second). Any time a fault condition is identified, the duty cycle may, for example, be set to its minimum duty cycle for the battery voltage. In a number of embodiments, the PUMP CONTROL procedure checks the battery voltage periodically (for example, once per minute) to set the appropriate maximum and minimum duty cycles. In the embodiment of the PULSE CHECK procedure set forth in the pseudocode of U.S. Pat. No. 6,092,992, microcontroller404determines if the average voltage across motor10is less than 1.4 volts after a start-up period of approximately 1.5 seconds if the temperature is greater than or equal to 5° C. If the temperature is less than 5° C., the determination is made after a period of approximately 2 seconds. If the motor signal is less than 1.4 volts after the start-up period, a fault is indicated. The start-up voltage threshold of 1.4 volts may be determined experimentally for a particular pump and motor combination. Pump and motor combinations may, for example, be tested over a range of load conditions, temperature conditions and battery voltages. Fault parameters or thresholds may, for example, be established by simulating various fault conditions. Various fault detection systems and methods may be used collectively or individually to detect pumping fault conditions in gas detection instruments. Blockage may, for example, be periodically simulated to test the continued operation of such systems and methods. FIG.16Cillustrates a system to effect control of pump406including motor406awhich drives pump406as described above. In the illustrated embodiment, motor406areceives energy from a battery system405via a switch mechanism such as a transistor switch using Pulse Width Modulation (PWM). In PWM, the battery voltage is generally pulsed on and off hundreds of times per second. The time duration or duty cycle of each pulse is varied to control the speed of motor406a. While the transistor switch is on, battery system405supplies power to motor406awhich energizes the windings of motor406aand causes motor406ato turn. While transistor switch is off, motor406acontinues to turn because of its momentum and acts like a generator to produce back electromotive force (emf). The energy (that is, the back emf) can be redirected back to motor406ausing a regeneration circuit including, for example, one or more diodes connected across motor406a. This technique is known as regeneration. The back emf can also be used to provide feedback to control motor406a. A motor signal proportional to a voltage across the windings of motor406awhile the transistor switch is in the off state is measured and used to control motor406a. There are a number of ways in which a motor signal proportional to the voltage across the windings during the off portion of the PWM cycle can be measured. For example, the approximate voltage at any defined instant during the off portion of each cycle can be measured. Further, the approximate average voltage developed across motor406aduring the off portion of the PWM cycle can be measured. In a number of embodiments, the approximate average voltage developed across motor406aduring both the off portion and the on portion of the PWM cycle is measured. Each of the above measurements is proportional to the voltage contributed by the regeneration phase of the cycle. The voltage contributed by the regeneration phase is, in turn, proportional to the speed of motor406a. Under light load conditions, motor406aruns at a relatively high speed and generates a high voltage. When the load on motor406aincreases, motor406aruns at a lower speed (assuming the energizing pulse has not changed) and the voltage decreases. In a number of embodiments, a microprocessor or microcontroller of processor system404measures the voltage decrease and then increases the pulse width (or duty cycle) proportionally to compensate for the load until the motor voltage is back to its normal operating value or within its normal operating range. When the load is removed, motor406awill speed up momentarily and increase the voltage. Processor system404adjusts the duty cycle until the voltage is again back to its normal operating value or range. By controlling the motor voltage, the speed of motor406a, and thereby the flow rate of pump406, are maintained in a relatively small operating range. Efficient motor control maximizes the life of battery system405. The normal operating conditions of motor406aunder light and heavy loads are preferably characterized to determine the maximum and minimum duty cycle required for motor406aover battery voltage changes and operating temperature changes normally experienced during use thereof. These maximum and minimum values may be used to determine normal operating limits for motor406aand to detect problems in the flow system such as a sample line failure or a motor failure. A clogged sample line or a stalled motor condition, for example, is detected by a low average motor voltage. A burned out motor winding or an open commutator circuit is detected by the absence of the regenerated voltage. A system and a method for detecting more marginal fault conditions, for example, caused by sudden changes in pneumatic loading may also be provided. Such sudden changes may occur, for example, when a liquid is inadvertently drawn into the free end of the sample line or when the sample line is restricted by a crushing force somewhere along its length. In one embodiment, the control system illustrated inFIG.16Cmeasures the rate of change in the value of the PWM required to maintain the average motor voltage constant. Once a predetermined center point or control point of average motor voltage is obtained, processor system404thereafter continuously adjusts the PWM to maintain the voltage constant and computes the rate of change in the PWM. The computed rate of change is continuously compared to an empirically determined normal, acceptable value of rate of change and any deviation in the computed rate greater than this acceptable rate is interpreted by processor system as a flow system failure or fault condition. In another embodiment processor system404causes a momentary shutdown of the PWM supply signal on a periodic basis and subsequently verifies the generation of an acceptable average motor voltage within a set time interval after the resumption of the PWM supply signal. This procedure is referred to as a PULSE CHECK procedure in connection withFIG.16A. The periodic shutdown may, for example, occur approximately every 15 seconds. This period is sufficiently frequent to monitor pump406and sample system performance, but not so frequent as to materially reduce the effective sample flow rate. The PWM shutdown period in this embodiment may, for example, be approximately 0.2 second. This shutdown period is sufficiently long to cause motor406ato stall (that is, to slow or stop) and to allow the checking of the acceleration of motor406aupon resumption of PWM within a predetermined interval of time. In a number of embodiments, the interval chosen for motor406to accelerate to a defined average voltage was approximately 1.5 seconds after the resumption of the PWM supply signal. While 1.5 seconds is an appropriate value around room temperatures, at lower temperatures more time may be allowed because of the slower acceleration of motor406arising from the “stiffness” of the mechanical components of pump406at such lower temperatures. Absent a marginal fault, motor406awill restart successfully (that is, within the defined time interval after the resumption of the PWM motor406awill again be regenerating an acceptable average voltage). A failure to “successfully” restart indicates a fault condition. For example, a marginal fault condition causing an excessive demand for motor torque upon restart is detected as a lower than normal average voltage at the end of the time interval and is interpreted by processor system404as a flow system failure. Testing the demand pump406for motor torque at a predetermined PWM provides a valuable check for a number of fault conditions. The foregoing description and accompanying drawings set forth representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope. | 112,777 |
11860176 | DETAILED DESCRIPTION In the following detailed description of the embodiments, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration, and not by way of limitation, specific embodiments in which the disclosure may be practiced. It is to be understood that other embodiments may be utilized and that logical, mechanical and electrical changes may be made without departing from the spirit and scope of the present disclosure. Certain terms will be used in this patent application, the formulation of which should not be interpreted to be limited by the specific term chosen, but as to relate to the general concept behind the specific term. The terms ‘sample’, ‘patient sample’ and ‘biological sample’ can refer to material(s) that may potentially contain an analyte of interest. The patient sample can be derived from any biological source, such as a physiological fluid, including blood, saliva, ocular lens fluid, cerebrospinal fluid, sweat, urine, stool, semen, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid, tissue, cultured cells, or the like. The patient sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous fluids, lysis or the like. Methods of treatment can involve filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents. A patient sample may be used directly as obtained from the source or used following a pretreatment to modify the character of the sample. In some embodiments, an initially solid or semi-solid biological material can be rendered liquid by dissolving or suspending it with a suitable liquid medium. In some embodiments, the sample can be suspected to contain a certain antigen or nucleic acid. The term ‘analyte’ can relate to a component of a sample to be analyzed, e.g., molecules of various sizes, ions, proteins, metabolites and the like. Information gathered on an analyte may be used to evaluate the impact of the administration of drugs on the organism or on particular tissues or to make a diagnosis. Thus, ‘analyte’ can be a general term for substances for which information about presence, absence and/or concentration is intended. Examples of analytes are e.g., glucose, coagulation parameters, endogenic proteins (e.g., proteins released from the heart muscle), metabolites, nucleic acids and so on. The term ‘analysis or ‘analytical test’ as used herein can encompass a laboratory procedure characterizing a parameter of a biological sample for qualitatively assessing or quantitatively measuring the presence or amount or the functional activity of an analyte. The term ‘reagent’ as used herein can refer to materials necessary for performing an analysis of analytes, including reagents for sample preparation, control reagents, reagents for reacting with the analyte to obtain a detectable signal, and/or reagents necessary for detecting the analyte. Such reagents may include reagents for isolating an analyte and/or reagents for processing a sample and/or reagents for reacting with an analyte to obtain a detectable signal and/or washing reagents and/or diluents. The terms ‘sample container’, ‘sample holder’ and ‘sample tube’ can refer to any individual container for storing, transporting, and/or processing a sample. In particular, the term without limitation can refer to a piece of laboratory glass- or plastic-ware optionally comprising a cap on its upper end. The container can comprise an opening for dispensing/aspirating liquid into or out of the vessel. The opening may be closed by a cap, a breakable seal or like suitable means for closing the opening in a liquid-tight manner. Sample tubes, e.g., sample tubes used to collect blood, often comprise additional substances such as clot activators or anticoagulant substances, which can have an impact on the processing of the sample. Consequently, different tube types typically can be adapted for pre-analytical and analytical requirements of a particular analysis, e.g., a clinical chemistry analysis, a hematological analysis or a coagulation analysis. A mix up of sample tube types can make samples unusable for analysis. To prevent errors in the collection and handling of samples, the sample caps of many tube manufacturers can be encoded according to a fixed and uniform color scheme. Some sample tubes types, in addition, or alternatively, can be characterized by particular tube dimensions, cap dimensions, and/or tube color. A dimension of a tube can comprises e.g., its height, its size and/or further characteristic shape properties. Sample containers can be identified using identification tag(s) attached thereto. The term ‘identification tag’ as used herein can refer to an optical and/or radio frequency based identifier that allows the identifier tag to be uniquely identified by a corresponding identification tag reader. The ‘identification tag’ shall comprise—but is not limited to—a barcode, a quick response (QR) code or a radio frequency identification (RFID) tag. The term ‘sample carrier’ as used herein can refer to any kind of holder configured to receive one or more sample tubes and configured to be used for transporting sample tube(s). Sample carriers may be of two major types, single holders and sample racks. A ‘single holder’ can be a type of sample carrier configured to receive and transport a single sample tube. Typically, a single holder can be provided as a puck, i.e., a flat cylindrical object with an opening to receive and retain a single sample tube. A ‘sample rack’ can be a type of sample carrier, typically made of plastics and/or metal, adapted for receiving, holding and transporting a plurality of sample tubes, e.g., five or more sample tubes e.g., disposed in one or more rows. Apertures, windows or slits may be present to enable visual or optical inspection or reading of the sample tubes or of the samples in the sample tubes or of a label, such as a barcode, present on the sample tubes held in the sample rack. The term ‘laboratory instrument’ as used herein can encompass any apparatus or apparatus component operable to execute one or more processing steps/workflow steps on one or more biological samples and/or one or more reagents. The expression ‘processing steps’ thereby can refer to physically executed processing steps such as centrifugation, aliquotation, sample analysis and the like. The term ‘instrument’ can cover pre-analytical instruments, post-analytical instruments as well as analytical instruments. The term ‘analyzer’/‘analytical instrument’ as used herein can encompass any apparatus or apparatus component configured to obtain a measurement value. An analyzer can be operable to determine via various chemical, biological, physical, optical or other technical procedures a parameter value of the sample or a component thereof. An analyzer may be operable to measure said parameter of the sample or of at least one analyte and return the obtained measurement value. The list of possible analysis results returned by the analyzer can comprise, without limitation, concentrations of the analyte in the sample, a digital (yes or no) result indicating the existence of the analyte in the sample (corresponding to a concentration above the detection level), optical parameters, DNA or RNA sequences, data obtained from mass spectrometry of proteins or metabolites and physical or chemical parameters of various types. An analytical instrument may comprise units assisting with the pipetting, dosing, and mixing of samples and/or reagents. The analyzer may comprise a reagent-holding unit for holding reagents to perform the assays. Reagents may be arranged for example in the form of containers or cassettes containing individual reagents or group of reagents, placed in appropriate receptacles or positions within a storage compartment or conveyor. It may comprise a consumable feeding unit. The analyzer may comprise a process and detection system whose workflow can be optimized for certain types of analysis. Examples of such analyzers are clinical chemistry analyzers, coagulation chemistry analyzers, immunochemistry analyzers, urine analyzers, nucleic acid analyzers, used to detect the result of chemical or biological reactions or to monitor the progress of chemical or biological reactions. The term ‘pre-analytical instrument’ as used herein can encompass any apparatus or apparatus component that can be configured to perform one or more pre-analytical processing steps/workflow steps comprising—but not limited to—centrifugation, resuspension (e.g., by mixing or vortexing), capping, decapping, recapping, sorting, tube type identification, sample quality determination and/or aliquotation steps. The processing steps may also comprise adding chemicals or buffers to a sample, concentrating a sample, incubating a sample, and the like. The term ‘post-analytical instrument’ as used herein can encompass any apparatus or apparatus component that can be configured to perform one or more post-analytical processing steps/workflow steps comprising—but not limited to—sample unloading, transport, recapping, decapping, temporary storage/buffering, archiving (refrigerated or not), retrieval and or disposal. The term ‘sample transportation system’ as used herein encompasses any apparatus or apparatus component that is configured to transport sample carriers (each holding one or more sample containers) between laboratory instruments. In particular, the sample transportation system is a one dimensional conveyor-belt based system, a two-dimensional transportation system (such as a magnetic sample carrier transport system) or a combination thereof. The term ‘laboratory middleware’ as used herein can encompass any physical or virtual processing device configurable to control a laboratory instrument/or system comprising one or more laboratory instruments in a way that workflow(s) and workflow step(s) can be conducted by the laboratory instrument/system. The laboratory middleware may, for example, instruct the laboratory instrument/system to conduct pre-analytical, post analytical and analytical workflow(s)/workflow step(s). The laboratory middleware may receive information from a data management unit regarding which steps need to be performed with a certain sample. In some embodiments, the laboratory middleware might be integral with a data management unit, may be comprised by a server computer and/or be part of one laboratory instrument or even distributed across multiple instruments of the analytical laboratory. The laboratory middleware may, for instance, be embodied as a programmable logic controller running a computer-readable program provided with instructions to perform operations. A ‘data storage unit’ or ‘database’ can be a computing unit for storing and managing data such as a memory, hard disk or cloud storage. This may involve data relating to biological sample(s) to be processed by the automated system. The data management unit may be connected to an LIS (laboratory information system) and/or an HIS (hospital information system). The data management unit can be a unit within or co-located with a laboratory instrument. It may be part of the laboratory middleware. Alternatively, the database may be a unit remotely located. For instance, it may be embodied in a computer connected via a communication network. The term ‘communication network’ as used herein can encompass any type of wireless network, such as a WiFi™, GSM™, UMTS or other wireless digital network or a cable based network, such as Ethernet™ or the like. In particular, the communication network can implement the Internet protocol (IP). For example, the communication network can comprise a combination of cable-based and wireless networks. An ‘analytical laboratory’ as used herein can comprise a laboratory middleware operatively coupled to one or more analytical; pre- and post-analytical laboratory instruments wherein the laboratory middleware can be operable to control the instruments. In addition, the laboratory middleware may be operable to evaluate and/or process gathered analysis data, to control the loading, storing and/or unloading of samples to and/or from any one of the analyzers, to initialize an analysis or hardware or software operations of the analysis system used for preparing the samples, sample tubes or reagents for said analysis and the like. In particular, the instruments of an analytical laboratory and the laboratory middleware can be interconnected by a communication network. A ‘test order’ as used herein can encompass any data object, computer loadable data structure, modulated data representing such data being indicative of one or more processing steps to be executed on a particular biological sample. For example, a test order may be a file or an entry in a database. A test order can indicate an analytical test if, for example, the test order can comprise or can be stored in association with an identifier of an analytical test to be executed on a particular sample. A ‘STAT sample’/‘urgent sample’ can be a sample which can need to be processed and analyzed very urgently as the analysis result may be of life-crucial importance for a patient. STAT or urgent samples can be identified either by data stored on an identifier tag attached to a sample container holding the biological sample and/or by data comprised by and/or associated with the test order indicative of an urgency/priority level of the respective test order. The urgency/priority level of a test order may be indicated as binary option (e.g., urgent respectively normal) and/or as a scale (e.g., 10 very urgent, 7 normal, 5 least urgent test order). Additionally, or alternatively, a STAT/urgent sample may be identified by a particular type of sample container, by a particular type of sample container cap, by a particular color of sample container cap and/or a particular type/color/format of an identifier tag attached to the sample container. Embodiments herein disclosed can address the need for an analytical laboratory and a method of operating an analytical laboratory, which can prevent overloading/underutilization of laboratory instruments by determining an effective flow rate of the laboratory instruments and dynamically reacting to the deviations of the effective flow rate by controlling the load limit of each instrument. Embodiments of the disclosed computer implemented method of operating an analytical laboratory comprising a laboratory middleware communicatively connected to a plurality of laboratory instruments configured to process biological samples can comprise the steps of setting a load limit for each laboratory instrument at a value equal to a maximum instrument capacity of the respective laboratory instrument by the laboratory middleware and dispatching biological samples to laboratory instrument(s) at a dispatch rate not greater than the instrument load limit by the laboratory middleware. The biological samples can be dispatched to those laboratory instrument(s), which can be configured to carry out at least one test order corresponding to the respective biological sample. The method can also comprise the steps of receiving and identifying biological samples by the laboratory instruments and, upon identifying a biological sample, sending test order queries to the laboratory middleware by each laboratory instrument. The test order query(s) comprising data identifying the biological sample. In response to the test order queries, the laboratory middleware can transmit test orders to the laboratory instruments corresponding to the biological samples identified in the respective test order queries. According to some embodiments disclosed herein, the laboratory middleware can retrieve the test orders corresponding to a biological sample from a data storage unit based on said data identifying the biological sample. The laboratory middleware can monitor a query rate of the plurality of laboratory instruments in order to determine an effective flow rate corresponding to each laboratory instrument. The query rate can be defined as a number of distinct test order queries received from a particular laboratory instruments in a set period of time, such as per minute, hour, and so on. Since the laboratory instruments send the test queries at a time when they are ready to process the biological sample(s), the query rate can be a good indication of the effective processing capacity of the respective laboratory instrument at that time. If the effective flow rate of the first laboratory instrument is lower than the dispatch rate to the first laboratory instrument, the laboratory middleware can decrease the load limit of a first laboratory instrument of the plurality of laboratory instruments. The load limit can be decreased to ensure that no backlog of unprocessed samples accumulates at the laboratory instrument, causing even further overloading of the instrument. On the other hand, if the effective flow rate of the first laboratory instrument is greater than or equal to the dispatch rate to the first laboratory instrument, the laboratory middleware can increase the load limit for the first laboratory instrument. Embodiments disclosed herein can be advantageous since adjusting the load limit of laboratory instruments as a reaction to their effective flow rate can avoid overloading, or underutilization. Furthermore, determining the effective flow rate by the laboratory middleware based on the test order queries received from the laboratory instruments can be advantageous as it can be devoid of any assumptions of performance and can be implemented even without any change to the existing laboratory instruments. According to further embodiments disclosed herein, the laboratory middleware can increase or decrease the load limit of the first laboratory instrument using continuously modulated control such as, for example, a proportional-integral-derivative PID, a proportional-integral PI, a proportional-derivative PD, a proportional or an integral control algorithm. Such embodiments can be particularly advantageous since continuously modulated control can be configured to keep the effective flow rate as close as possible to the maximum instrument capacity, quickly reacting to deviations without overreacting. In addition to controlling (i.e. increasing or decreasing) the load limit, further embodiments disclosed herein can react to deviations of the effective flow rate from the dispatch rate by performing load balancing between laboratory instruments and/or buffering biological sample(s) to temporarily reduce the load on an otherwise overloaded instrument. Further embodiments disclosed herein can address the further need for an analytical laboratory and method of operating an analytical laboratory wherein an overloading of the entire analytical laboratory can be prevented. As mentioned above, even if one would assume a theoretically perfect load distribution between laboratory instruments in an analytical laboratory, without some control of the input of the analytical laboratory as a whole, there can still be a risk that the analytical laboratory can become overloaded if the inflow of biological samples is higher than the overall processing capacity. Therefore, embodiments disclosed herein addressing this issue can further comprise the step of masking one or more of the plurality of laboratory instruments, wherein masking can comprise preventing one or more of the plurality of laboratory instruments from receiving biological sample(s), in particular biological sample(s) having at least one associated test order which the first laboratory instrument is configured to carry out. According to embodiments disclosed herein, preventing one or more of the plurality of laboratory instruments from receiving biological sample(s) can comprise preventing (physically) even the loading of the respective biological sample(s) and/or automatically unloading the biological sample(s), e.g., into an error output. Such embodiments can be advantageous as they can limit the inflow of biological samples into the analytical laboratory, thereby preventing that the overall analytical laboratory is overloaded, including buffer and archiving capacity of the laboratory instruments. Further embodiments disclosed herein can relate to ensuring the analytical laboratory can still receive and process urgent biological samples while still controlling the overall load of the analytical laboratory. In order to achieve this, according to further embodiments, masking of laboratory instruments can be performed with respect to all laboratory instruments, other than one or more laboratory instruments reserved for receiving biological samples of high priority. Referring initially toFIG.1,FIG.1shows a highly schematic block diagram of an embodiment of the disclosed analytical laboratory1. As shown on the block diagram ofFIG.1, embodiments of the disclosed analytical laboratory1for processing biological sample(s) can comprise a plurality of laboratory instruments10AI,10PRE,10POST and a laboratory middleware20communicatively connected by a communication network. The plurality of laboratory instruments10AI,10PRE,10POST can be configured to execute processing steps on the biological samples according to instructions from the laboratory middleware20. All laboratory instruments10AI,10PRE,10POST can be collectively referred to using the reference numeral10. The pre-analytical instruments10PRE comprised by the analytical laboratory1may be one or more from the list comprising: an instrument for centrifugation of samples, a capping-, decapping- or recapping instrument, aliquoter, a buffer to temporarily store biological samples or aliquots thereof. The post-analytical instruments10POST comprised by the analytical laboratory1may be one or more from the list comprising: a recapper, an unloader for unloading a sample from an analytical system and/or transporting the sample to a storage unit or to a unit for collecting biological waste. According to various embodiments of the disclosed analytical laboratory1, the plurality of laboratory instruments10AI,10PRE,10POST may be identical or different instruments such as clinical- & immunochemistry analyzers, coagulation chemistry analyzers, immunochemistry analyzers, urine analyzers, nucleic acid analyzers, hematology instruments and the like. The laboratory middleware20can be configured to control the analytical laboratory1to carry out the steps of one or more of the methods herein disclosed and can be communicatively connected to the data storage unit22. As shown onFIG.1, the analytical laboratory1can further comprise a sample transportation system10TRS interconnecting the plurality of laboratory instruments10AI,10PRE,10POST. According to embodiments disclosed herein, the sample transportation system10TRS can be a one-dimensional conveyor-belt based system. According to further embodiments disclosed (but not illustrated), the sample transportation system10TRS can be a two-dimensional transportation system (such as a magnetic sample carrier transport system). The analytical laboratory1can be configured to carry out the method according to the embodiments disclosed herein. Turing now toFIGS.2-5, embodiments of the disclosed method of operating an analytical laboratory shall be described with reference to the figures. As shown onFIG.2, in a first preparatory step100, a load limit can be set for each laboratory instrument10. The load limit can initially be set at a value equal to a maximum instrument capacity of the respective laboratory instrument10. According to embodiments disclosed herein, the maximum instrument capacity can be set by a vendor, manufacturer, and/or operator, optionally considering a safety margin. The maximum instrument capacity as well as the load limit may be expressed as a number of biological samples a laboratory instrument10can process in a given time frame, such as, for example, samples per hour/day and the like. Achieving an effective processing rate of the laboratory instruments10as close as possible to the maximum instrument capacity is the goal of the optimization by the laboratory middleware20. Once the load limit is set, in step102, the laboratory middleware20can dispatch biological samples to laboratory instrument(s)10at a dispatch rate not greater than the instrument load limit. If the number of biological sample(s) in the analytical laboratory1overall that need processing by the respective laboratory instrument10is lower than the load limit, then, of course, the laboratory middleware20can dispatch biological sample(s) at a rate lower than the load limit. The biological samples can be dispatched to those laboratory instrument(s)10, which are configured to carry out at least one test order corresponding to the respective biological sample. Additionally, according to embodiments disclosed herein, the laboratory middleware20can check whether respective laboratory instrument10has all resources (such as consumables, reagents, quality control) available and ready to process the biological sample according to the corresponding test order. Thereafter, not illustrated on the flowchart ofFIG.2for clarity, the laboratory instruments10can receive and identify the biological sample(s) dispatched thereto. Upon identifying the biological samples, each laboratory instrument10can send test order queries to the laboratory middleware20, the test order query comprising data identifying the biological sample. In other words, the laboratory instruments10can ask the laboratory middleware what test to perform on the received biological sample(s). In response to the test order queries, the laboratory middleware20can transmit test orders to the laboratory instruments10corresponding to the biological samples identified in the respective test order queries. A test order can comprise data indicative of one or more processing steps to be carried out on the biological sample. According to embodiments disclosed herein, the test orders can be retrieved from a data storage22such as, for example, a database internal or communicatively connected to the laboratory middleware20. The laboratory instruments10can then process the biological sample(s) according to the test orders sent to them by the laboratory middleware20. The sequence of the laboratory instruments10receiving/identifying biological samples and querying the laboratory middleware20, the laboratory middleware20replying with the test order, and the laboratory instruments10processing the biological samples can be repeated in the analytical laboratory1. Parallel thereto, in a step104, the laboratory middleware20can monitor the rate at which the plurality of laboratory instruments10query the laboratory middleware20for test orders (referred to hereafter as query rate). Since the laboratory instruments10cannot process biological samples without a test order, the query rate can be a direct and reliable indication of the rate at which the laboratory instrument10processes biological samples at a given time. Hence, by monitoring the query rate, in a step106, the laboratory middleware20can determine an effective flow rate corresponding to each laboratory instrument10, the effective flow rate being indicative of the rate at which the laboratory instrument10processes biological samples. The laboratory middleware20can then compare the effective flow rate of each laboratory instrument10with the dispatch rate of biological samples to that laboratory instrument10(referred to hereafter as first laboratory instrument10). If the effective flow rate of a first laboratory instrument10is lower than the dispatch rate to the first laboratory instrument10, the laboratory middleware20, in a step109, can decrease its load limit (of the first laboratory instrument10). In other words, if the laboratory middleware20determines that the first laboratory instrument10is not able to process its workload (dispatched samples), it can reduce its load limit to avoid overloading the instrument. On the other hand, if the effective flow rate of the first laboratory instrument10is greater than or equal to the dispatch rate to the first laboratory instrument10, the laboratory middleware20, in a step108, can increase the load limit for the first laboratory instrument10. The method and the system disclosed herein can be advantageous since adjusting the load limit of laboratory instruments10as a reaction to their effective flow rate can avoid overloading, or underutilization. Furthermore, determining the effective flow rate by the laboratory middleware based on the test order queries received from the laboratory instruments10can be advantageous as it can be devoid of any assumptions of performance and can be implemented even without any change to the existing laboratory instruments10. Some embodiments of how the middleware20can determine the amount the load limit can be increased/decreased will be described with reference to the sequence ofFIGS.3A-C. FIG.3Ashows a simulation of a current prior art performance in one particular scenario where a laboratory instrument is processing biological samples at its maximum instrument capacity for a considerable amount of time. The line310illustrates the dispatch rate to the laboratory instrument. The line320illustrates the effective flow rate of the laboratory instrument. The horizontal lines correspond to theoretical and physical limits of such laboratory instruments. With the current prior art laboratory middleware, the performance output (effective flow rate) would become degraded over time because the laboratory instruments are sometimes not able to process biological samples at such a constant dispatch rate. This situation can become even more critical when a laboratory instrument needs to be temporarily stopped (for replacing a reagent cassette for example), since biological samples would accumulate and a backlog would arise, which could overload the laboratory instrument. FIG.3B, shows the effect of the disclosed method comprising increasing respectively decreasing the load limit of a laboratory instrument10as a reaction to its effective flow rate. As illustrated in this figure, proactively decreasing the load limit of an instrument can prevent overloading, allowing the instrument to return closer to its maximum capacity. On the other hand, increasing the load limit once the instrument is again able to process samples at the rate they are dispatched can prevent underutilization of the instrument. According to embodiments disclosed herein, the laboratory middleware20can increase or decrease the load limit of the first laboratory instrument10using continuously modulated control such as, for example, a proportional-integral-derivative PID, a proportional-integral PI, a proportional-derivative PD, a proportional or an integral control algorithm. A continuously modulated control continuously (or quasi-continuously) can calculate an error value e(t) as the difference between a desired set point (SP) and a measured process variable (PV) and can apply a correction based on proportional, integral, and derivative terms (denoted P, I, and D respectively). The error value e can be calculated by the laboratory middleware20as the difference between the effective flow rate and the maximum instrument capacity. From the variation of the error value e over time, an error curve e(t) can be determined. In order to bring the effective flow rate of a laboratory instrument as close as possible to its maximum instrument capacity, the laboratory middleware20can increase/decrease the load limit by a correction value determined as a weighted sum of:A proportional term P—The proportional term P can be calculated as proportional to the error value e and can be indicative of the magnitude of the error value e. The proportional response can be adjusted by multiplying the error by a proportional gain.An integral term I—The integral term I can be calculated as an integral of the error curve e(t) over a period of time t and can be indicative of the magnitude and duration of the error value e. In other words, the contribution from the integral term I can be proportional to both the magnitude of the error value e and the duration of the error value e. The integral in a PID controller can be the sum of the instantaneous error over time and gives the accumulated offset that should have been corrected previously. The accumulated error can then be multiplied by the integral gain and added to the controller output. The advantage of the integral term I can be that it can accelerate the return of the performance (effective flow rate) of the process towards its target (maximum instrument capacity) and can eliminate the residual steady-state error that occurs with a pure proportional controller.A derivative term D—The derivative term D can be calculated as a derivative of the error curve e(t) over a period of time t and can be indicative of a rate of change of the error value e. The derivative of the process error can be calculated by determining the slope of the error over time and multiplying this rate of change by the derivative gain. The magnitude of the contribution of the derivative term D to the overall control action is termed the derivative gain. Derivative action can predict system behavior and thus can improve settling time and stability of the system. It can be noted that according to particular embodiments, one or more of the proportional gain; the integral gain; and/or the derivative gain may be also zero. According to further embodiments disclosed herein, the one or more of the proportional gain; the integral gain; and/or the derivative gain can be refined in view of the response of the system, namely the change of effective flow rate as a response to a change in the load limit. Turning now toFIGS.4A-D, further embodiments of the disclosed method will be described. FIG.4Ashows a first page of the multi-page flowchart showing steps100through109(as described above) and off-page connectors A to C, each off-page connector being related to particular embodiments of actions taken by the laboratory middleware20in response to the effective flow rate of laboratory instrument(s)10deviating from their respective load limits (the effective flow rate of the first laboratory instrument10) is lower than the corresponding dispatch rate). FIG.4Bshows the second page of the multi-page flowchart illustrating steps from off-page connector A. In order to (re) distribute the workload between laboratory instruments10(load balancing—step110), the laboratory middleware20can determine a second laboratory instrument10of the plurality of laboratory instruments10(other than the first laboratory instrument10) configured to carry out the same test order corresponding to the respective biological sample as the first laboratory instrument10. Having determined an alternative instrument to process the biological sample(s), in a step110a, the laboratory middleware20can increase the load limit of the second laboratory instrument10by the difference between the effective flow rate and the dispatch rate of the first laboratory instrument10. To prevent the second laboratory instrument10from being overloaded, the laboratory middleware20can increase the load limit of the second laboratory instrument10up to a value not greater than its maximum instrument capacity. Additionally, or alternatively, in a step110b, the laboratory middleware20can adjust load balancing rule(s) of the laboratory middleware20so to decrease the proportion of biological samples dispatched to the first laboratory instrument10and increase the proportion biological samples dispatched to the second laboratory instrument10. A load balancing rule can define the proportion of biological samples sent to each laboratory instrument10PRE,10AI,10POST configured to carry out a particular test order. Thereafter, in a step110c, the laboratory middleware20can redirect samples from the first laboratory instrument10to the second laboratory instrument10at a rate equal to the difference between the effective flow rate and the dispatch rate of the first laboratory instrument10. Additionally, or alternatively, the laboratory middleware20can dispatch biological samples to the first laboratory instrument10and/or to the second laboratory instrument10according to the load balancing rule (as adjusted in step110b). According to embodiments disclosed herein, in redirecting the biological samples (load balancing), the laboratory middleware20can also take into consideration a transportation time of the biological sample(s) to the laboratory instrument10the sample is redirected to. The transportation time can be the time required (estimated) to transport the biological sample(s) from the first laboratory instrument10to the second laboratory instrument10either manually and/or by an automated sample transportation system10TRS. The calculation/estimation of the transportation time can be based on data indicative of a layout of the sample transportation system10TRS and/or data indicative of an effective transportation capacity/availability of the sample transportation system10TRS or a specific transportation route of the sample transportation system10TRS from the first to the second laboratory instrument. Overall, in optimizing the processing of biological sample(s), the laboratory middleware20can monitor and control the load of the sample transportation system10TRS similarly to other laboratory instruments10, namely monitoring its effective flow rate and adjusting its load limit (in this case transportation capacity) to avoid overloading and/or underutilization of the sample transportation system10TRS. In such a way, the overall turn-around-time TAT of the respective biological sample(s) can be significantly improved by ensuring the biological sample(s) are transported to the laboratory instruments10as efficiently as possible. FIG.4Cshows the third page of the multi-page flowchart illustrating steps from off-page connector B. As illustrated on this figure, alternatively, or additionally, to load balancing (step110), if the effective flow rate of the first laboratory instrument10is lower than the corresponding dispatch rate, the laboratory middleware20can buffer biological samples to temporarily reduce the workload of the laboratory instruments10. In a first step, the laboratory middleware20can determine whether any laboratory instrument10(referred hereafter as third laboratory instrument) has available buffer capacity. Buffering may be provided either by laboratory instrument dedicated for temporarily storing biological samples and/or by laboratory instruments10, which have available temporary storage space for biological sample(s) fulfilling the requirements (temperature, humidity) for sample buffering. If there is available buffer capacity, in a step112a, the laboratory middleware20can dispatch biological samples to the third laboratory instrument10having available buffer capacity. After dispatching biological sample(s) for buffering, the laboratory middleware20can keep monitoring the effective flow rate of the first laboratory instrument10and—in a step112b—can dispatch biological samples from the third laboratory instrument10to the first laboratory instrument10as soon as the effective flow rate of the first laboratory instrument10is equal to or greater than the corresponding dispatch rate. This way, biological sample(s) can be kept in a buffer only as long as needed. Similarly to load balancing, the laboratory middleware20can also take into consideration a transportation time of the biological sample(s) to the third laboratory instrument10for buffering. In this way, it can be avoided that biological sample(s) are dispatched for buffering (temporary storage) for periods of time potentially shorter than the time it can take the sample transportation system10TRS to transport the samples for buffering. Such embodiments can be advantageous since waste of both buffering and transportation capacity can be prevented. FIG.4Dshows the fourth page of the multi-page flowchart illustrating steps from off-page connector C.FIG.4Dillustrates various methods of a process called instrument masking. Instrument masking, in general, can refer to the process of hiding a particular laboratory instruments10from other instruments, as if it would not be available; would be offline; and/or would not exist. According to embodiments disclosed herein, instrument masking can be ordered into two main categories: destination masking and input specific masking. Destination masking can refer to the process of preventing one or more of the plurality of laboratory instruments10from sending biological sample(s) to the first laboratory instrument10(the destination). According to a first embodiment of destination masking (step114a), referred to as overall destination specific masking, masking can comprise preventing one or more of the plurality of laboratory instruments10from sending any biological sample(s) to the first laboratory instrument10. In a further embodiment of destination masking, referred to as test specific destination masking—step114b, masking the first laboratory instrument10can comprise preventing one or more of the plurality of laboratory instruments10from sending any biological sample(s) having at least one associated test order which the first laboratory instrument10is configured to carry out. Instrument masking can address the need for an analytical laboratory1and method of operating an analytical laboratory1wherein an overloading of the entire analytical laboratory1can be prevented. According to embodiments disclosed herein, preventing one or more of the plurality of laboratory instruments10from receiving biological sample(s) can comprise preventing (physically) even the loading of the respective biological sample(s) and/or automatically unloading the biological sample(s), e.g., into an error output. Such embodiments can be advantageous as they can limit the inflow of biological samples into the analytical laboratory1, thereby preventing that the overall analytical laboratory1is overloaded, including buffer and archiving capacity of the laboratory instruments10. The second category of instrument masking, input specific masking, can relate to ensuring that the analytical laboratory1can still receive and process urgent biological samples while still controlling the overall load of the analytical laboratory1. In order to achieve this, according to further embodiments, masking of laboratory instruments10can be performed with respect to all laboratory instruments10, other than one or more laboratory instruments10reserved for receiving biological samples of high priority. Biological samples may be identified as urgent either by a data marking the biological sample as urgent (STAT sample), the data being read from an identifying label attached to a sample carrier and/or from a data storage unit22, in particular, as part of the corresponding test order. Alternatively, or additionally, a particular sample tube can identify the biological sample(s) contained therein as urgent. Alternatively, or additionally, any biological sample(s) loaded into a particular laboratory instrument may be designated as urgent, in such case, the respective laboratory instrument being reserved for urgent samples only. Within input specific masking, two embodiments can be distinguished:1) Overall input specific masking114c, wherein one or more of the plurality of laboratory instruments10other than the first laboratory instrument10and other than one or more laboratory instruments10reserved for receiving biological samples of high priority are prevented from receiving any biological sample(s).2) Test and input specific masking114d, wherein one or more of the plurality of laboratory instruments10other than the first laboratory instrument10and other than one or more laboratory instruments10reserved for receiving biological samples of high priority are prevented from receiving biological sample(s) having at least one associated test order which the first laboratory instrument10is configured to carry out. In order to provide an overview of various embodiments of managing the workload and resources of an analytical laboratory1by the laboratory middleware20,FIG.5shows a flowchart illustrating load limit control, load balancing, sample buffering as well as instrument masking processes. Turning now toFIGS.6-9, particular embodiments of the laboratory instruments10PRE,10POST,10AI are described. FIG.6shows a pre-analytical laboratory instrument10PRE comprising a sample container sorting unit14configured to sort sample containers30holding biological samples into sample racks40, each sample rack40being identified by a rack identifier of a rack tag42attached to the sample rack40, the pre-analytical laboratory instruments10PRE being further configured to transmit signals to the laboratory middleware associating the sample identifier(s) ID of sorted sample containers30with the sample rack identifier(s) of the corresponding sample rack(s)40. For embodiments where a pre-analytical laboratory instrument10PRE sorts sample containers30into sample racks40, one or more analytical laboratory instruments can be further configured to read the rack identifier Rack-ID from the rack tag42and transmit the rack identifier Rack-ID to the laboratory middleware with the test query. FIG.7shows a further embodiment of a pre-analytical laboratory instrument10PRE, comprising an aliquoting unit16configured to prepare aliquots of biological sample(s) from the sample container(s)30and provide each of the aliquots with a sample identifier ID on an identifier tag32by an identifier tag writer60. FIG.8shows an embodiment of an analytical laboratory instrument10AI, comprising an analytical unit18configured to carry out an analytical test to measure the presence, absence and/or concentration of at least one analyte in the biological sample. The analytical laboratory instrument10AI can perform analytical test(s) of the biological sample in response to the test order(s). FIG.9shows an embodiment of a post-analytical laboratory instrument10POST comprising a sample storage unit19. The post-analytical laboratory instrument10AI can be configured to store respectively retrieve sample containers30into respectively from the sample storage unit19. The query by post-analytical laboratory instrument(s)10POST to the laboratory middleware for a processing order can comprise a container to store respectively retrieve into respectively from the sample storage unit19. Correspondingly, when queried by a post-analytical laboratory instrument10POST, the laboratory middleware can transmit data indicative of a sample container30to be retrieved from the sample storage unit19. In response to the data indicative of a sample container30to be stored respectively retrieved, the post-analytical laboratory instrument10POST can store respectively retrieves the sample container30from the sample storage unit19. Further disclosed is a computer program product comprising instructions which, when executed by a laboratory middleware20of an analytical laboratory1, can cause the analytical laboratory1to perform the steps of any one of the methods disclosed herein. Thus, specifically, one, more than one or even all of method steps as disclosed herein may be performed by using a computer or a computer network (such as a cloud computing service) or any suitable data processing equipment. As used herein, a computer program product can refer to the program as a tradable product. The product may generally exist in any format, such as in a downloadable file, on a computer-readable data carrier on premise or located at a remote location (cloud). The computer program product may be stored on a non-transitory computer-readable data carrier; a server computer as well as on transitory computer-readable data carrier such as a data carrier signal. Specifically, the computer program product may be distributed over a data network. Furthermore, not only the computer program product, but also the execution hardware may be located on premise or in a remotely, such as in a cloud environment. Further disclosed and proposed is a non-transitory computer-readable storage medium comprising instructions which, when executed by a laboratory middleware20of an analytical laboratory1, can cause the analytical laboratory1to perform the steps of any one of the methods disclosed herein. Further disclosed and proposed is a modulated data signal comprising instructions, which, when executed by a laboratory middleware20of an analytical laboratory1, can cause the analytical laboratory1to perform the steps of any one of the methods disclosed herein. It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed embodiments or to imply that certain features are critical, essential, or even important to the structure or function of the claimed embodiments. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure. Having described the present disclosure in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the disclosure defined in the appended claims. More specifically, although some aspects of the present disclosure are identified herein as preferred or particularly advantageous, it is contemplated that the present disclosure is not necessarily limited to these preferred aspects of the disclosure. | 50,004 |
11860177 | DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for implementing the invention will be described. An operation mode used in an example refers to various modes such as a method of performing control on an object to be transported such as an examination object rack carried into an apparatus, a method of performing display when a certain condition is satisfied, and whether or not the apparatus and a host communicate with each other. The operation modes include a mode in which the apparatus executes or does not execute predetermined control (the control includes the control of transport of the examination object rack, the control of notification, the control of display, and the like) automatically when a certain condition is satisfied, and a mode in which the apparatus automatically executes control different from that when the certain condition is satisfied. In addition, regarding an operation mode switching rack, since it is not assumed that a general examination object is loaded thereon, unlike a rack for a general examination object. Accordingly, the operation mode switching rack may have a shape and structure that do not allow a general examination object to be loaded thereon, but will be called a rack in this specification for convenience of description. In addition, the operation mode switching rack is a member for notifying the apparatus of the switching of an operation mode, and may have a structure capable of being carried into the apparatus. Since the operation mode switching rack has a structure capable of being carried into the apparatus, the operation mode switching rack is a member having at least the same longitudinal and lateral lengths of the bottom surface as those of a general examination object rack. Example 1 In a first embodiment, an example in which an operation mode is switched in an operation mode switching rack will be described. Here, an example of the operation mode is an automatic reexamination mode. The first embodiment will be described with reference toFIGS.1to8showing the example. FIG.1is a schematic diagram illustrating the entire configuration of an automatic analyzer according to an embodiment of the invention. An automatic analysis system (automatic analyzer) according to this embodiment includes an examination object rack insertion unit1, an ID reading unit2, a transport line3, an examination object rack standby unit4, analysis modules5,6, and7, an examination object rack recovery unit8, and an overall management computer9. The examination object rack insertion unit1is a portion that inserts a plurality of examination object racks that respectively hold a plurality of examination objects (samples). The analysis modules5,6, and7are disposed along the transport line3and are removably connected to the transport line3. The number of analysis modules can be arbitrarily set, and a case of three analysis modules is described in this embodiment. The transport line3transports the examination object rack to the analysis modules5,6, and7in response to an analysis request from the examination object rack insertion unit1or transports the examination object rack to the examination object rack standby unit4that holds an examination object having been analyzed in the analysis modules5,6, and7, or transports an examination object rack, for which an analysis request has not been given, to the examination object rack recovery unit8. In addition, the automatic analysis system includes an overall management computer9that performs necessary control within the examination object rack insertion unit1, the ID reading unit2, the transport line3, the examination object rack standby unit4, and the examination object rack recovery unit8. An operation unit10for inputting necessary information and a display unit11that displays an analysis result are further connected to the overall management computer9. In addition, the overall management computer9is provided with a storage unit that stores an operation mode of the automatic analyzer, and a control unit that controls the apparatus on the basis of the operation mode stored in the storage unit. In addition, the control unit can switch an operation mode, and apply the switched operation mode to the apparatus. The examination object held by the examination object rack includes an examination object ID indicating attribute information (a reception number, a patient name, a requested analysis item, and the like) regarding the examination object, and the examination object rack includes a rack ID indicating rack identification information such as a rack number. Here, the rack ID means an identifier for uniquely identifying an individual rack, and examples of a representative identifier include a bar code and RFID. In addition, the rack is also provided with a hole for rack identification so that the rack can be uniquely determined from information regarding the presence and absence of a hole. The presence and absence of a hole can be identified by a transmissive or reflective sensor or the like, and this hole also corresponds to an identifier. For example, the apparatus performs conversion into a binarized number or a decimal number by using a combination of two values of the presence and absence of a hole, and thus it is possible to recognize the converted number as rack identification information such as a rack number. The examination object rack disposed in the examination object rack insertion unit1is transported by the transport line3. When the examination object rack is moved to the transport line3, an examination object ID and an examination object rack ID are read by the ID reading unit2and are transmitted to the overall management computer9. The overall management computer9determines in which analysis module the requested analysis item is executed, on the basis of the attribute information. Next, the analysis modules5,6, and7will be described with reference toFIG.14.FIG.14is a perspective view illustrating the entire configuration of the analysis modules. A biochemical automatic analyzer will be described as an example. In the reaction disk101, a plurality of reaction containers102each having a mixture of an examination object (sample), such as blood or urine, and a reagent are arranged on the circumference. A plurality of reagent bottles110can be mounted on the circumference in the reagent disk109. A transport line117is installed in the vicinity of the reaction disk101, and the transport line moves an examination object rack116on which examination object containers115, such as a test tube, which accommodate a sample are loaded. Reagent dispensing mechanisms107and108that are rotatable and movable in a vertical direction are installed between the reaction disk101and the reagent disk109, and include a reagent probe107a. A syringe for reagent118is connected to the reagent probe107a. A sample dispensing mechanism111which is rotatable and movable in a vertical direction is installed between the reaction disk101and the transport line117, and includes a sample probe111a. A sample syringe119is connected to the sample probe111a. The sample probe111amoves while drawing circular arcs centering on the rotation axis and performs the suction and discharging of an examination object from the examination object container115to the reaction container102. A cleaning mechanism103, a light source, a spectrophotometer104, stirring mechanisms105and106, the reagent disk109, and the transport line117are disposed in the vicinity of the reaction disk101, and a cleaning pump120is connected to the cleaning mechanism103. Cleaning tanks113,130,131,132, and133are disposed on the operation range of the reagent dispensing mechanisms107and108, the sample dispensing mechanism111, and the stirring mechanisms105and106. The examination object container115including an examination object is loaded on the examination object rack116and is carried by the transport line117. In addition, each mechanism is connected to a controller121, and the controller121controls each mechanism. A mixed liquid of an examination object and a reagent within the reaction container102is irradiated with light emitted from the light source. The emitted light is received by the spectrophotometer104, and the controller121calculates the concentration of a predetermined component contained in the examination object from the amount of light (the amount of transmitted light or scattered light of the mixed liquid). Meanwhile, a reagent varies depending on an analysis item. In this manner, biochemical automatic analysis is performed. FIG.2illustrates a screen for setting an examination object rack ID. Examination object type setting201is an area in which the range of a rack ID for examination object measurement is set with respect to five types such as a blood serum and urine. In this example, the range can be set for each type in a rack for a general examination object and a rack for an urgent examination object. Rack IDs having numbers in the 50000s are used in the rack for a general examination object, and rack IDs having numbers in the 40000s are used in the rack for an urgent examination object. Among these ranges, the range of a rack ID to be used is set for each type of examination object. An automatic reexamination mode ON rack202is an area in which an automatic reexamination-validated rack ID is defined, and an automatic reexamination mode OFF rack203is an area in which an automatic reexamination-invalidated rack ID is defined. A maximum of four racks can be set for each of the racks202and203. An operator performs setting so that the rack IDs do not overlap each other. A rack ID which is set by the operator's pressing-down of a registration button204is stored in a storage unit of the overall management computer9. Meanwhile, it is possible to stop the setting of rack by pressing down a cancellation button205, and the operator can move out of the screen. In this manner, the automatic reexamination-validated rack ID and the automatic reexamination-invalidated rack ID are stored in the apparatus, and the examination object rack having a set rack ID serves as an operation mode switching rack. Accordingly, the operator can set any examination object rack as an operation mode switching rack. Meanwhile, the automatic reexamination means that the apparatus automatically detects that a measurement result for a predetermined measurement item has a value falling outside a reference value, and analyzes the measurement item again. FIG.3illustrates an example of the installation of an examination object rack installed in the rack insertion unit1. In this example, it is possible to install an examination object container for a maximum of five examination objects per examination object rack. In the rack insertion unit1, racks having an examination object installed therein are installed in order in which the racks are desired to be measured. This is an example in a case where a plurality of examination object racks302to be measured in an automatic reexamination mode and an examination object rack304not desired to be measured in the automatic reexamination mode are provided. In general, in a case where operation is performed in the automatic reexamination mode, racks are installed in the examination object rack insertion unit1in order of an automatic reexamination mode ON rack301, the plurality of examination object racks302to be measured in the automatic reexamination mode, an automatic reexamination mode OFF rack303, the examination object rack304not desired to be measured in the automatic reexamination, and an automatic reexamination mode ON rack305. At this time, more preferably, when a mark for distinction from an examination object rack (302,304) for measurement is attached to the automatic reexamination mode ON racks301and303and the automatic reexamination mode OFF rack302, it is easy to confirm the racks for the automatic reexamination mode before the insertion of the racks into the apparatus, and it is possible to prevent an erroneous operation. When measurement is started from an operation screen of the operation unit10after the racks are installed in the insertion unit, the state of the apparatus transitions from a standby state to a measurement state. The racks installed in the rack insertion unit1are transported by the transport line3in order of the racks301,302,303,304, and305, and are read by the ID reading unit2so that rack IDs and examination object IDs of examination objects installed in the racks are recognized. Meanwhile, when an initial state is set to be automatic reexamination validation in advance in a standby state, the automatic reexamination mode ON rack301is not necessary. The rack301is read by the ID reading unit2, and thus the apparatus recognizes that racks to be subsequently carried thereinto are racks in which an automatic reexamination mode is valid, and applies an automatic reexamination validation mode. After a while, the rack303is read by the ID reading unit2, and thus the apparatus recognizes that racks to be subsequently carried thereinto are racks in which an automatic reexamination mode is invalid, and applies an automatic reexamination invalidation mode. In addition, after a while, the rack305is read by the ID reading unit2, and thus the apparatus recognizes that racks to be subsequently carried thereinto are racks in which an automatic reexamination mode is valid, and applies an automatic reexamination validation mode as described above. In this manner, it is possible to apply an automatic reexamination invalidation mode to only the rack304. FIG.4is a diagram illustrating a flow of rack ID identification processing of an automatic reexamination switching rack. In step401, the ID reading unit2reads a rack ID which is transported. In step402, the control unit determines the type of rack from the read rack ID and rack ID information which is set in the rack setting screen ofFIG.2. When the rack ID indicates an automatic reexamination mode ON rack, the control unit sets an automatic reexamination mode to be in an on-state in step403. Thereafter, in step405, the rack is transported to the rack recovery unit. When the rack ID indicates an automatic reexamination mode OFF rack, the control unit sets the automatic reexamination mode to be in an off-state in step404. Thereafter, in step405, the rack is transported to the rack recovery unit. In step402, when the rack ID indicates a rack for a general examination object or a rack for an urgent examination object, processing of a first measurement rack ofFIG.5is executed. Meanwhile, the rack does not have an examination object container, which is a measurement object, mounted thereon, and thus is transported to the rack recovery unit without stopping by in any of the analysis modules5,6, and7. FIG.5is a diagram illustrating a flow of rack processing during first measurement. In step502, the control unit confirms the automatic reexamination mode determined inFIG.4in units of examination object racks recognized. In a case where the automatic reexamination mode is turned on, the control unit causes the storage unit to store a fact that rack automatic reexamination of a measurement rack is valid in rack measurement management information for managing the measurement rack, in step511. In this example, the storage may be performed in units of racks, or automatic reexamination information may be stored on the basis of examination object measurement information managed in units of examination objects installed in the rack. In a case where the automatic reexamination mode is turned off, the control unit causes the storage unit to store a fact that the rack automatic reexamination of the rack is invalid, in step512. In step503, it is determined whether or not there is a measurement request item with respect to all examination objects installed in the examination object racks. In a case where there is a request item with respect to the examination object racks, a rack transport schedule for determining to which analysis module and in what order the examination object racks are to be transported on the basis of the request item is created, in step504. In step505, the examination object racks are transported to necessary analysis modules in order on the basis of the rack transport schedule. Examination objects of the examination object racks transported to the respective analysis modules are measured for necessary request items in the analysis modules. After the transport of the examination objects to all of the analysis modules required for measurement is terminated, the control unit confirms automatic reexamination information of the examination object racks in step506. In a case where the automatic reexamination is turned on, the control unit transports the rack to the rack standby unit through the transport line and causes the rack to standby in the rack standby unit until a first measurement result is output, in step507. In a case where the automatic reexamination information is turned off, the control unit transports the rack to the rack recovery unit without causing the rack to standby in the rack recovery unit, in step508. The rack transported to the rack recovery unit can be taken out from the apparatus by an operator. FIG.6is a diagram illustrating a flow of rack processing during reexamination measurement. When a measurement result corresponding to one examination object is output, a reexamination request is created on the basis of the measurement result in step602. In a case where rack automatic reexamination of the examination object is turned off in step603, the rack has been already recovered, and thus the processing is terminated (step604). In a case where the rack automatic reexamination is turned on, the control unit determines in step605whether all measurement results have been output (or whether measurement has been terminated) for the examination object mounted on the rack. In a case where all measurement results have not been output for the examination object of the rack, the rack continues standing by in the rack standby unit in step605. In a case where all measurement results have been output for the examination object of the rack, the control unit determines in step607whether or not there is a reexamination request for all examination objects of the rack. Here, the control unit confirms whether or not measurement results of respective items fall within a reference value, and determines that there is a reexamination request in a case where it is determined that even one item falls outside the reference value. In a case where there is no reexamination request, the rack is transported from the rack standby unit to the rack recovery unit in step610. In a case where there is a reexamination request, a rack transport schedule for determining to which analysis module and in what order the examination object racks are to be transported on the basis of the reexamination request item is created, in step608. In step609, the examination object racks are transported to necessary analysis modules in order on the basis of the rack transport schedule. The examination objects of the examination object racks transported to the respective analysis modules are measured for necessary request items in the analysis modules. FIG.7is a diagram illustrating an automatic reexamination state display screen which is displayed on the display unit. Reference numeral701denotes automatic reexamination mode display, and the control unit outputs the state of automatic reexamination to an area which is displayed at all times like the state of the apparatus and a clock display. The automatic reexamination mode display is set to be automatic reexamination ON display when an automatic reexamination ON rack is recognized, and is set to be automatic reexamination OFF display when an automatic reexamination OFF rack is recognized. Thereby, an operator can previously confirm an automatic reexamination mode of an examination object rack to be inserted next. Since this display is switched for every detection of an operation mode switching rack in a measurement state, it is possible to confirm the present automatic reexamination mode in real time. Meanwhile, in a configuration in which automatic reexamination ON or OFF can be set in a standby state as initial setting, the initial setting may be displayed as the state of automatic reexamination, regardless of whether or not the operation mode switching rack has been recognized. Reference numeral702indicates the state of validation or invalidation of automatic reexamination for each examination object mounted on a rack during measurement. The validation and invalidation states are displayed on the basis of rack automatic reexamination information. An examination object ID or a rack ID and the validation or invalidation of automatic reexamination are displayed in association with each other, and thus it is possible to confirm whether or not an examination object can be taken out immediately after the examination object or the rack is transported to the analysis module. That is, when the invalidation of automatic reexamination is displayed, the operator can recognize that the corresponding examination object or rack can be immediately taken out. As described so far, the operation mode switching rack can switch between a mode in which the examination object rack stands by in the examination object rack standby unit until a measurement result in the analysis module is output in a case where the automatic reexamination is valid and a mode in which the examination object rack is recovered to the examination object rack recovery unit without standing by in the examination object rack standby unit in a case where the automatic reexamination is invalid. As described above, the first embodiment has been described. The examination object rack is defined as an operation switching rack, and it is possible to notify the apparatus of an operation switching timing by the operation switching rack by using an ID reading unit of the related art. Accordingly, it is possible to perform switching between the validation and invalidation of an automatic reexamination mode in a measurement state without transitioning from the measurement state to a standby state as in the related art. In addition, in the apparatus having a configuration in which a rack ID is set for each type of examination object, it is not necessary to perform range setting for a rack ID by multiplying the types of examination objects and the validation or invalidation of automatic reexamination together, which does not result in the operator's erroneous arrangement of the examination objects which is caused by the complication of range setting of a rack ID. In addition, operation is dynamically switched by the operation switching rack, but the present operation mode is displayed on the display unit. Thus, the operator can operate an operation mode with respect to a desired examination object rack without fail. Example 2 A second embodiment will be described. An operation mode in this embodiment is a data abnormal value check. Here, the data abnormal value check refers to a check regarding whether or not a measurement result is an abnormal value during the output of the measurement result in an analysis module. For example, the data abnormal value check is not performed on an examination object, such as a dialysis patient examination object, which has a data abnormal value at all times, and thus it is possible to prevent wasteful automatic reexamination measurement and to shorten the time and prevent a wasteful use of the examination object. For this reason, it may be more useful in a case where a data abnormality check is not performed depending on a rack. An embodiment in such a case will be described. FIGS.8to10relate to an embodiment in which an operation mode switching rack is used for switching between the execution and non-execution of a data abnormal value check. FIG.8illustrates main portions of a screen for setting a rack ID of a switching rack of a data abnormal value check. The setting of the automatic reexamination mode inFIG.2being replaced with that inFIG.8is displayed as a rack setting screen. A data abnormal value check ON rack801is an area in which a data abnormal value check-validated rack ID is defined, and a data abnormal value check OFF rack802is an area in which an automatic reexamination-invalidated rack ID is defined. A maximum of four racks can be set for each of the racks801and802. An operator performs setting so that the rack IDs do not overlap each other. A rack ID which is set by the operator's pressing-down of a registration button204is stored in a storage unit of an overall management computer9. In a case where a measurement result is the execution of a data abnormal value check, racks of examination objects desired to be measured in a case of the execution of the data abnormal value check are inserted side by side into the apparatus in order behind the data abnormal value check ON rack. In a case where a measurement result is the non-execution of a data abnormal value check, racks of examination objects to be measured in a case of the non-execution of the data abnormal value check are inserted side by side into the apparatus in order behind the data abnormal value check OFF rack. FIG.9is a diagram illustrating a flow of rack ID identification processing of a switching rack of a data abnormal value check. In step811, an ID reading unit2reads a rack ID which is transported. In step812, a control unit determines the type of rack from the read rack ID and rack ID information which is set in the rack setting screen ofFIG.8. When the rack ID indicates a data abnormal value check ON rack, the control unit sets an abnormal value check mode to be in an on-state in step813. Thereafter, in step815, the rack is transported to a rack recovery unit. When the rack ID indicates a data abnormal value check OFF rack, the control unit sets the abnormal value check mode to be in an off-state in step814. Thereafter, in step815, the rack is transported to the rack recovery unit. Meanwhile, the rack does not have an examination object container, which is a measurement object, mounted thereon, and thus is transported to the rack recovery unit without stopping by in any of analysis modules5,6, and7. When the rack ID is a rack for a general examination object or a rack for an urgent examination object in step812, the control unit determines the data abnormal value check mode in units of recognized examination object racks in step816. In a case where the data abnormal value check mode is turned on, the control unit causes the storage unit to store a fact that a rack abnormal value check of a measurement rack is valid in rack measurement management information for managing the measurement rack, in step817. In this example, the storage may be performed in units of racks, or the validation or invalidation of an abnormal value check may be stored on the basis of examination object measurement information managed in units of examination objects installed in the rack. In a case where the data abnormal value check mode is turned off, the control unit causes the storage unit to store a fact that the rack abnormal value check is invalid, in step818. FIG.10is a diagram illustrating a flow of data abnormal value check processing during the output of a measurement result. When a measurement result for one item of an examination object of a rack is output (step851), it is determined in step852whether or not a rack abnormal value check is valid or invalid. When the rack abnormal value check is invalid, the control unit does not perform the data abnormal value check. That is, even when a measurement result for a predetermined item has a value falling outside a reference value, the measurement result is output without displaying an alarm indicating the abnormality of data. When the rack abnormal value check is valid, the control unit performs the data abnormal value check in step853. In a case where the output measurement result is abnormality (falling outside the reference value) in the data abnormal value check, a data alarm indicating data abnormality is added to the measurement result. In step854, the measurement result is registered, and a reexamination request is registered when an automatic reexamination mode is valid. This is because the reexamination request is registered on the basis of the data alarm indicating data abnormality in a case where the automatic reexamination mode is valid. Meanwhile, when the rack abnormal value check is invalid, a data alarm indicating data abnormality is not added, and thus a reexamination request is not registered. This is because the reexamination request is registered on the basis of the data alarm. When the reexamination request is registered for one or more items among the examination objects of the examination object racks after measurement for all of the examination object racks is completed, automatic reexamination measurement is performed. Any reexamination request is not registered, automatic reexamination is not performed, and the racks are recovered. As described so far, the operation mode switching rack can switch between a mode in which a data alarm is added to a measurement result in an analysis module in a case where a data abnormal value check is valid and the measurement result is abnormal and a mode in which a data alarm is not added to the measurement result in the analysis module in a case where the data abnormality check is invalid and the measurement result is abnormal. As described above, the second embodiment has been described. The examination object rack is defined as an operation switching rack, and it is possible to notify the apparatus of an operation switching timing by the operation switching rack by using an ID reading unit of the related art. Accordingly, it is possible to perform switching between the validation and invalidation of a data abnormal value check in a measurement state. Thereby, the data abnormal value check is invalidated even when an automatic reexamination mode is a valid operation mode, it is possible to prevent wasteful automatic reinspection and to shorten the time and prevent a wasteful use of the examination object with respect to an examination object, such as a dialysis patient examination object, which has a data abnormal value at all times. The same effects as those in the first embodiment are obtained in terms of preventing automatic reexamination. However, in a case of the second embodiment, a data alarm is not added, and thus it is possible to prevent an operator from performing reexamination and the like due to erroneous manual reinsertion into the apparatus in response to an alarm. Meanwhile, since a description is repeated, and thus the description will be omitted. However, also in this embodiment, it is also preferable to apply the display of validation or invalidation of the present data abnormal value check in real time and the display of association between an examination object ID or a rack ID and the validation or invalidation of a data abnormal value check, as inFIG.7. Example 3 A third embodiment will be described. An operation mode in this embodiment is a mode in which an apparatus performs inquiry about a measurement request item together with a high order host. For example, in a case where an examination object desired to be measured in accordance with a request item, defined in advance within an apparatus, and an examination object desired to be measured in accordance with a request item received from a high order host through a network are mixedly present, switching between the execution and non-execution of inquiry of a host request may be performed in accordance with an examination object rack. In this case, it is possible to eliminate a wasteful host inquiry or a processing time of a response received from a host with respect to the examination object to be measured in accordance with the request item defined in advance within the apparatus, and to improve a throughput of the measurement. In a case where the host does not include the corresponding examination object information with respect to a request inquiry by the apparatus, the host may not return a response to the apparatus, and the apparatus waits for measurement until the time-out of reception. Effectiveness is exhibited in such a case. FIGS.11to13relate to an embodiment in which an operation mode switching rack is used for switching between the execution and non-execution of high order host request inquiry. An overall management computer9is connected to a high order host system (hereinafter, also simply referred to as a host) through a network, and has a system configuration in which a request item is inquired from the high order host system about an examination object recognized by the apparatus, and the apparatus receives a measurement request item from the high order host system and performs measurement. FIG.11illustrates main portions of a screen for setting a rack ID of a switching rack of a host request inquiry. The setting of the automatic reexamination mode inFIG.2being replaced with that inFIG.11is displayed as a rack setting screen. A host request inquiry ON rack901is an area in which a host request inquiry-validated rack ID is defined, and a host request inquiry OFF rack902is an area in which a host request inquiry-invalidated rack ID is defined. A maximum of four racks can be set for each of the racks901and902. An operator performs setting so that the rack IDs do not overlap each other. A rack ID which is set by the operator's pressing-down of a registration button204is stored in a storage unit of the overall management computer9. In a case of examination objects desired to be measured in accordance with a request item designated by a host, racks of the examination objects desired to be measured in accordance with the request item designated by the host are inserted side by side into the apparatus in order behind a host inquiry ON rack. In a case of examination objects desired to be measured in accordance with a request item registered within the apparatus rather than a request received from the host, racks of the examination objects desired to be measured in accordance with the request item registered within the apparatus are inserted side by side into the apparatus in order behind a host inquiry OFF rack. FIG.12is a diagram illustrating a flow of rack ID identification processing of a switching rack of a host request inquiry. In step911, an ID reading unit2reads a rack ID which is transported. In step912, a control unit determines the type of rack from the read rack ID and rack ID information which is set in the rack setting screen ofFIG.11. When the rack ID indicates a host request inquiry ON rack, the control unit sets a host inquiry mode to be in an on-state in step913. Thereafter, in step915, the rack is transported to a rack recovery unit. When the rack ID indicates a host request inquiry OFF rack, the control unit sets the host inquiry mode to be in an off-state in step914. Thereafter, in step915, the rack is transported to the rack recovery unit. Meanwhile, the rack does not have an examination object container, which is a measurement object, mounted thereon, and thus is transported to the rack recovery unit without stopping by in any of analysis modules5,6, and7. When the rack ID is a rack for a general examination object or a rack for an urgent examination object in step912, the control unit determines the host inquiry mode in units of recognized examination object racks in step916. In a case where the host inquiry mode is turned on, the control unit stores a fact that a host inquiry of a measurement rack is valid (executed) in rack measurement management information for managing the measurement rack, in step917. In this example, the storage may be performed in units of racks, or the validation or invalidation of a host inquiry may be stored on the basis of examination object measurement information managed in units of examination objects installed in the rack. In a case where the host inquiry mode is turned off, the storage unit stores a fact that the host inquiry is invalid, in step918. FIG.13is a diagram illustrating a flow of host request inquiry processing in units of racks. In a case where an inserted rack is a general rack or an urgent rack (step951), a process of applying a request item to the number of examination objects mounted on the rack is performed (step952). In a case where host inquiry information of the rack is invalid in step953, a measurement request item is not inquired from a host. That is, measurement is executed in accordance with a request item registered within the apparatus. In a case where the rack host inquiry of the rack is valid, a request for the examination objects is inquired from the host in step954, and the request item received from the host is applied in step955, thereby performing measurement in accordance with the request item. Request information registered in advance through the screen or the like of the apparatus is applied to the examination objects in step956. At this time, in a case where the request item from the host is applied in step954, both the pieces of request information are applied. A flow thereof is performed on all of the examination objects mounted on the rack (step958). Thereafter, a rack transport schedule is created on the basis of the applied request information, and is transported to a target analysis module. In addition, also in a reexamination creating process (step602) after a first measurement result is output, a reexamination request is created through the request item application process ofFIG.13. For example, when there is a measurement request item added in response to a request inquiry from the host, a reexamination request is created on the basis of the added measurement request item also in the reexamination request. As described so far, the high order host system connected to the apparatus through a network is further included, the automatic analyzer inquires a measurement request item corresponding to an examination object container from the high order host system, and the corresponding measurement request item is received from the high order host system, whereby the measurement request item is measured in the analysis module. An operation mode is a mode regarding whether or not the measurement request item is inquired from the high order host system, and an operation mode switching rack is a rack that switches between the execution and non-execution of inquiry of the measurement request item from the high order host system. In addition, the operation mode switching rack can switch between a mode in which the inquiry from the high order host system is performed and a mode in which a measurement request item registered in the automatic analyzer is measured by the analysis module without performing inquiry from the high order host system. As described above, the third embodiment has been described. The examination object rack is defined as an operation switching rack, and it is possible to notify the apparatus of an operation switching timing by the operation switching rack by using an ID reading unit of the related art. Accordingly, it is possible to perform switching between the validation and invalidation of a request inquiry of a measurement item from the host in a measurement state. Thereby, in a case where a request item defined in advance within the apparatus and an examination object desired to be measured in accordance with a request item received from a high order host through a network are mixedly present, it is possible to perform operation by switching between the execution and non-execution of inquiry of a host request in accordance with an examination object rack and to give a request for a measurement item corresponding to conditions in a measurement state. As described above, the first to third embodiments have been described. As described in the embodiments, the invention includes a detection unit that detects an operation mode switching rack inserted into an insertion unit, and a control unit that switches an operation mode stored in a storage unit on the basis of the detection of the operation mode switching rack by the detection unit. The control unit applies the switched operation mode to an examination object rack transported from the insertion unit to a transport line after the operation mode switching rack. In the embodiments, an ID reading unit has been described as an example of the detection unit. However, the detection unit is not limited to the ID reading unit as long as the apparatus can recognize the operation mode switching rack. For example, the shape or color of an operation mode switching rack is made different from that of a normal examination object rack, and a shape or color sensor that detects the operation mode switching rack can also be used. In a case of a shape sensor, for example, the height of the operation mode switching rack is made larger or smaller than the height of the normal examination object rack, and it is possible to cause the apparatus to recognize the operation mode switching rack in accordance with the height. An example of the sensor is a height sensor including a plurality of light sources and a light receiving unit in a vertical direction. A distinction between the turn-on and turn-off of the operation mode switching rack can be made in accordance with two-stage heights. In addition, in a case of a color sensor, the color of the operation mode switching rack is made different from the color of the normal examination object rack, and it is possible to cause the apparatus to recognize the operation mode switching rack in accordance with the color. A distinction between the turn-on and turn-off of the operation mode switching rack can be made in accordance with two colors. In a case where such a shape or color sensor is used, it is possible to perform the same control as that of the ID reading unit as long as an area for setting height or color in a rack setting screen is provided. In addition, the detection unit reads an operation mode switching ID attached to a container mounted on an examination object rack instead of a rack ID attached to the rack, and thus the control unit may switch an operation mode stored in the storage unit. The operation mode switching ID is an identifier such as a barcode having unique numerals for switching an operation mode stored therein or RFID. In this case, the operation mode switching rack is an examination object rack on which a container having an operation mode switching ID attached thereto is mounted, and the detection unit detecting the operation mode switching rack can be considered to be a detection unit that detects the operation mode switching ID. That is, in this specification, a case where the operation mode switching rack is detected also includes a case where the ID of the container mounted on the rack is detected, in addition to a case where an actual rack is detected. However, it is useful in that the switching of an operation mode can be performed using a simple method without newly providing the shape or color sensor by using a rack ID. In addition, in the embodiments, a description has been given on the assumption that the operation mode switching rack is an operation mode-validated or invalided rack. However, the invention is not limited to a choice between validation and invalidation. For example, in a case of reexamination, the operation mode switching rack may be a rack that switches between a mode in which the reexamination is performed in the same analysis module as the first analysis module and a mode in which reexamination is performed in a different analysis module. In addition, it is preferable that the control unit switches the display of an operation mode displayed on a display unit on the basis of the detection of the operation mode switching rack by the detection unit. This is because the operator can perform operation while confirming the present operation mode. In addition, it is preferable that the control unit causes the storage unit to store the switched operation mode for each examination object rack or each examination object container mounted on the examination object rack with respect to the examination object rack transported after the operation mode switching rack. Thereby, it is possible to display an operation mode applied for each examination object rack or each examination object container. In addition, a description has been given of three types of operation modes of an automatic reexamination mode, a mode in which it is checked whether or not a measurement result has an abnormal value, and a mode regarding whether or not a measurement request item is inquired from a high order host system in a case where the high order host system is present, but is not limited thereto. Various applications can be made. In addition, in the embodiments, a description has been given of an example in which a distinction between a rack for a general examination object and a rack for an urgent examination object is made for the operation mode switching rack. However, two types of an operation mode switching rack for the rack for a general examination object and an operation mode switching rack for the rack for an urgent examination object may be used. In addition, in the embodiments, an ON rack and an OFF rack have been described. However, a switching rack that reverses a mode from ON to OFF and from OFF to ON may be registered in the apparatus. In addition, in the embodiments, the automatic analyzer including the plurality of analysis modules has been described as an example. However, the invention can also be applied to an automatic analyzer including a single analysis module. In addition, a description has been given of an example in which the analysis module is a biochemical automatic analyzer. However, the invention can also be applied in a case where the analysis module is an immunity automatic analyzer or a coagulation automatic analyzer. In addition, a description has been given of an example of a rack on which a plurality of examination object containers can be mounted. However, the invention can also be applied to a rack on which one examination object container is mounted. In addition, the invention described in claims is not limited to the embodiments, and includes various embodiments without departing from the scope of the invention. REFERENCE SIGNS LIST 1: EXAMINATION OBJECT RACK INSERTION UNIT (RACK INSERTION UNIT)2: ID READING UNIT (DETECTION UNIT)3: TRANSPORT LINE4: EXAMINATION OBJECT RACK STANDBY UNIT (RACK STANDBY UNIT)5,6,7: ANALYSIS MODULE8: EXAMINATION OBJECT RACK RECOVERY UNIT (RACK RECOVERY UNIT)9: OVERALL MANAGEMENT COMPUTER10: OPERATION UNIT11: DISPLAY UNIT | 47,227 |
11860178 | DETAILED DESCRIPTION A possible exemplary embodiment of a system according to the invention and also a method according to the invention for automatically closing sample vessels are explained hereafter on the basis of the figures. The system according to the invention is implemented in this case in a closing device, of which those details are illustrated in the figures which are important for the system according to the invention and its operation. Firstly, a closure cap10is shown inFIG.1, which is part of the system according to the invention. The closure cap10is designed as stackable having a convex outer side11and a concave inner side12. At a rear end13, the closure cap10is formed open, i.e., a further identically formed closure cap10can be set here with its convex outer side11so that it bears on the concave inner side12of the closure cap10shown. The closure cap10is closed by a base at a front end14. The closure cap10consists of a thin-walled material. This can be in particular a film material, such as a plastic film especially, wherein the closure cap10is then formed in particular by forming from a corresponding flat film section, for example, by deep-drawing. The diameter of the closure cap10tapers from the rear end13up to the front end14, wherein this takes place in steps here in three rough first steps15,16, and17, which are each in turn divided into smaller fine steps (no longer identified by reference signs in the figure, but still recognizable). In this case, the step located at the front end14, which is smallest in diameter, is used for centering the closure cap10during the insertion into an opening to be closed of a sample vessel. A peripheral collar18extends around the opening formed toward the inner side12there at the rear end13. FIG.2shows how a closure cap10according toFIG.1is seated in an opening O of a sample vessel PG, which is tubular here, and seals closed this opening O. The closure cap10is particularly suitable for sealing closed this opening O because it has a measure of flexibility due to its low wall thickness and can be inserted under tension into the opening O so that it deforms somewhat and thus exerts a pressure on the inner wall of the sample vessel PG. The stepped design of the outer side11of the closure cap10also assists for this purpose. Moreover, the stepped design of the outer side11and/or the diameter of the closure cap10has the result that sample vessels PG having different opening diameters can be closed using the same closure cap10. The sample vessel PG shown inFIG.2can be in particular a sample tube for medical laboratory samples, for example, for blood samples or the like. A further essential element of the system according to the invention is shown inFIG.3, namely a closure gripper20. This is arranged on a gripper arm21, which is linearly movable in the vertical and also horizontal directions, as will be explained in greater detail hereafter. The closure gripper20has, as may already be seen fromFIG.3, two diametrically opposing clamping jaws22, which are located in a release position in the position shown inFIG.3. The construction of the closure gripper20may be seen more accurately inFIG.4. This element is shown in a longitudinal section therein. It can be seen here that a centering piece23is arranged in the center of the closure gripper20, which is delimited at an end shown on top in the figure with a peripheral edge. It can also be seen here that the clamping jaws22are each rotatably mounted on an axis24and are fixed with an end25located beyond the axis24in a slot formed in a traction piece26. The traction piece26is arranged on a traction rod27and fixed thereon. In the position shown inFIG.4, the closure gripper20is plunged with the centering piece23into an uppermost closure cap10of a closure cap stack19, in which the identically shaped closure caps10are stacked one inside another in the manner according to the invention, and bears with a circumference on the inner side of this closure cap10. In this case, the peripheral collar18(not identified with a reference sign in the figure) abuts the peripheral edge at the edge of the centering piece23so that a stop is formed here. In this position, the traction piece26can be moved away from the centering piece23by moving the traction rod27, whereby the clamping jaws22are rotated and are pressed against the centering piece23, more precisely against the outer side of the closure cap10located on the centering piece23. The closure cap10is thus held and clamped. It is already shown here inFIG.5how the closure cap10can be lifted off of the closure cap stack19with the aid of a movement of the gripper arm21. An arrangement located in the closing device below a working position of the closure gripper20can be seen inFIG.6. A magazine30, which is formed like a drum magazine, can firstly be seen here. The magazine30contains, arranged distributed along its circumference around a center axis, for example, six or eight shafts31, in which closure cap stacks19can be accommodated. The magazine30is seated in this case on a spike32, which protrudes into a central opening of the magazine. The spike32has a protruding driver pin33, which is guided transversely through it, and which has a formfitting engagement with the magazine, so that the magazine30can be rotated by rotating the spike32. This takes place automatically and driven by a motor in operation of the closing device. The shafts31are open toward the outer side of the magazine30in a continuous slot. A lifting piece34can enter the respective shaft31through this slot and grasp a closure cap stack19at the base of the closure cap arranged lowermost. Via a toothed rack35and one or more gear wheels cooperating therewith and a drive driving them (not shown in greater detail), the lifting piece34can be moved in the vertical direction to thus transfer a closure cap stack19, as shown here inFIG.6, into a vertically arranged supply tube40. If a shaft31of the magazine30is completely emptied in this manner, the spike32is thus rotated until a further shaft31filled with a further closure cap stack19aligns with the supply tube40, so that a further closure cap stack19can be transferred using the lifting piece34into the supply tube40. A lifting piece guide36can also be seen inFIG.6. The magazine30is raised using this. When the lifting piece34begins, driven by the motorized drive, to move upward from a lowermost position, the lifting piece guide36, on which the lifting piece34bears in the lowermost position and locks it in a first position, is disengaged. The magazine30is then lifted by a spring (not shown in greater detail here) on the spike32by a travel distance of approximately 10 mm here and locked in a centering unit37(seeFIG.7). In this manner, a play in the and/or tolerances between the magazine(s)30can be compensated for. If the lifting piece34, for example, when a shaft31of the magazine30is completely empty, travels back into its lowermost position, it thus presses against the lifting piece guide36and the magazine30is pressed back downward against the force of the spring again and released from the locking with the centering unit37. In this case, a pressure spring plate38(seeFIG.7) assists, which also presses the magazine30downward into an unlocked position. A new shaft31having further closure caps10can then be rotated into position, for example, by rotation of the magazine30(a rotation of the spike32and thus the magazine30is only possible if the lifting piece34is located in its lowermost position and the centering unit37is thus released from the magazine30and unlocks it). A sample vessel PG, which has an opening still to be closed on an upper side, supplied to the closing device in a sample carriage PW, which can move independently on a path B, can also be seen inFIG.6. Furthermore, holding arms50can be seen, the function of which will be described in greater detail hereafter. FIG.7shows a similar situation asFIG.6once again, wherein a magazine30filled with closure cap stacks19in multiple shafts31is shown here, and also a closure cap stack19, which is arranged in the supply tube40. The lifting piece34, which is now located in a lowermost position and bears on the lifting piece guide36, and the toothed rack35can also be seen. FIG.8shows an enlarged illustration of a lower portion of the supply tube40having a threading opening for the closure cap stack19. A lowermost closure cap10in the closure cap stack19can be seen, which is held back in the supply tube40by two diagonally arranged spring plates41used for a first isolation of the closure caps10at the lower end of the supply tube40, so that a first isolation already takes place here. The arrangement of the spring plates41extending diagonally in relation to the longitudinal direction of the supply tube40is used for a better supply of the closure caps10. An upper portion of the supply tube40is also shown enlarged once again, in this case inFIG.9. It can be seen therein that flexible tabs42are fastened on one side using screws43and are placed extending diagonally in relation to the vertical and with the ends facing toward one another extending upward. These tabs42ensure an isolation of the uppermost closure cap10in the closure cap stack19. This is because the flexible tabs42press on the peripheral collar of the second-uppermost closure cap in the closure cap stack19and thus hold it back. The uppermost closure cap10is already detached to a certain extent from the closure cap stack19in this case, so that later it can be grasped more easily by the closure gripper and lifted off of the closure cap stack19. A detail of the closing device is shown once again enlarged inFIG.10, which contains a magazine store, in which multiple magazines30, four such magazines shown here, are arranged. The magazines stored therein each have multiple shafts31having closure cap stacks19arranged therein. If a magazine30having closure cap stacks19is completely emptied, a new magazine30can thus be supplied and the closure cap stacks19can be transported out of this magazine and the closure caps stacked therein can be processed. The magazine30can be formed in particular from a light material, for example, Styropore. A transfer of the magazines30from the magazine store into a working position, in which closure cap stacks19can be transferred from the magazine30into the supply tube40, can take place manually or also automatically in this case. Finally,FIG.11atoeschematically show a sequence for closing a sample vessel PG. At the beginning, as shown inFIG.11a, a sample vessel PG is supplied and moved into a working position. This takes place here by way of a self-propelled sample carriage PW, which independently transports the sample vessels PG in a path B. Holding arms50, which engage with holding jaws51on the sample vessel PG and fix it in its position, are provided for fixing the sample vessel PG. These holding arms50are already moved into a holding position inFIG.11a, so that they secure the sample vessels PG. The gripper arm21having the closure gripper20arranged thereon is lowered into a position in which an uppermost closure cap10of a closure cap stack located in the supply tube40can be grasped. The closure gripper20is introduced from the rear end into the closure cap10with the centering piece and engages on the inner side thereof on the wall thereof. The closure cap10is thus centered and also correctly positioned by the stops of the peripheral collar on the edge of the centering piece. In the position shown inFIG.11a, the clamping jaws22are still open. They are now closed, and the closure cap10is lifted off of the closure cap stack by moving the gripper arm21upward in the vertical direction. As shown inFIG.11b, a transportation also takes place in the horizontal direction at the same time, to transfer the closure cap10grasped using the closure gripper20to the sample vessel PG to be closed. The sample vessel PG is still fixed by the holding arms50. It is now shown inFIG.11chow the closure gripper20having the closure cap10fixed thereon is pressed against the sample vessel PG by lowering the gripper arm21to introduce the closure cap10into the opening to be closed. The clamping jaws22still also hold the closure cap10, which is now pressed and inserted in this way into the opening of the sample vessel PG to form a seal. For this purpose, pressure sensors and/or force sensors on the closure gripper20and/or on the gripper arm21control the movement distance or the drive force, respectively, during the insertion of the closure cap10into the opening of the sample vessel PG. If the closure cap10is correctly positioned in the opening and seals it closed, the clamping jaws22are opened by lowering the traction rod and the closure gripper20is released from the closure cap10by raising the gripper arm21, in particular the centering piece is freed from the opening at the rear end of the closure cap10. The sample vessel PG is now sealed closed and can be further processed. The holding arms50are now pivoted back accordingly, so that the holding jaws51release the sample vessel PG, which can now be transported further using the sample carriage PW, in particular to a sample archive. While a new sample vessel PG having opening O still to be closed can already be moved using a further sample carriage PW into the working position, as shown inFIG.11e, the gripper arm21having the closure gripper20travels back to the vertical upper end of the supply tube40, to accept a further closure cap10there, which now forms the uppermost closure cap of the closure cap stack arranged in the supply tube40, wherein the procedure for closing this further sample vessel PG begins again, as shown inFIG.11a. It is clear once again from the above description what a great advantage the system according to the invention and also the method according to the invention involve for the automatic closing of sample vessels. In addition to automatic handling and the option of securely closing sample vessels PG of different constructions, in particular having different opening diameters, the large quantity of uniform, stackable closure caps10is to be mentioned here, which can be stockpiled and processed in the system. A large number of sample vessels PG can thus be closed automatically, before a manual engagement is necessary, for example, for re-equipping with closure caps. In this case, the preceding exemplary embodiment is not to be understood as restricting the system according to the invention to this embodiment, but rather is merely used for explanation. A system according to the invention in its general form is defined in the following claims, as is a method according to the invention. LIST OF REFERENCE SIGNS 10closure cap11outer side12inner side13rear end14front end15step16step17step18collar19closure cap stack20closure gripper21gripper arm22clamping jaw23centering piece24axis25end26traction part27traction rod30magazine31shaft32spike33driver pin34lifting piece35toothed rack36lifting piece guide37centering unit38pressure spring plate40supply tube41spring plate42flexible tab43screw50holding arm514holding jawB pathO openingPG sample vesselPW sample carriage | 15,245 |
11860179 | DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments will be described with reference to the accompanying drawings. In the accompanying drawings, elements having the same functions may be represented by the same numbers or corresponding numbers. Although the accompanying drawings show embodiments and implementation examples based on the principle of the present disclosure, these are to understand the present disclosure and are never used to restrictively interpret the present disclosure. The description of the present specification is only a typical example and does not limit in any sense the claims or application examples of the present disclosure. The embodiments are sufficiently described in detail so that a person skilled in the art can carry out the present disclosure. However, other implementations and forms are possible, and it is necessary to understand that change of configurations/structures and replacement of various elements are possible without departing from the scope and spirit of the technical idea of the present disclosure. Therefore, the description below shall not be construed to be limited to the embodiments. First Embodiment FIG.1is an overall schematic diagram of an automatic analysis apparatus100according to a first embodiment. The automatic analysis apparatus100includes a control unit102, an input unit103, and a display unit104. The control unit102is a computer in charge of the entire operation of the automatic analysis apparatus100including a specimen dispensing mechanism107described later. The input unit103is an input apparatus, for example, a keyboard and a mouse, for inputting various operation instructions and various data. The display unit104is, for example, a liquid crystal display, a printer, or the like and is a device that can display various analysis results, operating states of apparatuses, presence or absence of occurrence of abnormality, and the like. The automatic analysis apparatus100further includes a specimen vessel105, a specimen vessel rack106, the specimen dispensing mechanism107, a reagent dispensing mechanism108, a reagent vessel109, a reaction vessel and dispensing tip accommodating unit110, and an incubator111. The specimen vessel105is a vessel that stores a specimen that is an object to be inspected. The specimen vessel rack106is a holding unit for holding the specimen vessel105. The specimen vessel rack106is transported to a suction unit106S described later by a transport mechanism not shown in the drawings. The specimen dispensing mechanism107is configured to be capable of holding a dispensing tip CP and sucking the specimen from the specimen vessel105and is also configured to be capable of discharging the sucked specimen to a reaction vessel RC. The reagent dispensing mechanism108is configured to be capable of sucking a reagent from the reagent vessel109and discharging the sucked reagent to the reaction vessel RC. As shown in the lower right ofFIG.1separated from the overall schematic diagram, the specimen dispensing mechanism107includes an arm1071that can rotate around a rotation shaft1070and a dispensing nozzle1072which is attached to a tip of the arm1071and configured to be capable of holding the dispensing tip CP. Although not shown in the drawings, the reagent dispensing mechanism108also includes an arm that can rotate around a rotation shaft and a nozzle attached to a tip of the arm. The reaction vessel and dispensing tip accommodating unit110is an accommodating unit that accommodates the reaction vessels RC and the dispensing tips CP. The reaction vessel RC is appropriately transported to the incubator111by a transport mechanism not shown in the drawings. The dispensing tip CP is appropriately transported to mounting unit114described later by a transport mechanism not shown in the drawings and thereafter attached to the specimen dispensing mechanism107in the mounting unit114. The incubator111has a temperature control mechanism and has a role to promote a reaction between the specimen and the reagent in the reaction vessel RC. The automatic analysis apparatus100further includes a sensor112, a disposal unit113, and a mounting unit114. The sensor112is a detector for detecting whether or not the dispensing tip CP is attached to (removed from) the dispensing nozzle1072of the specimen dispensing mechanism107as described later. Although a method of the sensor112is not limited to a specific method, as an example, an optical sensor can be used as the sensor112. However, the sensor112is not limited to an optical sensor, and a detection principle and the like of the sensor112do not matter as long as the sensor112can detect the presence of the dispensing tip CP. As an example, a transmission type photosensor or a reflection type photosensor can be employed as the optical sensor. The disposal unit113is a unit that removes a used dispensing tip CP from the specimen dispensing mechanism107and discards the dispensing tip CP after a specimen is injected into the reaction vessel RC. The mounting unit114is a mechanism for mounting an unused dispensing tip CP on the dispensing nozzle1072of the specimen dispensing mechanism107. The mounting unit114can have a system where the dispensing tip CP is pressed against the dispensing nozzle1072and the dispensing tip CP is crimped to the dispensing nozzle1072. However, the system does not matter as long as the dispensing tip CP is attached to the dispensing nozzle1072. Next, a positional relationship between a movement path Rm of the specimen dispensing mechanism107and other constituent elements will be described with reference toFIG.2. As described above, the specimen dispensing mechanism107includes the arm1071that can rotate around the rotation shaft1070and the dispensing nozzle1072that is attached to the arm1071. The specimen dispensing mechanism107is configured to be capable of being moved along the arc-like movement path Rm by the rotation of the arm1071. The movement path Rm is not limited to a curved line and may be a straight line. The curved line is not limited to an arc curve and may be, for example, an elliptic arc curve, a cycloidal curve, an asteroid curve, a spiral curve, or the like. The suction unit106S in the specimen vessel rack106, the disposal unit113, a discharging unit111P in the incubator111, and the mounting unit114are arranged along the movement path Rm. The suction unit106S, the disposal unit113, a discharging unit111P, and the mounting unit114are arranged along the movement path Rm, and thereby attachment, suction, discharge, and disposal operations of the dispensing tip CP are performed by the movement of the specimen dispensing mechanism107along the movement path Rm. A certain degree of freedom is allowed for the arrangement of the suction unit106S, the disposal unit113, a discharging unit111P, the mounting unit114, and the sensor112as described later. However, the sensor112is arranged so that the optical sensor112can detect the dispensing tip CP in a position between any two of the mounting unit114, the suction unit106S, the discharging unit111P, and the disposal unit113in the movement path Rm. In other words, the detection position of the optical sensor112located in a position farther than an end portion (inside) of the movement path Rm when viewed from at least one of the mounting unit114, the suction unit106S, the discharging unit111P, and the disposal unit113. By employing such an arrangement, it is possible to eliminate useless movement of the specimen dispensing mechanism107, and as a result, the throughput of the automatic analysis apparatus can be improved. In the example inFIG.2, the detection position of the optical sensor112is arranged in the center of the movement path Rm, the discharging unit111P and the mounting unit114are arranged on one side of the movement path Rm, and the disposal unit113and the suction unit106S are arranged on the other side of the movement path Rm. In other words, the sensor112is arranged to be capable of detecting the dispensing tip CP in a position between the disposal unit113and the discharging unit111P. This arrangement is particularly preferable from the viewpoint of minimizing useless movement of the specimen dispensing mechanism107. As shown in the flowchart inFIG.3, a specimen dispensing operation is performed in the order described below.(A) Mounting of the dispensing tip CP in the mounting unit114(step S1)(B) Suction of specimen in the suction unit106S (step S3)(C) Discharge of specimen in the discharging unit111P (step S5)(D) Disposal of specimen in the disposal unit113(step S7) In each of the operations of steps S1, S3, S5, and S7, the sensor112detects (checks) the presence or absence of the dispensing tip CP on the dispensing nozzle1072(steps S2, S4, S6, and S8). In the arrangement ofFIG.2, when viewed from the detection position of the sensor112, the mounting unit114and the discharging unit111P are arranged on one side of the movement path Rm and the suction unit106S and the disposal unit113are arranged on the other side of the movement path Rm. In the case of this arrangement, in any case of a movement from (A) to (B), a movement from (B) to (C), and a movement from (C) to (D), it is possible to pass through the detection position of the sensor112(without detouring the detection position of the sensor112) during the movement. Therefore, it is possible to minimize the movement of the specimen dispensing mechanism107for detecting the presence or absence of the dispensing tip CP, and the throughput can be improved accordingly. Further, in the example inFIG.2, the mounting unit114is arranged closer to (on a side closer to) the end portion of the movement path Rm than the suction unit106S, the discharging unit111P, and the disposal unit113. As described above, the mounting unit114is a unit where an unused dispensing tip CP is arranged. The mounting unit114is located close to the end portion of the movement path Rm, so that in the operations of the above (A) to (D), the (used) dispensing tip CP that has sucked a specimen does not pass through above the mounting unit114. Therefore, it is possible to avoid contamination of the unused dispensing tip CP arranged in the mounting unit114. An initial position of the specimen dispensing mechanism107(the dispensing nozzle1072) may be an arbitrary position. However, a position between the sensor112and the suction unit106S is suitable for the initial position. Alternatively, a standby position where the specimen dispensing mechanism107(the dispensing nozzle1072) stands by during a time other than a time of the specimen dispensing operation can be a position between the sensor112and the suction unit106S in the same manner as the initial position. By doing so, it is possible to detect the presence or absence of mounting of the dispensing tip CP on the dispensing nozzle1072before the dispensing nozzle1072moves from the initial position to the mounting unit114and the dispensing tip CP is mounted on the dispensing nozzle1072. Next, the specimen dispensing operation in the automatic analysis apparatus inFIG.2according to the flowchart inFIG.3will be described in detail. When a specimen dispensing instruction is inputted from the input unit103or the like through the control unit102, by a control signal from the control unit102, the dispensing nozzle1072of the specimen dispensing mechanism107starts moving from the initial position in a direction of an arrow A along the movement path Rm. The dispensing nozzle1072of the specimen dispensing mechanism107reaches the mounting unit114and is mounted with the dispensing tip CP (step S1). Thereafter, the dispensing nozzle1072moves in the opposite direction (direction of an arrow B) on the movement path Rm, passes through the discharging unit111P, the detection position of the sensor112, and the disposal unit113, reaches the suction unit106S, and sucks a specimen from the specimen vessel105in the suction unit106S (step S3). During a transitional period from step S1to step S3, when the dispensing nozzle1072passes through the detection position of the sensor112, the sensor112detects the presence or absence of mounting of the dispensing tip CP at the tip of the dispensing nozzle1072(step S2). When the mounting of the dispensing tip CP is detected, it is determined that the dispensing operation is normally performed, and the dispensing operation is continued. When the dispensing tip CP is not detected, it is determined that an abnormality occurs in the dispensing operation (the mounting of the dispensing tip CP fails), the operation of the specimen dispensing mechanism107is stopped, and an abnormality notification is displayed on the display unit104. After sucking the specimen in the suction unit106S, the specimen dispensing mechanism107returns again on the movement path Rm in the opposite direction (the direction of the arrow A), passes through the disposal unit113and the detection position of the sensor112, and reaches the discharging unit111P of the incubator111. In the discharging unit111P, the specimen dispensing mechanism107discharges the specimen into the reaction vessel RC (step S5). During a transitional period from step S3to step S5, when the dispensing nozzle1072passes through the detection position of the sensor112, the presence or absence of mounting of the dispensing tip CP at the tip of the dispensing nozzle1072is detected (step S4). When the mounting of the dispensing tip CP is detected, it is determined that the dispensing operation is normally performed, and the dispensing operation is continued. When the dispensing tip CP is not detected, it is determined that an abnormality occurs in the dispensing operation, the operation of the specimen dispensing mechanism107is stopped, and an abnormality notification is displayed on the display unit104. After discharging the specimen in the discharging unit111P, the dispensing nozzle1072of the specimen dispensing mechanism107moves again on the movement path Rm in the direction of the arrow B, passes through the sensor112, and then reaches the disposal unit113. In the disposal unit113, the dispensing tip CP is removed from the dispensing nozzle1072and the dispensing tip CP is discarded (step S7). During a transitional period from step S5to step S7, when the dispensing nozzle1072passes through the detection position of the sensor112, the presence or absence of mounting of the dispensing tip CP at the tip of the dispensing nozzle1072is detected (step S6). When the mounting of the dispensing tip CP is detected, it is determined that the dispensing operation is normally performed, and the dispensing operation is continued. When the dispensing tip CP is not detected, it is determined that an abnormality occurs in the dispensing operation, the operation of the specimen dispensing mechanism107is stopped, and an abnormality notification is displayed on the display unit104. When the disposal of the dispensing tip CP in the disposal unit113is completed, the specimen dispensing mechanism107returns to the standby position described above and stands by until the next dispensing instruction is received. Before transiting to this standby state, the specimen dispensing mechanism107passes through the detection position of the sensor112, so that the sensor112detects the presence or absence of the dispensing tip CP at the tip of the dispensing nozzle1072. When it is determined that the dispensing tip CP is absent, it is determined that the disposal of the dispensing tip CP is completed, and the dispensing operation is continued. When it is determined that the dispensing tip CP remains at the tip of the dispensing nozzle1072, it is determined that an abnormality occurs in the dispensing operation, the operation of the specimen dispensing mechanism107is stopped, and an abnormality notification is displayed on the display unit104. When a start instruction of the next specimen dispensing operation is received, the specimen dispensing mechanism107moves the dispensing nozzle1072from the standby position to the mounting unit114through the sensor112and the discharging unit111P. Hereinafter, the same procedure as described above will be repeated. In this way, in the apparatus inFIG.2, the mounting unit114, the suction unit106S, the discharging unit111P, and the disposal unit113are arranged as described above, so that the specimen dispensing mechanism107can pass through the detection position of the sensor112without detouring the detection position of the sensor112during a transition between steps S1, S3, S5, and S7described above. Therefore, it is possible to improve the throughput of the automatic analysis apparatus. The sensor112is provided outside the specimen dispensing mechanism107, and only one sensor112is provided, so that it is possible to reduce the cost. When the dispensing nozzle1072passes through the sensor112, if the sensor112cannot detect the dispensing tip CP even when the dispensing tip CP should have been mounted on the dispensing nozzle1072or if the sensor112detects mounting of the dispensing tip CP even when the dispensing tip CP should not have been mounted on the dispensing nozzle1072, It is possible to determine that an abnormality occurs, cause the display unit104to notify occurrence of abnormality, and stop the specimen dispensing operation. Second Embodiment Next, the second embodiment of the present invention will be described with reference toFIG.4.FIG.4is a schematic diagram showing an arrangement of a discharging unit111Pa, a disposal unit113a, a suction unit106Sa, a mounting unit114a, and a sensor112ain the automatic analysis apparatus100according to the second embodiment. The entire configuration of the apparatus is substantially the same as that inFIG.1except for the arrangement of the discharging unit111Pa, the disposal unit113a, the suction unit106Sa, the mounting unit114a, and the sensor112a, so that redundant description will be omitted. InFIG.4, the discharging unit111Pa, the disposal unit113a, the suction unit106Sa, the mounting unit114a, and the sensor112arespectively correspond to the discharging unit111P, the disposal unit113, the suction unit106S, the mounting unit114, and the sensor112of the first embodiment (FIG.2), so that redundant description will be omitted. In the second embodiment (FIG.4), the detection position of the optical sensor112ais arranged in the center of the movement path Rm, and this is the same as in the first embodiment (FIG.2). When viewed from the detection position of the sensor112a, the discharging unit111Pa and the mounting unit114aare arranged on one side of the movement path Rm, and the disposal unit113aand the suction unit106Sa are arranged on the other side of the movement path Rm. This is also the same as in the first embodiment. However, inFIG.4, the positional relationship between the disposal unit113aand the suction unit106Sa is reverse to that inFIG.2. Specifically, the disposal unit113ais arranged close to an end portion of the movement path Rm, and the suction unit106Sa is arranged more inside of the movement path Rm than the disposal unit113a. Also in the arrangement ofFIG.4, when the specimen dispensing operation (FIG.3) is performed, it is possible to eliminate useless movement of the specimen dispensing mechanism107and improve the throughput of the automatic analysis apparatus in the same manner as inFIG.2(the first embodiment). The details of the specimen dispensing operation in the second embodiment are substantially the same as that in the first embodiment, so that redundant description will be omitted. A moving distance of the specimen dispensing mechanism107when transiting from step S1to step S3in the second embodiment is shorter than that in the first embodiment. On the other hand, a moving distance of the specimen dispensing mechanism107when transiting from step S5to step S7in the second embodiment is longer than that in the first embodiment. However, as a whole, the throughput of the apparatus of the second embodiment is substantially the same as that of the first embodiment. Third Embodiment Next, the third embodiment of the present invention will be described with reference toFIG.5.FIG.5is a schematic diagram showing an arrangement of a discharging unit111Pb, a disposal unit113b, a suction unit106Sb, amounting unit114b, and a sensor112bin the automatic analysis apparatus100according to the third embodiment. The entire configuration of the apparatus is substantially the same as that inFIG.1except for the arrangement of the discharging unit111Pb, the disposal unit113b, the suction unit106Sb, the mounting unit114b, and the sensor112b, so that redundant description will be omitted. InFIG.5, the discharging unit111Pb, the disposal unit113b, the suction unit106Sb, the mounting unit114b, and the sensor112brespectively correspond to the discharging unit111P, the disposal unit113, the suction unit106S, the mounting unit114, and the sensor112of the first embodiment (FIG.2), so that redundant description will be omitted. In the third embodiment (FIG.5), the detection position of the optical sensor112bis arranged in the center of the movement path Rm, and this is the same as in the first and second embodiments (FIGS.2and4). When viewed from the detection position of the sensor112b, the discharging unit111Pb and the mounting unit114bare arranged on one side of the movement path Rm, and the disposal unit113band the suction unit106Sb are arranged on the other side of the movement path Rm. This is also the same as in the first embodiment. However, inFIG.5, the positional relationship between the mounting unit114band the discharging unit111Pb is reverse to that inFIGS.2and4. Specifically, the mounting unit114bis arranged in a position farther from an end portion of the movement path Rm than the discharging unit111Pb, and this is different from the examples inFIGS.2and4. When the mounting unit114bis located more inside of the movement path Rm than the discharging unit111Pb, the dispensing tip CP that has sucked a specimen can pass through above the mounting unit114bin the middle of the specimen dispensing operation. In this case, it can be considered that an unused dispensing tip CP mounted in the mounting unit114bis contaminated by the specimen and an accurate inspection is hindered. Therefore, in the third embodiment, this problem is solved by separately providing a mounting unit cover115bthat covers the mounting unit114b. As described later, during a mounting operation, the mounting unit cover115bis retreated from the mounting unit114b, and otherwise, the mounting unit cover115bis arranged so as to cover the mounting unit114b. As shown inFIG.6, the mounting unit cover115bincludes a rotation shaft1151and a shielding plate1152that rotates around the rotation shaft1151. The rotation shaft1151is arranged close to the mounting unit114band is configured to be able to be rotated by a motor not shown in the drawings. The shielding plate1152is set to a shielding position (solid line inFIG.6) that covers the mounting unit114bor a retreat position (dashed line inFIG.6) that does not cover the mounting unit114bby the rotation of the rotation shaft1151. A used dispensing tip CPo after the specimen dispensing mechanism107bsucks a specimen is held at the tip of the dispensing nozzle1072, and when such a used dispensing tip CPo passes through above the mounting unit114b, the shielding plate1152is set to the shielding position described above. Thereby, it is possible to prevent the specimen from leaking and falling from the used dispensing tip CPo to the mounting unit114band to prevent an unused dispensing tip CPn from being contaminated. In the example ofFIG.6, the mounting unit cover115bhas a structure that rotates around the rotation shaft, however, it is not limited thereto and the shielding plate1152may be slid by being driven by a solenoid or the like. Next, the specimen dispensing operation in the automatic analysis apparatus inFIG.5will be described in detail with reference to the flowchart inFIG.3. Here, as an example, it is assumed that the specimen dispensing mechanism107bis located in the initial position or the standby position between the disposal unit113band the suction unit106Sb and the next specimen dispensing instruction is waited. When the specimen dispensing instruction is inputted, the dispensing nozzle1072of the specimen dispensing mechanism107bstarts moving from the initial position or the standby position in a direction of an arrow A along the movement path Rm. The mounting unit cover115breceives the specimen dispensing instruction and moves to the retreat position. Instead that the mounting unit cover115breceives the specimen dispensing instruction and moves to the retreat position, it is possible to define that when the specimen dispensing mechanism107bis located in the initial position or the standby position, the specimen dispensing mechanism107bhas been moved to the retreat position. When the dispensing nozzle1072of the specimen dispensing mechanism107breaches the mounting unit114b, and the dispensing tip CP is mounted on the dispensing nozzle1072(step S1). Thereafter, the dispensing nozzle1072moves in the opposite direction (direction of an arrow B) on the movement path Rm, passes through the detection position of the sensor112band the disposal unit113b, reaches the suction unit106Sb, and sucks a specimen from the specimen vessel105in the suction unit106Sb (step S3). During a transitional period from step S1to step S3, when the dispensing nozzle1072passes through the detection position of the sensor112b, the sensor112bdetects the presence or absence of mounting of the dispensing tip CP at the tip of the dispensing nozzle1072(step S2). When the mounting of the dispensing tip CP is detected, it is determined that the dispensing operation is normally performed, and the dispensing operation is continued. When the dispensing tip CP is not detected, it is determined that an abnormality occurs in the dispensing operation, the operation of the specimen dispensing mechanism107bis stopped, and an abnormality notification is displayed on the display unit104. After step S1, the mounting unit cover115bmoves to the shielding position described above at any timing until a time point when the specimen dispensing mechanism107bpasses through the mounting unit114b. After sucking the specimen in the suction unit106Sb, the specimen dispensing mechanism107breturns again on the movement path Rm in the opposite direction (the direction of the arrow A), passes through the disposal unit113b, the detection position of the sensor112b, and above the mounting unit114bshielded by the mounting unit cover115b, and reaches the discharging unit111Pb of the incubator111b. Although the used dispensing tip CPo passes through above the mounting unit114b, the mounting unit114bis shielded by the mounting unit cover115b. Therefore, the unused dispensing tip CPn is prevented from being contaminated. In the discharging unit111Pb, the specimen dispensing mechanism107bdischarges the specimen into the reaction vessel RC (step S5). During a transitional period from step S3to step S5, when the dispensing nozzle1072passes through the detection position of the sensor112b, the presence or absence of mounting of the dispensing tip CP at the tip of the dispensing nozzle1072is detected (step S4). When the mounting of the dispensing tip CP is detected, it is determined that the dispensing operation is normally performed, and the dispensing operation is continued. When the dispensing tip CP is not detected, it is determined that an abnormality occurs in the dispensing operation, the operation of the specimen dispensing mechanism107bis stopped, and an abnormality notification is displayed on the display unit104. After discharging the specimen in the discharging unit111Pb, the dispensing nozzle1072of the specimen dispensing mechanism107bmoves again on the movement path Rm in the direction of the arrow B, passes through the mounting unit114band the sensor112b, and then reaches the disposal unit113b. Also in this stage, it is preferable that the mounting unit114bis covered by the mounting unit cover115b. In the disposal unit113b, the dispensing tip CP is removed from the dispensing nozzle1072and the dispensing tip CP is discarded (step S7). During a transitional period from step S5to step S7, when the dispensing nozzle1072passes through the detection position of the sensor112b, the presence or absence of mounting of the dispensing tip CP at the tip of the dispensing nozzle1072is detected (step S6). When the mounting of the dispensing tip CP is detected, it is determined that the dispensing operation is normally performed, and the dispensing operation is continued. When the dispensing tip CP is not detected, it is determined that an abnormality occurs in the dispensing operation, the operation of the specimen dispensing mechanism107bis stopped, and an abnormality notification is displayed on the display unit104. The operation after the dispensing tip CP has been discarded in the disposal unit113bis substantially the same as that in the embodiments described above. As described above, also in the third embodiment, the same effect as that of the embodiments described above can be obtained. In the third embodiment, although the mounting unit114bis located in the middle of the movement path Rm, it is possible to avoid contamination of the mounting unit114bbecause the mounting unit cover115bis provided. Fourth Embodiment Next, the fourth embodiment of the present invention will be described with reference toFIG.7.FIG.7is a schematic diagram showing an arrangement of a discharging unit111Pc, a disposal unit113c, a suction unit106Sc, amounting unit114c, and a sensor112cin the automatic analysis apparatus100according to the fourth embodiment. The entire configuration of the apparatus is substantially the same as that inFIG.1except for the arrangement of the discharging unit111Pc, the disposal unit113c, the suction unit106Sc, the mounting unit114c, and the sensor112c, so that redundant description will be omitted. InFIG.7, the discharging unit111Pc, the disposal unit113c, the suction unit106Sc, the mounting unit114c, and the sensor112crespectively correspond to the discharging unit111P, the disposal unit113, the suction unit106S, the mounting unit114, and the sensor112of the first embodiment (FIG.2), so that redundant description will be omitted. In the fourth embodiment (FIG.7), the detection position of the optical sensor112cis arranged in the center of the movement path Rm, and this is the same as in the first to the third embodiments. When viewed from the detection position of the sensor112c, the discharging unit111Pc and the mounting unit114care arranged on one side of the movement path Rm, and the disposal unit113cand the suction unit106Sc are arranged on the other side of the movement path Rm. This is also the same as in the first to the third embodiments. However, in the fourth embodiment (FIG.7), in the same manner as in the third embodiment (FIG.5), the mounting unit114bis arranged in a position farther from an end portion of the movement path Rm than the discharging unit111Pb. Therefore, a mounting unit cover115cthat covers the mounting unit114cis provided. The mounting unit cover115cmay be the same as the mounting unit cover115bof the third embodiment. In the same manner as in the second embodiment, the disposal unit113cis arranged at an end portion of the movement path Rm, and the suction unit106Sc is arranged inside of the movement path Rm. Also in the fourth embodiment, the same effect as that of the embodiments described above can be obtained. In the fourth embodiment, although the mounting unit114cis located in the middle of the movement path Rm in the same manner as in the third embodiment, it is possible to avoid contamination of the unused dispensing tip CPn of the mounting unit114cbecause the mounting unit cover115cis provided. In the first to the fourth embodiments described above, the sensor is located at the center of the movement path Rm, the mounting unit (114to114c) and the discharging unit (111P to111Pc) are located on one side of the movement path Rm, and the suction unit (106S to106Sc) and the disposal unit (113to113c) are located on the other side of the movement path Rm. The configurations of these embodiments can minimize useless movement of the specimen dispensing mechanism. However, the present invention is not limited to the first to the fourth embodiments. The arrangement of the mounting unit, the suction unit, the discharging unit, the disposal unit, and the sensor can be variously changed. Fifth Embodiment Next, the fifth embodiment of the present invention will be described with reference toFIGS.8and9.FIG.8is a schematic diagram showing an arrangement of a discharging unit111Pd, a disposal unit113d, a suction unit106Sd, a mounting unit114d, and a sensor112din the automatic analysis apparatus100according to the fifth embodiment. The entire configuration of the apparatus is substantially the same as that inFIG.1except for the arrangement of the discharging unit111Pd, the disposal unit113d, the suction unit106Sd, the mounting unit114d, and the sensor112d, so that redundant description will be omitted. InFIGS.8and9, the discharging unit111Pd, the disposal unit113d, the suction unit106Sd, the mounting unit114d, and the sensor112drespectively correspond to the discharging unit111P, the disposal unit113, the suction unit106S, the mounting unit114, and the sensor112of the first embodiment (FIG.2), so that redundant description will be omitted. The fifth embodiment is different from the embodiments described above. In the fifth embodiment, the discharging unit111Pd, the disposal unit113d, the suction unit106Sd, and the mounting unit114dare arranged along a straight line, and a specimen dispensing mechanism107dmoves a dispensing nozzle1075along the straight line. As shown inFIG.9as an example, in a movement mechanism including a movement rail1073and a block1074movable along the movement rail1073, the dispensing nozzle1075can be attached to one end of the block1074. Thereby, the dispensing nozzle1075can move along the straight-line movement path Rm. This is different from the embodiments described above where the dispensing nozzle is arcuately moved. The automatic analysis apparatus of the fifth embodiment is provided with the sensor112dthat detects the presence or absence of mounting of the dispensing tip CP on the dispensing nozzle1075that moves lineally. Here, the sensor112dincludes a laser light source112L that emits laser light along the movement path Rm and an optical sensor112darranged on an extended line of the movement path Rm. The laser light from the laser light source112L is blocked when the dispensing tip CP is attached to the dispensing nozzle1075. Thereby, the optical sensor112dcan detect the presence or absence of the mounting of the dispensing tip CP. The sensor112dmay be an image sensor arranged on the extended line of the movement path Rm instead of the laser light source and the optical sensor as shown inFIGS.8and9. The operation of the fifth embodiment can be performed similarly to the operations of the embodiments described above, so that its description is omitted here. An arrangement order of the discharging unit111Pd, the disposal unit113d, the suction unit106Sd, and the mounting unit114dcan be arbitrarily changed. Others FIGS.10A and10Bis a diagram illustrating combinations of the arrangement orders of the mounting unit114, the suction unit106S, the discharging unit111P, the disposal unit113, and the sensor112in the first to the fourth embodiments, and their evaluations (SP (superb) (best), EX (excellent) (better), GD (good) (good), or NG (no good) (poor)). InFIGS.10A and10B, A, B, C, D and E represent the mounting unit, the suction unit, the discharging unit, the disposal unit, and the sensor, respectively. For example, No.1shows an arrangement in which the mounting unit, the suction unit, the discharging unit, the disposal unit, and the sensor are arranged in this order from an end portion of the movement path. InFIGS.10A and10B, No.12, No.11, No.38, and No.46represent the first embodiment, the second embodiment, the third embodiment, and the fourth embodiment, respectively. These can minimize useless movement of the specimen dispensing mechanism, so that their evaluations are SP (best) or EX (better). The first and the second embodiments are preferable in particular because the mounting unit cover is not required in these embodiments. Among 60 combinations shown inFIGS.10A and10B, combinations in which the sensor (E) is located at an end portion of the movement path Rm (for example, No.1) have to move the specimen dispensing mechanism to the end portion of the movement path Rm every time step S1, S3, S5, or S7inFIG.3is completed, so that high throughput cannot be obtained. Therefore, combinations in which the sensor (E) is located at the end portion of the movement path Rm are determined to be NG (poor). In the other combinations (sensor), the sensor (E) is arranged in a position between any two of the mounting unit (A), the suction unit (B), the discharging unit (C), and the disposal unit (D). In such arrangements, after steps S1, S3, S5, or S7is completed, the specimen dispensing mechanism has to move (detour) to the sensor once or twice. However, as a whole, sufficiently high throughput can be obtained, so that the arrangements are determined to be GD (good). While the embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in other various forms, and can be variously omitted, replaced, and changed without departing from the scope of the invention. These embodiments and their modifications are included in the scope and gist of the invention and are also included in the inventions described in the claims and their equivalents. REFERENCE SIGNS LIST 100automatic analysis apparatus102control unit103input unit104display unit105specimen vessel106specimen vessel rack106to106Sd suction unit107to107dspecimen dispensing mechanism108reagent dispensing mechanism109reagent vessel110reaction vessel and dispensing tip accommodating unit111to111dincubator111P to111Pd discharging unit112doptical sensor112L laser light source112to112dsensor113to113ddisposal unit114to114dmounting unit115b,115cmounting unit cover1070rotation shaft1071arm1072,1075dispensing nozzle1151rotation shaft1152shielding plateCP dispensing tipRC reaction vesselRm movement path | 38,819 |
11860180 | DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS With reference toFIGS.3and4there is shown a digital dispense device10for accurately dispensing an amount of one or more fluids onto a substrate14from a fluid droplet ejection cartridge12that moves in a direction of arrow20across the substrate14. In some embodiments, the substrate14may be moved by a tray in a direction orthogonal to the direction of movement of the fluid droplet ejection cartridge12during a fluid deposition process. Accordingly, a rear opening24may be provided in the housing26of the digital dispense device for movement of the substrate therethrough. According to an embodiments of the disclosure, the digital dispense device10includes a removable maintenance fluid holder30that is removably disposed through an opening32in the digital dispense device10. In the embodiment illustrated inFIGS.3and4, the opening32is a side opening in the housing26of the digital dispense device10. As shown inFIG.3, the removable maintenance fluid holder is supported in the digital dispense device by a maintenance platform34in a maintenance area of the digital dispense device remote from the substrate14. During maintenance of the fluid droplet ejection cartridge12, fluid16is dispensed from the cartridge12into a fluid receptacle35or onto an absorbent pad36in the fluid receptacle35of the removable maintenance fluid holder30. As shown inFIG.3, the removable maintenance fluid holder30is supported in the maintenance area of the digital dispense device at a distance D2that is substantially less than the distance D1ofFIG.2. The distance D2may range from about 1 mm to about 2 mm or more. However, the smaller the gap, the lower the amount of misting of fluid during a maintenance procedure. In one embodiment, the removable maintenance fluid holder30has an L-shaped housing38(FIG.5) that includes the fluid receptacle35and a handle40distal from the fluid receptacle. In some embodiments, the fluid receptacle35has a single fluid containment area. In another embodiment, the fluid receptacle is segmented into two or more discrete non-staggered areas (areas A, B, and C shown for illustration purposes). In some embodiments, discrete non-staggered areas A, B, and C of the fluid receptacle may be separated by a plastic or metal partition between the segments to prevent fluids from mixing together in the fluid receptacle35. In some embodiments, as shown inFIG.6, an absorbent pad36may be removably or fixedly attached in the fluid receptacle5. Accordingly, any one or more of the discrete non-staggered segments of the fluid receptacle35may contain an absorbent pad36. Different types of absorbent pads36may be used in each of the discrete segments at the same time. During maintenance of the fluid droplet ejection cartridge12, the cartridge may be moved over the fluid receptacle35, or over one of the discrete areas A, B, or C of the fluid receptacle35so that fluid may be dispensed into one discrete area at a time until that area is saturated or filled with fluid, then a next segment or area may be used for fluid maintenance until all segments have been saturated or filled with fluid. At that point the removable maintenance fluid holder30may be removed from the device10and disposed of, such as in a biohazard collection bin. In some embodiments, fluid may be dispensed in one or more of the discrete areas A, B, or C of the fluid receptacle35while the fluid droplet ejection cartridge12is moving incrementally in the maintenance area of the digital dispense device10. In some embodiments, as shown inFIG.6, the absorbent pad36may be removable from the housing38and disposed of and the housing38reused. In this embodiment, the removable pad may be disposed in the fluid receptacle area of the housing38. FIG.7illustrates an alternative embodiment of the removable maintenance fluid holder50. In this embodiment, the removable maintenance holder50has an elongate housing52that includes fluid receptacle54that may be segmented as described above and a handle56that is distal from the fluid receptacle54. As in the previous embodiment, the entire removable maintenance fluid holder50may be disposable or an absorbent pad disposed in the fluid receptacle54may be removable from the housing52so that only the absorbent pad is disposed of. In another embodiment, illustrated inFIGS.8-10, the removable maintenance fluid holder30or50may be inserted and removed from the digital dispense device10through a front opening60in the housing unit62of the digital dispense device. Once the removable maintenance fluid holder30or50is inserted through the front opening60and a substrate14is disposed on a substrate holder64of a movable tray mechanism66, a power button68may be pushed to turn on the device10. As shown inFIG.10, a carriage translation mechanism70is used to move the fluid droplet ejection cartridge12over the substrate14during a fluid dispense procedure in the x-direction of arrow20or toward a maintenance area72for depositing fluid onto the removable maintenance fluid holder50. In the embodiments described herein, the removable maintenance housings may be made of a variety of materials including metals, plastics and ceramics. The absorbent pads may be made of absorbent fibers, felt pads and fibers treated with absorbent polymers. The materials for the absorbent pads are desirably selected from materials that are compatible with the fluids being ejected from the fluid droplet ejection cartridges. It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or can be presently unforeseen can arise to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they can be amended are intended to embrace all such alternatives, modifications variations, improvements, and substantial equivalents. | 7,150 |
11860182 | Corresponding reference numerals are used to indicate corresponding parts in the drawings. DETAILED DESCRIPTION The embodiments of the present disclosure described below are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art may appreciate and understand the principles and practices of the present disclosure. InFIG.1a silo10in the form of a so-called bunker silo is shown. The silo10comprises a floor12and side walls (not shown) which are generally all produced from concrete. The silo10is designed as a trench silo into which chopped foliage plants are introduced, distributed and compacted, as known in EP 3 403 487 A1 and EP 3 403 488 A1, the entire disclosures thereof being incorporated by reference herein. In addition, U.S. Pat. No. 10,617,054 is hereby incorporated by reference herein. The foliage plants may be harvested by a harvesting chain which consists of a harvesting vehicle in the form of a forage harvester and a towing vehicle with a trailer, or a loader wagon with a towing vehicle. The harvested crops are directly discharged from the trailer or loader wagon onto the silo10or initially deposited in a heap in the vicinity thereof and distributed on the silo10by a suitable vehicle or other means. The harvested crops are then compacted by a compaction vehicle28which is moved along travel paths over the silo10. In the embodiment inFIG.1, the compaction vehicle28is connected to a relatively wide compaction device36. If a separate compaction device36is not used, the compaction may be carried out simply by the wheels40,42of the compaction vehicle28alone. The chopped foliage plants, denoted hereinafter as silage16, may be supplied with an ensilage agent during harvesting or during or after storage in order to improve the fermentation. Additionally, after the compaction the silage16is covered at the top and toward the open sides of the silo10with a film in order to shield the silage from the oxygen of the ambient air. The compaction vehicle28is composed of a tractor32and the compaction device36in the form of a roller attached to the three-point hitch34thereof. The compaction device36could also be dispensed with or replaced by a ballast weight. The compaction is carried out in this case by the wheels40,42of the compaction vehicle28. Instead of a tractor32, a vibrating roller (single drum roller) or any other vehicle, for example, a snow groomer, could also be used as a compaction vehicle28. The tractor32comprises a load-bearing chassis38which is supported on steerable front wheels40and drivable rear wheels42, which are drive-connected to the crankshaft of an internal combustion engine44in a torque-proof manner. An electronic control device46(the computing power thereof also being able to be outsourced, whether to a computer which is remote, stationary or designed as a mobile device, for example, of the operator or the Cloud) is connected to an operator interface48which is assigned to the workstation of an operator in a cab50. The control device46is also connected in a signal-transmitting manner to an automatic steering and speed control device52, a tire pressure regulator54, a position determining device60and an actuator56(power lifter) for adjusting the position of the lower link arm of the three-point hitch34, and is configured to control this automatically. By the actuator56, the contact force of the compaction device36may be varied and the compaction device lifted away. When storing the silage16for the purpose of compaction, the compaction vehicle28is automatically moved by the control device46or steered by the driver along travel paths, i.e., generally driven to and fro parallel to the side walls of the silo10until a desired degree of compaction is achieved. Subsequently a layer of new silage16is applied and compacted again until the silo10is filled. In a further method, however, it might also be possible to fill the silo10gradually in the horizontal direction and then compact the silo when it is filled. A combination of the two methods is also conceivable. A possible automation of the compaction is disclosed in DE 10 2020 110 297 A1. (U.S. application Ser. No. 17/184,757 is incorporated by reference herein.) For measuring the density of the silage16, the compaction vehicle28is provided with a sensor arrangement14. The sensor arrangement14comprises a wheel20which is freely rotatably mounted on a hub32. The hub is mounted on a rocker arm22which is articulated at a pivot point30on a bracket18which in turn is fastened to the front side of the compaction vehicle28, for example, to a front weight or to a front three-point hitch, or is formed thereby. A pretensioning element26, the one end being articulated on the rocker arm22and the other end on a rigid fastening24, serves to pretension the rocker arm22and thus the wheel20during the compaction process with a predetermined force against the silage16and to lift away the rocker arm22and thus the wheel20upwardly into an inactive position when not in use. The rotational axes of the pivot point30, of the hub32(and thus of the wheel20) and the axes of articulation of the pretensioning element26are oriented horizontally and transversely to the forward direction of the compaction vehicle28which runs to the left inFIG.1. In contrast toFIG.1, rocker arms22could be arranged on both sides of the wheel20, i.e., the rocker arms22could be designed as a fork. The pretensioning element26is thus articulated on one or both rocker arms22. The pretensioning element26may be designed as a hydraulic or pneumatic cylinder and acted upon by the on-board hydraulic system or pneumatic system of the compaction vehicle28. For all embodiments of the sensor arrangement14a runner58could be used instead of the wheel20, as shown inFIG.4. In embodiments in which a compaction device36is dispensed with or when sufficient space is still present in spite of the attachment of a compaction device36to the rear side of the compaction vehicle28, the sensor arrangement14could also be fastened thereto, for example, to the rear three-point hitch34. FIG.2shows an enlarged schematic view of a first possible embodiment of a sensor arrangement14. The sensor arrangement14comprises a source62of a gaseous medium which is at an overpressure, i.e., a pressure which is greater than that of the ambient air. The medium is, in particular, compressed air which may be provided, for example, by the on-board pneumatic system of the compaction vehicle28(i.e., the pneumatic braking system thereof) or it is taken from a compressed gas bottle. The source62is connected to a valve64, the free diameter thereof being adjustable by an electromagnetic actuator, and the valve is electrically connected to an evaluation device72. The valve64on the outlet side is connected to a line66which at its lower end transitions into an opening68which faces the lower face of the wheel20and is adjacent thereto. A sensor70detects the volumetric flow rate in the line66and supplies the evaluation device72with a corresponding electrical signal which contains information regarding the volumetric flow rate in the line66and thus through the opening66. The line66, the sensor70and the opening68do not rotate with the wheel20when the wheel is moved over the silage16, but are rigidly coupled to the rocker arm22, so that during compaction mode the opening68is continuously aligned downwardly. As already mentioned above, the source62may be arranged on-board the compaction vehicle28which similarly applies to the valve64and the evaluation device72or as shown inFIG.2they are integrated spatially in the wheel20of the sensor arrangement14. The wheel20comprises a perforated lateral surface forming its periphery so that the gas from the end region of the line66adjacent to the silage16may flow through the lateral surface of the wheel20(forming the opening68) into the silage16. To the side, the wheel20may be connected by spokes (or closed walls) to the hub32. The hole size of the perforations74corresponds inFIGS.2and3approximately to the diameter of the end region of the line66adjacent to the silage16. The end region of the line66adjacent to the silage16may have a cross section of any shape, i.e., for example, rectangular, circular or oval. The cross section of the perforations74can be analogous in any way, for example, rectangular, circular or oval. In particular, the cross sections of the perforations74and of the end region of the line66adjacent to the silage16at least approximately coincide in terms of shape and dimensions, or the perforations74are dimensioned to be larger than the end region of the line66adjacent to the silage16. By the relatively large dimension of the hole size of the perforations74it is achieved that as few particles as possible of the silage16collect in the perforations74. However, it might also be conceivable to select the hole size of the perforations74to be smaller than the size of the end region of the line66adjacent to the silage16, in order to permit a continuous measuring mode in which the outflow of the gaseous medium from the line66is not regularly interrupted by the material of the wheel20which remains between the perforations74or the throttle action, which changes with the rotation of the wheel20and is dictated by the material of the lateral surface of the wheel20between the perforations74, is at least approximately averaged out over time. In the embodiment shown inFIGS.2and3in which the hole size of the perforations74corresponds approximately to the dimension of the end region of the line66adjacent to the silage16, the line66is regularly covered by the material of the wheel20which is located between the perforations74. In order to avoid this falsifying effect on the output signal of the evaluation device72, the rotational position of the wheel20may be detected by a rotary angle sensor76(also in the embodiment according toFIG.3). Using a known correlation stored in the evaluation device72between the rotational position of the wheel20and the position of the perforations74, the evaluation device72connected to the rotary angle sensor76may be configured to take into account the influence of the rotational position of the wheel20on the output signal, in particular since a measurement is carried out when a perforation74is precisely aligned with the lower end region of the line66. The mode of operation of the first embodiment shown inFIG.2is based on the density of the silage16influencing the flow behavior of the gaseous medium flowing downwardly out of the opening68. If the silage16is well compacted, it provides a significant flow resistance against the gaseous medium, but this flow resistance would be lower if the silage16were to be less well compacted. In order to be able to detect a representative variable for the aforementioned flow resistance, it is provided that during operation the evaluation device72adjusts the valve64such that (by electrically adjusting the actuator assigned to the valve64) the sensor70of the evaluation device72displays a predetermined value for the volumetric flow rate in the line66. If, therefore, the predetermined value of the volumetric flow rate is reached with a valve64opened to a relatively small extent, the silage16is compacted to a greater degree than if the valve64has to be opened further for producing the same volumetric flow rate. The actuating signal to the valve64or a pressure measured upstream or downstream of the valve64by a further sensor (not shown) is thus a measurement of the density of the silage16. The evaluation device72forwards the actuating signal or one of the pressure values (or a value dependent thereon, in particular calibrated to the density of the silage, for example, measured in kg/m3or a proportion of the harvested crops in the silage volume measured in %) to the control device46. The control device may display the determined value of the density on the operator interface48or instructions derived therefrom to the operator, from which it is possible to identify whether and where the silage has to be still recompacted, or may use this value for automatically activating the compaction vehicle32, as is disclosed in DE 10 2020 110 297 A1, the disclosure being incorporated by reference herein. (U.S. application Ser. No. 17/184,757 is incorporated by reference herein.) The sensor arrangement14may operate continuously or may be activated intermittently, for example, after covering defined distances, for example, every 0.5 m or carry out a measurement and provide an output signal precisely when the lower end region of the line66is aligned with a perforation74, which may be detected as described above using the rotary angle sensor76. In the second embodiment shown inFIG.3of the sensor arrangement14, elements which coincide or which are equivalent to the first embodiment are identified by the same reference numerals. The source62is illustrated here as a pressure accumulator but may be replaced by a source mentioned in connection withFIG.2. In the second embodiment, the valve64is a pressure relief valve and accordingly provides the medium at a predetermined pressure at its outlet. The medium flows from the valve64through the line66and through the opening68and finally into the silage16. The sensor70detects, as in the first embodiment, the volumetric flow rate of the medium through the line66. The evaluation device72is electrically connected to the sensor70and also to the control device46. The mode of operation of the second embodiment is similar to that of the first embodiment but does not operate at a constant flow of the medium as in the first embodiment but at a constant pressure and the volumetric flow rate, which is dependent on the flow resistance to which the gaseous medium is subjected in the silage16, is detected. The evaluation device72forwards the signal of the sensor70(or a value dependent thereon, in particular calibrated to the density of the silage) to the control device46which uses the value in the manner described relative to the first embodiment. In the third embodiment shown inFIG.4of the sensor arrangement14, elements which coincide or which are equivalent to the first or second embodiment are also identified by the same reference numerals. In the sensor arrangement according toFIG.4, the wheel20has been replaced by a skid58which is guided over the silage16by the rocker arm22ofFIG.1at a pressure defined by the pretensioning element26. The line66widens downstream of the source62in a continuous or stepwise manner and one respective sensor70, which is, however, designed as a pressure sensor, is assigned to a portion of the line66which has a different diameter from the portion of the line66assigned to the other sensor70. The line66tapers again at the lower end directly upstream and above the opening68, which however is optional. The mode of operation of the third embodiment is based on a measurement of the flow rate of the medium through the silage16—dependent on the density of the silage16—and thus through the line66. In the case of dense silage16, the flow rate through the line—at a constant pressure of the source62—is less than in the case of less dense silage since in the second case more medium will flow out through the opening68. The flow rate could be detected directly by a single sensor70designed as a speed sensor, which could also be used, for example, instead of the sensor70of the embodiment ofFIG.2or3detecting the volumetric flow rate, but in the embodiment ofFIG.4the flow rate is determined using a differential pressure measurement. To this end, both sensors70are designed as pressure sensors, as already mentioned above, and assigned to sections of the line66having different diameters and thus different flow rates and pressures. InFIG.4, therefore, a so-called differential pressure sensor is used for determining the flow rate. The evaluation device72connected to both sensors70accordingly determines the flow rate using the signals of both sensors70and the known diameters of the line66(for example, by using Bernoulli's principle) and on the basis thereof in turn determines a value dependent on the density of the silage16and forwards it to the control device46which uses the value in the manner described relative to the first embodiment. While embodiments incorporating the principles of the present disclosure have been disclosed hereinabove, the present disclosure is not limited to the disclosed embodiments. Instead, this application is intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains and which fall within the limits of the appended claims. | 17,003 |
11860183 | DETAILED DESCRIPTION The drawings may not be drawn to scale. PTAT circuits are useful in compensating for temperature dependent variations in electronic components. PTAT circuits are designed to exhibit a predictable (e.g., linear) response to changes in temperature over a wide range of temperatures. However, some circuits may exhibit an acceleration in temperature dependent variation at some threshold temperature, leading to potential runaway or cascade degradation in performance. Described herein is temperature dependent acceleration current source circuitry that, rather than generating a current that exhibits a constant variation responsive to temperature changes, outputs an “acceleration current” in response to a threshold temperature being reached. As used herein, “acceleration current” is shorthand for a current that is near nil until a threshold temperature and then exhibits a steep rise to a plateau current within a relatively narrow range of temperature with respect to the threshold temperature. During operation, until the threshold temperature is reached, the acceleration current is nil or negligible. In this manner, the acceleration current may be used compensate for a rapid acceleration in temperature dependent variations experienced by electronic components at differing temperatures. FIG.1Ais a block diagram depicting example temperature dependent acceleration current source circuitry110that outputs an acceleration current (I(temp)) in response to a threshold temperature being reached. The threshold temperature is controlled by a temperature selection input signal Tsel. The temperature dependent acceleration current source circuitry110is powered by an input voltage Vinwhich is used to generate the acceleration current. In one example, the temperature dependent acceleration current source circuitry110draws minimal current (e.g., on the order of picoamperes) when the ambient temperature is lower than the threshold temperature. FIG.1Bis a plot depicting acceleration current output by the temperature dependent acceleration current source circuitry110versus temperature. The acceleration current is negligible or nil until the threshold temperature TTHRESHis reached. In response to the threshold temperature being reached, the temperature dependent acceleration current source circuitry110outputs a rapidly increasing (responsive to temperature changes) acceleration current that plateaus within a narrow range of increasing temperature (e.g., an increase of 8 nanoamperes within a range of 18 degrees Celsius from TTHRESH). FIG.2is a block diagram depicting example temperature dependent acceleration current source circuitry210, which is an example of the temperature dependent acceleration current source circuitry110ofFIG.1. The temperature dependent acceleration current source circuitry210includes current source circuitry220and temperature sensing circuitry250. The temperature sensing circuitry250includes a first temperature sensitive device260configured to generate a first signal that varies according to a first rate responsive to changes in temperature. The temperature sensing circuitry250includes a second temperature sensitive device270configured to generate a second signal that varies according to a second rate responsive to changes in temperature. The difference between the first signal and the second signal depends on temperature due to the difference in temperature-related variation between the first and second signals. In one example, the first signal is a leakage current that is exhibited by or characteristic of the first temperature sensitive device and the second signal is a leakage current that is exhibited by or characteristic of the second temperature sensitive device. The first and second temperature sensitive devices may be designed or selected to exhibit leakage currents having temperature-related dependence with a predetermined ratio to control the operation of the temperature dependent acceleration current source circuitry210. For example the ratio of leakage currents is one factor that controls the threshold temperature current (seeFIG.1B, TTHRESH) at which the acceleration current (seeFIG.1B, I(temp)) is triggered. In one example the leakage current is source-drain leakage current including a weak inversion (sub-threshold) current and a punch-through current. Both of these components show a temperature dependency that can serve as the basis for triggering the acceleration current. The current source circuitry220includes differential trigger circuitry230and acceleration current source circuitry240. The differential trigger circuitry230is configured to generate a trigger signal based on a difference between the first signal and the second signal. In one example, the differential trigger circuitry230is configured to generate the trigger signal in response to a difference between a leakage current of the second temperature sensitive device270and a leakage current of the first temperature sensitive device260exceeding a threshold. The temperature at which the differential trigger circuitry generates the trigger signal can be controlled or selected by a temperature selection input signal Tsel. This feature will be described in more detail with reference toFIGS.3and5. The acceleration current source circuitry240is configured to output the acceleration current (I(temp)) in response to the trigger signal. FIG.3is a schematic diagram depicting example temperature dependent acceleration current source circuitry310, which is an example of the temperature dependent acceleration current source circuitries110,210ofFIGS.1and2, respectively. The temperature dependent acceleration current source circuitry310includes current source circuitry320and temperature sensing circuitry350. The temperature dependent acceleration current source circuitry includes three enable (EN) inputs that each responds to an enable signal to switch on associated EN MOS transistors to enable current flow in three separate branches. The temperature sensing circuitry350is an example of temperature sensing circuitry250ofFIG.2and includes resistors R1and R2, and first temperature sensitive device360and second temperature sensitive device370, which are examples of first temperature sensitive device260and second temperature sensitive device270, respectively. Current source circuitry320is an example of current source circuitry220ofFIG.2and includes transistors Q1and Q2, and differential trigger circuitry330and acceleration current source circuitry340, which are examples of differential trigger circuitry230and acceleration current source circuitry240, respectively. First temperature sensitive device360includes a transistor Q4, and temperature sensitive device370includes a transistor Q5. Differential trigger circuitry330includes trigger temperature selection circuitry335, a diode device332and a trigger device Q3. Trigger temperature selection circuitry335includes transistors Q6, Q7, and Q8. Diode device332includes transistors Q9, Q10, Q11, and Q12. Trigger device334(Q3) includes transistor Q3. Acceleration current source circuitry340includes transistors Q13, Q14, Q15, Q16, Q17, Q18, Q19, Q20, Q21, and Q22. Also illustrated is an input voltage source Vincoupled to an internal input voltage node301of temperature dependent acceleration current source circuitry310and an electrical ground305coupled to an internal “common” node303of temperature dependent acceleration current source circuitry310. In this example, transistors Q1-Q22are field-effect transistors (FET), and in particular metal-oxide semiconductor field-effect transistor (MOSFETs), and more in particular p-type MOSFETs, also referred to herein as p-channel or p-type MOSFETs (also referred to as PMOS transistors). Accordingly, transistor Q3is also referred to herein interchangeably as a “trigger MOS device.” Also, the example ofFIG.4includes devices and circuits implemented with PMOS transistors. FETs have a gate as a control terminal and a source and drain as first and second transistor terminals. In other examples, one or more of transistors Q1-Q22and transistors inFIG.4are n-channel or n-type MOSFETs (also referred to as NMOS transistors) or bipolar junction transistors (BJTs) with appropriate characteristics. BJTs have a base as a control terminal and a collector and emitter as first and second transistor terminals. As illustrated, the respective sources of transistors Q1, Q2, and Q13are coupled to input voltage node301. The respective sources of transistors Q6, Q7, and Q9are coupled to the drain of transistor Q1. The respective drains of transistors Q6, Q7, and Q9are coupled to the respective sources of transistors Q8and Q10. The respective drains of transistor Q8and Q10are coupled to the source of transistor Q11. The drain of Q11is coupled to the source of Q12and the drain of Q12is coupled to a gate of Q3and a source and gate of Q4. The drain of Q2is coupled to the source of Q3. The drain of Q3is coupled to the source and gate of Q5and the respective gates of Q13, Q14, Q15, Q16, Q17, Q18, Q19, Q20, and Q21. The drain of Q13is coupled to the source of Q14. The drain of Q14is coupled to the source of Q15. The drain of Q15is coupled to the source of Q16. The drain of Q16is coupled to the source of Q17. The drain of Q17is coupled to the source of Q18. The drain of Q18is coupled to the source of Q19. The drain of Q19is coupled to the source of Q20. The drain of Q20is coupled to the source of Q21. The drain of Q21is coupled to the source of Q22. The drain of Q22provides the acceleration current I(temp). The respective drains of Q4and Q5are coupled to the common node303. Trigger temperature selection circuitry335is illustrated as including three transistors. However, a different number of transistors may be included depending on desired power capacity or other factors. Diode device332is illustrated as including four transistors. However, a different number of transistors may be included depending on desired power capacity or other factors. Acceleration current source340is illustrated as including ten transistors. However, a different number of transistors may be included depending on desired power capacity or other factors. First temperature sensitive device360is illustrated as including a single transistor. However, a different number of transistors may be included depending on desired power capacity or other factors. Second temperature sensitive device370is illustrated as including a single transistor. However, a different number of transistors may be included depending on desired power capacity or other factors. First temperature sensitive device360exhibits a first leakage current (indicated generally as BIAS) that varies at a first rate depending on temperature. Second temperature sensitive device370exhibits a second leakage current (indicated generally by TRIG) that varies a second rate, higher than the first rate, depending on temperature. An example leakage current (e.g., the BIAS or TRIG current) ranges from about 100 to 600 picoamperes. The first temperature sensitive device360and the second temperature sensitive device370are coupled in parallel with one another between current source circuitry320and a common node303. The BIAS current (leakage current through first temperature sensitive device360) generates a BIAS voltage that is provided to the respective gates of the transistors of the diode device332Q9, Q10, Q11, and Q12and also to the gate of the trigger MOS transistor Q3. The operating region (e.g., saturation, triode, or cut-off) of the trigger MOS transistor Q3is determined by a difference between the BIAS voltage present at the gate of MOS transistor Q3and a TRIG voltage generated by the TRIG current (leakage current through second temperature sensitive device370) present at its drain. As temperature increases the BIAS current increases at a first rate dependent on the characteristics of the first temperature sensitive device360. Similarly, as temperature increases the TRIG current increases at a second rate dependent on the characteristics of the second temperature sensitive device370. Some example characteristics of the first temperature sensitive device360and the second temperature sensitive device370that control the first rate and second rate, respectively, are described with reference toFIG.4. In the following examples, the TRIG current increases at a faster rate than the BIAS current. The opposite relationship may also be used in other examples. The difference between the first and second rates causes the TRIG voltage to diverge from the BIAS voltage so that the difference between the TRIG voltage and BIAS voltage varies responsive to temperature changes. The TRIG current controls the drain-to-source voltage across the trigger MOS transistor Q3. When the TRIG current is close to the BIAS current the trigger MOS transistor Q3exhibits a low drain-to-source voltage and the trigger MOS transistor Q3operates in triode region. When the TRIG current becomes high enough (e.g., reaches the saturation current of trigger MOS transistor Q3) due to temperature induced leakage in the second temperature sensitive device370, the trigger MOS transistor Q3transitions from triode region to saturation region, leading to a rapid increase of the TRIG voltage. The drain of the trigger MOS transistor Q3(the TRIG voltage) is coupled to the gates of the MOS transistors of the acceleration current source340. When the trigger MOS transistor Q3operates in triode region, the drain voltage is insufficient to switch the staged MOS transistors of the acceleration current source340to an ON state, or simply ON. However, when the trigger MOS transistor operates in saturation region, the drain voltage becomes sufficient to turn the MOS transistors of the acceleration current source to ON (e.g., saturation region), allowing the acceleration current I(temp)to flow out of the acceleration current source circuitry340, at the drain of transistor Q22. The number of MOS transistors in the acceleration current source circuitry may be selected to control the level of the acceleration current. The trigger temperature selection circuitry335provides temperature selection signal (Tsel1,Tsel2) for selecting a threshold temperature. Each respective temperature selection input signal turns on one or more associated MOS transistors Q6, Q7, Q8of the diode device332. When Q6is activated by Tsel1a shorting path is created around device331(Q9). The shorting path “shorts” Q9and reduces the resistance of the diode device332. When Q7and Q8are activated by Tsel2, a shorting path is created around device331and device333(Q10), further reducing the resistance of the diode device332. Using a specific combination of temperature selection inputs, the BIAS current is adjusted up or down relative to the TRIG current. In this manner the point at which the difference between the BIAS and TRIG voltages becomes large enough to drive the trigger MOS transistor Q3into the saturation region can be adjusted. For example when both temperature selection signals have a high level neither device transistor Q9nor device Q10is shorted. When Tsel1is has a low level and Tsel2has a high level, device Q9is shorted. When Tsel1is has a high level and Tsel2is has a low level or both signals have low levels, both device Q9and Q10are shorted. Activating one of the shorting paths lowers the resistance of the diode device332which increases the BIAS voltage. Accordingly, the BIAS voltage, at lower temperatures, is further from the TRIG voltage than it would be with none of the shorting paths activated. This means that the difference between the BIAS and TRIG voltages becomes sufficient to saturate the trigger MOS transistor Q3at a lower temperature. In other words, when one or more shorting paths are activated the resistance of the diode device332is reduced, thereby leading to a decrease in the gate-to-source voltage of the trigger MOS transistor Q3. The reduction in gate-to-source voltage of the trigger MOS transistor Q3reduces the saturation current of the trigger MOS transistor Q3, which leads to a lower temperature trigger point. The shift in trigger point is because the leakage current of second temperature sensitive device370remains the same, while the current capability of the trigger MOS transistor Q3is reduced, leading to a triggering at a lower temperature. In general, the more shorting paths that are activated the lower the temperature at which the acceleration current is output by the acceleration current source340. FIG.4is a schematic diagram depicting example temperature dependent acceleration current source circuitry410, which is an example of the temperature dependent acceleration current source circuitry310ofFIG.3. The acceleration current source circuitry410includes current source circuitry320as earlier described. The acceleration current source circuitry410also includes temperature sensing circuitry450, which is an example of temperature sensing circuitry250ofFIG.2. Temperature sensing circuitry450includes resistors R1and R2, and first temperature sensitive device460and second temperature device470, which are examples of first temperature sensitive device460and second temperature sensitive device270, respectively. The first temperature sensitive device460includes six PMOS transistors Q23, Q24, Q25, Q26, Q27, and Q28. The drain of transistor Q12is coupled to the respective bodies of transistors Q23, Q24, Q25, Q26, Q27, and Q28. The respective drains of transistors Q23, Q24, Q25, Q26, Q27, and Q28are coupled to the common node303. The source of a one of the MOS transistors (e.g., Q28in the illustrated example) is coupled to the drain of transistor Q12. The respective sources of the remaining transistors (e.g., Q23, Q24, Q25, Q26, and Q27in the illustrated example) are left open (unconnected). The body leakage of the six PMOS transistors Q23, Q24, Q25, Q26, Q27, and Q28contributes to the leakage current BIAS. There is no leakage through the source-to-drain channel of the unconnected transistors Q23, Q24, Q25, Q26, and Q27and thus leakage through the source-to-drain channel of these transistors does not contribute to the leakage current BIAS. However, the leakage through the source-to-drain channel of the transistor Q28contributes to the leakage current BIAS. The second temperature sensitive device470includes six MOS transistors Q29, Q30, Q31, Q32, Q33, and Q34. The drain of transistor Q3is coupled to the respective bodies of transistors Q29, Q30, Q31, Q32, Q33, and Q34. The respective drains of transistors Q29, Q30, Q31, Q32, Q33, and Q34are coupled to the common node303. The respective sources of transistors Q29, Q30, Q31, Q32, Q33, and Q34are coupled to the drain of transistor Q3. The body leakage of the six PMOS transistors Q29, Q30, Q31, Q32, Q33, and Q34contributes to the leakage current TRIG. Leakage through the source-to-drain channel of transistors Q29, Q30, Q31, Q32, Q33, and Q34contribute to the leakage current TRIG. In this example, the number of PMOS transistors in the first temperature sensitive device460matches the number of PMOS transistors in the second temperature sensitive device470. The PMOS transistors Q23, Q24, Q25, Q26, Q27, and Q28of the first temperature sensitive device460are matched to the MOS transistors Q29, Q30, Q31, Q32, Q33, and Q34of the second temperature sensitive device470. Thus the leakage currents exhibited by both devices460,470to behave similarly or in the same way. Because the MOS transistors of the first temperature sensitive device460are matched to the MOS transistors of the second temperature sensitive device470, and all of the bodies of the MOS transistors in devices460,470are contributing to the leakage current, the contribution of body leakage current to the BIAS current and the TRIG current should be the same as temperature changes. Thus body leakage effects do not contribute to the temperature based variance in the differential between the BIAS and TRIG voltages and the body leakage effects are not a significant factor in triggering the output of the acceleration current. The sources of all of the MOS transistors Q29, Q30, Q31, Q32, Q33, Q34are coupled to the trigger MOS device Q3so that leakage through all of the source-to-drain channels of the MOS transistors Q29, Q30, Q31, Q32, Q33, Q34contributes to the leakage current TRIG. Thus, while only one source-to-drain leakage path (associated with MOS transistor Q28) in the first temperature sensitive device460contributes the BIAS current, six source-to-drain leakage paths (associated with MOS transistors Q29, Q30, Q31, Q32, Q33, Q34) in the second temperature sensitive device470contributes the TRIG current. This difference in source-to-drain leakage contributions between the devices460and470causes the first rate of change of BIAS current with respect to temperature to differ from the second rate of change of the TRIG current with respect to temperature. In other words, temperature dependent subthreshold and punch though source-to-drain leakage form the basis of operation for the temperature dependent acceleration current source circuitry410. A six to one ratio between the source coupled MOS transistors (e.g., having a source coupled to the diode device332) of the second temperature sensitive device470and the source coupled MOS transistor of the first temperature sensitive device460is used in the example. However, any other numbers of MOS transistors and numbers of source-coupled MOS transistors may be used that yield a desired ratio in the first rate of change of BIAS current with respect to temperature different from the second rate of change of the TRIG current with respect to temperature. This ratio is another factor that determines the threshold temperature for the temperature dependent acceleration current source circuitry410. FIG.5is a plot of various electrical signals generated during operation of the temperature dependent acceleration current source circuitry410ofFIG.4responsive to temperature changes. Curves labeled 0 illustrate the BIAS current and the TRIG current. The BIAS current does not vary as much as the TRIG current as the temperature increases. Curve labeled 1 illustrates the BIAS voltage generated by the leakage current through the first temperature sensitive device460which remains fairly constant across the temperature range. Curve labeled 2 represents the ratio TRIG/BIAS for the leakage currents. As the TRIG current varies more significantly than the BIAS current responsive to temperature changes, the ratio TRIG/BIAS grows. Curve labeled 3 depicts the TRIG voltage which determines whether the trigger MOS transistor Q3is in triode or saturation region. As the TRIG voltage increases with temperature, the trigger MOS transistor Q3approaches saturation until, at TTHRESH, the trigger MOS transistor Q3enters saturation region and the acceleration current begins to flow. Curve labeled 4 illustrates the acceleration current that is output by the temperature dependent acceleration current source circuitry410. Because the curve labeled 4 is in terms of negative current, the downward slope indicates an increase in output current. One application of the described temperature dependent acceleration current source circuitry is a watchdog timer circuit that, for example, triggers the refreshing of capacitors used in a sample-and-hold reference system of an ultra-low power buck boost converter. The refresh rate should increase as temperature increases due to the temperature effects on discharge rate for capacitors in the sample-and-hold reference system. FIG.6is a block diagram depicting an example watchdog timer circuit600that includes temperature dependent acceleration current source circuitry610, a current source682, a switch684, an integration capacitor686, and an integrator circuit688. In an example, the current source682is or includes a PTAT current source that provides a current that charges the integration capacitor686. The integrator circuit688generates an output pulse in response to the voltage across the integration capacitor686reaching a threshold. This output pulse serves as a watchdog trigger signal (WD_trigger) that may be used to trigger the refresh of capacitors in a sample-and-hold reference system. The output pulse also closes a reset switch684to drain the integration capacitor686and reset the watchdog timer circuit600. If the current from the current source682remains constant (indicating no change in ambient temperature), the rate at which the watchdog trigger signal is generated will remain constant because the time needed to charge the integration capacitor686is constant. As temperature increases, the current from the current source682increases, which increases the rate at which the watchdog trigger signal is generated because the integration capacitor686charges more quickly. It is possible that at some higher threshold temperature, the refresh rate needed for proper functioning of the sample-and-hold reference system increases in an accelerated manner that outpaces the increase in the watchdog trigger signal rate that would be caused by the increase in the current from the current source682. At lower temperatures and until the current threshold is reached the temperature dependent acceleration current source circuitry610does not output an acceleration current. However, at the threshold temperature, the temperature dependent acceleration current source circuitry610outputs an acceleration current that additionally charges the integration capacitor686along with the current682from the PTAT current source. The addition of the acceleration circuit significantly reduces the time it takes to charge the integration capacitor686and in turn significantly increases the rate of the watchdog trigger signal. This allows for compensation of a rapid change in the rate of temperature induced variation in component performance and avoids potential runaway or cascade failure scenarios. In one example, circuits310,410, and600are included on a single integrated circuit (IC) or piece of silicon. In other examples, various components of circuits310,410, and600may be included on different ICs or pieces of silicon. FIG.7Aillustrates a nominal simulation of a refresh time of the watchdog timer circuit600ofFIG.6responsive to temperature changes. Curve labeled 5 illustrates the refresh time (inverse of refresh rate) when the temperature dependent acceleration current source circuitry610is disabled. It can be seen that the refresh time decreases in an approximately linear fashion as the current682from the PTAT current source increases in an approximately linear fashion. Curve labeled 6 illustrates the refresh time when the temperature dependent acceleration current source circuitry610is enabled. At the threshold temperature, the refresh time drops significantly in response to the addition of the acceleration current output by the temperature dependent acceleration current source circuitry610to more quickly charge the integration capacitor686. FIGS.7B-7Cillustrate refresh times for respective temperature selection input combinations. Different process corners are illustrated as well to show the various in the threshold temperature caused by process variation.FIG.7Billustrates the refresh time for the watchdog timer circuit when the Tsel1is low and Tsel2is high, which results in a threshold temperature for a nominal process of around 60° C. A range of around 50° C. for threshold temperature is exhibited between strong and weak process corners.FIG.7Cillustrates the refresh time for the watchdog timer circuit when Tsel2is low, which results in a lower threshold temperature for a nominal process of around 50° C. A range of around 50° C. for threshold temperature is exhibited between strong and weak process corners.FIG.7Dillustrates the refresh time for the watchdog timer circuit when Tsel1and Tsel2are both high, which results in a higher threshold temperature for a nominal process of around 70° C. A range of around 50° C. for threshold temperature is exhibited between strong and weak process corners. FIG.8is a flow diagram depicting an example method800for providing an acceleration current. The method800may be performed, for example by circuits110,210,310,410,610ofFIGS.1A,2,3,4, and6, respectively. The method includes, at810, generating a first signal that varies responsive to temperature changes according to a first rate. The method includes, at820, generating a second signal that varies responsive to temperature changes according to a second rate. At830, a trigger signal is generated in response to a difference between the first signal and the second signal exceeding a threshold. An acceleration current is output in response to the trigger signal at840. In one example, the first signal is generated with a first temperature sensitive device that exhibits a first leakage current that varies responsive to temperature changes according to the first rate and the second signal is generated with a second temperature sensitive device that exhibits a second leakage current that varies responsive to temperature changes according to the second rate. The trigger signal corresponds to a drain current of a trigger MOS transistor having a gate coupled to the first temperature sensitive device and a drain coupled to the second sensitive device, wherein the trigger MOS transistor enters saturation region in response to a difference between the first leakage current and the second leakage current exceeding a threshold. The first leakage current may be generated with a first plurality of metal oxide semiconductor (MOS) transistors. A body of each MOS transistor in the first plurality conducts a respective portion of the first leakage current and sources of a first number of the MOS transistors in the first plurality conduct respective portions of the first leakage current while sources of a remainder of the first plurality of MOS transistors are open. The second leakage current may be generated with a second plurality of MOS transistors. A body of each MOS transistor in the second plurality conducts a respective portion of the second leakage current. Sources of a second number of the MOS transistors in the second plurality conduct respective portions of the second leakage current while sources of a remainder of the second plurality of MOS transistors are open. In one example the ratio of the first number to the second number is 1 to 6. In one example, a bias voltage is generated with a diode device, wherein the diode device is coupled in series with the first temperature sensitive device and is biased by the first leakage current. The diode device includes a third plurality of MOS transistors coupled in series, wherein each MOS transistor in the third plurality has a gate coupled to the bias voltage. The bias voltage is provided to the gate of trigger MOS transistor. In one example, the bias voltage is adjusted by selectively activating one or more MOS transistors that form shorting paths around respective one or more of the MOS transistors in the third plurality. As described above, providing a temperature dependent acceleration current source enables compensation for rapid changes in temperature dependent performance of electronic devices that occur at a particular temperature within the operating range. The methods are illustrated and described above as a series of acts or events, but the illustrated ordering of such acts or events is not limiting. For example, some acts or events may occur in different orders and/or concurrently with other acts or events apart from those illustrated and/or described herein. Also, some illustrated acts or events are optional to implement one or more aspects or embodiments of this description. Further, one or more of the acts or events depicted herein may be performed in one or more separate acts and/or phases. In some embodiments, the methods described above may be implemented in a computer readable medium using instructions stored in a memory. In the description and in the claims, the terms “including” and “having” and variants thereof are intended to be inclusive in a manner similar to the term “comprising” unless otherwise noted. Unless otherwise stated, “about,” “approximately,” or “substantially” preceding a value means +/−10 percent of the stated value. In another example, “about,” “approximately,” or “substantially” preceding a value means +/−5 percent of the stated value. IN another example, “about,” “approximately,” or “substantially” preceding a value means +/−1 percent of the stated value. The term “couple”, “coupled”, “couples”, and variants thereof, as used herein, may cover connections, communications, or signal paths that enable a functional relationship consistent with this description. For example, if device A generates a signal to control device B to perform an action, in a first example device A is coupled to device B, or in a second example device A is coupled to device B through intervening component C if intervening component C does not substantially alter the functional relationship between device A and device B such that device B is controlled by device A via the control signal generated by device A. Moreover, the terms “couple”, “coupled”, “couples”, or variants thereof, includes an indirect or direct electrical or mechanical connection. A device that is “configured to” perform a task or function may be configured (e.g., programmed and/or hardwired) at a time of manufacturing by a manufacturer to perform the function and/or may be configurable (or re-configurable) by a user after manufacturing to perform the function and/or other additional or alternative functions. The configuring may be through firmware and/or software programming of the device, through a construction and/or layout of hardware components and interconnections of the device, or a combination thereof. Although not all separately labeled in the FIGS., components or elements of systems and circuits illustrated therein have one or more conductors or terminus that allow signals into and/or out of the components or elements. The conductors or terminus (or parts thereof) may be referred to herein as pins, pads, terminals (including input terminals, output terminals, reference terminals, and ground terminals, for instance), inputs, outputs, nodes, and interconnects. As used herein, a “terminal” of a component, device, system, circuit, integrated circuit, or other electronic or semiconductor component, generally refers to a conductor such as a wire, trace, pin, pad, or other connector or interconnect that enables the component, device, system, etc., to electrically and/or mechanically connect to another component, device, system, etc. A terminal may be used, for instance, to receive or provide analog or digital electrical signals (or simply signals) or to electrically connect to a common or ground reference. Accordingly, an input terminal or input is used to receive a signal from another component, device, system, etc. An output terminal or output is used to provide a signal to another component, device, system, etc. Other terminals may be used to connect to a common, ground, or voltage reference, e.g., a reference terminal or ground terminal. A terminal of an IC or a PCB may also be referred to as a pin (a longitudinal conductor) or a pad (a planar conductor). A node refers to a point of connection or interconnection of two or more terminals. An example number of terminals and nodes may be shown. However, depending on a particular circuit or system topology, there may be more or fewer terminals and nodes. However, in some instances, “terminal”, “node”, “interconnect”, “pad”, and “pin” may be used interchangeably. Modifications are possible in the described examples, and other implementations are possible, within the scope of the claims. | 36,043 |
11860184 | DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS FIG.1shows a top view of a micromechanical structure1, including a substrate2and a seismic mass3movable with respect to substrate2. Seismic mass3is connected with the aid of two main springs5to substrate2via an anchoring element52, main springs5being designed as torsion springs. A first direction11and a second direction12essentially perpendicular to first direction11define a main extension plane of substrate2. A third direction13is perpendicular to the main extension plane. Seismic mass3includes a first sub-mass31and a second sub-mass32, the sub-masses31,32being situated on different sides of torsion spring51. During an acceleration of seismic mass3in third direction13, one of the sub-masses31,32moves toward substrate2and the other of the sub-masses32,31moves away from substrate2. First sub-mass31moves toward substrate2and second sub-mass32moves away from substrate2when micromechanical structure1is accelerated in third direction13. First sub-mass31moves away from substrate2and second sub-mass32moves toward substrate2when micromechanical structure1is accelerated opposite to third direction13. FIG.2shows a cross section of micromechanical structure1fromFIG.1at the section line designated as AA′ inFIG.1. Detection means4are provided for detecting a deflection of seismic mass3, detection means4including an electrode structure including first electrodes41mounted at seismic mass3and including second electrodes42mounted at substrate2. First electrodes41and second electrodes42have an essentially two-dimensional extension in first direction11and in second direction12. FIG.3shows a cross section of micromechanical structure1fromFIG.1at the section line designated as BB′ inFIG.1. Moreover, micromechanical structure2has a graduated stop structure6including a first spring stop61, a second spring stop62, and a fixed stop63. First spring stop61and fixed stop63are apparent inFIG.2, while second spring stop62and fixed stop63are apparent inFIG.3. First spring stop61and second spring stop62are situated one behind the other in second direction12. Stop structure6is designed in such a way that, initially, first spring stop61comes into mechanical contact during a movement of at least one portion of seismic mass3in third direction13beyond an operating range; thereafter, second spring stop62comes into mechanical contact during a further movement of at least one portion of seismic mass3in third direction13; and thereafter, fixed stop63comes into mechanical contact during a further movement of at least one portion of seismic mass3in third direction13. At an overload of micromechanical structure1, due to which the operating range is exited, first spring stop61may initially impact. Energy is then stored in first spring stop61, which may subsequently be given off again to seismic mass3. If micromechanical structure1is further loaded, second spring stop62may impact. It may be provided that first spring stop61is further compressed, so that energy is now also stored in first spring stop61as well as in second spring stop62, which may be subsequently given off again to seismic mass3. First spring stop61, second spring stop62, and fixed stop63are situated between seismic mass3and substrate2. In one exemplary embodiment, first spring stop61includes a first stop spring71and second spring stop62includes a second stop spring72. A first spring stiffness of first stop spring71is less than a second spring stiffness of second stop spring72and the first spring stiffness is greater than a third spring stiffness of main spring5, i.e., torsion spring51. First stop spring71and the second stop spring72are situated in the portion of first spring stop61and second spring stop62, respectively, mounted at seismic mass3. In one exemplary embodiment, as shown inFIGS.1through3, main spring5includes a torsion spring51, detection means4being provided for detecting a rotary deflection of seismic mass3about a rotation axis14, rotation axis14being situated in second direction12. In one exemplary embodiment, as shown inFIGS.1through3, fixed stop63has a greater distance to torsion spring51than second spring stop62with respect to first direction11. Moreover, first spring stop61has a greater distance to torsion spring51than second spring stop62with respect to first direction11. In third direction13, a vertical distance of fixed stop63is greater than the vertical distances of fixed stops61,62. Moreover, a first functional layer81, a second functional layer82, and a third functional layer83are shown inFIGS.2and3, substrate2being situated in first functional layer81and seismic mass3, together with torsion spring51, being situated in third functional layer83. Detection means4and graduated stop structure6are situated completely in second functional layer82in this exemplary embodiment. FIG.4shows a cross section of an alternative embodiment of seismic mass1, which corresponds to seismic mass1fromFIGS.1through3, provided that no differences are described in the following. A top view of this micromechanical structure1would correspond toFIG.1. The cross section is guided through section line designated as AA′. First stop spring71is situated in the portion of first spring stop61mounted at the substrate. FIG.5shows a cross section of the alternative embodiment of seismic mass1fromFIG.4at the section line designated as BB′. Second stop spring72is situated in the portion of second spring stop62mounted at the substrate. Alternatively, one of spring stops61,62may also include a stop spring71,72situated at substrate2and one of spring stops61,62may include a stop spring71,72mounted at seismic mass3. InFIGS.2through5, two first spring stops61and two second spring stops62are shown in each case, first spring stops61being situated on various sides of torsion spring51and second spring stops62also being situated on various sides of torsion spring51. This means that a first spring stop61is situated at first sub-mass31and a first spring stop61is situated at second sub-mass32, and a second spring stop62is situated at first sub-mass31and a second spring stop62is situated at second sub-mass32. Only one first spring stop61and only one second spring stop62may also be provided in each case. Fixed stop63is situated only on one side of torsion spring51, at first sub-mass31in each case. Alternatively or additionally, it may also be provided to situate a fixed stop63at second sub-mass32. FIG.6shows a view of a seismic mass3of a micromechanical structure1from below, i.e., as viewed from substrate2. In this exemplary embodiment, stop springs71,72are situated within seismic mass3. Micromechanical structure1fromFIG.6corresponds to above-described micromechanical structures1, provided that no differences are described in the following. FIG.7shows a cross section of micromechanical structure1fromFIG.6at the section line designated as CC′. First stop springs71of first spring elements61are situated within third functional layer83in a first recess33of seismic mass3in each case. A first projection64of first spring stop61projects over the seismic mass. FIG.8shows a cross section of micromechanical structure1fromFIG.6at the section line designated as DD′. Fixed stop63is situated between seismic mass3and substrate2. FIG.9shows a cross section of micromechanical structure1fromFIG.6at the section line designated as EE′. Second stop springs72of second spring elements62are situated within third functional layer83in a second recess34of seismic mass3in each case. A second projection65of second spring stop62projects over the seismic mass. In the exemplary embodiment fromFIGS.6through9, fixed stop63has a smaller distance to torsion spring51than second spring stop62with respect to first direction11. Moreover, second spring stop62has a smaller distance to torsion spring51than first spring stop61with respect to first direction11. Moreover, spring stops61,62and fixed stop63are designed in such a way that the stops, in a rest position, have an identical base distance in each case. Due to this embodiment, it may be achieved that first spring stop61initially comes into mechanical contact during a movement of first sub-mass31or of second sub-mass32toward substrate2, i.e., in third direction13, beyond an operating range; thereafter, the second spring stop62comes into mechanical contact during a further movement; and thereafter, fixed stop63comes into mechanical contact during a further movement. FIG.10shows a top view of a micromechanical structure1, which also includes a substrate2, a seismic mass3, and main springs5. Seismic mass3is connected to substrate2with the aid of main springs5via an anchoring area52. A first direction11and a second direction12essentially perpendicular to first direction11define a main extension plane of substrate2. FIG.11shows a cross section of micromechanical structure1fromFIG.10. Detection means4for detecting a deflection of seismic mass3are not represented, although they may be situated as described above. Micromechanical structure1has a graduated stop structure6including a first spring stop61, a second spring stop62, and a fixed stop63. Stop structure6is designed in such a way that first spring stop61initially comes into mechanical contact during a movement of seismic mass3in a third direction13, which is perpendicular to first direction11and to second direction13, beyond an operating range. Thereafter, second spring stop62comes into mechanical contact during a further movement of seismic mass3in third direction13. Thereafter, fixed stop63comes into mechanical contact during a further movement of seismic mass3in third direction13. In contrast to the embodiments fromFIGS.1through9, the measuring principle of micromechanical structure1fromFIGS.10and11is therefore based on a complete deflection of seismic mass3in third direction13and not on a rotation of seismic mass3about rotation axis14. Moreover, it may be provided in this exemplary embodiment, as represented inFIG.11, that fixed stop63has a greater base distance than second spring stop62with respect to third direction13and second spring stop62has a greater base distance than first spring stop61with respect to third direction13. As a result, initially first spring stop61, then second spring stop62, and then fixed stop63come into mechanical contact during a movement of seismic mass3in third direction13beyond the operating range. FIG.12shows a micromechanical sensor9including one of the described micromechanical structures1and an optional control chip91for evaluating the measuring signals of the detection means. Although the present invention was described with reference to the preferred exemplary embodiments, the present invention is not limited to the described examples and other variations therefore may be derived by those skilled in the art without departing from the scope of protection of the present invention. | 10,942 |
11860185 | DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments of the invention will be described with reference to the drawings. The invention is not limited by the embodiments described below, and modes to which the invention can be applied are not limited to the following embodiments. In the description of the drawings, the same reference numerals are given to the same elements. First Embodiment System Configuration FIG.1is a block diagram illustrating a configuration of a positioning system1000according to a first embodiment. The positioning system1000is used by being equipped on a vehicle and measures a position of the vehicle. As the vehicle, any vehicle may be used as long as it is moving on the ground which is a substantially horizontal surface such as a bicycle, a motorcycle, a four-wheel automobile including a truck and a bus, an agricultural machine such as a tractor, a movable construction machine such as a bulldozer or a shovel car, and there is no particular limitation. As illustrated inFIG.1, the positioning system1000includes a global positioning system (GPS) module1002which is a satellite positioning receiver, an inertial measurement unit (IMU)100which is an inertia measurement device, and a computation unit1004. The GPS module1002receives a GPS satellite signal transmitted from a GPS satellite and measures GPS positioning information including a date and time, a position, a speed, and an attitude represented by latitude and longitude based on the received GPS satellite signal. A satellite positioning receiver may be a receiver of a global navigation satellite system (GNSS), and may be not a GPS but a satellite positioning receiver using other satellite positioning systems such as a global navigation satellite system (GLONASS), GALILEO, a beidou navigation satellite system (Beidou) or the like. The IMU100is a sensor unit including an angular velocity sensor110and an acceleration sensor120. The angular velocity sensor110is a sensor that measures an angular velocity in a sensor coordinate system that is a three-dimensional orthogonal coordinate system associated with the IMU100, and is also referred to as a gyro sensor. The angular velocity sensor110includes an X-axis angular velocity sensor112which detects an angular velocity around the X-axis and outputs the detected angular velocity as a first angular velocity signal, a Y-axis angular velocity sensor114which detects an angular velocity around the Y-axis and outputs the detected angular velocity as a second angular velocity signal, and a Z-axis angular velocity sensor116which detects an angular velocity around the Z-axis and outputs the detected angular velocity as a third angular velocity signal. The acceleration sensor120measures acceleration in the sensor coordinate system which is the same as that of the angular velocity sensor110and is a three-dimensional orthogonal coordinate system associated with the IMU100. The acceleration sensor120includes an X-axis acceleration sensor122which detects acceleration in the X-axis direction and outputs the detected acceleration as a first acceleration signal, a Y-axis acceleration sensor124which detects acceleration in the Y-axis direction and outputs the detected acceleration as a second acceleration signal, and a Z-axis acceleration sensor126which detects acceleration in the Z-axis direction and outputs the detected acceleration as a third acceleration signal. The computation unit1004calculates a position of the positioning system1000using the GPS module1002and measured data of the IMU100. For example, the inertial navigation computation using the measured data of the IMU100is performed to calculate the position of the positioning system1000. When GPS positioning information is output from the GPS module1002, the position calculated by the inertial navigation computation is corrected using the GPS positioning information. This because although positioning information obtained by the GPS module1002is higher in accuracy than the position obtained by the inertial navigation computation using the measured data of the IMU100, an output interval (positioning interval) of the GPS positioning information is longer than an output interval of the measured data of the IMU100. For example, the IMU100outputs measured data every 10 milliseconds, and the GPS module1002outputs GPS positioning information every one second. Sensor Coordinate System The positioning system1000is fixedly equipped on the vehicle so that a sensor coordinate system which is a three-dimensional orthogonal coordinate system associated with the IMU100satisfies a predetermined relationship with respect to a direction of the vehicle.FIG.2is a diagram for explaining a relationship between the sensor coordinate system and a moving direction of a vehicle1100.FIG.2illustrates an example in which the positioning system1000is equipped on a four-wheel automobile which is an example of the vehicle1100. The X-axis of the sensor coordinate system is the front and rear direction of the vehicle1100, and the forward direction (straight advancing direction) is the positive direction of the X-axis. Further, the Y-axis of the sensor coordinate system is the left and right direction of the vehicle1100, and the right direction is the positive direction of the Y-axis. The Z-axis of the sensor coordinate system is a direction orthogonal to the X-axis and the Y-axis, and the down direction of the vehicle1100is the positive direction of the Z-axis. In the first embodiment, since the vehicle1100moves in a substantially horizontal plane, the XY-plane becomes a moving plane of the vehicle, and a Z-axis positive direction of the Z-axis matches the direction of gravity. The attitude of the vehicle1100is represented by a roll angle around the X-axis, a pitch angle around the Y-axis, and a yaw angle around the Z-axis. Further, since the vehicle1100moves in a substantially horizontal plane, the roll angle which is the attitude corresponds to inclination in the left and right direction of the vehicle1100, the pitch angle corresponds to inclination in the front and rear direction of the vehicle1100, the yaw angle corresponds to conversion or an azimuth in the moving direction of the vehicle1100. Angular Velocity Sensor As the characteristics of the first embodiment, the angular velocity sensor110is configured so that a bias error (output error at stationary condition) B and the Allan variance BIS representing bias stability have the following characteristics. That is, the Z-axis angular velocity sensor116is configured to have “higher accuracy” than the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114. (A) Bias Error The X-axis angular velocity sensor112, the Y-axis angular velocity sensor114, and the Z-axis angular velocity sensor116are configured so that bias errors of Bx [deg/sec], By [deg/sec], and Bz [deg/sec] satisfy the following expressions (1a) to (1c) when the Bx [deg/sec] is a bias error of an output signal of the X-axis angular velocity sensor112, the By [deg/sec] is a bias error of an output signal of the Y-axis angular velocity sensor114, and the Bz [deg/sec] is a bias error of the output signal of the Z-axis of the Z-axis angular velocity sensor116. P≤(V/Bz)×(1−cos(Bz×T) (1a) Bz<Bx(1b) Bz<By(1c) In the expression (1a), “T” is a positioning interval (second) of the GPS module1002, and is an interval at which a positioning result is output from the GPS module1002to the computation unit1004. “P” is a position error [m] caused by a bias error B of the output signal of the angular velocity sensor110while a vehicle1100moves for T seconds at the moving speed V [m/sec]. FIG.3is a diagram for explaining a position error P.FIG.3is a view in which the mobile object1100is viewed from above, that is, illustrates a view of the XY-plane in the sensor coordinate system. The actual movement direction is indicated by the solid line as an original movement direction. Since the forward direction of the vehicle1100is the positive direction of the X-axis, the actual moving direction is also the positive direction of the X-axis. It is assumed that a position M1of the vehicle1100at the time t1is known and set a position of the vehicle1100at the time t2as “M2”, which is the position calculated by the inertial navigation computation using the measured data of the IMU100. The distance between the position M2and the original advancing direction is the position error P caused by the bias error B of the output signal of the angular velocity sensor110while the vehicle1100moves from the time t1to the time t2. Although the position calculated by the inertial navigation computation based on the measured data of the IMU100deviates from the original moving direction, this positional deviation is caused by an error in the attitude based on the measured data of the IMU100. Specifically, in the first embodiment, since the vehicle1100moves in a substantially horizontal plane, the positional deviation is caused by an error in the yaw angle which is an attitude. In the inertial navigation computation, the attitude is calculated by integrating the angular velocity which is the output signal of the angular velocity sensor110with respect to time. That is, a roll angle around the X-axis calculated by integrating the output signal of the X-axis angular velocity sensor112with respect to time, and a pitch (Pitch) angle around the Y-axis is calculated by integrating the output signal of the Y-axis angular velocity sensor114with respect to time, and the yaw angle around the Z-axis calculated by integrating the output signal of the Z-axis angular velocity sensor116. However, since the output signal of the angular velocity sensor110includes the bias error B, the calculated attitude also includes an error. Further, since the output signal of the angular velocity sensor110is integrated with respect to time, the error included in the calculated attitude increases with the lapse of time. On the other hand, the attitude can also be calculated from the output signal of the acceleration sensor120. Specifically, the acceleration sensor120constantly detects gravitational acceleration G. That is, the X-axis acceleration sensor122detects an X-axis direction component of gravitational acceleration G, the Y-axis acceleration sensor124detects a Y-axis direction component of the gravitational acceleration G, and the Z-axis acceleration sensor126detects a Z-axis direction component of the gravitational acceleration G. For that reason, by combining gravitational acceleration components of the respective axes based on the output signals of the respective axes of the acceleration sensor120at a stationary state, it is possible to calculate the direction of the gravitational acceleration G with respect to the sensor coordinate system, that is, the attitude of the vehicle1100which is the attitude of the sensor coordinate system in real space. Since a bias error is included also in the output signal of the acceleration sensor120, an error is included also in the attitude calculated from the output signal of the acceleration sensor120. However, the error included in the attitude calculated from the output signal of the acceleration sensor120is not an error which increases with the lapse of time but is a stable error with time. Accordingly, by complementarily using the attitude calculated from the output signal of the acceleration sensor120, it is possible to calculate the attitude with stable accuracy with time regardless of the bias error B of the output signal of the angular velocity sensor110. However, as in the first embodiment, in the case where the vehicle1100moves on a substantially horizontal plane, the Z-axis of the sensor coordinate system is substantially coincident with the gravitational acceleration direction. For that reason, even if the yaw angle of the vehicle1100changes, the Z-axis direction component of the gravitational acceleration G hardly changes. Accordingly, with respect to the yaw angle, supplementation by the output signal of the acceleration sensor120is almost impossible or extremely difficult. As illustrated inFIG.3, in a case where the vehicle1100moves on a substantially horizontal plane, positional deviation calculated by the inertial navigation computation using the measured data of the IMU100is caused by the error of the yaw angle which is the attitude. Although the yaw angle is calculated from the output signal of the Z-axis angular velocity sensor116, as the bias error Bz of the output signal of the Z-axis angular velocity sensor116is integrated, the error of the yaw angle increases with the lapse of time. FIG.4is a diagram for explaining calculation of the position by the inertial navigation computation. It is assumed that the vehicle1100linearly moves on a substantially horizontal plane at the moving speed V [m/sec] Further, the bias error of the Z-axis angular velocity sensor116is assumed as Bz [deg/sec]. When the position of the vehicle1100is calculated by inertial navigation computation using the measured data of the IMU100assuming that the position M1of the vehicle1100at the time t1is known, since the error of the yaw angle which is the attitude increases with the lapse of time, a trajectory of the position draws a locus gradually moving away from the original moving direction which is the linear direction. In the inertial navigation computation, it is repeated to obtain the yaw angle from the output signal of the Z-axis angular velocity sensor116and calculate the position at the next time t assuming that the vehicle has moved at the moving speed V in the direction of the yaw angle are repeated, at each predetermined minute time Δt. The error Δθ[deg] of the yaw angle occurring during the minute time Δt[second] is expressed by the following expression (2). Δθ=Bz×Δt(2) Then, the position error Δp [m] occurring during the minute time Δt[second] is expressed by the following expression (3). Δp=V×T×sin Δθ (3) By integrating the position error Δp from the time t1to the time t2(=t1+T) after T seconds with respect to time, the position error P[m] that occurs when moving at the moving speed V[m/sec] during the T seconds from the time t1to the time t2is obtained by the following expression (4). P=(V/Bz)×(1−cos(Bz×T) (4) As described above, the position calculated by the inertial navigation computation can be corrected by using high accurate GPS positioning information output at each positioning interval T from the GPS module1002. For that reason, it is sufficient that the position error P occurring during the positioning interval T is at least equal to or less than the predetermined allowable maximum position error Pp. The extent of the allowable maximum position error Pp is determined depending on the purpose of use of the vehicle1100and the like. Accordingly, the expression (1a) expressing that the position error P calculated by the expression (4) becomes equal to or less than the predetermined allowable maximum position error Pp is a conditional expression to be satisfied by the bias error Bz of the Z-axis angular velocity sensor116. Since the bias error Bx of the X-axis angular velocity sensor112and the bias error By of the Y-axis angular velocity sensor114can be supplemented by the output signal of the acceleration sensor120as described above, accuracy of the bias errors Bx and By may be lower than that of the bias error Bz of the Z-axis angular velocity sensor116. However, since it is undesirable to have an accuracy as low as to detect that the vehicle1100in the horizontal state has rolled over, the bias error Bx of the X-axis angular velocity sensor112and the bias error By of the Y-axis angular velocity sensor114are conditional on satisfying the following expressions (5a) and (5b). T×Bx<90[degrees] (5a) T×By<90[degrees] (5b) Subsequently, consider specific examples of the bias errors Bx, By, and Bz of the angular velocity sensor110. Specifically, as a vehicle, assume an agricultural machine that is controlled to be automatically operated, a movable construction machine, and a transportation work vehicle. In recent years, in order to realize automatic operation control in the agricultural machine, the construction machine, and the like, accuracy of centimeter order is required for the measurement position of the positioning system1000equipped on the agricultural machine, the construction machine, and the like. That is, in a case of automatic operation control of the agricultural machine such as a rice planting machine, the construction machine such as a shovel car, the transportation work vehicle such as a forklift truck, and the like, although the moving speed is a low speed of about 15 km/h or less, the accuracy of the position is very important from the purpose of its use. The bias errors Bx, By, and Bz of the angular velocity sensor110necessary for responding to this positional accuracy requirement are obtained. That is, the error of the yaw angle which is the attitude and a position error caused by the error of this yaw angle were calculated assuming that the moving speed V of the vehicle1100is 15 [km/h], which is the general moving speed of agricultural machine, the construction machine, and the transportation work vehicle. For calculation, seven types of Z-axis angular velocity sensors116having bias errors Bz of 0[deg/hour], 360[deg/hour], 570[deg/hour], 760[deg/hour], 890[deg/hour], 1010[deg/hour], and 1140[deg/hour], respectively, were assumed. FIG.5is a graph illustrating a relationship between the elapsed time and an error of the yaw angle which is the attitude, andFIG.6is a graph illustrating a relationship between the elapsed time and a position error. The position error is obtained from the bias error Bz and the moving speed V according to the expression (4). As illustrated inFIG.5, since the yaw angle is obtained by integrating the output signal of the Z-axis angular velocity sensor116with respect to time, the error of the yaw angle increases in proportion to the elapsed time. Also, even if the elapsed time is the same, the larger the bias error Bz, the larger the error of the yaw angle. Accordingly, as illustrated inFIG.6, the position error also increases as the elapsed time increases. Even if the elapsed time is the same, as the bias error Bz increases, the error of the yaw angle increases, so that the position error also increases. Generally, the positioning interval T of the GPS module1002is 1 [second]. This positioning interval T is a time interval during which GPS positioning information is not output and corresponds to a period of time during which position correction based on GPS positioning information cannot be performed. In order to allow the position accuracy to satisfy accuracy of centimeter order, it is necessary to set the position error during the positioning interval T to 10 cm or less. Accordingly, according toFIG.6, in order to make the elapsed time equal to or less than 100 mm (=10 cm) at the time point of 1 [second], which is the positioning interval T of the GPS module1002, the bias error Bz of the Z-axis angular velocity sensor116needs to be 570[deg/hour] or less. There is no problem even if the bias error Bx of the X-axis angular velocity sensor112and the bias error By of the Y-axis angular velocity sensor114exceed 1140[deg/hour] greater than the 570[deg/hour]. That is, the bias error Bz of the Z-axis angular velocity sensor116needs to be smaller than the bias error Bx of the X-axis angular velocity sensor112and the bias error By of the Y-axis angular velocity sensor114. Specifically, the bias error Bz is sufficient to satisfy the following expressions (6a) to (6c)). Bx>1140[deg/hour] (6a) By>1140[deg/hour] (6b) Bz<570[deg/hour] (6c) According to this, it can be said that it is desirable that the bias error Bz is desirably 50% or less (0.5≈570/1140) of the bias errors Bx and By as illustrated in the following expressions (7a) and (7b). Bz<0.5×Bx(7a) Bz<0.5×By(7b) (B) Bias Stability (Allan Variance) Next, it is assumed that the vehicle equipped with the positioning system1000is, for example, an agricultural machine or a construction machine of which operation is automatically controlled. It is assumed that the agricultural machine and the construction machine are used in an environment where GPS satellite signals cannot be received due to multipath and the like by surrounding buildings, forests, and the like. Matters that that accuracy related to long-term stability of the required measuring position can be secured even in such an environment will be described below. Specifically, the X-axis angular velocity sensor112, the Y-axis angular velocity sensor114, and the Z-axis angular velocity sensor116are configured so that the Allan variances of BISx[deg/hour], BISy[deg/hour], and BISz[deg/hour] satisfy the following expressions (8a) and (8b) when the BISx[deg/hour] is the Allan variance of an output signal of the X-axis angular velocity sensor112, the BISy[deg/hour] is the Allan variance of an output signal of the Y-axis angular velocity sensor114, and the BISz[deg/hour] is the Allan variance of the output signal of the Z-axis of the Z-axis angular velocity sensor116. BISz<0.5×BISx (8a) BISz<0.5×BISy (8b) Since 1/f noise (fluctuation) that determines bias stability is not modeled in estimation means of the bias error B such as the Kalman filter, that is, the 1/f noise is not eliminated, this causes an increase in an attitude error due to accumulation of bias errors over a long period of time. FIG.7is a graph illustrating an example of an Allan variance curve, and illustrates three types of angular velocity sensors having different characteristics. In general, the Allan variance σ draws a curve in which it decreases as the time constant (averaging time) t increases, and then converges to a fixed value. This fixed value is taken as the Allan variance BIS representing bias stability of the first embodiment. In the example ofFIG.7, the Allan variance BIS is exemplified for three types of angular velocity sensors with the Allan variance BIS being 2.5[deg/hour], 5[deg/hour], and 10[deg/hour]. Subsequently, consider specific examples of Allan variance BISx, BISy, and BISz of the angular velocity sensor110. Similarly to the consideration of the specific example of the bias error Bz, assume the agricultural machine, the construction machine, and the transportation work vehicle of which operations are automatically controlled as the vehicle. Then, the error of the yaw angle which is the attitude and the position error is calculated assuming that the moving speed V of the vehicle is 15 [km/h], which is a high speed when the agricultural machine, the construction machine, and the transportation work vehicle perform a work by automatic operation. For the calculation, three types of Z-axis angular velocity sensors116with the Allan variance BISz being 2.5[deg/hour], 5[deg/hour], and 10[deg/hour] were assumed. FIG.8is a graph illustrating a relationship between the elapsed time and the error of the yaw angle which is the attitude, andFIG.9is a graph illustrating a relationship between the elapsed time and the position error. As illustrated inFIG.8, the error of the yaw angle increases as the elapsed time increases. Also, even if the elapsed time is the same, the larger the Allan variance BISz is, the larger the error of the yaw angle becomes. Accordingly, as illustrated inFIG.9, the position error also increases as the elapsed time increases, and even if the elapsed time is the same, the larger the Allan variance BISz is, the larger the position error becomes. The position error due to Allan variance becomes a problem in a case where the bias error B of the angular velocity sensor110is accumulated over a relatively long period of time. Matters that the assumed agricultural machine and construction machine are used in an environment in which GPS satellite signals due to multipath or the like by surrounding buildings, forests or the like cannot be received or reception signals are weak signals, and matters that the work transportation vehicle is used indoors where it cannot receive the GPS satellite signal are assumed. Therefore, consider securing a position error of approximately 15 [cm] that can be determined as an excessive error for automatic operation in a case where a GPS satellite signal is not received for 30 seconds. According toFIG.9, in order to make the position error 15 cm (=150 mm) or less at the time when the elapsed time is 30 seconds, the Allan variance BISz of the Z-axis angular velocity sensor116needs to be 2.5[deg/hour]. Then, the Allan variance BISx of the X-axis angular velocity sensor112and the Allan variance BISy of the Y-axis angular velocity sensor114do not cause a problem even if the Allan variances BISx and BISy are 5[deg/hour] or more which is larger than 2.5 [deg/hour]. That is, the Allan variance BISz of the Z-axis angular velocity sensor116needs to be smaller than the Allan variance BISx of the X-axis angular velocity sensor112and BISy of the Y-axis angular velocity sensor114. Specifically, the following expressions (9a) to (9c) may be satisfied. BISx≥5[deg/hour] (9a) BISy≥5[deg/hour] (9b) BISz≤2.5[deg/hour] (9c) According to this, as illustrated in the expressions (8a) and (8b) described above, it can be said that the Allan variance BISz is desirably 50% (0.5=2.5/5) or less of the Allan variances BISx and BISy. Operational Effect As such, in the first embodiment, the Z-axis angular velocity sensor116is configured such that the bias error B and the Allan variance BIS are smaller than those of the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114. That is, the Z-axis angular velocity sensor116is configured to have “higher accuracy” than the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114. Specifically, the Z-axis angular velocity sensor116has the bias error Bz which is smaller than the bias errors Bx and By of the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114and is a value that makes the position error during the movement of the vehicle mounted with the positioning system1000for T[seconds], which is the positioning interval of the GPS module1002, equal to or less than the predetermined allowable maximum position error Pp. With this configuration, the position error due to the positioning system1000can be made to be equal to or less than the allowable maximum position error Pp. The Allan variance BISz of the Z-axis angular velocity sensor116is smaller than the Allan variances BISxz and BISy of the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114. With this configuration, even if the GPS satellite signal cannot be received for a predetermined period of time, it is possible to set the position error due to the positioning system1000within the predetermined allowable range. Second Embodiment Next, a second embodiment will be described. Hereinafter, differences from the first embodiment will be mainly described, and the same reference numerals are given to the same constituent elements as in the first embodiment, and redundant description thereof will be omitted. The second embodiment is an embodiment of a sensor unit that is the IMU100in the first embodiment. Outline of Sensor Unit FIG.10is a perspective view for explaining a state of fixing a sensor unit160to a mounted surface19according to a second embodiment.FIG.11is a perspective view of the sensor unit160when viewed from the mounted surface19side ofFIG.10. First, an outline of the sensor unit160according to the second embodiment will be described. InFIGS.10and11, the sensor unit160is an inertial measurement unit (IMU) which is an inertia measurement device and detects the attitude and behavior (inertial momentum) of a moving body (mounted device) such as a car or a moving body such as a robot. The sensor unit160includes a plurality of inertial sensors, for example, a triaxial acceleration sensor120which measures acceleration acting in directions of three axes orthogonal to each other and a three-axis angular velocity sensor110which measures an angular velocity acting around each axis. The sensor unit160is a rectangular parallelepiped having a planar shape of a rectangle, and includes screw holes2as fixing portions formed in the vicinity of two apexes positioned in the diagonal direction of the rectangle. The sensor unit160is used in a state of being fixed on the mounted surface19of a mounted object (device) such as an automobile through two screws5in the two screw holes2. The shape described above is an example, and it is possible to reduce the size of the sensor unit to a size that can be mounted on, for example, various wearable electronic devices, smart phones, digital cameras, and the like by selection of parts and design change. As illustrated inFIG.11, an opening4is formed on the surface of the sensor unit160when viewed from the mounted surface side. A plug type (male) connector10is disposed inside (inside) the opening4. In the connector10, a plurality of pins are arranged side by side. A connector (not illustrated) of a socket type (female) is connected to the connector10from the mounted device, and transmission and reception of electrical signals such as power supply to the sensor unit160and output of detection data detected by the sensor unit160are performed between the sensor unit160and the mounted device. Configuration of Sensor Unit FIG.12is an exploded perspective view of the sensor unit160when viewed in the same direction asFIG.11. Subsequently, a configuration of the sensor unit160will be described in detail with reference mainly toFIG.12while appropriately combiningFIG.10andFIG.11. As illustrated inFIG.12, the sensor unit160is configured with an outer case1, an annular cushioning material6, a sensor module7, and the like. In other words, a configuration in which the sensor module7is mounted on the inside3of the outer case1with an annular cushioning material6interposed therebetween is adopted. The sensor module7is configured with an inner case8and a circuit board9. In order to make the description easier to understand, although the outer case and the inner case are used as the part names, the outer case and the inner case may be referred to as a first case and a second case. The outer case1is a pedestal from which aluminum is cut out into a box shape. The material thereof is not limited to aluminum, but other metals such as zinc and stainless steel, a resin, a composite material of a metal and a resin, or the like may be used. The outer shape of the outer case1is a rectangular parallelepiped having a planar shape of a rectangle similarly to the overall shape of the sensor unit160described above, and the screw holes2are respectively formed in the vicinity of two apexes positioned in the diagonal direction of the square. An example in which the outer shape of the outer case1is a rectangular parallelepiped having a planar shape of a rectangle and a box shape without a lid is described, but is not limited thereto. The planar shape of the outer shape of the outer case1may be a polygon such as a hexagon or an octagon, and corners of the apex portion of the polygon may be chamfered, each side may be curved, or the outer shape may be circular. Configuration of Circuit Board FIG.13is a perspective view of the circuit board9. The configuration of the circuit board9on which a plurality of inertial sensors are mounted will be described below. The circuit board9is a multilayer substrate having a plurality of through-holes formed therein, and a glass epoxy substrate is used. The invention is not limited to the glass epoxy substrate, but may be a rigid substrate capable of mounting a plurality of inertial sensors, electronic components, connectors and the like. For example, a composite substrate or a ceramic substrate may be used. On the surface of the circuit board9(surface on the side of the inner case8), a connector10, a multi-axis inertial sensor17in which three-axis angular velocity sensors and three-axis acceleration sensors are accommodated, and a high-accuracy angular velocity sensor18, and the like are installed. The connector10is a plug type (male) connector, and includes two rows of connection terminals in which a plurality of pins are disposed at an equal pitch. The number of terminals may be appropriately changed according to design specifications. The high-accuracy angular velocity sensor18is a gyro sensor for detecting an angular velocity of one axis in the Z-axis direction which is the direction of gravity. In the vehicle on which the sensor unit160is mounted, when a preset straight advancing direction of the vehicle is set as an X-axis, a gravitational direction of the vehicle is set as a Z-axis, and an axis orthogonal to the X-axis and the Z-axis set as a Y-axis, the sensor unit160functions as a Z-axis angular velocity sensor which detects an angular velocity around the Z-axis and outputs an angular velocity signal around the Z-axis, and calculates the yaw (YAW) angle around the Z-axis of the vehicle based on the angular velocity signal around the Z-axis. As a preferable example of the high-accuracy angular velocity sensor18, a resonance frequency change type crystal gyro sensor in which quartz crystal is used as a material and which measures an angular velocity from a Coriolis force applied to a vibrating object is used. Further, the high-accuracy angular velocity sensor18is not limited to the quartz crystal-crystal gyro sensor, but may be a multi-gyro sensor in which a plurality of electrostatic capacitance change type silicon-micro electro mechanical systems (Si-MEMS) angular velocity sensors are connected in a multi-connected manner. The multi-axial inertial sensor17includes an X-axis angular velocity sensor112which detects an angular velocity around the X-axis and outputs a first angular velocity signal, a Y-axis angular velocity sensor114which detects an angular velocity around the Y-axis and outputs a second angular velocity signal, a Z-axis angular velocity sensor116which detects an angular velocity around the Z-axis and outputs a third angular velocity signal, an X-axis acceleration sensor122which detects acceleration in the X-axis direction and outputs a first acceleration signal, a Y-axis acceleration sensor124which detects acceleration in the Y-axis direction and outputs a second acceleration signal, and a Z-axis acceleration sensor126which detects acceleration in the Z-axis direction, and outputs a third acceleration signal. Here, since the high-accuracy angular velocity sensor18installed on the circuit board9by itself functions as the Z-axis angular velocity sensor116which detects the angular velocity around the Z-axis and outputs the angular velocity signal around the Z-axis, the Z-axis angular velocity sensor is not necessarily mounted, for the multi-axis inertial sensor17. In the case where the Z-axis angular velocity sensor is mounted on the multi-axis inertial sensor17, the high-accuracy angular velocity sensor18and the Z-axis angular velocity sensor may share the functional role as appropriate according to design specifications and the like. As the acceleration sensor120mounted on the multi-axis inertial sensor17, an electrostatic capacitance change type Si-MEMS acceleration sensor (acceleration sensor) capable of measuring (detecting) acceleration in the X-axis direction, the Y-axis direction, and the Z-axis direction with one device (one chip) is used. That is, the acceleration sensor120mounted on the multi-axis inertial sensor17includes the X-axis acceleration sensor122which detects acceleration in the X-axis direction and outputs the first acceleration signal, and the Y-axis acceleration sensor124which detects acceleration in the Y-axis direction and outputs the second acceleration signal, and the Z-axis acceleration sensor126which detects acceleration in the Z-axis direction and outputs the third acceleration signal. The acceleration sensor120is not limited to this capacitance change type Si-MEMS acceleration sensor, and it suffices if the acceleration sensor120is a sensor capable of detecting acceleration. For example, the acceleration sensor120may be a frequency change type quartz crystal acceleration sensor, a piezo-resistive type acceleration sensor, or a heat detection type acceleration sensor, or the acceleration sensor120may have a configuration in which one acceleration sensor is provided for each axis like the high-accuracy angular velocity sensor18described above. A control IC11is mounted on the back surface (surface on the outer case1side) of the circuit board9. The control IC11is a micro controller unit (MCU), and incorporates a storing unit including a nonvolatile memory, an A/D converter, and the like and controls each unit of the sensor unit160. In the storing unit, a program defining the sequence and contents for detecting acceleration and angular velocity, a program for digitizing detected data to be incorporated into packet data, accompanying data, and the like are stored. On the circuit board9, a plurality of other electronic components are mounted. Third Embodiment Next, a third embodiment will be described. Hereinafter, differences from the first and second embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those in the first and second embodiments, and redundant description will be omitted. The third embodiment is an embodiment of a quartz crystal gyro sensor element which is the high-accuracy angular velocity sensor18in the second embodiment. Configuration of High Accuracy Angular Velocity Sensor In the high-accuracy angular velocity sensor18of the third embodiment, by adopting quartz crystal (SiO2) as the material, a high Q value is obtained from high crystallinity of the quartz crystal and characteristics in which impedance characteristic and frequency temperature characteristic are stable over a wide temperature range can be exhibited. In general, the Q value of the quartz crystal is approximately 30,000, whereas the Q value of the Si-MEMS constituting the capacitance type angular velocity sensor element is approximately 5,000, which is extremely low and is one-sixth of the Q value of quartz. Since the Q value of quartz crystal is much higher than the Q value of Si-MEMS, in the high-accuracy angular velocity sensor18made of quartz crystal, vibration characteristics in which the amplitude is large even at a drive voltage lower than the drive voltage of Si-MEMS and noise is small are obtained and thus, excellent characteristics having a large S/N ratio compared with that of the Si-MEMS are obtained. FIG.14is a schematic plan view illustrating a quartz crystal gyro sensor element200of the third embodiment. The quartz crystal gyro sensor element200is formed using quartz crystal, which is a piezoelectric material, as a base material. The quartz crystal has an X-axis called an electric axis, a Y-axis called a mechanical axis, and a Z-axis called an optical axis. The quartz crystal gyro sensor element200is cut along a plane defined by the X-axis and Y-axis orthogonal to the quartz crystal axis and processed into a flat plate shape, and has a predetermined thickness in the Z-axis direction orthogonal to the plane. The predetermined thickness is appropriately set depending on an oscillation frequency (resonance frequency), external size, workability, and the like. It should be noted that the X-axis, the Y-axis, and the Z-axis described in the third embodiment indicate the electric axis, the mechanical axis, and the optical axis which are crystal axes of quartz crystal, and have different meanings from the X-axis, the Y-axis, and the Z-axis in the sensor coordinate system which is a three-dimensional orthogonal coordinate system associated with the IMU100described in the first embodiment. Further, the flat plate constituting the quartz crystal gyro sensor element200can allow an error of a cutting angle from the quartz crystal to some extent for each of the X-axis, the Y-axis, and the Z-axis. For example, it is possible to use the flat plate which is rotated in a range of 0 degrees to 2 degrees around the X-axis and cut out. This also applies to the Y-axis and the Z-axis. The crystal gyro sensor element200is formed by etching (wet etching or dry etching) using the photolithography technique. A plurality of crystal gyro sensor elements200can be taken out from one quartz crystal wafer. As illustrated inFIG.14, the quartz crystal gyro sensor element200has a so-called double T type configuration. The quartz crystal gyro sensor element200includes a base portion210positioned at the center portion and a pair of detection vibration arms211aand211bone of which extending linearly along the Y-axis from the base portion210in the plus direction of the Y-axis and the other of which extending linearly along the Y-axis from the base portion210in the minus direction of the Y-axis. Further, the quartz crystal gyro sensor element200includes a pair of connection arms213aand213bone of which extends linearly along the X-axis from the base portion210in the plus direction of the X-axis and the other of which extends linearly along the X-axis from the base portion210in the minus direction of the X-axis so as to be orthogonal to the detection vibration arms211aand211b. Further, the quartz crystal gyro sensor element200includes a pair of drive vibration arms214aand214band a pair of drive vibration arms215aand215b, and in the pair of drive vibration arms214aand214b, one of the drive vibration arms extends linearly along the Y-axis from the tip end side of a connection arms213ain the plus direction of the Y-axis and the other of the drive vibration arms extends linearly along the Y-axis from the tip end side of the connection arms213ain the minus direction of the Y-axis so as to be parallel to the detection vibration arm211a, and in the pair of drive vibration arms215aand215b, one of the drive vibration arms extends linearly along the Y-axis from the tip end side of a connection arm213bin the plus direction of the Y-axis and the other of the drive vibration arms extends linearly along the Y-axis from the tip end side of the connection arm213bin the minus direction of the Y-axis so as to be parallel to the detection vibration arm211b. In the crystal gyro sensor element200, detection electrodes (not illustrated) are formed on the detection vibration arms211aand211band drive electrodes (not illustrated) are formed on drive vibration arms214a,214b,215a, and215b. In the quartz crystal gyro sensor element200, a detecting vibration system for detecting the angular velocity is configured with the detection vibration arms211aand211band a drive vibration system for driving the quartz crystal gyro sensor element200is configured with the connection arms213aand213band the drive vibration arms214a,214b,215a, and215b. In addition, weight portions212aand212bare formed at the tip end portions of the detection vibration arms211aand211b, respectively, and weight portions216a,216b,217a, and217bare formed at the tip end portions of the drive vibration arms214a,214b,215a, and215b, respectively. With this configuration, the quartz crystal gyro sensor element200is reduced in size and improved in detection sensitivity of angular velocity. The detection vibration arms211aand211binclude the weight portions212aand212b, and the drive vibration arms214a,214b,215a,215binclude weight portions216a,216b,217a, and217b. Furthermore, in the crystal gyro sensor element200, four beams220a,220b,221a, and221bextend from the base portion210. The beam220aextends from the outer edge of the base portion210between the connection arm213aand the detection vibration arm211a. The beam220bas a first beam extends from the outer edge of the base portion210between the connection arm213bpositioned on the plus side from the base portion210in the X-axis direction and the detection vibration arm211apositioned on the plus side from the base portion210in the Y-axis direction. The beam221aextends from the outer edge of the base portion210between the connection arm213aand the detection vibration arm211b. The beam221bas a second beam extends from the outer edge of the base portion210between the connection arm213bpositioned on the plus side from the base portion210in the X-axis direction and the detection vibration arm211bpositioned on the plus side from the base portion210in the Y-axis direction. The beam220bis configured to include a first folded portion220cincluding a first extending portion220b1extending from the base portion210along the X-axis in the plus direction of the X-axis, a second extending portion220b2extending from the tip end portion of the first extending portion220b1along the Y-axis in the plus direction of the Y-axis, and a third extending portion220b3extending from the tip end portion of the second extending portion220b2along the X-axis in the minus direction of the X-axis. The beam221bis configured to include a second folded portion221cincluding a fourth extending portion221b1extending from the base portion210along the X-axis in the plus direction of the X-axis, a fifth extending portion221b2extending from the tip end portion of the fourth extending portion221b1along the Y-axis in the minus direction of the Y-axis, and a sixth extending portion221b3extending from the tip end portion of the fifth extending portion221b2along the X-axis in the minus direction of the X-axis. Each of the beams220a,220b,221a, and221bof the crystal gyro sensor element200is rotationally symmetric with respect to the center of gravity G of the quartz crystal gyro sensor element200. Specifically, the beam220aand the beam221bare in a rotationally symmetrical shape with the center of gravity G of the quartz crystal gyro sensor element200as the rotation center, and the beam221aand the beam220bare in a rotationally symmetrical shape around the center of gravity G of the quartz crystal gyro sensor element200. With this configuration, a folded portion220dwhich is in a rotationally symmetric shape with respect to the second folded portion221cis formed in the beam220a, and a folded portion221dwhich is in a rotationally symmetric shape with respect to the first folded portion220cis formed in the beam221a. The tip end portions of the beams220aand220bare connected to a support portion222positioned on the plus side from the detection vibration arm211ain the Y-axis direction and extending along the X-axis, and the tip end portions of the beams221aand221bare connected to a support portion223positioned on the minus side from the detection vibration arm211bin the Y-axis direction and extending along the X-axis. It is preferable from the viewpoint of balance that the support portion222and the support portion223are in a rotationally symmetrical shape with the center of gravity G of the quartz crystal gyro sensor element200as the rotation center. The quartz crystal gyro sensor element200is supported by the support portions222and223being fixed to a support table or the like which will be described later. Fourth Embodiment Next, a fourth embodiment will be described. Hereinafter, differences from the first to third embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those in the first to third embodiments, and redundant description thereof will be omitted. The fourth embodiment is an embodiment of a physical quantity sensor which is the high-accuracy angular velocity sensor18in the second embodiment. A gyro sensor (physical quantity sensor)300as an example of the electronic device illustrated inFIGS.15,16, and17includes a quartz crystal gyro sensor element32as a function element for detecting the angular velocity, a first support base material39aand a second support base material39bconstituting a support portion for supporting the quartz crystal gyro sensor element32, and a package35for collectively accommodating support portions divided into the quartz crystal gyro sensor element32, the first support base material39a, and the second support base material39b. The quartz crystal gyro sensor element32is, for example, the quartz crystal gyro sensor element200in the third embodiment. The package35includes a base36and a lid37joined to the base36. Fifth Embodiment Next, a fifth embodiment will be described. Hereinafter, differences from the first to fourth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to fourth embodiments, and redundant description thereof will be omitted. The fifth embodiment is an embodiment of a physical quantity sensor which is the high-accuracy angular velocity sensor18in the second embodiment. A physical quantity sensor400illustrated inFIG.18is a Si-MEMS type angular velocity sensor element capable of measuring an angular velocity Oz around the Z-axis. As illustrated inFIG.18, a shape of an element portion404is symmetrical with respect to an imaginary straight line α. The element portion404includes drive portions41A and41B disposed on both sides of the imaginary straight line α. The drive portion41A includes a tooth-shaped movable drive electrode411A and a fixed drive electrode412A which is formed in a tooth shape and disposed to be in mesh with the movable drive electrode411A. Similarly, the drive portion41B includes a tooth-shaped movable drive electrode411B and a fixed drive electrode412B which is formed in a tooth shape and disposed to be in mesh with the movable drive electrode411B. The fixed drive electrode412A is positioned outside (farther from the imaginary straight line a) than the movable drive electrode411A, and the fixed drive electrode412B is positioned outside (side farther from the imaginary straight line α) than the movable drive electrode411B. Each of the fixed drive electrodes412A and412B is joined to the upper surface of a mount221and is fixed to the substrate402. Each of the movable drive electrodes411A and411B is electrically connected to a wiring73and each of the fixed drive electrodes412A and412B is electrically connected to a wiring74. In addition, the element portion404includes four fixed portions42A disposed around the drive portion41A and four fixed portions42B disposed around the drive portion41B. Each of the fixing portions42A and42B is joined to the upper surface of the mount and is fixed to the substrate402. The element portion404includes four drive springs43A connecting the fixed portions42A and the movable drive electrode411A and four drive springs43B connecting the fixed portions42B and the movable drive electrode411B. The drive springs43A are elastically deformed in the X-axis direction so that displacement of the movable drive electrode411A in the X-axis direction is permitted and the driving springs43B are elastically deformed in the X-axis direction so that displacement of the movable drive electrode411B in the X-axis direction is permitted. When a drive voltage is applied between the movable drive electrodes411A and411B and the fixed drive electrodes412A and412B through the wirings73and74, electrostatic attractive forces are generated between the movable drive electrode411A and the fixed drive electrode412A and between the movable drive electrode411B and the fixed drive electrode412B, and the movable drive electrode411A vibrates in the X-axis direction while elastically deforming the drive spring43A in the X-axis direction and the movable drive electrode411B vibrates in the X-axis direction while elastically deforming the drive spring43B in the X-axis direction. Since the drive portions41A and41B are disposed symmetrically with respect to the imaginary straight line a, the movable drive electrodes411A and411B vibrate in opposite phases in the X-axis direction so as to repeat approaching and separating from each other. For that reason, vibration of the movable drive electrodes411A and411B is canceled, and vibration leakage can be reduced. Hereinafter, this vibration mode is also referred to as a drive vibration mode. In the physical quantity sensor400of the fifth embodiment, although an electrostatic drive method is used in which the drive vibration mode is excited by electrostatic attractive force is applied, a method of exciting the drive vibration mode is not particularly limited, and for example, a piezoelectric drive method, an electromagnetic drive method using a Lorentz force of a magnetic field, or the like can also be applied. Further, the element portion404includes detection portions44A and44B disposed between the drive portions41A and41B. The detection portion44A includes a movable detection electrode441A having a plurality of electrode fingers disposed in a tooth shape and fixed detection electrodes442A and443A disposed to be in mesh with electrode fingers of the movable detection electrode441A provided with a plurality of electrode fingers disposed in a tooth shape. The fixed detection electrodes442A and443A are arranged side by side in the Y-axis direction, and the fixed detection electrode442A is positioned on the plus side in the Y-axis direction and the fixed detection electrode443A is positioned on the minus side in the Y-axis direction with respect to the center of the movable detection electrode441A. In addition, a pair of the fixed detection electrodes442A and a pair of fixed detection electrodes443A are arranged so as to sandwich the movable detection electrode441A from both sides in the X-axis direction. The movable detection electrode441A has mass different from that of the movable drive electrode411A. In the fifth embodiment, the mass of the movable detection electrode441A is larger than the mass of the movable drive electrode411A. However, the mass of the movable detection electrode441A is not limited thereto, and the mass of the movable detection electrode441A may be equal to the mass of the movable drive electrode411A, or may be smaller than the mass of the movable drive electrode411A. Similarly, the detection portion44B includes a movable detection electrode441B having a plurality of electrode fingers disposed in a tooth shape and fixed detection electrodes442B and443B disposed to be in mesh with electrode fingers of the movable detection electrode441B provided with a plurality of electrode fingers disposed in a tooth shape. The fixed detection electrodes442A and443B are arranged side by side in the Y-axis direction, and the fixed detection electrode442B is positioned on the plus side in the Y-axis direction and the fixed detection electrode443B is positioned on the minus side in the Y-axis direction with respect to the center of the movable detection electrode441B. In addition, a pair of the fixed detection electrodes442B and a pair of fixed detection electrodes443B are arranged so as to sandwich the movable detection electrode441B from both sides in the X-axis direction. The movable detection electrode441B has mass different from that of the movable drive electrode411B. In the fifth embodiment, the mass of the movable detection electrode441B is larger than the mass of the movable drive electrode411B. However, the mass of the movable detection electrode441B is not limited thereto, and the mass of the movable detection electrode441B may be equal to the mass of the movable drive electrode411B, or may be smaller than the mass of the movable drive electrode411B. The movable detection electrodes441A and441B are electrically connected to the wiring73, respectively, the fixed detection electrodes442A and443B are electrically connected to the wiring75, respectively, and the fixed detection electrodes443A and442B are electrically connected to the wiring76, respectively. When the physical quantity sensor400is driven, an electrostatic capacitance Ca is formed between the movable detection electrode441A and the fixed detection electrode442A and between the movable detection electrode441B and the fixed detection electrode443B, and an electrostatic capacitance Cb is formed between the movable detection electrode441A and the fixed detection electrode443A and between the movable detection electrode441B and the fixed detection electrode442B. Further, the element portion404includes two fixed portions451and452disposed between the detection portions44A and44B. The fixed portions451and452are respectively bonded to the upper surface of the mount and fixed to the substrate402. The fixed portions451and452are arranged in the Y-axis direction and spaced apart from each other. In the fifth embodiment, the movable drive electrodes411A and411B and the movable detecting electrodes441A and441B are electrically connected to the wiring73via the fixed portions451and452. In addition, the element portion404includes four detection springs46A for connecting the movable detection electrode441A and the fixed portions42A,451, and452, and four detection springs46B for connecting the movable detection electrode441B and the fixed portions42B,451, and452. The detection springs46A are elastically deformed in the X-axis direction so that displacement of the movable drive electrode441A in the X-axis direction is permitted and the detection springs46A are elastically deformed in the Y-axis direction so that displacement of the movable drive electrode441A in the Y-axis direction is permitted. Similarly, the detection springs46B are elastically deformed in the X-axis direction so that displacement of the movable drive electrode441B in the X-axis direction is permitted, and the detection springs46B are elastically deformed in the Y-axis direction so that displacement of the movable drive electrode441B in the Y-axis direction is permitted. Further, the element portion404includes a reverse phase spring47A disposed between the drive portion41A and the detection portion44A and connecting the movable drive electrode411A and the movable detecting electrode441A and a reverse phase spring47B disposed between the drive portion41B and the detection portion44B and connecting the movable drive electrode411B and the movable detecting electrode441B. The reverse phase spring47A is elastically deformed in the X-axis direction so that the movable detection electrode441A can be displaced in the X-axis direction with respect to the movable drive electrode411A. Similarly, the reverse phase spring47B is elastically deformed in the X-axis direction so that the movable detection electrode441B can be displaced in the X-axis direction with respect to the movable drive electrode411B. When the Si-MEMS type angular velocity sensor element is driven with a drive voltage (for example, 1.8 V) equivalent to that of the quartz crystal gyro sensor element, stable vibration characteristics cannot be obtained, so that it is necessary to further generate a bias voltage (for example, 15 V) of ten and several V using a bias generation circuit for a drive voltage (for example, 1.8 V) and drive the Si-MEMS type angular velocity sensor element. However, since noise generated by the bias generation circuit is increased, a signal to noise ratio (S/N ratio) cannot be increased, and it is difficult to obtain low noise electrical characteristics as in the quartz gyro sensor element. Sixth Embodiment Next, a sixth embodiment will be described. Hereinafter, differences from the first to fifth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to fourth embodiments, and redundant description thereof will be omitted. The sixth embodiment is an embodiment of a physical quantity detection device which is the high-accuracy angular velocity sensor18in the second embodiment. In a physical quantity detection device300of the sixth embodiment, a plurality of physical quantity detection elements302are connected in a multi-connected manner. A physical quantity detection element302is, for example, the physical quantity sensor400in the fifth embodiment. In the physical quantity detection device300configured as described above, since a plurality of physical quantity detection elements302are electrically connected to terminals XP and XN of a physical quantity detection circuit through terminals307(for output signals) and terminals308(ground (GND) terminals), when it is assumed that the number of the physical quantity detection elements302is M and physical quantity components included in signals output from M physical quantity detection elements302are s1X, s2X, . . . , sMX, respectively, the physical quantity component SXincluded in the signals input from the terminals XP and XN is expressed by the following expression (10). SX=s1X+s2X+ . . . +sMX(10) Structures of the M physical quantity detection elements302are the same, and when it is assumed that s1X≈s2X= . . . ==SMX=sXin the expression (10), the expression (10) is transformed as illustrated in the following expression (11). SX=M·sX(11) On the other hand, there is no correlation between white noise components simultaneously output from the M physical quantity detection elements302through the terminals307and308of the physical quantity detection circuit. Accordingly, when it is assumed that the white noise components included in the signals output from the M physical quantity detection elements302are n1X, n2X, . . . , nMX, a white noise component NXincluded in the signals input from the terminals XP and XN of the physical quantity detection is represented by the following expression (12). NX=(n1X)2+(n2X)2+…+(nMX)2(12) Structures of the M physical quantity detection elements302are the same, and when it is assumed that (n1X)2≈(n2X)2= . . . =(nMX)2=(nX)2in the expression (12), the expression (12) is transformed as illustrated in the following expression (13). NX=√{square root over (M)}·nX(13) By dividing the expression (11) by the expression (13), the following expression (14) is obtained. SXNX=M·sXnX(14) In the expression (14), the signal to noise ratio of the signal input from the terminals XP and XN of the physical quantity detection circuit is IM times (for example, twice if M=4) the S/N ratio of the output signal of each of the M physical quantity detection elements302. Accordingly, according to the physical quantity detection device300of the sixth embodiment, the S/N ratio of the output angular velocity signal is improved. According to a mounting form illustrated inFIG.19, since the plurality of physical quantity detection elements302are mounted on a common substrate360, the distance between the adjacent physical quantity detection elements302can be reduced. Also, since wirings361,362, and363are provided on the substrate360, the distance between each physical quantity detection element302and the wirings361,362, and363is reduced, which is advantageous for miniaturizing the physical quantity detection device300. According to the mounting form illustrated inFIG.20and the mounting form illustrated inFIG.21, since the plurality of containers310on which the plurality of physical quantity detection elements302are mounted are stacked, a disposition area of the plurality of physical quantity detection elements302becomes small, and the physical quantity detection device300can be miniaturized. Furthermore, according to the mounting form illustrated inFIG.20and the mounting form illustrated inFIG.21, there is no need to provide wirings for electrically connecting the terminals307and308to the terminals XP and XN on a dedicated wiring substrate, which is advantageous for miniaturizing the physical quantity detection device300. Accordingly, the high-accuracy angular velocity sensor18according to the sixth embodiment is configured by connecting the plurality of physical quantity detection elements302as a plurality of Si-MEMS type angular velocity sensor elements as described above, thereby making it possible to improve the S/N ratio of the angular velocity signal output from the high-accuracy angular velocity sensor18. In the sixth embodiment, although the high-accuracy angular velocity sensor18which is the Z-axis angular velocity sensor116is described, similarly, the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114can be configured as the physical quantity detection device300in which the physical quantity detection elements302which are a plurality of sensor elements are connected in a multi-connected manner. In this case, the sensor element may be, for example, a gyro sensor element in the seventh embodiment which will be described later. By setting the number Ngz of the sensor elements constituting the Z-axis angular velocity sensor116to be larger than the number Ngx of the sensor elements constituting the X-axis angular velocity sensor112and the number Ngy of the sensor elements constituting the Y-axis angular velocity sensor114, the Z-axis angular velocity sensor116can be made “high accuracy” as compared with the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114. For example, it is preferable to set the number Ngz of the sensor elements constituting the Z-axis angular velocity sensor116to a value larger than 2 elements, that is, 3 or more sensor elements. As the number of sensor elements increases, statistical computation and numerical analysis such as utilizing an average value and median value becomes possible, resulting in “high accuracy”. Seventh Embodiment Next, a seventh embodiment will be described. Hereinafter, differences from the first to sixth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those in the first to sixth embodiments, and redundant description thereof will be omitted. The seventh embodiment is an embodiment of gyro sensor elements mounted on the multi-axis inertial sensor17in the second embodiment, which are the X-axis angular velocity sensor112and the Y-axis angular velocity sensor114in the first embodiment. A gyro sensor element500illustrated inFIG.22is an angular velocity sensor capable of detecting an angular velocity around the X-axis. The gyro sensor element500illustrated inFIG.22has two structures50(50aand50b) and two fixed detection portions59(59aand59b) aligned in the Y-axis direction. The two structures50aand50bare configured symmetrically in the vertical direction towardFIG.22, and have configurations similar to each other. Each structure50includes a mass portion51, a plurality of fixed portions52, a plurality of elastic portions53, a plurality of drive portions54(movable drive electrodes), a plurality of fixed drive portions55and56(fixed drive electrodes), detection portions571and572(movable detection electrodes), and a plurality of beams58. The mass portion51is integrally formed including the drive portions54, a frame573, the detection portions571and572, and the beams58. That is, the detection portions571and572are in a shape included in the mass portion51. The outer shape of the mass portion51is a quadrilateral frame shape in a plan view when seen in the Z-axis direction (hereinafter, simply referred to as “a plan view”), and includes the drive portion54, the frame573, the detection portions571and572as described above. Specifically, the outer shape of the mass portion51is configured with a pair of portions extending in parallel to each other in the Y-axis direction and a pair of portions connecting end portions of the pair of portions and extending parallel to each other along the X-axis direction. Four fixed portions52are provided for one structure50, and each fixed portion52is fixed to the substrate. In addition, each of the fixing portions52is disposed outside the mass portion51in a plan view, and in the seventh embodiment, each of the fixing portions52is disposed at a position corresponding to each corner portion of the mass portion51. In the illustration, the fixed portion52positioned on the −Y-axis side of the structure50aand the fixed portion52positioned on the +Y-axis side of the structure50bare used as a common fixed portion. Four elastic portions53are provided in this embodiment with respect to one structure50, and each elastic portion53connects a portion of the mass portion51and the fixed portion52in a plan view. In the seventh embodiment, the elastic portions53are connected to the corner portions of the frame573of the mass portion51, but are not limited thereto, and may be positioned at any position as long as the mass portion51can be displaced with respect to the fixed portion52. InFIG.22, a configuration in which the mass portion51can be displaced in the Y-axis direction is adopted. In the illustration, each of the elastic portions53has a meandering shape in a plan view and includes a first portion extending along the X-axis direction and a second portion extending along the Y-axis direction. The shape of the drive portion54is not limited to the illustrated shape as long as a configuration in which the drive portion54is elastically deformable in a desired driving direction (Y-axis direction in the seventh embodiment). Eight drive portions54are provided for one structure50, and each drive portion54is connected to a portion of the mass portion51extending along the Y-axis direction. Specifically, the four drive portions54are positioned on the +X side of the mass portion51, and the remaining four drive portions54are positioned on the −X side of the mass portion51. Each drive portion54has a tooth shape including a trunk portion extending from the mass portion51in the X-axis direction and a plurality of branch portions extending from the trunk portion in the Y-axis direction. Eight fixed drive portions55and eight fixed drive portions56are provided for each structure50, respectively, and respective fixed drive portions55and56are fixed to the upper surface23of the substrate described above. In addition, the fixed drive portions55and56have tooth shapes corresponding to the drive portion54, and are provided so as to sandwich the driving portion54therebetween. Each of the detection portions571and572is a plate-shaped member having a rectangular shape in a plan view, which is disposed inside the mass portion51and is connected to the mass portion51by the beam58. The detection portions571and572can rotate (displace) around a rotation axis J4, respectively. The fixed detection portion59(fixed detection electrode) faces the detection portions571and572. Further, the fixed detection portion59is separated from the detection portions571and572. In addition, the mass portion51, the elastic portion53, the drive portion54, a portion of the fixed drive portion55, a portion of the fixed drive portion56, the detection portions571and572, and the beam58having the configuration described above are provided above the substrate and are separated from the substrate2. The structure50as described above is collectively formed by patterning a conductive silicon substrate doped with impurities such as phosphorus and boron by etching. As the constituent material of the fixed detection portion59, for example, aluminum, gold, platinum, indium tin oxide (ITO), ZnO (zinc oxide), or the like can be used. Although not illustrated, the fixed portion52, the fixed drive portion55, the fixed drive portion56, the fixed detection portion59a, and the fixed detection portion59bare electrically connected to wirings and terminals (not illustrated), respectively. These wirings and terminals are provided on a substrate, for example. The configuration of the gyro sensor element500has been briefly described as above. A gyro sensor element500having such a configuration can detect the angular velocity ωx as follows. First, when a drive voltage is applied between the drive portion54and the fixed drive portions55and56included in the gyro sensor element500, an electrostatic attractive force periodically changing in intensity occurs between the fixed drive portions55and56and the drive portion54. With this configuration, each drive portion54vibrates in the Y-axis direction with elastic deformation of each elastic portion53. In this case, the plurality of drive portions54included in the structure50aand the plurality of drive portions54included in the structure50bvibrate (drive vibration) in opposite phases in the Y-axis direction. When the angular velocity (Ox is applied to the gyro sensor element500in a state where the drive portion54vibrates in the Y-axis direction as described above, the Coriolis force acts and the detection portions571and572are displaced around a rotation axis J4. In this case, the detection portions571and572included in the structure50aand the detection portions571and572of the structure50bare displaced in opposite directions. For example, when the detection portions571and572included in the structure50aare respectively displaced in the +Z-axis direction, the detection portions571and572included in the structure50bare respectively displaced in the −Z-axis direction. Further, when the detection portions571and572included in the structure50aare respectively displaced in the −Z-axis direction, the detection portions571and572included in the structure50bare respectively displaced in the +Z-axis direction. As the detection portions571and572displace (detect vibration) in this manner, a distance between the detection portions571and572and the fixed detection portion59changes. As the distance changes, the electrostatic capacitance between the detection portions571and572and the fixed detection portion59changes. The angular velocity (Ox applied to the gyro sensor element500can be detected based on the amount of change in the electrostatic capacitance. Although the X-axis angular velocity sensor112is described, the same applies to the Y-axis angular velocity sensor114. Eighth Embodiment Next, an eighth embodiment will be described. Hereinafter, differences from the first to seventh embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to seventh embodiments, and redundant description thereof will be omitted. The eighth embodiment is an embodiment of a physical quantity sensor mounted on the multi-axis inertial sensor17in the second embodiment, which corresponds to the X-axis acceleration sensor122and the Y-axis acceleration sensor124in the first embodiment. A physical quantity sensor600illustrated inFIG.23is an acceleration sensor capable of detecting acceleration Ax in the X-axis direction. Such a physical quantity sensor600includes a base portion602and an element portion604which is provided in the base portion602and measures acceleration Ax (physical quantity) in the X-axis direction. The element portion604includes a fixed electrode64attached to the base portion602, a movable member65displaceable in the X-axis direction (a first direction which is a detection axis direction of a physical quantity) with respect to the base portion602, and movable electrodes66provided on the movable member65. The fixed electrode64includes a first fixed electrode641and a second fixed electrode642arranged side by side along the Y-axis direction (a second direction which is a direction crossing the detection axis (orthogonal to the detection axis in the eighth embodiment)). The first fixed electrode641includes a first stem portion643and a plurality of first fixed electrode fingers645which are provided on both sides of a first stem portion643in the Y-axis direction (second direction) and of which a longitudinal direction is along the second direction. In addition, the second fixed electrode642includes a second stem portion644and a plurality of second fixed electrode fingers646which are provided on both sides in the Y-axis direction (second direction) from the second stem portion644and of which a longitudinal direction is along the second direction. The movable electrode66includes a first movable electrode661and a second movable electrode662arranged side by side along the Y-axis direction (second direction). At least a portion of the first movable electrode661includes a plurality of first movable electrode fingers663which are positioned on both sides of the first stem portion643in the Y-axis direction (second direction), of which the longitudinal direction is along the second direction, and which face the first fixed electrode fingers645in the X-axis direction (first direction). At least a portion of the second movable electrode662includes a plurality of second movable electrode fingers664which are positioned on both sides of the second stem portion644in the Y-axis direction (second direction), of which the longitudinal direction is along the second direction, and which face the second fixed electrode fingers646in the X-axis direction (first direction). With such a configuration, the first and second fixed electrode fingers645and646and the first and second movable electrode fingers663and664can be respectively shortened while maintaining the electrostatic capacitance between the first movable electrode finger663and the first fixed electrode finger645and the electrostatic capacitance between the second movable electrode finger664and the second fixed electrode finger646sufficiently large. For that reason, the physical quantity sensor600in which the electrode fingers645,646,663, and664are hard to be broken and which has excellent impact resistance is obtained. Although the X-axis acceleration sensor122is described, the same applies to the Y-axis acceleration sensor124. Ninth Embodiment Next, a ninth embodiment will be described. Hereinafter, differences from the first to eighth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those in the first to eighth embodiments, and redundant description thereof will be omitted. The ninth embodiment is an embodiment of the physical quantity sensor mounted on the multi-axis inertial sensor17in the second embodiment, which is the Z-axis angular velocity sensor116in the first embodiment. FIG.24is a schematic plan view of a physical quantity sensor700of a ninth embodiment. A movable body720includes a first movable member720aand a second movable member720b. The movable body720includes the first movable member720aon one side in a direction orthogonal to a rotation axis, the second movable member720bon the other side in the direction orthogonal to the rotation axis, and a fifth beam and a sixth beam connecting the first movable member720aand the second movable member720b, in a plan view, with the rotation axis as a boundary, and an opening portion726is disposed between the fifth beam and the sixth beam in a plan view, and the third beam connects the first beam and the fifth beam, and the fourth beam connects the second beam and the sixth beam. The first movable member720ais positioned on one side (−X-axis direction side in the illustrated example) of a support axis Q in a plan view (viewed from the Z-axis direction). The second movable member720bis positioned on the other side (+X-axis direction side in the illustrated example) of the support axis Q in a plan view. In a case where acceleration in the vertical direction (for example, gravitational acceleration) is applied to the movable body720, rotational moment (moment of force) is generated in each of the first movable member720aand the second movable member720b. Here, in a case where rotational moment (for example, counterclockwise rotational moment) of the first movable member720aand rotational moment of the second movable member720b(for example, clockwise rotational moment) are balanced, inclination of the movable body720does not change and acceleration cannot be detected. Accordingly, the movable body720is designed so that the rotational moment of the first movable member720aand the rotational moment of the second movable member720bare not balanced and the movable body720has a predetermined inclination when the acceleration in the vertical direction is applied. In the physical quantity sensor700, by disposing the support axis Q at a position deviated from the center (center of gravity) of the movable body720(by making the distances from the support axis Q to the tip ends of the first movable member720aand the second movable member720bdifferent), the first movable member720aand the second movable member720bhave different masses. That is, the mass of the movable body720is different between one side (first movable member720a) and the other side (second movable member720b) with the support axis Q as a boundary. In the illustrated example, the distance from the support axis Q to an end face723of the first movable member720ais greater than the distance from the support axis Q to an end face724of the second movable member720b. In addition, the thickness of the first movable member720ais equal to the thickness of the second movable member720b. Accordingly, the mass of the first movable member720ais larger than the mass of the second movable member720b. As such, since the first movable member720aand the second movable member720bhave different masses, when the acceleration in the vertical direction is applied, the rotational moment of the first movable member720aand the rotational moment of the second movable member720bcannot be balanced. Accordingly, when acceleration in the vertical direction is applied, it is possible to cause the movable body720to have a predetermined inclination. Although not illustrated, by disposing the support axis Q at the center of the movable body720and making the thicknesses of the first movable member720aand the second movable member720bdifferent from each other, the first movable member720aand the second movable member720bmay have different masses from each other. Even in such a case, when the acceleration in the vertical direction is applied, a predetermined inclination can be generated in the movable body720. The movable body720is provided apart from a substrate702. The movable body720is provided above a recessed portion11. A gap is provided between the movable body720and the substrate702. With this configuration, the movable body720can swing. The movable body720includes a first movable electrode721and a second movable electrode722which are provided with the support axis Q as a boundary. The first movable electrode721is provided on a first movable member720a. The second movable electrode722is provided on a second movable member720b. The first movable electrode721is a portion of the movable body720that overlaps with a first fixed electrode750in a plan view. The first movable electrode721forms an electrostatic capacitance C1between the first movable electrode721and the first fixed electrode750. That is, an electrostatic capacitance C1is formed by the first movable electrode721and the first fixed electrode750. The second movable electrode722is a portion of the movable body720that overlaps with the second fixed electrode752in a plan view. The second movable electrode722forms an electrostatic capacitance C2between the second movable electrode722and a second fixed electrode752. That is, the electrostatic capacitance C2is formed by the second movable electrode722and the second fixed electrode752. In the physical quantity sensor700, since the movable body720is made of a conductive material (silicon doped with impurities), the movable electrodes721and722are provided. That is, the first movable member720afunctions as the first movable electrode721and the second movable member720bfunctions as the second movable electrode722. The electrostatic capacity C1and the electrostatic capacity C2are configured to be equal to each other, for example, in a state where the movable body720is horizontal. The positions of the movable electrodes721and722change according to movement of the movable body720. Depending on the positions of the movable electrodes721and722, the electrostatic capacitances C1and C2change. A predetermined potential is applied to the movable body720through a support portion730. In the movable body720, a through-hole725penetrating the movable body720is formed. With this configuration, it is possible to reduce the influence (air resistance) of air when the movable body720swings. A plurality of through-holes725are formed. In the illustrated example, a planar shape of the through-hole725is a square. The movable body720is provided with an opening portion726penetrating the movable body720. The opening portion726is provided on the support axis Q in a plan view. In the illustrated example, the planar shape of the opening portion726is a rectangle. A support portion730is provided on the substrate702. The support portion730is positioned in an opening portion726. The support portion730supports the movable body720. The support portion730includes a first fixed portion, a second fixed portion, a first beam41, a second beam42, a third beam43, and a fourth beam44. The first fixed portion and the second fixed portion are fixed to the substrate702. The first fixed portion and the second fixed portion are provided so as to sandwich the support axis Q in a plan view. In the illustrated example, the first fixing portion is provided on the −X-axis direction side of the support axis Q, and the second fixing portion is provided on the +X-axis direction side of the support axis Q. Tenth Embodiment Next, a tenth embodiment will be described. Hereinafter, differences from the first to ninth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to ninth embodiments, and redundant description thereof will be omitted. The tenth embodiment is an embodiment of the multi-axis inertial sensor17in the second embodiment. Next, the physical quantity sensor according to the tenth embodiment will be described with reference toFIGS.25and26.FIG.25is a plan view illustrating a schematic configuration of a physical quantity sensor800according to the tenth embodiment. For convenience of explanation,FIG.25illustrates a state in which a resin package is seen through.FIG.26is a cross-sectional view illustrating a schematic configuration of the physical quantity sensor800according to the tenth embodiment. In the following description, three axes orthogonal to each other will be described using the X-axis, the Y-axis, and the Z-axis. Also, for the sake of convenience of explanation, the surface on the +Z-axis direction side, which is the sensor element side, is referred to as an upper surface and the surface on the opposite side to the −Z-axis direction is referred to as a lower surface, in a plan view when viewed in the Z-axis direction. As illustrated inFIGS.25and26, the physical quantity sensor800according to the tenth embodiment can be used as a six-axis sensor including a three-axis acceleration sensor capable of independently measuring accelerations in the X-axis direction, the Y-axis direction, and the Z-axis direction and a three-axis angular velocity sensor capable of independently measuring angular velocities in the X-axis direction, the Y-axis direction, and the Z-axis direction. Such a physical quantity sensor800includes a frame871, an integrated circuit (IC)840as a circuit element disposed on the frame871, and an acceleration sensor element820and an angular velocity sensor element830as sensor elements disposed one on each side of the IC840in the X direction in a plan view in the Z-axis direction, and a resin package884covering these constituent parts. The frame871is attached to a circuit board872through a joining member (not illustrated). The acceleration sensor element820and the angular velocity sensor element830are attached to the upper surface of the frame871through a resin adhesive material818as a joining material. Further, the IC840is attached to the upper surface of the frame871through an adhesive layer841. In the tenth embodiment, the frame871corresponds to the substrate to which the acceleration sensor element820and the angular velocity sensor element830are attached. The IC840includes, for example, a drive circuit for driving the acceleration sensor element820and the angular velocity sensor element830, a detection circuit for detecting acceleration in each of the X-axis, Y-axis, and Z-axis directions based on a signal from the acceleration sensor element820, a detection circuit for detecting an angular velocity in each of the X-axis, Y-axis, and Z-axis directions based on a signal from the angular velocity sensor element830, and an output circuit for converting signals from the respective detection circuits into predetermined signals and outputting the signals, and the like. The IC840includes a plurality of electrode pads (not illustrated) on its upper surface, and electrode pads are electrically connected to connection terminals875and877provided on the circuit board872through bonding wires874and876. The other electrode pads are electrically connected to terminals878of the acceleration sensor element820through bonding wires879. The other electrode pads are electrically connected to terminals881of the angular velocity sensor element830through bonding wires882. With this configuration, the IC840can control the acceleration sensor element820and the angular velocity sensor element830. The acceleration sensor element820and the angular velocity sensor element830are attached to the frame871by a resin adhesive material818. On the lower surface of the circuit board872, a plurality of external terminals885are provided. The plurality of external terminals885correspond to the connection terminals875and877provided on the upper surface of the circuit board872, respectively, and are electrically connected through internal wiring (not illustrated) or the like. Eleventh Embodiment Next, an eleventh embodiment will be described. Hereinafter, differences from the first to tenth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to tenth embodiments, and redundant description thereof will be omitted. The eleventh embodiment is an embodiment of a vehicle positioning device. FIG.27is a block diagram illustrating the overall system of a vehicle positioning device3000according to an eleventh embodiment.FIG.28is a diagram illustrating an action of the vehicle positioning device3000illustrated inFIG.27. A vehicle positioning device3000illustrated inFIG.27is a device which is used by being mounted on a vehicle and performs positioning of the vehicle. The vehicle is not particularly limited, and may be any of a bicycle, an automobile (including a four-wheeled automobile and a motorcycle), a train, an airplane, a ship, and the like, but in the eleventh embodiment, the vehicle is described as a four-wheeled automobile. The vehicle positioning device3000includes an inertia measurement device3100(IMU), a computation processing unit3200, a GPS reception unit3300, a receiving antenna3400, a position information acquisition unit3500, a position synthesis unit3600, a processing unit3700, a communication unit3800, and a display3900. As the inertia measurement device3100, for example, the IMU100in the first embodiment described above can be used. The inertia measurement device3100includes a triaxial acceleration sensor3110and a triaxial angular velocity sensor3120. The computation processing unit3200receives acceleration data from the acceleration sensor3110and angular velocity data from the angular velocity sensor3120, performs inertial navigation computation processing on these data, and outputs inertial navigation positioning data (data including acceleration and attitude of the vehicle). The GPS reception unit3300receives a signal (GPS carrier wave, satellite signal on which position information is superimposed) from the GPS satellite via the receiving antenna3400. Further, the position information acquisition unit3500outputs GPS positioning data representing the position (latitude, longitude, altitude), speed, direction of the vehicle positioning device3000(vehicle) based on the signal received by the GPS reception unit3300. The GPS positioning data also includes status data indicating a reception state, a reception time, and the like. Based on inertial navigation positioning data output from the computation processing unit3200and the GPS positioning data output from the position information acquisition unit3500, the position synthesis unit3600calculates the position of the vehicle, more specifically, the position on the ground where the vehicle is traveling. For example, even if the position of the vehicle included in the GPS positioning data is the same, as illustrated inFIG.28, if the attitude of the vehicle is different due to the influence of inclination of the ground or the like, the vehicle is traveling at different positions on the ground. For that reason, it is impossible to calculate an accurate position of the vehicle with only GPS positioning data. Therefore, the position synthesis unit3600calculates the position on the ground where the vehicle is traveling, using inertial navigation positioning data (in particular, data on the attitude of the vehicle). This determination can be made comparatively easily by computation using a trigonometric function (inclination θ with respect to the vertical direction). The position data output from the position synthesis unit3600is subjected to predetermined processing by the processing unit3700and displayed on the display3900as a positioning result. Further, the position data may be transmitted to the external device by the communication unit3800. The vehicle positioning device3000has been described as above. As described above, such a vehicle positioning device3000includes the inertia measurement device3100, the GPS reception unit3300(reception unit) that receives a satellite signal on which position information is superimposed from a positioning satellite, the position information acquisition unit3500(acquisition unit) that acquires position information of the GPS reception unit3300based on the received satellite signal, the computation processing unit3200(computation unit) that computes the attitude of the vehicle based on the inertial navigation positioning data (inertia data) output from the inertia measurement device3100, and the position synthesis unit3600(calculation unit) that calculates the position of the vehicle by correcting position information based on the calculated attitude. With this configuration, the effect of the inertia measurement device3100which is the IMU100can be achieved, and the vehicle positioning device3000with high reliability can be obtained. In the above description, although description is made by using the global positioning system (GPS) as a satellite positioning system, other global navigation satellite system (GNSS) may be used. For example, one or more of satellite positioning systems among satellite positioning systems such as European geostationary-satellite navigation overlay service (EGNOS), quasi zenith satellite system (QZSS), global navigation satellite system (GLONASS), GALILEO, beidou navigation satellite system (Bei Dou) may be used. Also, a stationary satellite type satellite-based augmentation system (SBAS) such as wide area augmentation system (WAAS) or European geostationary-satellite navigation overlay service (EGNOS) may be utilized in at least one of the satellite positioning systems. Twelfth Embodiment Next, a twelfth embodiment will be described. Hereinafter, differences from the first to eleventh embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to eleventh embodiments, and redundant description thereof will be omitted. The twelfth embodiment is an embodiment of an electronic device. FIG.29is a perspective view illustrating an electronic device according to the twelfth embodiment. A smartphone1200(mobile phone) illustrated inFIG.29is one to which the electronic device according to the invention is applied. In the smartphone1200, the sensor unit160in the second embodiment, and the control circuit1210(control unit) that performs control based on detection signals output from the sensor unit160are incorporated. Detection data (angular velocity data) measured by the sensor unit160is transmitted to the control circuit1210, and the control circuit1210can recognize the attitude and behavior of the smartphone1200from the received detection data, change a display image displayed on the display unit1208, generate an alarm sound or sound effect, or drive the vibration motor to vibrate the main body. Such a smartphone1200(electronic device) has the sensor unit160and the control circuit1210(control unit) that performs control based on detection signals output from the sensor unit160. Thirteenth Embodiment Next, a thirteenth embodiment will be described. Hereinafter, differences from the first to twelfth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to twelfth embodiments, and redundant description thereof will be omitted. The thirteenth embodiment is an embodiment of an electronic device. FIG.30is a perspective view illustrating an electronic device according to the thirteenth embodiment. A digital still camera1300illustrated inFIG.30is an example of the electronic device. The digital still camera1300includes a case1302, and a display1310is provided on the back surface of the case1302. The display1310is configured to perform display based on the image capturing signal by a charge coupled device (CCD), and functions as a finder that displays the subject as an electronic image. A light receiving unit1304including an optical lens (image capturing optical system), the CCD, and the like is provided on the front side (the back side in the figure) of the case1302. When a photographer confirms the subject image displayed on the display1310and presses a shutter button1306, the image capturing signal of the CCD at that time is transferred and stored in the memory1308. In the digital still camera1300, the sensor unit160of the second embodiment and a control circuit1320(control unit) that performs control based on detection signals output from the sensor unit160are incorporated. The sensor unit160is used for camera shake correction, for example. Such a digital still camera1300(electronic device) includes the sensor unit160in the second embodiment and the control circuit1320(control unit) that performs control based on detection signals output from the sensor unit160. For that reason, the effect of the sensor unit160can be achieved, and high reliability can be exhibited. In addition to the personal computer and mobile phone and the digital still camera, the electronic device of the thirteenth embodiment can be applied to, for example, a smartphone, a tablet terminal, a clock (including smart watch), an ink jet type discharging device (for example, an ink jet printer), a laptop personal computer, a TV, a wearable terminals such as HMD (head mounted display), a video camera, a video tape recorder, a car navigation device, a pager, an electronic datebook (including a datebook with communication function), an electronic dictionary, a calculator, an electronic game machines, a word processor, a work station, a videophone, a security TV monitor, an electronic binoculars, a POS terminal, medical equipment (for example, electronic clinical thermometer, blood pressure monitor, blood glucose meter, electrocardiogram measurement device, ultrasonic diagnostic device, electronic endoscope), a fish finder, various measuring instruments, mobile terminal base station equipment, instruments (for example, instruments of vehicles, aircraft, and ships), a flight simulator, a network server, and the like. Fourteenth Embodiment Next, a fourteenth embodiment will be described. Hereinafter, differences from the first to thirteenth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to thirteenth embodiments, and redundant description thereof will be omitted. The fourteenth embodiment is an embodiment of a portable electronic device. FIG.31is a plan view illustrating a portable electronic device according to the fourteenth embodiment.FIG.32is a functional block diagram illustrating a schematic configuration of the portable electronic device illustrated inFIG.31. A watch type activity meter1400(active tracker) illustrated inFIG.31is a wristwatch device which is a type of the portable electronic device. The activity meter1400is attached to a part (subject) such as the user's wristwatch by a band1401. The activity meter1400includes a display1402for digital display and can perform wireless communication. The sensor unit160in the second embodiment is incorporated in the activity meter1400as an acceleration sensor1408for measuring acceleration and an angular velocity sensor1409for measuring angular velocity. The activity meter1400includes a case1403in which the acceleration sensor1408and the angular velocity sensor1409are accommodated, a processing unit1410which is accommodated in the case1403and is for processing output data from the acceleration sensor1408and the angular velocity sensor1409, the display1402accommodated in the case1403, and a translucent cover1404covering the opening of the case1403. A bezel1405is provided outside the translucent cover1404. A plurality of operation buttons1406and1407are provided on the side surface of the case1403. As illustrated inFIG.32, the acceleration sensor1408measures acceleration in each of the three axis directions which intersect (ideally orthogonal to) each other, and outputs a signal (acceleration signal) according to the magnitude and direction of the detected three-axis acceleration. An angular velocity sensor1409measures angular velocity in each of the three axis directions intersecting (ideally orthogonal to) each other, and outputs a signal (angular velocity signal) according to the magnitude and direction of the detected three-axis angular velocity. In the liquid crystal display (LCD) constituting the display1402, depending on various detection modes, for example, position information using a GPS sensor1411and a geomagnetic sensor1412, exercise information such as the amount of movement, the amount of exercise using the acceleration sensor1408and the angular velocity sensor1409, biometric information such as a pulse rate using a pulse sensor1413or the like, and time information such as current time, and the like are displayed. The environmental temperature using a temperature sensor1414can also be displayed. A communication unit1415performs various controls for establishing communication between a user terminal and an information terminal (not illustrated). The communication unit1415is configure to include a transceiver compatible with the short range wireless communication standard such as, for example, a Bluetooth (registered trademark) (including BTLE: Bluetooth Low Energy), Wireless Fidelity (Wi-Fi) (registered trademark), Zigbee (registered trademark), near field communication (NFC), ANT+ (registered trademark) or the like, and a connector compatible with a communication bus standard such as the universal serial bus (USB) or the like. The processing unit1410(processor) is constituted by, for example, a micro processing unit (MPU), a digital signal processor (DSP), an application specific integrated circuit (ASIC), or the like. The processing unit1410executes various processing based on the program stored in a storing unit1416and a signal input from an operation unit1417(for example, operation buttons1406and1407). Processing by the processing unit1410includes data processing for each output signal of the GPS sensor1411, the geomagnetic sensor1412, a pressure sensor1418, the acceleration sensor1408, the angular velocity sensor1409, the pulse sensor1413, the temperature sensor1414, and the clocking unit1419, display processing for causing the display1402to display an image, sound output processing for causing a sound output unit1420to output sound, communication processing for performing communication with the information terminal via the communication unit1415, and Power control processing for supplying power from a battery1421to each unit, and the like. Such an activity meter1400can have at least the following functions.1. Distance: Measure the total distance from the start of measurement with highly accurate GPS function.2. Pace: Display a current running pace from pace distance measurement.3. Average speed: Calculate an average speed and display the average speed from the start of running to the present.4. Altitude: Measure and display altitude with GPS function.5. Stride: Measure and display the stride even in a tunnel where GPS radio waves do not reach.6. Pitch: Measure and display the number of steps per minute.7. Heart rate: The heart rate is measured and displayed by the pulse sensor.8. Gradient: Measure and display the gradient of the ground in training and trail runs in the mountains.9. Auto lap: Automatically perform lap measurement when running for a fixed distance set in advance or for a fixed time.10. Exercise consumption calorie: Display calorie consumption.11. Step count: Display the total number of steps from the start of the exercise. Such an activity meter1400(portable electronic device) includes the physical quantity sensors such as the acceleration sensor1408and an angular velocity sensor1409, the case1403accommodating the physical quantity sensors, the processing unit1410which is accommodated in the case1403and performs processing output data from the physical quantity sensor, the display1402accommodated in the case1403, and the translucent cover1404covering the opening portion of the case1403. As described above, the activity meter1400includes the GPS sensor1411(satellite positioning system), and can measure a moving distance and a movement trajectory of the user. For that reason, a highly convenient activity meter1400can be obtained. The activity meter1400can be widely applied to a running watch, a runner's watch, a runner's watch for multiple sports such as duathlon and triathlon, an outdoor watch, and a GPS watch on which a satellite positioning system such as the GPS is mounted. Fifteenth Embodiment Next, a fifteenth embodiment will be described. Hereinafter, differences from the first to fourteenth embodiments will be mainly described, and the same reference numerals are given to the same constituent elements as those of the first to fourteenth embodiments, and redundant description thereof will be omitted. The fifteenth embodiment is an embodiment of a vehicle. FIG.33is a perspective view illustrating a configuration of an automobile which is an example of a vehicle in the fifteenth embodiment. As illustrated inFIG.33, the sensor unit160in the second embodiment is incorporated in an automobile1500, and for example, the attitude of a vehicle body1501can be detected by the sensor unit160. The detection signal of the sensor unit160is supplied to a vehicle body attitude control device1502as an attitude control unit for controlling the attitude of the vehicle body and the vehicle body attitude control device1502can measure the attitude of the vehicle body1501based on the signal, control hardness of the suspension according to the detection result, and control brakes of the individual wheels1503. In addition, the sensor unit160can be widely applied to an electronic control unit (ECU) such as a keyless entry, an immobilizer, a car navigation system, a car air conditioner, an anti-lock braking system (ABS), an air bag, a tire pressure monitoring system (TPMS), an engine control, a control device for inertial navigation for automatic operation, a battery monitor of a hybrid vehicle or an electric vehicle, and the like. In addition to the examples described above, the sensor unit160can be used for attitude control of a biped walking robot and a train, remote control of a radio control airplane, a radio control helicopter, a drone, and the like, or attitude control of an autonomous flying object, attitude control of an agricultural machine, a construction machine, and the like, for example. As described above, in realizing attitude control of various vehicles, the sensor unit160and respective control units (not illustrated) are incorporated. Since such a vehicle includes the sensor unit160and the control unit (not illustrated) in the second embodiment, the vehicle has excellent reliability. Sixteenth Embodiment A sixteenth embodiment is an embodiment that allows automatic operation in the vehicle1500of the fifteenth embodiment. An advanced driver assistance systems (ADAS) locator used for the automatically operated vehicle1500illustrated inFIG.33includes, in addition to an inertial sensor including a sensor module1610, a global navigation satellite system (GNSS) receiver, and a map database storing map data. The ADAS locator measures a traveling position of the vehicle in real time by combining a positioning signal received by the GNSS receiver and a measurement result of the inertial sensor. The ADAS locator reads the map data from the map database. An output from the ADAS locator including the sensor module1610is input to an automatic operation control unit1620. The automatic operation control unit1620controls at least one of acceleration, braking, and steering of the vehicle1500based on the output (including a detection signal from the sensor module1610) from the ADAS locator. FIG.34is a block diagram illustrating a system1600related an advanced driver assistance systems (ADAS) locator. A switcher1630switches execution or non-execution of automatic operation in the automatic operation control unit1620based on change in the output (including change in the detection signal from the sensor module1610) from the ADAS locator. The switcher1630outputs a signal for switching from execution of the automatic operation to non-execution of the automatic operation to the control unit1620, for example, in a case of abnormality in which detection capability of the sensor (including the sensor module1610) in the ADAS locator is deteriorated. | 110,721 |
11860186 | DETAILED DESCRIPTION OF THE EMBODIMENT Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present invention pertains may easily practice. However, the present invention may be implemented in various different forms, and is not limited to exemplary embodiments described herein. In addition, in the drawings, portions unrelated to the description will be omitted to obviously describe the present invention, and similar reference numerals will be used to describe similar portions throughout the specification. In addition, throughout the present specification, when any one part is referred to as being “connected to” another part, it means that any one part and another part are “directly connected to” each other or are “electrically connected to” or “indirectly connected to” each other with the other part interposed therebetween. Throughout the present specification, when any member is referred to as being positioned “on”, “at upper portion”, “at upper end”, “under”, “at lower portion”, and “at lower end” another member, it includes not only a case in which any member and another member are in contact with each other, but also a case in which the other member is interposed between any member and another member. Through the present specification and claims, unless explicitly described otherwise, “comprising” any components will be understood to imply the inclusion of other components rather than the exclusion of any other components. The terms “about”, “substantially”, and the like used throughout this specification mean figures corresponding to manufacturing and material tolerances specific to the stated meaning and figures close thereto, and are used to prevent unconscionable abusers from unfairly using the present invention of figures precisely or absolutely described to aid the understanding of the present invention. The term “˜step” or “˜step of” used throughout this specification does not mean “˜step for”. In the present specification, the term “unit” includes a unit realized by hardware, a unit realized by software, and a unit realized by both the hardware and software. Further, one unit may be realized by two or more hardware, and two or more units may be realized by one hardware. In the present specification, some of the operations or functions described as performed by a terminal, an apparatus, or a device may be performed instead in a server connected to the corresponding terminal, apparatus, or device. Similarly, some of the operations or functions described as being performed by a server may be performed in a terminal, an apparatus, or a device connected to the corresponding server. Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention relates to a fall detection apparatus and method and relates to an algorithm for detecting a fall using an acceleration tilt angle and a triangle feature before an impact occurs using an inertial sensor. Hereinafter, a fall detection apparatus (hereinafter referred to as “the present apparatus”) according to an embodiment of the present invention will be described. The fall detection apparatus1is an apparatus that detects a fall by receiving acceleration and angular velocity values from an inertial sensor (not illustrated) attached to a user, and the inertial sensor and the fall detection apparatus1can be connected through a network. At this time, the network refers to a connection structure capable of exchanging information between each node, such as a plurality of terminals and servers. Examples of such a network include a 3rd generation partnership project (3GPP) network, a long term evolution (LTE) network, a world interoperability for microwave access (WIMAX) network, Internet, a local area network (LAN), a wireless local area network (wireless LAN), a wide area network (WAN), a personal area network (PAN), a Bluetooth network, a satellite broadcasting network, an analog broadcasting network, a digital multimedia broadcasting (DMB) network, and the like, but are not limited thereto. FIG.1is a schematic configuration diagram of a fall detection apparatus according to an exemplary embodiment of the present invention. Referring toFIG.1, the fall detection apparatus1according to the present invention may include an acceleration measurement unit10, an angular velocity measurement unit11, an acceleration value determination unit20, an angular velocity value determination unit21, an angle value determination unit22, a triangle feature calculation unit30, and the fall detection unit40. However, since the present apparatus1ofFIG.1is only an example of the present invention, according to various exemplary embodiments of the present invention, the present apparatus1may also be configured differently fromFIG.1. The fall detection apparatus1is implemented as a single device as illustrated inFIG.1, and may receive acceleration and angular velocity measured by a sensor, determine a triangle feature based on acceleration and angular velocity, determine to be a fall when the acceleration, the angular velocity, and the triangle feature satisfy conditions of a first reference value to a third reference value determined in advance to detect a fall of a user, and output information corresponding to the detected fall. The sensor may be worn on a user's body such as a waist and may measure the acceleration and the angular velocity to transmit data on the acceleration and the angular velocity to the fall detection apparatus1. The sensor can include an inertial sensor, and may be, for example MPU-9150 (Invensens®, USA). In addition, the sensor may be worn on a user's waist constituted by any one of a ring type, a band type, and a belt type. Such a sensor may transmit and receive fall detection information using short-range wireless communication with a mobile communication terminal. An example of the short-range wireless communication may be Bluetooth. The Bluetooth means a standard that enables real-time communication in both directions by wirelessly connecting computers, mobile communication terminals, and other various electric and electronic apparatuses for short distance. On the other hand, in another exemplary embodiment, the fall detection apparatus1may include a mobile communication terminal (not illustrated). Here, a mobile communication terminal H is a mobile communication apparatus in which portability and mobility are guaranteed, and examples thereof may include all types of handheld-based wireless communication devices such as personal communication system (PCS), global system for mobile communication (GSM), personal digital cellular (PDC), personal handyphone system (PHS), personal digital assistant (PDA), international mobile telecommunication (IMT)-2000, code division multiple access (CDMA)-2000, W-code division multiple access (W-CDMA), a wireless broadband Internet (Wibro) terminal, a smart phone, a smart pad, a tablet PC, and the like. In relation to this, information corresponding to a fall may be sound information and vibration information, in which the sound information may be output using a mobile communication terminal and a speaker module, and the vibration information may be output using a vibrator of the mobile communication terminal. Hereinafter, each configuration of the fall detection apparatus1will be described in detail with reference toFIG.1. The acceleration measurement unit10may measure first to third accelerations corresponding to a user. The acceleration measurement unit10may include a three-axis acceleration sensor. First, the acceleration sensor refers to a sensor that detects a change in speed per unit time, and detects dynamic forces such as acceleration, vibration, and shock. In the 3-axis acceleration sensor, the acceleration sensors are located in each of the 3-axis x, y, and z axes, and the absolute direction of the sensor can be measured using the acceleration values generated in the x, y, and z axes based on the gravitational acceleration. That is, each of the first to third accelerations measured by the acceleration measurement unit10may be x-axis acceleration, y-axis acceleration, and z-axis acceleration. The angular velocity measurement unit11may measure first and second angular velocities corresponding to a user. The angular velocity measurement unit11may include a 3-axis gyro sensor. First, the gyro sensor is also called a gyroscope, and refers to a sensor that detects angular velocity and senses rotational inertia. The 3-axis gyro sensor may acquire an angular velocity value at which an object rotates in a unit time because the gyro sensor is located in each of three directions of the x, y, and z axes. At this time, rotation about the x-axis is called roll, rotation about the y-axis is called pitch, and rotation about the z-axis is called yaw. That is, each of the first and second angular velocities measured by the angular velocity measurement unit11may be a pitch angular velocity and a roll angular velocity. Here, the acceleration measurement unit10and the angular velocity measurement unit11may be implemented as the above-described sensor (not illustrated). The acceleration value determination unit20may determine the acceleration value for the user based on at least one of the first to third accelerations measured by the acceleration value measurement unit11described above. Specifically, the acceleration value determination unit20may calculate the acceleration value through a square root of a sum of squares of each of the first to third accelerations. Hereinafter, an example in which the acceleration value determination unit20determines the acceleration value based on Equation 1 will be described. ACCSVM=√{square root over (ACCx2+ACCy2+ACCz2)} [Equation 1] The acceleration value determination unit20uses the x-axis acceleration ACCx(first acceleration), the y-axis acceleration ACCy(second acceleration), and the z-axis acceleration ACCz(third acceleration) to obtain the acceleration value (ACCSVM). The angular velocity value determination unit21may determine the angular velocity value for the user based on at least one of the first and second angular velocities measured by the angular velocity value measurement unit11described above. Specifically, the angular velocity value determination unit21may calculate the angular velocity value through a square root of a sum of squares of each of the first and second angular velocities. Hereinafter, an example in which the angular velocity value determination unit21determines the angular velocity value based on Equation 2 will be described. ωSVM=√{square root over (ωPitch2+ωRoll2)} [Equation 2] Referring to Equation 2, the angular velocity value determination unit21may use a pitch angular velocity ωPitchand a roll angular velocity ωRollto obtain an angular velocity value (ωSVM). In addition, the angular velocity value determination unit21may use a yaw angle which is the z-axis acceleration. The angle value determination unit22may determine the angle value for the user based on at least one of the first to third accelerations measured by the acceleration value measurement unit11described above. Hereinafter, an example in which the angle value determination unit22determines the angle value based on Equation 3 will be described. DegSaggital=tan-1ACCzACCy×180πDegFrontal=tan-1ACCxACCy×180π[Equation3] Referring to Equation 3, the angle value determination unit22may use ACCxwhich is the acceleration of the x-axis, ACCywhich is the acceleration of the y-axis, and ACCzwhich is the acceleration of the z-axis to obtain angle values (DegSaggital, DegFrontal). The triangle feature calculation unit30may calculate a triangle feature using the first to third accelerations. FIG.2is a diagram illustrating a definition of a triangle feature according to an exemplary embodiment of the present invention (FIG.2A) and a change in the triangle feature according to a vertical angle of a user (FIG.2B). Referring toFIG.2, the triangle feature may mean an area of a triangle formed by a sum of vectors of the x-axis acceleration and the z-axis acceleration and the y-axis acceleration (FIG.2A). The triangle feature may increase until the vertical angle of the user reaches 45°, and may then decrease (FIG.3B). That is, while the user is standing, the x-axis acceleration ACCx, the y-axis acceleration ACCy, and the z-axis acceleration ACCzmay be 0 g, 1 g, and 0 g, respectively, and accordingly, the triangle feature may be 0. After the user falls, the x-axis acceleration ACCx, the y-axis acceleration ACCy, and the z-axis acceleration ACCzmay be 0 g, 0 g, and 1 g, respectively, and accordingly, the triangle feature may be 0. In addition, when the vertical angle of the user is 45°, the X-axis acceleration ACCx, the y-axis acceleration ACCy, and the z-axis acceleration ACCzmay be 1/√{square root over (2)} g and 1/√{square root over (2)}g, respectively, and accordingly, the triangle feature may be 0.25. The fall detection unit40may detect the fall of the user based on at least one of the acceleration value, the angular velocity value, the angle value, and the triangle feature. FIG.3is a diagram schematically illustrating an algorithm performed in the fall detection unit according to an exemplary embodiment of the present invention. Referring toFIG.3, when the acceleration measurement unit10and the angular velocity measurement unit11generate the x-axis acceleration, the y-axis acceleration, the z-axis acceleration, the pitch value, the roll value, and the triangle feature, the fall detection unit40may detect the fall of the user by considering a comparison result between an acceleration value and the first reference value. At this time, when the acceleration value is smaller than the first reference value, the fall of the user may be detected. When the acceleration value is greater than or equal to the first reference value, it may be determined that the user does not fall (‘activities of daily living (ADLs)’ inFIG.3). When the fall of the user is detected through the comparison result between the acceleration value and the first reference value, the fall of the user may be detected by further considering the comparison result between the angular velocity value and the second reference value. At this time, when the angular velocity value is greater than the second reference value, the fall of the user may be detected. When the angular velocity value is smaller than or equal to the second reference value, it may be determined that the user does not fall (‘activities of daily living (ADLs)’ inFIG.3). When the fall of the user is detected through the comparison result between the angular velocity value and the second reference value, the fall of the user may be detected by further considering the comparison result between the triangle feature and the second reference value. At this time, when the triangle feature is greater than the third reference value, the fall of the user may be detected. When the triangle feature is smaller than or equal to the third reference value, it may be determined that the user does not fall (‘activities of daily living (ADLs)’ inFIG.3). That is, when the acceleration value determined by the acceleration value determination unit20is smaller than the first reference value, the angular velocity value determined by the angular velocity value determination unit21is greater than the second reference value, and the angle value determined by the angle value determination unit22is greater than the third reference value, the fall detection unit40may detect the fall of the user when all of these cases are satisfied. Meanwhile, the first reference value to the third reference value may be determined based on the user information. As an example of the first reference value to the third reference value, the first reference value may be 0.9 g, the second reference value may be 47.3°/s, and the third reference value may be 0.19. Here, the user information may include at least one of an age, a gender, a body dimension, and a weight. The fall detection apparatus1may output information corresponding to a fall detected by a sensing unit (not illustrated) using the user information as an input value. Specifically, the sensing unit (not illustrated) may generate the input values to correspond to each of the age, gender, body dimension, and weight, and differently generate output information according to the comparison value generated based on the output reference value allocated to each input value. Specifically, the fall detection apparatus1may include a sensing unit (not illustrated). The sensing unit may generate input values for each user information. For example, the sensing unit may designate an age value, a gender value, a body dimension value, and a weight value for the user information as the above-described input values. The sensing unit may generate output reference values allocated to each input value. For example, the sensing unit may set an age reference value, a gender reference value, a body dimension reference value, and a body weight reference value. Therefore, the sensing unit may differently generate the output information by comparing the input reference value with the output reference value allocated to the input value. For example, the fall accident may be more fatal to a user of the age of 50 to 70 than a user of the age of 20 to 30, so when a fall of a user of the age of 20 to 30 is detected, a sound of 50 to 60 dB may ring and when a fall of a user of the age of 50 to 70 is detected, a sound of 100 to 110 dB may ring. Here, the output information may be output through at least one of sound, image, data, signal, or light. Hereinafter, the operation flow of the present invention will be briefly described based on the details described above. FIG.4is an operation flowchart for a fall detection method according to an exemplary embodiment of the present invention. The fall detection method illustrated inFIG.4may be performed by the fall detection apparatus1described above. Therefore, even if omitted, the description of the fall detection apparatus1may be similarly applied to the description of the fall detection method. In the fall detection method according to the exemplary embodiment of the present invention, first, first to third accelerations corresponding to a user may be measured (S401). Next, it is possible to measure first to third angular velocities corresponding to the user (S402). Next, the acceleration value for the user may be determined based on at least one of the first to third accelerations (S403). Next, the angular velocity value for the user may be determined based on at least one of the first and second angular velocities (S404). Next, the angle value for the user may be determined based on at least one of the first to third accelerations (S405). Next, the triangle feature may be calculated using the acceleration value (S406). Next, the fall of the user may be detected based on at least one of the acceleration value, the angular velocity value, the angle value, and the triangle feature (S407). In the above description, steps S401to S407may be further divided into additional steps or combined into fewer steps, according to the implementation embodiment of the present invention. Also, some steps may be omitted if necessary, and the order between the steps may be changed. The fall detection method according to the embodiment of the present invention may be implemented in a form of program commands that may be executed through various computer means and may be recorded in a computer-readable recording medium. The computer-readable recording medium may include a program command, a data file, a data structure or the like, alone or a combination thereof. The program commands recorded in the computer-readable recording medium may be especially designed and configured for the present invention or be known and usable by those skilled in a field of computer software. Examples of the computer-readable recording medium may include a magnetic medium such as a hard disk, a floppy disk, or a magnetic tape; an optical medium such as a compact disk read only memory (CD-ROM) or a digital versatile disk (DVD); a magneto-optical medium such as a floptical disk; and a hardware device specially configured to store and execute program commands, such as a ROM, a RAM, a flash memory, or the like. Examples of the program commands include a high-level language code capable of being executed by a computer using an interpreter, or the like, as well as a machine language code made by a compiler. The above-described hardware device may be constituted to be operated as one or more software modules to perform an operation according to the present invention, and vice versa. In addition, the above-described fall detection method may also be implemented in the form of a computer program or application executed by a computer, stored in a recording medium. The above description of the present invention is for illustrative purposes, and those skilled in the art to which the present invention pertains will understand that it is possible to be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-mentioned embodiments are exemplary in all aspects but are not limited thereto. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form. It is to be understood that the scope of the present invention will be defined by the claims rather than the above-mentioned description and all modifications and alternations derived from the claims and their equivalents are included in the scope of the present invention. | 22,125 |
11860187 | DETAILED DESCRIPTION The specific embodiments of the present invention will be further described below in conjunction with the drawings and technical solutions. FIG.2shows the numerical simulation process diagram, wherein (a) shows the deformation solution model established in ABAQUS, (b) shows the deformation contour of cell, (c) shows the change of the normal force of the conical AFM probe with the compression depth, and (d) shows the comparison between the simulation results and the fitted results of Sneddon model.FIG.3shows the relative error of the elastic modulus of cells fitted by Sneddon model when changing the curvature radius of the cone at the tip and compression depth of conical AFM probe with fixed half angle.FIG.4shows: when r/d is taken as the abscissa, the relation between r/d and relative error δ is approximately linear.FIG.5shows that polynomial function is used to fitted the errors which will occur when Sneddon model is used to fit the elastic modulus of cells. The probes with different shape parameters are pressed into different depths. Where, P is the normal force of the AFM probe, a is the half angle of the AFM probe, r is the curvature radius of the AFM probe at the tip, and d is the compression depth of the AFM probe. δ is the relative error between the cell elastic modulus fitted by Sneddon model and the simulation results.FIG.6shows the verification of the modified model used in the elastic modulus test of human osteosarcoma cells. The force displacement curves of human osteosarcoma cells tested by AFM probe are fitted by Sneddon model and modified model, respectively, then the fitting results of the two formulas are compared.FIG.7shows the validation of the modified model in the elastic modulus test of PVA hydrogels. Sneddon model and the modified model are used to fit the force displacement curves of PVA hydrogel tested by AFM probe and the fitting results are compared with the macroscopic results obtained by the macroscopic compression test. (a) Comparison with the absolute value of the elastic modulus of hydrogels, where the shaded part is the elastic modulus obtained from the macro test. (b) Comparison with the relative ratios of elastic modulus of hydrogels where the relative ratio of the elastic modulus of the hydrogel is the elastic modulus measured by the AFM divided by the elastic modulus measured by the macroscopic test. Example 1 (1) Firstly, the model of conical AFM probe and cell was established in ABAQUS, as shown inFIG.2(a). The shape parameters of conical AFM probe were set. The shape parameters were designed by changing the half angle of the cone α and the curvature radius of the cone at the tip r. The compression depth d was set as 600 nm, the half angle of the cone α was set as 60°, the curvature radius of the cone at the tip r was set as 20 nm, 30 nm, 40 nm, 50 nm, 60 nm. (2) Under the action of the external force of the AFM probe, the cells deform along the shape of the AFM probe, and the contact area between the cells and the AFM probe gradually increases, which is a problem of nonlinear contact and large deformation. ABAQUS can solve complex nonlinear problems, so ABAQUS is selected to simulate the deformation of cell under external force. The deformation response under the action of external force was simulated in ABAQUS, as shown inFIG.2(b). The relationship between the normal force of the conical AFM probe and the compression depth was extracted, as shown inFIG.2(c). The grid convergence analysis was carried out to verify the effectiveness of the algorithm. The simulation results are shown inFIG.2(d). (3) The simulated relationship between the normal force and the compression depth of a conical AFM probe was compared with Sneddon model to calculate the relative error in the elastic modulus of cells based Sneddon model fitting. (4) Based on the linear relationship between r/d and relative error δ discovered during data consolidation, as shown in theFIG.4, polynomial function is used to fit the errors which will occur when Sneddon model is used to fit the elastic modulus of cells, as shown in theFIG.5, where the probes with different shape parameters are pressed into different depths. (5) Firstly, the Sneddon model is modified by the fitting function of relative error to obtain the modified formula. Secondly, the force displacement curves of human osteosarcoma cells (MG63) were measured using AFM probes with two shape parameters. The Sneddon model and the modified formula were used to fit the elastic modulus of the cells, respectively. By comparison, it is found that the elastic modulus fitted by the Sneddon model increases significantly with the decrease of the compression depth, while the elastic modulus fitted by the modified formula changes marginally with the change of the compression depth, as shown inFIG.6. Then, the force displacement curves of PVA hydrogels were tested with AFM probes of two shapes. Sneddon model and modified formula were used to fit the elastic modulus of hydrogels, respectively, and then compared with the macroscopic elastic modulus of hydrogels obtained from macroscopic compression test on the universal testing machine. By comparison, it is found that there is an error between the elastic modulus fitted by the Sneddon model and the elastic modulus obtained by the macro test, and the smaller the compression depth is, the greater the error is. The elastic modulus fitted by the modified formula agrees well with the elastic modulus obtained by the macro test, and the error is independent on the compression depth, as shown inFIG.7. The above experiments on PVA hydrogel and human osteosarcoma cells verify the accuracy of the fitting function obtained in the third step. The above implementation example only demonstrates the implementation method of the invention and shall not be construed as limiting the scope of the patent for the invention. It should be noted that a number of transformations and improvements may be made by a person skilled in this field without deviating from the conception of the invention, which are within the scope of protection of the invention. | 6,164 |
11860188 | DESCRIPTION OF THE INVENTION Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. FIG.3is a diagram schematically showing a binary state scanning probe microscope according to an example of the present invention, andFIG.4is a schematic diagram showingFIG.3in more detail. As shown, a scanning probe microscope (SPM) of the present invention includes a probe100, a driving unit200, a voltage applying unit300, a data unit400, and an imaging unit500. The probe100scans a surface of a sample S, the driving unit200includes a piezo actuator210moving the probe100with respect to the sample S, the voltage applying unit300applies a voltage to the sample S, the data unit400converts an electrical signal applied to each electrode120into a data signal, and the imaging unit500generates a surface image of the sample S. First, the probe100according to the present invention will be described in detail.FIG.5shows a probe according to an example of the present invention, andFIG.6shows a conductive tip in a probe. As shown, the probe100of the present invention includes a substrate110, a plurality of electrodes120and a tip array130. The substrate110provides a space in which the conductive tips130are seated, and may be, for example, a glass substrate. The plurality of electrodes120may be formed on the substrate, and specifically, may be formed by coating a metal, which is a conductive material, on the substrate110. The tip array130is provided on the substrate110and includes a plurality of conductive tips135respectively connected to the plurality of electrodes120, and in this case, the conductive tips135may form an array. An array refers to a form in which the conductive tips135are arranged in a predetermined pattern. For example, the conductive tips135may have a quadrangular shape and are arranged in an n×m (n and m are natural numbers of 1 or 2 or greater) array. The conductive tips135may be arranged in a 2×2 form as shown inFIG.5(a). However, the present invention is not limited thereto, and the conductive tips135may be arranged in a circular shape having concentric circles as a whole. At this time, the respective conductive tips135may have the same size and shape as each other, but even if the size and shape (e.g., a position of a tip portion of each of the conductive tips) have a predetermined deviation, information obtained therefrom may be synchronized with each other by software to be corrected, so that an interval between the conductive tips135or an interval patterns formed by arranging the conductive tips135may not be physically equal to each other. Here, each conductive tip135of the present invention may be compressible and relaxed. That is, the probe of the present invention scans the surface of the sample, and as described below, when the probe descends, the conductive tip touches the surface of the sample, and in this state, when the probe continues to descend, the probe may be pressed by the surface of the sample and the conductive tip may be compressed. Thereafter, when the probe changes directions and ascends, the conductive tip may relax to an initial shape thereof. As shown inFIG.6, each conductive tip135of the present invention may include an elastic portion136formed therein and a metal layer137formed on a surface of the elastic portion136. The elastic portion136may be formed of a material having elasticity, for example, an elastomer material, and specifically, may be an elastomer manufactured by curing or crosslinking a curable polymer resin. A specific example of the elastomer may be a cured polydimethylsioxane (PDMS) material. Also, the elastic portion136may have a pyramid shape. The conductive tip135of the present invention may be formed of the metal layer137formed by coating a surface of the elastic portion136formed of an elastic material and having a pyramid shape with a metal, and here, the metal layer137may include at least one of Au, Ag, Cr, Mo, Al, Ti, Cu, Ni, Pt, Pd, Rh, and W and may be formed of a single layer or a multi-layer. FIG.7is a view schematically showing that a conductive tip of the present invention is compressed and deformed. As such, in the conductive tip135of the present invention, the elastic portion136formed therein is formed of an elastic material, so that the conductive tip135may be compressed in contact with the sample S to be deformed, and thereafter, the conductive tip135may be separated from the sample S and relaxed to be returned to its initial shape. Also, since the metal layer137is formed on the surface of the elastic portion136, the surface of the sample S may be scanned using an electrical signal as described below. FIG.8is a graph showing the degree of irreversible deformation according to the properties of the elastic portion and the metal layer in the conductive tip of the present invention.FIG.8(a)shows the degree of irreversible deformation according to a crosslinking ratio of PDMS and a thickness of the metal layer137,FIG.8(b)is a graph showing comparison between crosslinking ratios of PDMS as 5:1 and 30:1,FIG.8(c)is an image of a sample S generated using a probe in which a crosslinking ratio of PDMS is 5:1, andFIG.8(d)is an image of a sample S generated using a probe in which a crosslinking ratio of PDMS is 30:1. The crosslinking ratio of PDMS is a weight ratio of a curable PDMS resin (PDMS base)/curing agent, and the crosslinking ratio of PDMS may be adjusted according to a weight ratio of the curable PDMS resin and the curing agent. For example, as the content of the curing agent increases as compared to the curable PDMS resin, the crosslinking ratio may increase to manufacture a hard elastic portion, and as the content of the curing agent decreases, a soft elastic portion may be manufactured. Accordingly, the crosslinking ratio of PDMS, X:1, which will be described below, refers to the weight ratio of the curable PDMS resin and the curing agent, unless otherwise defined, which means that a soft elastic portion is manufactured as X increases. As shown inFIG.8, as the crosslinking ratio of PDMS and the thickness of the metal layer137(that is, a coating thickness of the metal) decrease, elasticity of the conductive tip135increases, thereby reducing the degree of irreversible deformation, and as the crosslinking ratio of PDMS and the thickness of the metal layer137increase, plasticity of the conductive tip135increases, thereby increasing the degree of irreversible deformation. As shown inFIGS.8(c) and8(d), it can be seen that resolution of the sample image is high when the crosslinking ratio of PDMS is 5:1, whereas the resolution of the sample image is low when the crosslinking ratio of PDMS is 30:1. FIG.9is an SEM image after compression relaxation of a conductive tip according to various examples of the present invention. As shown, the shape of the conductive tip135after compression relaxation may be appropriately restored by appropriately adjusting the crosslinking ratio of PDMS and the thickness of the metal layer137. For example, after the conductive tip135is compressed and relaxed one or more times, a height h1of the conductive tip may be 90% or more, specifically 95% or more, compared to an initial height h0of the conductive tip. For a more preferred example, after the conductive tip135is compressed and relaxed100times or more, the height h1of the conductive tip may be 90% or more of the initial height h0of the conductive tip. In this manner, resolution of the sample image may be adjusted by appropriately adjusting a recovery rate of the conductive tip. Here, the initial height corresponds to a height of the conductive tip in an initial state in which the conductive tip is not compressed and relaxed. If the elasticity of the conductive tip135is too large, the conductive tip135may not be restored to its original state after being compressed. If the plasticity of the conductive tip135is too large, the conductive tip135itself may be damaged when the conductive tip135is compressed, so the conductive tip135cannot be returned to its original state, and thus, the tip portion of the conductive tip135may become dull and the resolution of the sample image may be lowered. Therefore, it is preferable for the conductive tip135to have an appropriate degree of irreversible deformation by appropriately adjusting the thickness of the crosslinking ratio of PDMS and the thickness of the metal layer137. Here, if the ratio of the curing agent to a main material of PDMS (curable PDMS resin) exceeds 20:1, the recovery rate of the conductive tip may decrease, as shown, and thus, the ratio of the main material of PDMS to the curing agent may be between 5:1 and 20:1. That is, the elastic portion136of the conductive tip135may be manufactured from a curing reaction of the curable PDMS resin and the curing agent in a weight ratio of 5:1 to 20:1, preferably 7:1 to 18:1, more preferably 10:1 to 15:1. In addition, if the thickness of the metal layer exceeds 50 nm, the recovery rate of the conductive tip may be similarly reduced, so the thickness of the metal layer may be 50 nm or less, preferably 40 nm or less, more preferably 30 nm or less, and may be 1 nm or more, preferably 5 nm. At this time, the numerical range according to the combination of each numerical upper limit and lower limit should also be interpreted as being included in the thickness of the metal layer according to the present invention. However, the present invention is not limited thereto, and in an actual experiment, a metal layer having a thickness of 100 nm may be used depending on the purpose thereof, and of course, the thickness of the metal layer may be designed to be 50 nm or more in consideration of the elasticity of the elastic portion. FIG.10is a view showing a method of manufacturing a probe according to an exemplary embodiment of the present invention. The probe100of the present invention may be formed by etching a pattern of a tip using a mask (registry) on a silicon substrate to generate a silicon mold etched in a pyramid shape, stacking elastic elastomer (as a specific example, PDMS) on a glass substrate110, generating a PDMS casting including a bottom portion131and a plurality of elastic portions136disposed on the bottom portion using the silicon mold generated before, appropriately patterning a resist on the PDMS casting in consideration of an electrode line to be connected to the conductive tip, and depositing and coating a metal on the PDMS casting on which the resist is patterned. The deposition-coating of the metal may be performed by known deposition processes such as chemical vapor deposition, atomic layer deposition, and physical vapor deposition, but is not limited thereto. As described above, according to the present invention, there is an advantage in that a plurality of conductive tips135may be manufactured at once in a simple process, and in this case, since the metal is deposited on the tip casting on which the resist is patterned, the plurality of electrodes120may be simultaneously formed on the substrate110at the same time when the metal layer137is applied to the surface of the elastic portion136. Thus, since the plurality of electrodes120may be formed by the same process as the process of forming the metal layer137on the surface of the elastic portion136, the probe100may be simply manufactured. In the probe100manufactured as described above, the tip array130may further include the bottom portion131, and the conductive tips135may each be disposed on the bottom portion131. Here, the bottom portion131and the elastic portion136of the conductive tip may be integrally formed as shown in (4) ofFIG.10, and each of the plurality of electrodes120may include a bottom portion surface metal layer121formed by applying a metal to the surface of the bottom portion131. Here, the bottom portion surface metal layer121, which is formed by deposition-coating a metal on the PDMS casting as described above, may be formed simultaneously when the metal layer137of the conductive tip135is formed, and accordingly, the bottom portion surface metal layer121and the metal layer137of the conductive tip135connected to each electrode120may be integrally formed. As described above, compared with the probe of the SPM of the related art including a cantilever and an optical system as essential components, the probe of the present invention does not need a separate cantilever and optical system, so that a configuration thereof is very simple, an overall packaging size may be significantly reduced, it is easy to manufacture the probe, and manufacturing costs may be significantly reduced. Furthermore, since the probe includes a plurality of conductive tips, a large area of the sample may be scanned at the same time, so that scanning throughput may be significantly increased, and since the conductive tip may be freely arranged according to the shape of the sample, the types of samples that can be scanned with the probe may be expanded. Hereinafter, a binary state SPM according to an exemplary embodiment of the present invention including the probe described above will be described in detail. Referring back toFIGS.3and4, as described above, the SPM10of the present invention includes the probe100, the driving unit200, the voltage applying unit300, the data unit400, and the imaging unit500, and although not shown separately, may further include a controller for controlling each component, and may further include a position sensor250measuring a position of a piezo actuator. The driving unit200moves the probe100relative to the sample, and may include a piezo actuator210that is a driving element moving the probe200. The piezo actuator210may move in a direction Z perpendicular to the sample S and a direction XY horizontal to the sample S, and the probe100may be mounted on the piezo actuator210and may be moved in a vertical direction and a horizontal direction with respect to the sample S. Here, as a more specific example, the piezo actuator210of the present invention may include an xy piezo actuator210-1moving a state on which the sample S is mounted in a horizontal direction and a z piezo actuator210-2, on which the probe100is mounted, moving the probe100in a vertical direction, but the configuration of such a piezo actuator may be freely changed, and, hereinafter, the xy piezo actuator210-1and the z piezo actuator210-2will be collectively referred to as a piezo actuator, without being distinguished from each other. The voltage applying unit300applies a voltage to the sample, and when the sample S is a conductive material, the voltage applying unit300may apply a voltage directly to the sample S, and when the sample S is not a conductive material, that is, an insulating material, a surface of the sample S may be coated with a metal, a conductive material, to form a conductive layer on the surface of the sample, and then, a voltage may be applied to the conductive layer on the surface of the sample S. A magnitude of the voltage applied to the sample S by the voltage applying unit300may be about 10 to 100 mV, and may be normally applied with a magnitude of 40 mV. The data unit400converts an electrical signal applied to each electrode120into a data signal, and may generate a data signal by sampling the electrical signal applied to each electrode120. Here, the electrical signal may correspond to a current or a voltage signal applied to each electrode, and in particular, a voltage signal may be used in the present invention. FIGS.11and12show an operating principle of a microscope according to an example of the present invention. As shown, the probe100is moved in a vertical downward direction with respect to the sample S by the piezo actuator210, and each of the conductive tips135of the probe100comes into contact with the surface of the sample S. At this time, since a voltage is applied to the sample S by the voltage applying unit300, the conductive tip135and an electrode120′ electrically connected to the corresponding conductive tip form a closed circuit and a voltage is applied to the electrode120the moment the conductive tip comes into contact with the surface of the sample S. Thereafter, the piezo actuator210moves downward by a set distance and then moves upward again, so that the conductive tip135is separated from the surface of the sample S and no voltage is applied thereto. That is, when the conductive tip135comes into contact with the surface of the sample S according to the downward movement of the probe100, a voltage is applied to the electrode120′ connected to the corresponding conductive tip135, and when the conductive tip135is separated from the surface of the sample S according to the upward movement of the probe (i.e., in the case of non-contact), a voltage is short-circuited with respect to the electrode120′ connected to the corresponding conductive tip135. Whether a voltage is applied to the electrode120′ generated by whether the conductive tip135and the surface of the sample S are in contact with each other may be transmitted to the data unit400and converted into a data signal of 1 or 0 by the data unit400. That is, when a voltage is applied to each electrode120(i.e., when each conductive tip and the sample surface are in contact with each other), the data unit400may generate a binary contact data signal of 1, and when no voltage is applied to each electrode120(i.e., when each conductive tip and the sample surface are not in contact with each other), the data unit400may generate a binary contact data signal of 0, and the data unit400may transmit the generated binary contact data signal to the imaging unit500. FIG.13is a diagram illustrating an operation path of a probe according to an exemplary embodiment of the present invention, illustrating a relationship between a movement path of the probe100and a voltage applied to the electrode120′ connected to the conductive tip135. The sample S may be divided into a lower region L and a higher region H, and the blue line inFIG.13indicates a relative movement path of the probe100with respect to the sample S, and the red line ofFIG.13indicates a voltage applied to the electrode120connected to the conductive tip135. As shown, the probe100, that is, the conductive tip135, moves downward along a path {circle around (1)}-{circle around (3)} and comes into contact with a surface (−0.9 μm point) of the lower region L of the sample S at a point {circle around (2)} of the downward movement path. In this case, as shown, the conductive tip135and the surface of the sample S come into contact at the point {circle around (2)}, so that a voltage is applied to the conductive tip135and the electrode120′. Thereafter, the conductive tip135moves upward along the {circle around (4)} path, the voltage becomes 0 the moment the conductive tip135is separated from the surface of the sample S. In this path, a time point at which a voltage is applied to the conductive tip135corresponds to a moment of about 2 seconds, and a time point at which the conductive tip135is separated from the surface of the sample S corresponds to a moment of about 3 seconds, and thus, it can be seen that a voltage is applied to the conductive tip135for a time interval of a total of 1 second. Thereafter, the conductive tip135moves in the horizontal direction on the surface of the sample S along a path {circle around (5)} to be located above the higher region H of the sample S, moves downwardly along a path {circle around (6)}-{circle around (8)}, and comes in contact with a surface (−0.3 μm point) of the higher region H of the sample S at point {circle around (7)} of the downward movement path. At this time, as shown, at point {circle around (7)}, the conductive tip135comes into contact with the surface of the sample S, so that a voltage is applied to the conductive tip135and the electrode120′, and it can be seen that a corresponding time point corresponds to about 4 seconds.FIGS.14and15are views showing a contact process between the conductive tip and the sample surface. As shown, it can be seen that a voltage is applied to the conductive tip135and the electrode120′ when the conductive tip135comes into contact with the sample S surface and a voltage is not applied when the conductive tip135is separated from the sample surface. In this manner, information on the voltage applied to each electrode120may be sampled by the data unit400, a binary contact data signal converted into 1 and 0 may be transmitted to the imaging unit500, and the imaging unit500may generate a surface image of the sample S using the received sampling data including 1 and 0, that is, a binary contact data signal. Here, the imaging unit500may be provided with information on a time point at which voltage starts to be applied to each electrode120from the data unit400(a time point of 2 seconds according to the example described above) and information on a time interval from the time point to an end point at which a voltage is not applied to each electrode120(a time interval of 1 second with the end point of 3 seconds according to the example described above), and the imaging unit500may generate a surface image of the sample S based on the information. More specifically, the imaging unit500may generate a surface image of the sample with reference to a vertical position (displacement) of the piezo actuator obtained through the piezo actuator position sensor described above. For the example described above, when it is assumed that vertical scanning of two points with a 600 nm step difference in the sample is performed from a point 0.3 μm higher than the higher point H, contact occurs at the higher point H when the vertical position of the piezo actuator is −0.3 μm and contact occurs at the lower point L when the vertical position of the piezo actuator is −0.9 μm, so a step difference of 600 nm may be obtained by comparing displacements of the two points. Meanwhile, in the present invention, information on a vertical movement speed vz-piezoof the piezo actuator210and information on a sampling rate frateof the binary contact signal may be used to obtain maximum resolution of sample measurement. Specifically, when having a specific speed (e.g., 100 μm/s) and a specific sampling rate (e.g., 100 kHz), the resolution of the microscope according to the present invention may be estimated as vz-piezo/frate. FIG.16is a surface image of a sample obtained through a microscope of the present invention, illustrating a topography image of a sample having a square pattern of 16×16 μm2with a height difference of 600 nm.FIG.17is a line profile of the sample ofFIG.16, and it can be seen that a line obtained by a binary-state probe microsopy (BSPM) of the present invention and a line obtained by the conventional single-tip AFM match well. FIG.18is a view illustrating scanning a surface of a sample using a 2×2 array probe according to an example of the present invention, in whichFIG.18(a)schematically illustrates overlapped scanning regions of respective conductive tips, andFIG.18(b)illustrates a composite image of samples obtained from the respective conductive tips. As shown, since the probe includes a plurality of conductive tips, the probe may scan a large region of the sample simultaneously, thereby significantly increasing scanning throughput, and since the overlapped scanning regions between the respective conductive tips are well synchronized, a clear surface image of the sample may be obtained. FIG.19is a test chart image for each Siemens having a thickness of 32 nm.FIG.19(a)is an SEM image, andFIG.19(b)shows an image obtained using the BSPM of the present invention. As shown, it can be seen that the SEM image and the BSPM image match well. FIG.20is a diagram illustrating scanning of a multilayer graphene sample surface using a 100×1 array probe according to an example of the present invention. As shown, parallel scanning may be performed on each region of a sample using 100 conductive tips. In this case, it can be seen that even a height difference of 13 nm may be distinguished on the surface of the sample. FIG.21is a diagram illustrating scanning a periodic table pattern sample surface using a 100×1 array probe according to an example of the present invention. As shown, parallel scanning may be performed on each region of a sample using 100 conductive tips, and it can be seen that even a difference in length of 780 nm may be distinguished on the surface of the sample. As described above, in the SPM of the present invention, as a large-area scanning may be performed on a sample using a probe equipped with a plurality of conductive tips, scanning throughput may be significantly increased, and unlike the multi-probe SPM of the related art that detects a continuous physical interaction of all probes, the present invention may configure a multi-probe structure very simply by recognizing only a contact/non-contact state of a conductive tip and a sample. In addition, according to the present invention, positions of all conductive tips are moved and tracked using a single piezo actuator, thereby minimizing an increase in complexity of a measurement system occurring when the number of conductive tips increases, and since a cantilever and an optical system are eliminated, compared with the existing imaging method requiring micromachining for a cantilever structure in the multi-probe SPM of the related art, the imaging method of the present invention is simpler. Furthermore, the present invention may be applicable to semiconductor microprocess monitoring and high-speed screening of nanomaterials, which conventional SPM has not been applied to. In particular, 2D materials, which are researched as next-generation electronic materials, have physical properties significantly changed according to the number of atomic layers, so large-area photographic measurement is essential, which can be applied to product manufacturing processes using 2d materials. As set forth above, compared with the probe of the SPM of the related art including a cantilever and an optical system as essential components, the probe of the present invention does not need a separate cantilever and optical system, so that a configuration thereof is very simple, an overall packaging size may be significantly reduced, it is easy to manufacture the probe, and manufacturing costs may be significantly reduced. Furthermore, since the probe includes a plurality of conductive tips, a large area of the sample may be scanned at the same time, so that scanning throughput may be significantly increased, and since the conductive tip may be freely arranged according to the shape of the sample, the types of samples that can be scanned with the probe may be expanded. Although the embodiments of the present disclosure have been described above with reference to the accompanying drawings, a person skilled in the art to which the present disclosure pertains will understand that the present disclosure may be implemented in any other specific forms without changing the technical spirit or essential features thereof. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. DETAILED DESCRIPTION OF MAIN ELEMENTS 10: binary state SPM100: probe110: substrate120: a plurality of electrodes121: bottom portion surface metal layer120: electrode connected to each conductive tip130: tip array131: bottom portion135: conductive tip136: elastic portion137: metal layer200: driving unit210: piezo actuator300: voltage applying unit400: data unit500: imaging unit | 27,695 |
11860189 | DETAILED DESCRIPTION Referring now to the drawings, various probe devices are shown for measuring attributes and/or applying power to an electronic circuit under test. Probe device100a, shown inFIGS.1-3A, can have rotatable probe body140that allows a user to extend the conductive tip152of probe connector150into areas which are difficult to reach into with a straight probe body. A user may apply the conductive probe tip152to a conductive surface of an electronic circuit under test where the conductive surface is blocked by an obstruction that cannot accommodate the straight probe device100ashown inFIG.2from reaching. The user may then rotate probe body140relative to main body130as shown inFIG.3Ato change the orientation of the probe connector150relative to the orientation of the main axis of the probe body and/or handle so that the conductive probe tip152can reach behind the obstruction to contact the conductive surface of the electronic circuit under test. Probe device200a, shown inFIGS.4-6A, can have an inductive clamp270that could be used to indirectly measure current flowing through a wire that is disposed within an aperture defined by clamp270. The user could simply depress lever262to move hinged core272as shown inFIG.6Ato open up inductive clamp270. The user may then move probe device200asuch that a wire, such as wire275, associated with an electronic circuit under test is disposed within aperture273, defined by hinged core272and stationary core274. The user could then release lever262to close inductive clamp270about wire275to easily and conveniently measure current through the wire. Another probe device400is shown inFIGS.8-12, having a main body410and a probe body430coupled together by a joint body420and a pin440. The probe body430may be rotatable relative to the joint body420along an axis452. The main body410may be rotatable relative to the joint body420along an axis454. As shown inFIG.13, a user may rotate the probe body430relative to the main body410to allow the user to easily grip the main body410and view the user interface display412while moving the conductive probe tip432to touch a conductive test site of an electronic circuit under test, such as conductive test sites522and524. Alternatively, the user may rotate the main body410while holding the probe body430to ensure that the user may view the user interface display412when using the probe device400. Aspects of these, and other, embodiments are described in further detail below. Referring now toFIGS.1-7, probe device100ais shown inFIGS.1-3Ahaving a main body130and a probe body140. Main body130comprises a grip160while probe body140comprises a probe connector150, allowing a user of probe device100ato easily apply probe connector150to any location on an electronic device to test (not shown) without needing to use a second hand. Grip160comprises a gripping surface with ergonomic features, such as indents162configured to receive fingers of a user's hand and improve friction force between a user's hand (not shown) and grip160. While grip160is shown as having indents162, grip160could be shaped in any suitable way to allow for a user's fingers to easily grip the surface of grip160. Grip160could be made of any suitable material, such as a rubber, a thermoplastic, or a metal, but preferably comprises a non-conductive material having a higher elasticity than a non-conductive shell139of main body130, allowing for a user's hand to slightly compress grip160during gripping to increase the friction force between the user's fingers and grip160. For example, grip160may comprise a rubber having less than 1 GPa while the shell139may comprise a thermoplastic having more than 10 GPa. Probe device100aalso has a power connector120, a ground connector110, and a probe connector150, allowing probe device100ato alternatively apply either power or ground to probe connector150. Power connector120is configured to couple to a power source. For example, in one embodiment power connector120could comprise a 6-foot wire that terminates in a male plug configured to plug into a female A/C outlet. In another embodiment, power connector120could comprise a 3-foot wire that terminates in an insulated alligator clip configured to electronically couple to a positive terminal of a car battery (or other DC power source) or a cigarette plug for power. Ground connector110is configured to couple to a ground source, such as a conductive body via an alligator clip at the end of an 18-inch wire of ground connector110or a male digital multimeter (DMM) jack. Probe connector150is configured to be applied to a conductive surface of an electronic device via conductive tip152. Probe connector150comprises a conductive core that ends in a sharpened conductive tip152and comprises an insulation sleeve that limits the exposed conductive surface of probe connector150. While power connector120is shown as a wire having a plug, ground connector110is shown as a wire having an alligator clip, and probe connector150is shown as a post having a beveled conductive tip, each of power connector120, ground connector110, and probe connector150could be configured in any suitable manner to electronically couple to an appropriate power source, ground source, and conductive surface, respectively. Both power connector120and ground connector110are preferably configured to couple to a power source and ground source, respectively, in a manner that holds them in place without any external force needing to be applied by a user, such that they stay coupled while a user moves probe device100awith his/her hand, which could apply some lateral forces to any wires of power connector120and ground connector110. This allows a user to freely move probe connector150around an electronic device without needing to worry about decoupling either power connector120or ground connector110. Contemplated coupling mechanisms include biased clamps, mating indents and detents, male and female plugs, and threaded connections. Probe connector150is preferably plug engageable with to a female outlet (not shown) of probe body140and is held in place within probe body140using friction force, such as a mating elastic indent/detent that holds an end (not shown) of probe connector150in place within the female outlet of probe body140. Such receptacle jacks could be similar to those used for a DMM jack. In some embodiments, probe connectors of different shapes and/or sizes could be plugged into the female outlet to allow for different types of probe connectors to be coupled to probe body140. For example, probe connectors that are J-shaped, angled, offset, thin, or flexible could be interchanged with probe connector150to plug into the same outlet. While ground connector110and power connector120are shown here as coupled to main body130and probe connector150is shown here as coupled to probe body140, the terminals of probe device100acould be coupled to any suitable portion of probe device100ato allow for electricity to flow from the conductive surface of the terminal to the internal circuitry of probe device100a. User interface130ais disposed on a top surface of main body130. User interface130ais preferably placed on an opposite side of grip160on probe device100ato allow for a user's fingers to grip the surface of grip160while the user's thumb is positioned to manually regulate the operation of elements of user interface130a. This allows a user to manipulate any switches of user interface130awithout needing to move the user's fingers from grip160or move probe device100ain any manner to displace probe connector150in order to interact with user interface130a. In some embodiments, another user interface could be positioned opposite user interface130a, for example a trigger that activates a light, such as light146. In such an embodiment, the user could activate the trigger with a forefinger while simultaneously regulating operation of user interface130a. User interface130ahas a breaker reset button131, a beeper button132, a main switch133, a voltage switch134, a display135, a positive light136, and a negative light137. Main switch133is shown as a 3-position rocker switch having an overmold rubber cover that allows a user to push forward to a forward position (or power position) to switch main switch133to a positive applied voltage mode, backward to a rear position (or ground position) to switch main switch133to a negative, or ground, applied voltage mode, and to the center position (or measure position) to switch main switch133to a voltage measure mode. Main switch133is preferably biased to return to the measure position when no force is placed on main switch133(e.g. when a user releases a thumb from main switch133) so that probe device100a, by default, measures attributes of the electronic circuit under test. Main switch133could be configured to be biased to return to the measure position using any suitable means, for example by using a spring or a resiliently deformable flange. Preferably, main switch133is configured such that main switch133is only in one position at a time, which prevents probe device100afrom being placed in a mode that can both apply power and measure voltage or current simultaneously. Referring to the exemplary embodiment having three positions (a power position, a ground position, and a measure position) above, when main switch133is in the power position, a processor (not shown) in probe device100apreferably transmits power from power supply connector120to probe connector152. The amount of power applied can be controlled by manipulation of voltage switch134, which is shown as controlling the power output between 3 volts, 5 volts, and 12 volts. When a user moves voltage switch134from the 3-volt position to the 12-volt position, the amount of voltage applied to probe connector150when main switch133is pushed to the power position is changed from 3 volts to 12 volts. Preferably display135does not show any numerical values while main switch133is in the power position, allowing a user to quickly note that probe device100ais not in measurement mode with a quick glance at display135. While voltage switch134is shown as being configured to allow a user to switch the power output between three different modes, more or fewer switching modes, such as two modes or five modes, could be used in alternative embodiments. Probe device100ais configured to display voltage values on display135when probe device100ais in measure mode. Display135is configured to display voltage that is measured by probe connector150. For example, when main switch133is in the measure position and conductive tip152probe connector150is applied to a conductive surface of an electronic circuit under test, display135will display the measured voltage on display135. As noted previously, when main switch133is in the measure position, no power is applied to probe connector150from probe device100a. In preferred embodiments, this measured voltage will continue to be shown on display135until a user moves main switch133to another position, or until power connector120is unplugged from a power source. In this way, if a user is not able to see display135when probe connector150is applied to an electronic device and main switch is in the measure position, the user can later look at display135to see what the measured voltage is. In some embodiments, the measured voltage may change over time, such as when a conductive surface has a periodic voltage shift, or when a user applies probe tip152to many different conductive surfaces while main switch133is in a measure position. In this embodiment, a memory (not shown) of probe device100acould save a series of measured voltages at different periods of time and could replay those voltages in a loop on display135. The periods of time could be preprogrammed or could be set by a user via a separate connection (e.g. a Bluetooth wireless connection or a USB connection). For example, a user or an admin could set probe device100ato save 5 seconds of voltage measurements, one for every 0.1 seconds, or 10 seconds of voltage measurements, one for every 0.5 seconds. In other embodiments, a user could save measured voltages, for example by pushing down on main switch133(i.e. towards grip160) while main switch133is in the measure position. Probe device100acould also be configured to activate positive light136when main switch133is in the power position and activate negative light137when main switch133is in the ground position, rapidly informing a user of probe device100awhen probe connector150is safe to touch. When power is not applied to probe connector150, for example when main switch133is in the measure position, probe device100acould be configured to activate positive light136when probe connector150measures a non-zero voltage, and could be configured to activate negative light137when probe connector150measures a ground charge. Again, this rapidly informs a user when probe connector150is safe to touch. Positive light136is preferably a red LED to provide a warning notification while negative light137is preferably a greed LED to provide a safety notification, although other colors and/or types of light are contemplated. Beeper button132provides a way for a user to activate or deactivate a beeper that activates when probe connector150is not safe to touch, for example when a positive voltage is measured via probe connector150when probe device100ais in measure mode, or when a positive voltage is applied to probe connector150when probe device100ais in a power mode. The beeper speaker preferably deactivates when probe connector150is safe to touch, for example when a ground voltage is measured via probe connector150when probe device100ais in measure mode or when a ground voltage is applied to probe connector150when probe device100ais in a ground mode. In this manner, the speaker (not shown) could act similarly to positive light136and negative light137, providing an auditory signal that probe connector150is safe or not safe to touch in addition to the visual signal of positive light136and negative light137. Breaker reset button131provides a way for a user to reset an internal fuse (not shown) of probe device100a. The internal fuse shuts off power between power connector120and electronic components of probe device100awhen a voltage or a current exceeds a predefined threshold to prevent electronic components from being damaged. When a user presses breaker reset button131, the internal circuit breaker is reset, allowing power to flow from power connector120to the internal circuitry of probe device100a. A user of probe device100amay attempt to apply probe connector150to conductive terminals that are difficult to reach, such as terminals behind an obstacle or at an awkward angle from where a user is. In these situations, it is useful to alter an angle between the probe axis174of probe body140relative to the main axis172main body130, such as in the configuration shown inFIG.3A. Main body130has a main axis172running along its major length shown inFIG.2, while probe body140has a probe axis174running along its major length shown inFIG.3A. InFIG.2, main axis172is shown as substantially parallel to probe body174while inFIG.3A, main axis172is shown as angled at substantially a 60-degree angle to probe body174. As used herein, two axis that are substantially parallel to one another are parallel to one another within 1, 2, 3, 4, or 5 degrees from one another. Joint142is shown here as a rotational condyloid joint having an ovoid cross-sectional rotation surface. A user can rotate probe body140relative to main body130by twisting probe body140in a clockwise or counter-clockwise direction to rotate probe body140between a first configuration shown inFIG.2and a second configuration shown inFIG.3Aabout rotational axis182. Preferably, main body130and probe body140are configured to have mating indents and detents that hold the two bodies in place relative to one another when in the first configuration and in the second configuration. While joint142is shown as a rotational condyloid joint, any suitable joint or pivot point could be used to allow main body130and probe body140to rotate relative to one another, such as a hinge joint, a pivot joint, a ball and socket joint, or a saddle joint. Likewise, while joint142allows a user to rotate main body130and probe body140between only two different angles relative to one another, joint142could be configured to allow main body130and probe body140to rotate to many different angles relative to one another, for example a parallel angle, a 30 degree angle, a 60 degree angle, a 90 degree angle, a 120 degree angle, and so on and so forth. While only two bodies—main body130and probe body140—are shown coupled to one another via a joint142, probe device100acould comprise any number of components that rotate relative to one another in any number of ways in alternative embodiments. Probe device embodiments having a plurality of joints may be useful to thread a probe device through a complex passageway. Portions of probe device100aare shown as having a non-conductive rubber overmold having an elastic tensile strength (e.g. less than 1 GPa) used to improve friction tension for when a user grips the overmold areas, and to decrease abrasive damage that could be caused by a user brushing up against a nonelastic surface. Preferably, main switch133has a rubber overmold to assist a user who moves main switch133from one position to another position, breaker reset button131and beeper button132have rubber overmolds to assist a user to push the buttons to activate a breaker or activate/deactivate a beeper, and portions of probe body140have rubber overmold144to assist a user to rotate probe body140relative to main body130. A probe light146is preferably mounted on an end of probe body140to illuminate an area that probe connector150is applied to. Probe light146is preferably a white, bright LED, although any color and/or type of light could be used in alternative embodiments. Probe light146preferably activates when a user pushes or pulls on main switch133, and deactivates after a threshold time period has passed, such as 1-2 minutes. While probe light146is shown as coupled to an end of probe body140, probe light could be coupled to any portion of probe device100ain alternative embodiments, and could be even mounted to a stiff arm that could be aimed by a user by manipulating the stiff arm. Activation of probe light146could be triggered in any suitable manner, for example by an accelerometer that detects a minimum threshold movement of probe device100a, or by an index-finger trigger located on grip160of probe device100a. FIG.3Bshows an alternative probe device100bhaving first joint142and second joint192. Second joint192comprises a second condyloid joint that rotates a first portion of main body130arelative to a second portion of main body130babout a rotational axis184. A user could rotate the second portion of main body130brelative to probe body140about first joint142and rotational axis182and rotate the first portion of main body130arelative to the second portion of main body130babout second joint192and rotational axis184to offset conductive probe tip152from the first portion of main body130a. This could be useful in scenarios where a user needs to snake conductive probe tip152just to the side of an obstacle. FIG.3Cshows another alternative probe device100chaving a first joint142and a second joint194. Second joint194comprises another condyloid joint that rotates a first portion of main body130crelative to a second portion of main body130dabout rotational axis186. Similar to probe device100b, a user of probe device100ccould rotate the second portion of main body130drelative to probe body140about first joint142and rotational axis182and rotate the first portion of main body130crelative to the second portion of main body130dabout second joint194and rotational axis186to offset conductive probe tip152from the first portion of main body130a. This could be useful in scenarios where a user needs to snake conductive probe tip152around an obstacle in a U-shaped manner to wrap around the obstacle. While a condyloid joint is shown inFIGS.3B and3C, any other suitable joint could be used to alter a shape of probe device100bor100cto allow conductive probe tip152to access previously inaccessible areas. An alternative probe device200ais shown inFIGS.4-6A, where the main body230and the probe body240are formed of a single contiguous piece. Probe device200ais similar to probe device100a, in that probe device200aalso has a power connector220, a ground connector210, a probe connector250, and a grip260opposite a user interface230a, wherein user interface230ahas a breaker reset button231, a beeper button232, a main switch233, a voltage switch234, a display235, a positive light236, and a negative light237, which each function similarly to their corresponding parts on probe device100a. However, probe device200ahas an induction clamp270that could be used to inductively measure a current in wire275clamped by induction clamp270. Induction clamp270comprises a clamp that inductively measures current via an inductive coil. Typical induction clamps use a hinged core with a compensation winding wrapped around a portion of the core, where the opening of the clamp acts as a magnetic field sensor within an air gap. Any suitable induction clamp could be used. Induction clamp270comprises a stationary core274embedded within a bottom section of probe body240and comprises a hinged core272controlled by lever262on grip260. When a user's fingers (e.g. a user's forefingers or index and middle fingers) pull on lever262, hinged core272opens, as shown inFIG.6A, allowing the user to dispose induction clamp270around wire275for indirectly measuring a current through wire275. Lever262is preferably biased, for example using a spring, to open when force is applied to lever262, allowing a user to close hinged core272by simply releasing a compressive force on lever262. User interface230acomprises a current switch238that is configured to allow a user to activate an inductive current-measuring mode, and to alter the amount of currents measured by the probe device. Both the voltage-measuring mode and the current-measuring mode are preferably modes wherein probe200adoes not apply any voltage to probe connector250. Here, current switch238is shown as a switch that switches probe device230between three modes—an off mode, a 1 mV/A mode, and a 10 mV/A mode. More or less modes could be used in other embodiments. When current switch238is switched to off mode, probe device230is configured to measure volts flowing through probe connector250only, allowing probe device230to act similarly to probe device130. Display235is configured to display a VOLT indicator, which indicates to a user that probe device230is in volt measurement mode. When current switch238is switched to 1 mV/A mode, probe device230is configured to measure a first range of amps flowing through probe connector250(e.g. 5 A to 300 Arms). In 1 mV/A mode, display235displays an AMP indicator, which indicates to a user that probe device230is in current measurement mode. When current switch238is switched to 10 mV/A mode, probe device230is configured to measure a second range of amps flowing through probe connector250(e.g. 50 A to 3000 Arms). In 10 mV/A mode, display235also displays an AMP indicator, indicating to a user that probe device230is in current measurement mode. Clamp270is preferably disposed adjacent to probe connector250, which allows a user to easily manipulate the position of probe connector250by manipulating grip260with only one hand. User interface230ais disposed to be slightly recessed from an upper surface of main body230, allowing portions of the user interface, such as breaker reset button231, beeper button232, and main switch233to remain below an upper plane280of probe device200a. This allows a user to set probe device200adown on the side of user interface230awithout danger of contact with a table surface or a floor surface accidentally interacting with user interface230a(e.g. pressing any buttons or pushing any switches of user interface230a). The upper surface of main body230preferably has a recess282to form legs284and286, which further assist in allowing a user to place probe200aon a surface with the upper surface of main body230facing down without accidentally activating any of the buttons or switches on user interface230a. FIG.6Bshows an alternative probe device200bhaving a joint292along a top edge of probe body240. Joint292represents another condyloid joint that allows probe connector250to rotate at a substantially 90-degree angle relative to the orientation shown inFIG.6B, allowing conductive probe tip252to reach around obstacles to contact conductive surfaces of an electronic circuit under test that probe device200bwas previously unable to reach. While a condyloid joint is shown, any other suitable joint could be used to alter a shape of probe device200bto allow conductive probe tip252to access previously inaccessible areas. FIG.7shows a logical schematic300of a probe device having a power connector310, a ground connector320, a power interface330, a fuse340, a processor350, memory355, a user interface360, a voltage measurement device370, current measurement device375, a power transformer380, and a probe connector390. In some embodiments, a current measurement device375could be used to measure current flowing through probe connector390. In other embodiments, such as with probe device200a, a current measurement device375could be used to indirectly measure current flowing through wire275through inductive clamp395. Power interface330interfaces with both the ground connector320and the power connector310to receive power from a power source coupled to power connector310and to receive ground from a ground source coupled to ground connector320. Power interface330also provides power to components of the probe device, such as processor350, memory355, measurement370, and power transformer380, via fuse340. If processor350detects a power surge in one of the connected devices, or if fuse340detects a power surge from power connector310, a command could be sent to fuse340to break a power connection between power interface330and any of processor350, memory355, voltage measurement device370, power transformer380, and/or current measurement device375and prevent the power surge from damaging any of the electronic components of the probe device. When a user transmits a break command from user interface360(e.g. via break button131or break button231), processor350could receive the command and send a reset command to fuse340to reset the fuse and allow power to flow again from power interface330. Processor350executes software instructions saved on memory355to control the various electrical components of the probe device and to process commands transmitted by user interface360. For example, where user interface360transmits a command to processor350to operate in voltage measurement mode, processor350could transmit a command to voltage measurement device370to measure a voltage via probe connector390and output that measured voltage amount to a display of user interface360. Where user interface360transmits a command to processor350to operate in current measurement mode to measure current flowing through probe connector390, processor350could transmit a command to current measurement device375to measure current flowing through probe connector390and outputs that measured current amount to a display of user interface360. Where user interface360transmits a command to processor350to operate in current measurement mode to measure current flowing through inductive clamp395, processor350could transmit a command to current measurement device375to inductively measure current from wire275within inductive clamp395and outputs that measured current amount to a display of user interface360. In some embodiments, the measured attributes could temporarily be saved on memory355to be output to a display of user interface360over a period of time, or at a later time. Where user interface360transmits a command to processor350to operate in power mode at a specified voltage (e.g. 3V, 5V, or 12V), processor350could transmit a command to power transformer380to transmit the specified voltage from power connector310to probe connector390. When the probe device is in power mode, processor350could also activate a positive light of user interface360(e.g. positive light136of probe device100a), and could transmit a command to user interface360to deactivate the display that displays a measured voltage. Where user interface360transmits a command to processor350to operate in ground mode, processor350could transmit a command to power transformer380to transmit the ground voltage from ground connector320to probe connector390. When the probe device is in ground mode, processor350could also activate a negative light of user interface360(e.g. negative light137of probe device100a), and could transmit a command to user interface360to deactivate the display that displays a measured voltage. Where user interface360transmits a command to processor350to activate a speaker when voltage is flowing through probe connector390(e.g. because power transformer380is transmitting voltage from power connector310, because voltage measurement device370detects a voltage from probe connector390, or because current measurement device375detects a current from probe connector390), then processor350could transmit a buzzing noise to a speaker of user interface360when a voltage or current is detected to be flowing through probe connector390. In preferred embodiments, the probe device is configured such that voltage measurement device370and power transformer380cannot be activated simultaneously. This is to ensure that the device does not apply and measure voltage or current simultaneously. Using the disclosed embodiments, a user could easily use a probe device, such as probe device100aor probe device200a, to apply power, apply ground, measure voltage, or measure current of an electronic device all with just one hand. For example, a user could use probe device100ato measure voltage of an electronic device by coupling power connector120to a power source, such as by plugging a male plug into a female A/C outlet, and by coupling ground connector110to a ground source, such as a conductive body of the electronic device. The user could then ensure that main switch133is in the measure position, for example by using a thumb to manipulate main switch133, or by not applying any force to main switch133in embodiments where main switch133is biased to always return to the measure position when no force is applied to main switch133. When main switch133is in the measure position, the user could then manipulate grip160to position the conductive tip152of probe device100ato any conductive surface of the electronic circuit under test to determine how much voltage is that conductive surface has as compared to ground. Probe device100awould then measure the voltage, and display the measured voltage on display135, illuminate the positive light136if the measured voltage is greater than zero, and illuminate the negative light137if the measured voltage is zero. If the user is in a position where they cannot see display135, the user could push beeper button132to activate the beeper, instructing probe device100ato make an audible beep if the measured voltage is greater than zero, and not make an audible beep if the measured voltage is zero. If the conductive surface of the electronic circuit under test is in an area that cannot accommodate the full length of probe device100a, the user could rotate probe body140relative to main body130by grabbing probe body140with one hand and main body130with another hand and twisting to move probe device100afrom the first configuration shown inFIG.2to the second configuration shown inFIG.3A. This way, conductive tip152could be more easily maneuvered behind obstacles or around short passages that cannot accommodate the full length of probe device100a. In some embodiments where a user might need to use a probe connector of a different shape or size, the user could unplug probe connector150from probe body140and could plug a new probe connector into the female socket. Should a user wish to temporarily apply power or ground to an electronic circuit under test (e.g. if the user wishes to activate the electronic device but it is not plugged in, or if the user wishes to discharge the electronic device to be able to move it to a second location), the user could use their thumb to push main switch133to the positive position, or pull main switch133to the negative position. When main switch133is in the positive position, probe device100acould activate the positive warning light136, and could activate a beeper if the beeper has been turned on. When main switch133is in the negative position, probe device could activate the negative warning light137. If a power surge activates an internal fuse of probe device100a, the probe device could break a circuit between the power from power connector120and one or more electrical components of probe device100a, and the user could press breaker reset button131to reset the internal fuse. All of the essential operations of probe device100acan be performed easily with one hand once the user has connected power connector120, ground connector110, and has set probe device100ato the proper straight or angled configuration. A user could also use probe device200ato perform similar functions, such as measuring voltage, applying power, applying ground, and measuring voltage, and could also use probe device200ato inductively measure current of a wire, such as wire275. The user could first use their thumb to push current switch238from the off position to one of the active positions, such as 10 mV/A, which would trigger probe device200ato alter display235from displaying the word VOLT to displaying the word AMP as an indicator that probe device200ais now in current-measurement mode. The user could then use their fingers to pull on lever262and open hinged core272. Once hinged core272is open, the user could move probe device200ato wrap either stationary core274or hinged core272around wire275under test and release lever262to close hinged core272. Probe device200awould then indirectly measure current in wire275via induction, which would then be displayed on display235. Referring now toFIGS.8-12, a probe device400may have a main body410, a joint body420, and a probe body430. As shown inFIG.11, the joint body420may have an aperture426, which may be configured to receive the shaft411of the main body410shown inFIG.9.FIG.8shows the joint body420mounted on the main body410when the shaft411is received by the aperture426. The shaft411of the main body410may have an indent415(seeFIG.9) that circumscribes the outer circumference of the shaft411to mate with the projection427(seeFIG.11) of the aperture426which circumscribes the inner circumference of the aperture. Such a configuration may help to ensure that the joint body420stays on the main body as the as the main body410rotates relative to the joint body420along the axis454shown inFIG.8. Thus, the mechanical, rotary connection between the aperture426of the joint body420and the shaft411of the main body410may collectively form a joint that allows the probe body430and the main body410to rotate relative to one another about the axis454. While the mechanical connection between the joint body420and the main body410forms a rotary joint, other types of joints may be used between the joint body420and the main body410may be used, such as a hinge joint, a pivot joint, a ball and socket joint, a saddle joint, or a condyloid joint. While the shaft411is shown having the indentation415sized to mate with the projection427, other rotational coupling mechanisms may be used to facilitate a rotary joint mechanical coupling between the shaft411and the aperture426, such as a ball bearing mechanism or a friction fit. Moreover, an opposing configuration is contemplated such as an indentation in the aperture426that is sized and disposed to mate with a projection formed in the shaft411. Allowing the joint body420to rotate about the shaft411along the axis454shown inFIG.8allows a user to rotate the probe body430relative to the main body410about the axis454to ensure that a user may easily manipulate the probe body430while yet maintaining a direct view of the display412. As shown inFIG.11, the joint body420may also have a left knuckle422and a right knuckle424sized and disposed to receive the pin440ofFIG.12via the apertures417and419, respectively. In addition, the probe body430(seeFIG.10) may also have a knuckle438having an aperture425sized and disposed to receive the pin440. As shown inFIG.8, the knuckle438of the probe body430may be disposed between the left knuckle422and the right knuckle424of the joint body, and the pin440may be inserted through the apertures of the left knuckle422and right knuckle424of the joint body420and the aperture of the knuckle438of the probe body430. The pin440may be retained in the knuckles422,424via a set screw442and be friction fit. In such a configuration, the probe body430may rotate relative to the joint body420along the axis452shown inFIG.8. The pin440may then be locked in place within the knuckles422,424, and438by tightening the set screw442shown inFIGS.8and12. Thus, the mechanical, hinged connection between the knuckles422and424of the joint body420, the knuckle438of the probe body430, and the pin440may collectively form a joint that allows the probe body430and the main body410to rotate relative to one another about the axis452. While the mechanical connection between the joint body420and the probe body430forms a hinge joint, other joints between the joint body420and the probe body430may be used, such as a rotary joint, a pivot joint, a ball and socket joint, a saddle joint, or a condyloid joint. It should be understood that the pin440may also have another set screw installed on the opposite side on knuckle422. The pin440may also have ringed detents441and443that may be sized to mate with the ringed indents421and423of the left knuckle422and the right knuckle424, respectively, when set screws, such as the set screw442, are tightened. Other coupling mechanisms may be used to facilitate movement about hinged mechanical coupling, such as a detent in the knuckles and a matching indent in the pin or ball bearing joints. While a rotary joint is shown as being used to allow the main body410to rotate relative to the joint body420along the axis454and a hinge joint is shown as being used to allow the probe body430to rotate relative to the joint body420along the axis452, any suitable joint that allows one body to rotate relative to the other body may be used, such as a ball and socket joint, a saddle joint, a condyloid joint, a hinge joint, a rotary joint or a pivot joint. While a single joint body420is shown as interposed between the main body410and the probe body430to provide two separate axis of rotation, a plurality of rotary joints may be interposed between the main body410and the probe body430to allow more than two axes of rotation between the main body410and the probe body430, which would enable the probe body430to be oriented in any number of configurations relative to the main body410, allowing such a probe device to snake through any number of obstacles during use. As shown inFIG.9, the shaft411of the main body410may also have a port413that allows one or more cables, such as the cable431of the probe body430shown inFIG.10, to plug into the probe body430and provide an electrical connection between the probe body430and the main body410. The cable431may be routed through the aperture426of the joint body420and connected to the electronics located in the main body410via the port413. Allowing at least one cable431of the probe body430to plug into the port413allows for electronic communication between the probe body430and the main body410. The cable431may have a plurality of wires that allow electronic communication between the probe body430and the main body410. For example, a first wire in the cable431may be used to transmit a signal from a processor of the main body to a heating circuit of the probe body430to activate a heating circuit, while a second wire in the cable431may be used to close a circuit between the ground terminal404and the conductive probe tip432. Since the main body410only rotates 360°, the cable431does not get tangled as the probe body430rotates relative to the main body410. As shown inFIG.8, the probe device400may have a ground cable402with a ground terminal404that may be configured to couple to a ground source, such as the frame of a car. The ground terminal404acts as a conductive ground connector that may be used to measure a metric of a device under test. For example, when a user touches the conductive probe tip432of the probe device400to a conductive test site of a device under test, the processor of the probe device400may calculate a voltage between the probe tip432and the ground terminal404, or calculate a current between the probe tip432and the ground terminal404at a specified resistance. The probe device400may also have a power cable406with a positive terminal407and a negative terminal408that may be coupled to positive and negative terminals of a power source, such as a car battery or cigarette lighter. The power cable406may be used to deliver power to portions of the probe device400, such as the processor, the probe tip432, or to a heating circuit of the probe body410. The conductive probe tip432may extend from an insulated shaft433that may comprise an insulated surface made of a material that both conductively insulates and thermally insulates the surface from the core of the shaft433—for example a rubber or silicone. As shown inFIGS.9and10, the main body410and the probe body430may have one or more user interfaces, such as a display412, a voltage switch418, a main switch416, a positive light434(e.g., light emitting diode), a negative light435, and a breaker reset button436, which may function similarly to the display235, voltage switch234, main switch233, positive light236, negative light237, and breaker reset button231of the user interface230ofFIG.4, respectively. A processor may be disposed within the walls of the main body410to transmit signals via the cable431to the probe body430, such as signals to activate or deactivate the positive light434and negative light435, or signals to activate a heating element685(seeFIG.14) in the probe body430. The main body410may also have a soldering switch414, which may be used to transmit a signal to the processor to activate a heating element of the probe body430. The heating element may be in heat conduction to the probe tip432so that, when the heating element heats up, the probe tip heats up too as well. A signal from the processor may be transmitted to a heating element687(seeFIG.14) of the probe body430via the cable431to heat the probe tip432to a hot enough temperature to melt solder, for example to a temperature at least 350° F., 365° F., 375° F. or 400° F. The insulated shaft433may comprise an insulated material that thermally insulates the surface from the core of the shaft433—for example a rubber or silicone. Such insulation preferably insulates the surface temperature of the probe shaft to be at most 200° F. when the probe tip432is heated to a temperature of 400° F. via the heating element687. As shown inFIG.13, the probe device400may be used to test a device under test500without needing a user510to maneuver their hand512around obstacles to touch the conductive probe tip432conductive test sites, such as conductive test sites522and524. The user510may rotate the probe body430relative to the main body410along the axis452shown inFIG.8to allow the probe body430to fold along multiple planes of rotational freedom. The user510may also rotate the probe body430relative to the main body along the axis454shown inFIG.8to allow the probe body430to rotate about the main body410along 360 degrees of rotational freedom. Allowing multiple axes of rotational freedom allows the user510to hold the main body410using their hand512while looking flat at the user interface display412while rotating the conductive probe tip432in an appropriate direction to still touch conductive test sites522and524. The main body410may remain stationary in the user's hand512while the probe body430may be configured to rotate along multiple non-parallel planes, such as the X, Y, and/or Z planes. The first and second axes452,454are shown as being perpendicular to each other. However, it is also contemplated that the first and second axes452,454may be at other angles relative to each other such as between 45 to 90 degrees. A user may use the probe device400to perform any suitable metric measurement of a device under test, such as an electric load continuity test, voltage measurement or a current measurement. An electric load continuity test may be conducted, for example, to determine if there exists a bad connection in a circuit. For example, both of the conductive test sites522and524may be located on a common bus of a circuit for a device under test that is connected to a power supply that provides 12.2 V. A user may configure the probe device400to be in voltage measurement mode using the voltage switch418(shown inFIG.9) and view the current voltage flowing through the conductive probe tip432on the display412when testing the device under test500inFIG.13. The user may touch the conductive probe tip432to the conductive test site522and to the conductive test site524, and the user may then view the display412to measure the voltage at both conductive test sites. The user may expect to see a reading of 12.2V for both measurements, as both the conductive test site522and the conductive test site524are located on the same bus connected to the power supply that provides 12.2 V. If the measured voltage at each of the conductive test sites differs by more than 0.5 V (e.g. the measurement at the conductive test site522is 12.2V, while the measurement at the conductive test site524is 11.5V), the user may then understand that there is a bad connection between the conductive test sites522and524. The minimum threshold voltage drop used to determine if a circuit is bad may vary, for example a user may determine that a circuit is bad if the measured voltage at the conductive test sites vary by more than 0.1 V or 2 V. Such an electric load continuity test may also be performed on any number of conductive surfaces that share a common electrical bus, such as a wire or a multiplexor. FIG.14shows a logical schematic600of a probe device having a power connector610, a ground connector620, a power interface630, a fuse640, a processor650, memory655, a user interface660, a voltage measurement device670, current measurement device675, a power transformer680, a probe connector690, and a probe connector heating element687. Power interface630interfaces the power connector610to receive power from a power source coupled to power connector610and provides power to components of the probe device, such as processor650, memory655, measurement670, and power transformer680, via fuse640. The processor650may be configured to detects a power surge in one of the connected devices, or the fuse640may be configured to detect a power surge from power connector610, and, in response, trigger a command to fuse640to break a power connection between the power interface630and any of processor650, memory655, voltage measurement device670, power transformer680, and/or current measurement device675, thereby preventing the power surge from damaging any of the electronic components of the probe device. When a user transmits a break command from the user interface660(e.g. via the breaker reset button436ofFIG.9), the processor650may be configured to, in response to receiving the command, send a reset command to fuse640to reset the fuse and allow power to flow again from power interface630. The ground connector620may be conductively coupled to a bus that allows any of the processor650, the voltage measurement device670, and/or the current measurement device675to perform measurements against the ground source, such as a measurement of a voltage at the probe connector690as compared to the ground source, or a current flowing through the probe connector690to the ground source. The processor650may be configured to execute software instructions saved on memory655to control the various electrical components of the probe device and to process commands transmitted by user interface660. For example, the where user interface660transmits a command to processor650to operate in voltage measurement mode, the processor650may transmit a command to voltage measurement device670to measure a voltage via probe connector690and output that measured voltage amount to a display of user interface660. Such a configuration allows a user to perform a continuity test on the electronic load of an electronic device under test by connecting the probe device to the ground via the ground connector620, and then by touching the probe tip695to a plurality of conductive test sites that share the same conductive bus such as a wire. For example, using the probe device400inFIG.8, a user may attach the ground terminal404to a ground source, such as the body of a car. The user may then grip the handle417of the main body410to direct the conductive probe tip432to two or more conductive test sites that share the same conductive bus and look at the display412to determine the measured voltage at each conductive test site. If there is a bad connection between conductive test sites that share the same conductive node, the circuit's voltage may drop more than 0.5 V between one conductive test site and another (e.g. the user expects 12V but sees 11.2 V), which may be detected by the voltage measurement device670and sent to the user interface660for monitoring by a user. Where the user interface660transmits a command to processor650to operate in current measurement mode to measure current flowing through probe connector690, the processor650may be configured to transmit a command to current measurement device675to measure current flowing through probe connector690and output that measured current amount to a display of user interface660. The processor650may be configured to temporarily save the measured attributes to the memory355to be output to a display (e.g. display454ofFIG.8) of the user interface660over a period of time, or at a later time. Where user interface660transmits a command to processor50(e.g, user applies a force to the main switch416on the main body410) to operate in power mode at a specified voltage (e.g. 3V, 5V, or 12V), the processor650may be configured to transmit a command to power transformer680to transmit the specified voltage from power connector610to probe connector690. When the probe device is in power mode, the processor650may be configured to also activate an LED of user interface660(e.g. positive light434of probe body430inFIG.10), and may be configured to transmit a command to user interface660to deactivate the display (e.g. the display412on the main body410ofFIG.9) that displays a measured voltage. Where user interface660transmits a command to processor650to operate in ground mode, the processor650may be configured to transmit a command to power transformer680to transmit the ground voltage from ground connector620to probe connector690. When the probe device is in ground mode, the processor650may be configured to also activate a negative light of user interface360(e.g. negative light435of the probe body430inFIG.10), and may be configured to transmit a command to user interface660to deactivate the display (e.g. the display412of the main body410ofFIG.9) that displays a measured voltage. Where the user interface660transmits a command to processor650to activate a speaker when voltage is flowing through probe connector690(e.g. in response to detecting that power transformer680is transmitting voltage from power connector610, in response to detecting that the voltage measurement device670detects a voltage from probe connector690, or in response to detecting that the current measurement device675detects a current from probe connector690), then processor650may be configured to transmit a buzzing noise to a speaker of the user interface660when a voltage or current is detected to be flowing through the probe connector690. The user interface660may be configured to transmit a command to the processor650to activate a heating element, such as via activation of the soldering switch414inFIG.9. Upon receiving a trigger from the user interface660, the processor650may be configured to transmit a signal to activate the heating circuit685, such as by transmitting current through a resistor, to heat up the probe connector heating element687. Allowing the probe connector to be heated allows a user to temporarily change a probe device, such as the probe device400inFIG.8, to act as a temporary welding hand, or a soldering hand, by providing a heated wand in the form of the heated probe tip432so the user may solder some wire in case it's needed. The probe device may be configured such that the modules cannot be activated simultaneously, for example the voltage measurement device670and power transformer680, the current measurement device675and the power transformer680, or the heating circuit685and the voltage measurement device670. Such a configuration ensures that the device does not damage components, or apply power and measure voltage simultaneously. Using the disclosed embodiments, a user could easily use a probe device, such as probe device400, to apply power, apply ground, measure voltage, measure current, or apply solder to a conductive surface of an electronic device all with just one hand while easily viewing and accessing controls and the display of the main body410, as seen inFIG.13. The above description is given by way of example, and not limitation. Given the above disclosure, one skilled in the art could devise variations that are within the scope and spirit of the invention disclosed herein, including various ways of manufacturing and using probe devices. Further, the various features of the embodiments disclosed herein can be used alone, or in varying combinations with each other and are not intended to be limited to the specific combination described herein. Thus, the scope of the claims is not to be limited by the illustrated embodiments. | 54,854 |
11860190 | DESCRIPTION OF EMBODIMENTS (A) First Embodiment Hereinafter, a first embodiment of a probe unit according to the present disclosure will be described in detail with reference to the drawings. (A-1) Configuration of First Embodiment FIG.2is an overall configuration diagram showing the overall configuration of a probe unit according to the first embodiment. FIG.1is a partially enlarged view of a portion A inFIG.2. InFIG.2, a probe unit1according to the first embodiment includes a main body11, a coaxial connector12, a high-frequency conducting path13, a plurality of contactors14(14ato14c), and a plurality of pedestals15. When the configuration common to the contactors14ato14c, for example, is described, the contactors14ato14care written and described as, for example, “the contactors14”; when the individual configurations of the contactors14ato14c, for example, are described, the contactors14ato14care written and described as, for example, “the contactor14a”, “the contactor14b”, and “the contactor14c”. The same applies to the other constituent elements. The probe unit1is, for example, a high-frequency probe that is used when an inspection of the electrical characteristics of a high-frequency circuit as an object to be inspected is made. The probe unit1is also called a probe head. A case where the probe unit1includes three contactors14and is a ground-signal-ground (GSG) type high-frequency probe is illustrated. The probe unit1is not limited thereto, however, and can also be applied to a high-frequency probe with a high-speed transmission line, such as a GS type high-frequency probe with two contactors14and a GSGS type or GSSG type high-frequency probe with four contactors14. The probe unit1is connected to a tester (not shown inFIG.2) via a coaxial cable and can make electrical contact with an electrode terminal of the object to be inspected. For example, at the time of an inspection, in the probe unit1, the coaxial connector12receives an input of an electrical signal from the tester, the high-frequency conducting path13relays the electrical signal to each contactor14, and each contactor14supplies the electrical signal to the electrode terminal of the object to be inspected with which the contactor14is in electrical contact. Moreover, the probe unit1provides the tester with an electrical signal output from the object to be inspected supplied with the electrical signal. This allows the tester to make an inspection of the electrical characteristics of the object to be inspected. The main body11includes a base portion111and a supporting portion112. The supporting portion112of the main body11supports the coaxial connector12and the high-frequency conducting path13in such a way that they are inclined in order to allow the tip of each contactor14joined to the high-frequency conducting path13to reliably make electrical contact with the electrode terminal of the object to be inspected. The coaxial connector12is connected to the coaxial cable connected to the tester. For example, as illustrated inFIG.2, the coaxial connector12is fixed to a fixture with the position of the coaxial connector12being inclined and is attached to the supporting portion112of the main body11. The high-frequency conducting path13is an electric circuit that relays an electrical signal to the coaxial connector12and each contactor14, and a coaxial semirigid cable, for example, can be applied to the high-frequency conducting path13. One end of the high-frequency conducting path13is connected to the coaxial connector12and the other end is joined to the plurality of contactors14. A part of the other end of the high-frequency conducting path13is cut to form a level end face (also referred to as a “joint surface”)131in order to make the contactors14level. The plurality of contactors14are joined to the end face131of the high-frequency conducting path13. The contactor14makes electrical contact with the electrode terminal of the object to be inspected and is a ground line or a signal line of the high-frequency probe. The contactor14is formed of a conductive material. One end of the contactor14is joined to the pedestal15and joined to the end face131of the high-frequency conducting path13with the pedestal15being placed therebetween, and the other end has, on the underside thereof, a contact portion16that makes electrical contact with the electrode terminal of the object to be inspected. As described above, the contactor14is a cantilever type probe (contactor) that is supported on the end face131of the high-frequency conducting path13with the pedestal15being placed therebetween and has a uniform thickness. Moreover, the contactor14has a uniform board thickness and has an approximately triangular shape in a plan view, with the width thereof decreasing in the longitudinal direction toward the side where the object to be inspected is located. It is to be noted that the shape of the contactor14is not limited to this shape. The contact portion16is a portion that makes electrical contact with the electrode terminal of the object to be inspected and is formed of a conductive material. In this embodiment, a case where the contact portion16is a circular cylinder is illustrated; the shape of the contact portion16is not limited thereto and may be a triangular pyramid, a pyramid or the like. The pedestal15is a component that is interposed between the contactor14and the end face131of the high-frequency conducting path13and is formed of a conductive component such as a nickel alloy. The pedestal15maintains the free length of the contactor14, which is a cantilever, when the contactor14is joined to the high-frequency conducting path13. It can be said that the pedestal15is a free length adjusting component that adjusts the free length of the contactor14. Moreover, the pedestal15can make dynamic physical quantities (for example, pressure, stress, shearing force, and moment of force) that develop in the plurality of contactors14nearly equal when the contactors14make contact with the electrode terminals of the object to be inspected, which allows stable contact to be made and improves measurement quality. The pedestal15is provided for each contactor14. The pedestal15is a component having a uniform thickness and, though not limited to a particular shape, the planar shape of the pedestal15may be a rectangle, a square or the like. Next, a joint structure of the contactor14that is joined to the end face131of the high-frequency conducting path13will be described using the drawings. FIG.3Ais a side view of the joint structure of the contactor14according to the first embodiment andFIG.3Bis a bottom view thereof.FIG.4Ais a side view showing a joint structure of a conventional contactor94andFIG.4Bis a bottom view thereof. FIGS.5A and5Bare explanatory diagrams explaining the manner in which contactors94ato94cmake contact with electrode terminals5ato5cof an object to be inspected. InFIGS.5A and5B, a joint surface of a high-frequency conducting path93to which the contactors94ato94care joined is illustrated in an abstract manner. As illustrated inFIGS.4A and4B, conventionally, an end face931of the high-frequency conducting path93and the contactor94have been directly joined together by a technique such as brazing, ultrasonic bonding, resistance welding, or laser welding. The contactor94is a cantilever type probe that is supported on the end face931of the high-frequency conducting path93. Therefore, for example, as illustrated inFIG.5A, even when the electrode terminals5of the object to be inspected vary in height, the contactors94are elastically deformed and can make contact with the electrode terminals5with stability (seeFIG.5B). For example, as shown inFIG.5B, when the electrode terminal5bis higher than the other electrode terminals5aand5c, greater pressure develops in the contactor94bwhen the contactor94bis brought into contact with the electrode terminal5bthan in the other contactors94aand94c, and the contactor94bis elastically deformed more greatly than the other contactors94aand94c. The contactor94is a microscopic structure fabricated by micro electromechanical systems (MEMS) or the like; therefore, when the contactor94is joined to the high-frequency conducting path93, the contactor94is sometimes joined to a position that deviates from the designed joint position. When the contactor94is joined to a position that deviates from a joint position in the high-frequency conducting path93, the free length of the contactor94changes, which affects the amount of pressure that develops in the contactor94at the time of contact. This makes it impossible to make stable contact with the electrode terminal of the object to be inspected and can affect measurement quality. For this reason, in the first embodiment, the pedestal15is provided such that the free length of the contactor14is equal to the design value even when the contactor14is joined to a position that deviates from the design value of a joint position in the high-frequency conducting path13. As shown inFIG.3A, the pedestal15is interposed between the high-frequency conducting path13and the contactor14. When the free length of the contactor14is designed and the contactor14and the pedestal15are joined together, the contactor14and the pedestal15are joined together such that the length from the position of an end face151of the pedestal15to the position of the contact portion16is equal to the designed free length in the longitudinal direction of the contactor14. That is, the contactor14and the pedestal15are joined together such that the length from the position of the end face151of the pedestal15to the position of the contact portion16is equal to the design value. Then, the contactor14is joined to the high-frequency conducting path13by joining together the pedestal15provided in the contactor14and the end face131of the high-frequency conducting path13. By joining the contactor14to the high-frequency conducting path13with the pedestal15interposed therebetween as described above, the free length of the contactor14can maintain the design value, which makes it possible to keep the pressure of the contactor14at a value that approximates the design value, make stable contact with the electrode terminal, and improve measurement quality. FIGS.6A to6Cshow models in which the contactor94is joined to a position that deviates from a joint position in the high-frequency conducting path93and stress analysis diagrams at the time of overdrive. The joint surface of the high-frequency conducting path93to which the contactor94is joined is illustrated in an abstract manner also in the stress analysis diagrams ofFIGS.6A to6C. As shown inFIG.6B, when the contactor94is joined to a position that does not deviate from the design value (for example, the value of joint deviation=±0 mm), the stress analysis diagram of the contactors94at the time of overdrive reveals that nearly equal pressure develops in the three contactors94. Moreover, the amount of the pressure of each contactor94is also a value close to the design value (the designed pressure value). FIG.6Ashows a stress analysis diagram observed when the contactor94was joined to a position that deviates from the design value in a −X direction (for example, the value of joint deviation=−0.1 mm). In this case, since the free length of the contactor94is shorter than the design value, the amount of the pressure of each contactor94at the time of overdrive is greater than the amount (the designed pressure value) of the pressure of each contactor94ofFIG.6B. FIG.6Cshows a stress analysis diagram observed when the contactor94was joined to a position that deviates from the design value in a +X direction (for example, the value of joint deviation=+0.1 mm). In this case, a comparison with the amount of the pressure of each contactor94ofFIG.6Breveals that the amount of the pressure of each contactor94ofFIG.6Cis smaller than the amount (the designed pressure value) of the pressure of each contactor94ofFIG.6B. As shown inFIGS.6A to6CandFIG.7, joining the contactor94at a position deviated from the design value causes a change in the free length of the contactor94, which affects the amount of the pressure of the contactor94. FIGS.8A to8Cshow joint models in which the pedestal15provided in the contactor14is joined to the high-frequency conducting path13and stress analysis diagrams at the time of overdrive in the first embodiment. InFIG.8B, when the contactor14provided with the pedestal15is joined to the high-frequency conducting path13, the contactor14is joined to a position that does not deviate from the design value (for example, the value of joint deviation=±0 mm). In this case, since the free length of the contactor14is equal to the design value, the amount of pressure that develops in each contactor14at the time of overdrive is a value close to the design value. Moreover, even when the contactor14is joined to a position that deviates from the design value in the −X direction (for example, the value of joint deviation=−0.1 mm) as inFIG.8Aand even when the contactor14is joined to a position that deviates from the design value in the +X direction (for example, the value of joint deviation=+0.1 mm) as inFIG.8C, the free length of the contactor14does not change and can maintain the design value. Thus, as shown inFIG.9, since it is possible to maintain the free length of each contactor14as specified in the design irrespective of a joint position, the amount of pressure that develops in each contactor14at the time of overdrive is a value close to the design value. This makes it possible to make reliable and stable contact with the electrode terminal and also improves measurement quality. As described earlier, by interposing the pedestal15between the contactor14and the high-frequency conducting path13when joining the contactor14to the high-frequency conducting path13, it is possible to maintain the free length of the contactor14as specified in the design. The contactor14and the pedestal15can be joined together and the pedestal15and the high-frequency conducting path13can be joined together using a technique such as brazing, ultrasonic bonding, resistance welding, or laser welding. Moreover, when each of the plurality of contactors14is joined to the end face131of the high-frequency conducting path13, the impedance (output impedance) on the side where the high-frequency conducting path13is located and the impedance (input impedance) on the side where the plurality of contactors14are located are designed to be equal to each other. For example, in order to prevent a standing wave, which interferes with transmission of a high-frequency signal, from being generated by reflection, impedance matching is performed such that the input and output impedance has an impedance value of 50Ω, for example. The characteristic impedance Z0in a high-frequency probe that transmits a high-frequency signal has characteristics expressed by formula (1). Z0=√{square root over (L/C)} (1) In formula (1), C represents capacitance and L represents inductance. The capacitance C is proportional to permittivity cy and a line width and is inversely proportional to a space. For characteristic impedance matching, the contactors14are made to have a uniform thickness and a uniform “line width”. Furthermore, to make a “space” uniform, the contactor14aand the contactor14bare joined such that the gap between the contactor14aand the contactor14bhas the designed gap length value w1. Likewise, the contactor14band the contactor14care joined such that the gap between the contactor14band the contactor14chas the designed gap length value w2. Moreover, inFIG.3B, the width (the length in a Y direction) of the pedestal15aand the pedestal15ccorresponding to the contactor14aand the contactor14c, which are ground lines, is designed to be larger than the width of the contactor14aand the contactor14c. Furthermore, the width of the pedestal15bcorresponding to the contactor14b, which is a signal line, is designed to be nearly equal to the width of the contactor14b. (A-2) Effects of First Embodiment As described above, according to the first embodiment, by providing the pedestal in the contactor such that the length from the position of the pedestal end face to the position of the contact portion is equal to the designed free length in the longitudinal direction of the contactor, it is possible to join the contactor to the high-frequency conducting path without a change in the free length of the contactor, which makes it possible to make stable contact and also improves measurement quality. (B) Second Embodiment Next, a second embodiment of the probe unit according to the present disclosure will be described in detail with reference to the drawings. (B-1) Configuration of Second Embodiment A probe unit according to the second embodiment is written as a probe unit1A and a contactor according to the second embodiment is written as a contactor24or the like. The basic configuration of the probe unit1A of the second embodiment is the same as the configuration of the probe unit1ofFIG.2of the first embodiment. Therefore, a description is given usingFIG.2of the first embodiment also in the second embodiment. FIG.10Ais a partially enlarged view of a portion A of the probe unit1A of the second embodiment inFIG.2andFIG.10Bis a bottom view of the portion A. In the probe unit1A of the second embodiment, the structure of the contactor24is different from that of the contactor14of the first embodiment. Thus, in the second embodiment, the structure of the contactor24will be the focus of what is described in detail. As in the case of the first embodiment, in the probe unit1A of the second embodiment, a pedestal15is provided for each contactor24, and a high-frequency conducting path13and the contactor24are joined together with the pedestal15being interposed therebetween. As shown inFIG.10A, as in the case of the first embodiment, the contactor24(24a,24b,24c) is a ground line or a signal line of a high-frequency probe. Moreover, the contactor24is formed of a conductive material and formed so as to have a uniform thickness. Furthermore, the contactor24is supported on an end face131of the high-frequency conducting path13with the pedestal15being placed therebetween. The contactor24has a hole formed in a central area thereof so as to pass therethrough in a thickness direction (a direction orthogonal to an arrangement direction of each contactor24), and this hole is also referred to as a space area241. The reason why the space area241is formed in the contactor24will be given below. It is necessary to match the impedance of the probe unit1A to that of the high-frequency conducting path13. For example, in the first embodiment, impedance matching is achieved by making the contactors14have a uniform thickness and making uniform the air gap between the contactor14a(or the contactor14c) which is a ground line and the contactor14bwhich is a signal line. However, when a probe is designed with priority given to impedance matching, problems might arise such as too high or too low pressure (for example, stress, shearing force, moment of force or the like) that develops in the contactor. For this reason, in the second embodiment, by forming the space area passing through the contactor24in a direction in which the contactor24is deformed, the amount of pressure that develops in the contactor24is adjusted without a change in the thickness of the signal line and the ground line (the thickness of the contactor24) and the air gap length between the signal line and the ground line (between the contactors24). The characteristic impedance Z0has characteristics expressed by formula (1) described earlier. As described earlier, in formula (1), the capacitance C is proportional to permittivity εy and a line width and is inversely proportional to a space. Consequently, the characteristic impedance Z0converges to the capacitance C and the inductance L and is influenced by the electrical characteristics of a component material, the width of the contactor24, the thickness of the contactor24, and the degree of proximity of the signal line and the ground line (the air gap length between them). In the second embodiment, the contactors24have a uniform thickness and the air gap length between the signal line and the ground line is also uniform. Thus, the space area241is provided so as to make uniform the width of an edge portion242and an edge portion243, each being a part of the contactor24surrounding the space area241. A description is given by taking the structure of the contactor24aofFIG.10Bas an example. The contactor24bwhich is the signal line and the contactor24cwhich is the ground line also have the same structure. In the example ofFIG.10B, the edge portion242aand the edge portion243aare end portions of the contactor24a, which is nearly triangular in a plan view in a width direction (the Y direction). In other words, the edge portion242aand the edge portion243aare located at both ends of the space area241ain a horizontal direction (the Y direction). Moreover, the width of the edge portion242aand the edge portion243ain the width direction is uniform in free length. As described above, by providing the space area241in each contactor24, it is possible to achieve impedance matching while adjusting the pressure of each contactor24without changing the thickness of each contactor24and the air gap between the signal line and the ground line. (B-2) Effects of Second Embodiment As described above, according to the second embodiment, in addition to the effects described in the first embodiment, the following effects can be obtained. According to the second embodiment, by forming the space area in the signal line and the ground line while maintaining the uniform thickness of the signal line and the ground line and the uniform length of an air gap between the signal line and the ground line, it is possible to adjust impedance while adjusting the pressure that develops in the contactor at the time of contact. (C) Other Embodiments While various modifications have been mentioned in the above-described first and second embodiments, the present disclosure can also be applied to the following modifications.(C-1) In the second embodiment, a case where the space area241is a nearly triangular elongated hole in accordance with the shape of the contactor24has been illustrated; the shape of the space area241is not limited thereto. Moreover, two lines of space areas may be placed in the longitudinal direction of the contactor24; in that case as well, the width of an edge portion which is present between the two lines of space areas of the contactor is uniform.(C-2) In the embodiments described above, a case where the contactor has a nearly triangular shape in a plan view has been illustrated; the shape of the contactor is not limited to a particular shape.(C-3) In the embodiments described above, an example in which the pedestal is joined to the contactor has been described; the contactor may be integrally formed with the pedestal by MEMS or the like. REFERENCE SIGNS LIST 1and1A probe unit5(5ato5c) electrode terminal11main body111base portion112supporting portion12coaxial connector13high-frequency conducting path131end face14(14ato14c) and24(24ato24c) contactor15(15ato15c) pedestal151end face16contact portion241space area242aedge portion243aedge portion | 23,514 |
11860191 | DETAILED DESCRIPTION OF THE DISCLOSURE Various exemplary embodiments of the present disclosure are described in detail below with reference to the drawings. It should be noted that unless otherwise stated, the relative arrangement of components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present disclosure. The following description of at least one exemplary embodiment is actually illustrative only, and in no way serves as any limitation to the present disclosure and application or use thereof. The technology and apparatus known in the art may not be discussed in detail, but when appropriate, the technology and apparatus should be regarded as part of the description. In all examples shown and discussed herein, any specific value should be interpreted as exemplary only, rather than as a limitation. Therefore, other examples of the exemplary embodiment may have different values. It should be noted that similar reference numerals and letters indicate similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further discussed in the subsequent drawings. It should be noted that, in the embodiments, a display module used in conjunction with the probe module provided by the present disclosure is used for exemplary description, but it does not mean that the probe module provided by the present disclosure can only be applied to the crimp-connection testing of the display modules, liquid crystal modules or other display devices. It is not easy for the probe module to be assembled and it is not convenient for a pin in the module to be replaced due to the structure limitation of the existing probe module. In order to solve the problem, the present disclosure provides a probe module. Referring toFIGS.1-5, the probe module includes a body1having a hollow cavity; a floating plate2located at the bottom of the body1; and a probe assembly3located on the side of the body1which is far away from the floating plate2. The probe assembly3includes a cover plate31connected to and fixed on the top of the body1; a mould core32connected to and fixed on the bottom surface of the cover plate31and located in the hollow cavity, and the mould core32comprises pin grooves321; and blade pins33located in the hollow cavity and in one-to-one correspondence to the pin grooves321, and each blade pin is limited and fixed in a corresponding pin groove321by a limiting member. The floating plate2comprises pin holes21in one-to-one correspondence to electrical contact terminals331of the blade pins33. The floating plate2is configured to be floatable relative to the body1in an extension direction of the blade pin33and the electrical contact terminals331of the blade pins33may protrude from a surface of the floating plate2which is far away from the body through the pin holes21(that is to say, the electrical contact terminals331of the blade pins33may be inserted into the pin holes21from the surface of the floating plate2close to the body1and protrude from the surface of the floating plate2which is far away from the body1). In the probe module provided by the present disclosure, the floating plate2and the probe assembly3are fixed on opposite two sides of the body1respectively, and the blade pins33are fixed in the pin grooves321by the limiting member, so that the probe assembly3formed by fixing the cover plate31, the mould core32and the blade pins33with each other constitutes an integral structure. Accordingly, the probe assembly as a whole can be replaced or the single blade pin in the probe assembly can be replaced when a blade pin is damaged. During replacement of the probe assembly, the assembly accuracy of the floating plate and the body which are both components of the probe module is not affected, thus ensuring that the blade pins can be in one-to-one correspondence to the pin holes on the floating plate after the replacement of the probe assembly. Therefore, there is no need to further adjust the assembly accuracy, thus improving the working efficiency of replacing the pin of the probe module, facilitating pin maintenance and increasing the stability of a testing process. In some embodiments, the present disclosure provides implementations for fixing the mould core32, the blade pins33and the cover plate31together. In one embodiment, referring toFIG.2,FIG.3andFIG.4, the probe assembly3further comprises a mould core fixing member34located in the hollow cavity, and the mould core fixing member34is sleeved outside the mould core32and is connected and fixed to the cover plate31; the mould core fixing member34comprises recessed step portions341which form the limiting member. Further, the blade pin33comprises a limiting edge332corresponding to the step surfaces of the step portions341, and the blade pin33is limited and fixed in the pin groove321of the mould core32by means of cooperation between the step surfaces of the step portions341and the limiting edge332of the blade pin33. On one hand, the mould core fixing member34can enclose the blade pins33to prevent the blade pins from dropping out of the pin groove. On the other hand, the recessed step portion341of the mould core fixing member34can firmly integrate the mould core32with the cover plate31at the bottom of the mould core32to form an integral structure, and by using the limiting member formed by the step portions341, the blade pins33are limited and fixed in the pin groove321by means of the cooperation between the step surfaces of the step portions341and the limiting edge332. It can be understood that there is a space left between the step portions341so that the electrical contact terminals331of the blade pins33may protrude from the surface of the floating plate2which is far away from the body1through the pin holes21(that is to say, the electrical contact terminals331of the blade pins33may be inserted into the pin holes21from the surface of the floating plate2close to the body1and protrude from the surface of the floating plate2which is far away from the body1). In one embodiment, at least one positioning column4and at least one positioning hole5matching the positioning column4are provided between the mould core fixing member34and the cover plate31. In an embodiment, the body1comprises a limiting portion which is formed by inward extension of the inner wall of the hollow cavity and configured to firmly fix the mould core fixing member34between the body1and the cover plate31. In the present disclosure, first, an initial assembly and positioning of the blade pins are realized by means of the mould core, and then a complete positioning of the blade pins is realized by means of the fixing of the mould core and the cover plate and the fixing of the mould core fixing member and the cover plate. The at least one positioning column and the at least one positioning hole matching each other between the mould core fixing member and the cover plate can ensure the assembly accuracy of the mould core fixing member and the cover plate. It can be understood that the at least one positioning column can be disposed on the cover plate or on the mould core fixing member and the at least one positioning hole opposite to the positioning column can be disposed on the mould core fixing member or the cover plate correspondingly. The at least one positioning column can be a separate member for which it is convenient to be subsequently replaced and assembled. However, it is merely illustrative but not limited in the present disclosure. In some embodiments, the floating plate2is connected to and fixed on the bottom surface of the body1by means of equal-height screws6. At least one elastic member7is disposed between the floating plate2and the body1and the acting force direction of the at least one elastic member7is along the axial direction of the equal-height screws6. The equal-height screw6comprises a screw connection portion at the head end for connecting and fixing the floating plate2to the body1and a guide portion at the rear end of the screw connection portion for guiding the floating of the floating plate2. In addition, the elastic member7can be a spring or other elastic elements with the elastic reset ability, which is not limited herein. If the elastic member7is a spring, the spring can be arranged between the floating plate2and the bottom of the body1. In another example, in order to ensure that the spring is subjected to uniform force and does not deform, the spring may be sleeved outside the equal-height screw to maintain the floating stability of the floating plate. Referring toFIG.5, an enclosing wall22surrounding the pin holes21is further disposed on the surface of the floating plate2far away from the body1. When the probe module is connected and fixed to a test structure to perform crimp-conduction for B2B and FPC of a display module, the enclosing wall22can provide a pre-positioning function to ensure that the electronic product to be tested is located directly under the blade pins, so that an electrical contact terminals of several blade pins can be in one-to-one correspondence to the electrical contact terminals of the product and thus accurately and stably crimp-connected to the electrical contact terminals to form electrical connections. In one embodiment, referring toFIG.2, at least one positioning pin8and at least one positioning pin hole matching the positioning pin8are provided between the body1and the cover plate31, to ensure the assembly accuracy of the integral structure formed of the body and the probe assembly. In one embodiment, referring to the structure shown in FIGS., the cover plate31comprises positioning pin holes311corresponding to the head ends333of the blade pins33which is far away from the electrical contact terminals331, to further ensure the positioning accuracy when assembling the blade pins33. In the embodiments as shown inFIGS.1-5, in order to avoid collision between the floating plate2and the mould core32or the mould core fixing member34that might cause shaking of the blade pins33and thus affect the connection accuracy of the blade pins33, the floating plate2does not contact the mould core32or the mould core fixing member34when the floating plate2is located at an extreme position close to the body1(i.e. the nearest position to the body1). In practical applications, the probe module provided by the present disclosure is connected and fixed to the test structure to perform a crimp-connection testing on the B2B and FPC of the display module. In an initial stage, the floating plate is in a bounced status, that is, the floating plate is located at a position far away from the body. In the stage, the electrical contact terminals of the blade pins are hidden in the floating plate to protect the blade pins. In the testing stage, the probe module is crimp-connected to the B2B or FPC, the floating plate is retracted under force and moves to the body. Hence, the electrical contact terminals of the blade pins are exposed from the surface of the floating plate which is far away from the body and contacts the B2B or FPC, so that signal conduction may be realized and then the testing may be completed by means of other device. It is not easy for the probe module to be assembled and it is not convenient for a pin in the module to be replaced due to the structure limitation of the existing probe module. In order to solve the problem, the present disclosure further provides another probe module different from that disclosed in the first embodiment. Referring toFIGS.6-9, the probe module includes a body1having a hollow cavity; a floating plate2located at the bottom of the body1, and a probe assembly3located on the side of the body1which is far away from the floating plate2. The probe assembly3includes a cover plate31connected to and fixed on the top of the body1; a mould core32connected to and fixed on the bottom surface of the cover plate31and located in the hollow cavity, and the mould core32comprises pin grooves321; and blade pins33located in the hollow cavity and in one-to-one correspondence to the plurality of pin grooves321, and each blade pin is fixed in the corresponding pin groove321by a limiting member. The floating plate2comprises pin holes21corresponding to electrical contact terminals331of the blade pins33. The floating plate2is configured to be floatable relative to the body1in an extension direction of the blade pin33and the electrical contact terminals331of the blade pins33may protrude from the surface of the floating plate2which is far away from the body1through the pin holes21(that is to say, the electrical contact terminals331of the blade pins33may be inserted into the pin holes21from the surface of the floating plate2close to the body1and protrude from the surface of the floating plate2which is far away from the body1). Similar to that of the first embodiment, in the probe module provided by the present disclosure, the floating plate and the probe assembly are fixed on opposite two sides of the body1respectively, and the pin grooves matching the blade pins are disposed on the mould core, so that the plurality of blade pins can be mounted in the pin grooves in one-to-one correspondence, thus ensuring the assembly effectiveness. Since the shape of the pin groove is consistent with the shape of the blade pin, the overall structure is more compact, thus increasing the stability of a testing process. In this embodiment, compared with the probe module in the first embodiment, each blade pin includes a limiting edge332, and the limiting member is formed on the mould core32. The blade pin33is limited and fixed in the pin groove321of the mould core32by means of the cooperation between the limiting member and the limiting edge332. In the probe module provided by the present disclosure, the floating plate2and the probe assembly3are fixed on opposite two sides of the body1respectively, and the blade pins33are fixed in the pin grooves321by the limiting member, so that the probe assembly3formed by fixing the cover plate31, the mould core32and the blade pins33with each other constitutes an integral structure. Accordingly, the probe assembly as a whole can be replaced when a blade pin is damaged. During replacement of the probe assembly, the assembly accuracy of the floating plate and the body which are both components of the probe module is not affected, thus ensuring that the blade pins can be in one-to-one correspondence to the pin holes on the floating plate after the replacement of the probe assembly. Therefore, there is no need to further adjust the assembly accuracy, thus improving the working efficiency of replacing the pin of the probe module, facilitating pin maintenance and increasing the stability of a testing process. In some embodiments, referring toFIG.8, the mould core32comprises a body portion provided with the plurality of pin grooves321and a limiting surface322formed on the lower surface of the body portion which forms the limiting member, so that the limiting edge332of the blade pin33can be hung on the inner surface of the limiting surface322, thus realizing the fixing of the blade pins33in the pin grooves321by the limiting member. It can be understood that the limiting surface322should be provided with openings through which the electrical contact terminals331of the blade pins33pass, so that the electrical contact terminal331of the blade pin33may protrude from the surface of the floating plate2which is far away from the body1through the pin holes21(that is to say, the electrical contact terminals331of the blade pins33may be inserted into the pin holes21from the surface of the floating plate2close to the body1and protrude from the surface of the floating plate2which is far away from the body1). In one embodiment, the blade pin comprises a limiting edge and the limiting member is formed in the pin groove of the mould core. The blade pin is fixed in the pin groove of the mould core by means of cooperation between the limiting member and the limiting edge. In one embodiment, the mould core comprises a body portion provided with the plurality of pin grooves and a projection structure in each pin groove. The projection structure is formed by protrusion of the body portion of the mould core and the projection structure forms the limiting member, so that the limiting edge of the blade pin can be hung on the projection structure, thus realizing the fixing of the blade pin in the pin groove by the limiting member. On the basis of the above structure, in some embodiments, at least one positioning column and at least one positioning hole matching the positioning column are provided between the mould core3and the cover plate31. In this embodiment, first, an initial assembly and positioning of the blade pins33are realized by means the pin grooves321of the mould core32, and then a complete positioning of the blade pins is realized by means of the fixing of the mould core32and the cover plate31and the cooperation between the limiting edge332of the blade pin33and the limiting surface322of the mould core32. The positioning column and positioning hole matching each other between the mould core32and the cover plate31can ensure the assembly accuracy of the mould core32and the cover plate31. It can be understood that the positioning column can be disposed on the cover plate or on the mould core and the positioning hole opposite to the positioning column can be disposed on the mould core or the cover plate correspondingly. The positioning column can be a separate member for which it is convenient to be subsequently replaced and assembled. However, it is merely illustrative but not limited in the present disclosure. In one embodiment, the floating plate2is connected to and fixed on the bottom surface of the body1by means of equal-height screws6. At least one elastic member7is disposed between the floating plate2and the body1and the acting force direction of the elastic member7is along the axial direction of the equal-height screws6. The equal-height screw6comprises a screw connection portion at the head end for connecting and fixing the floating plate2to the body1and a guide portion at the rear end of the screw connection portion for guiding the floating of the floating plate2. In addition, the elastic member7can be a spring or other elastic elements with the elastic reset ability, which is not limited herein. If the elastic member7is a spring, the spring can be arranged between the floating plate2and the bottom of the body1. In another example, in order to ensure that the spring is subjected to uniform force and does not deform, the spring may be sleeved outside the equal-height screw to maintain the floating stability of the floating plate. Referring toFIG.9, an enclosing wall22surrounding the pin holes21is further disposed on the surface of the floating plate2far away from the body1. When the probe module is connected and fixed to a test structure to perform crimp-conduction for B2B and FPC of a display module, the enclosing wall22can provide a pre-positioning function to ensure that an electronic product to be tested is located directly under the blade pins, so that the electrical contact terminals of several blade pins can be in one-to-one correspondence to the electrical connection terminals of the product and thus accurately and stably crimp-connected to electrical contact terminals to form electrical connections. In some embodiments, referring toFIG.7, at least one positioning pin8and at least one positioning pin hole matching the positioning pin8are provided between the body1and the cover plate31, to ensure the assembly accuracy of the integral structure formed of the body and the probe assembly. In one embodiment, referring to the structure shown in FIGS., the cover plate31comprises a positioning pin holes311corresponding to the head ends333of the blade pins33which is far away from the electrical contact terminals331, to further ensure the positioning accuracy when assembling the blade pins33. In addition, in order to avoid collision between the floating plate and the mould core that might cause shaking of the blade pins and thus affect the connection accuracy of the blade pins, in some embodiments, the floating plate2does not contact the mould core32when the floating plate2is located at an extreme position close to the body1. In practical applications, the probe module provided by the present disclosure is connected and fixed to the test structure to perform a crimp-connection testing on the B2B and FPC of the display module. In an initial stage, the floating plate is in a bounced status, that is, the floating plate is located at a position far away from the body. In the stage, the electrical contact terminals of the blade pins are hidden in the floating plate to protect the blade pins. In the testing stage, the probe module is crimp-connected to the B2B or FPC, the floating plate is retracted under force and moves to the body. Hence, the electrical contact terminals of the blade pins are exposed from the surface of the floating plate which is far away from the body and contacts the B2B or FPC, so that signal conduction may be realized and then the testing may be completed by means of other device. The above embodiments of the present disclosure are merely examples provided to illustrate the present disclosure, and are not intended to limit the implementation of the present disclosure. Other changes or modifications in different forms on the basis of the above description may be made. It is impossible to list all the implementations herein. Any changes or modifications derived from the embodiments of the present disclosure still fall within the protection scope of the present disclosure. | 21,881 |
11860192 | DETAILED DESCRIPTION OF THE INVENTION Contents of the description below merely exemplify the principle of the present disclosure. Therefore, those of ordinary skill in the art may implement the theory of the present disclosure and invent various apparatuses which are included within the concept and the scope of the present disclosure even though it is not clearly explained or illustrated in the description. Furthermore, in principle, all the conditional terms and embodiments listed in this description are clearly intended for the purpose of understanding the concept of the present disclosure, and one should understand that this present disclosure is not limited the exemplary embodiments and the conditions. The above described objectives, features, and advantages will be more apparent through the following detailed description related to the accompanying drawings, and thus those of ordinary skill in the art may easily implement the technical spirit of the present disclosure. The embodiments of the present disclosure will be described with reference to cross-sectional views and/or perspective views which schematically illustrate ideal embodiments of the present disclosure. For explicit and convenient description of the technical content, thicknesses and widths of members and regions in the figures may be exaggerated. Therefore, variations from the shapes of the illustrations as a result, for example, of manufacturing techniques and/or tolerances, are to be expected. In addition, a limited number of holes are illustrated in the drawings. Thus, the embodiments should not be construed as limited to the particular shapes of regions illustrated herein but are to include deviations in shapes that result, for example, from manufacturing. In describing various embodiments, the same reference numerals will be used throughout different embodiments and the description to refer to the same or like elements or parts. In addition, the configuration and operation already described in other embodiments will be omitted for convenience. Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. FIG.1is a view schematically illustrating a probe card100according to an embodiment of the present disclosure. In this figure, for convenience of description, the number and size of a plurality of probes80are illustrated exaggeratedly. Depending on the structure of installing the probes80on a space transformer ST and the structure of the probes80, the probe card100may be divided into a vertical type probe card, a cantilever type probe card, a micro-electro-mechanical system (MEMS) probe card100. In the present disclosure, as an example, a vertical type probe card100is illustrated to describe a coupling structure between the space transformer ST and other peripheral parts. The type of the probe card in which the coupling structure between the space transformer ST of the present disclosure and other peripheral parts is implemented is not limited thereto, and may be the MEMS probe card and the cantilever type probe card. FIG.1illustrates a contact state of electrode pads WP of a wafer W. A test for electrical characteristics of semiconductor devices is performed by approaching the wafer W to the probe card100having the plurality of probes80on a wiring board, and bring the respective probes80into contact with corresponding electrode pads WP on the wafer W. After the probes80reach positions where the probes80are brought into contact with the electrode pads WP, the wafer W may be further lifted by a predetermined height toward the probe card100. This process may be overdrive. As illustrated inFIG.1, the probe card100according to the present disclosure may include the space transformer ST made of an anodic oxide film101, and including a vertical wiring2, a horizontal wiring3connected to the vertical wiring2, and a probe connection pad130electrically connected to each of the plurality of probes80; and a coupling member150having a first end150afixed to a surface of the space transformer ST and a second end150bcoupled to the circuit board160provided above the space transformer ST. In this case, the coupling member150may be configured as a bolt, but is not limited thereto. As illustrated inFIG.1, the circuit board160may be provided above the space transformer ST, and the probe head1on which the plurality of probes80are provided may be provided below the space transformer ST. In other words, the space transformer ST may be located between the circuit board160and the probe head1. The space transformer ST may be coupled to peripheral parts by the coupling member150. With such a structure, the space transformer ST coupled to the circuit board160by the coupling member150may be electrically connected thereto by an interposer170interposed between the circuit board160and the space transformer ST. Specifically, a first interposer connection pad110may be provided on an upper surface of the space transformer ST, and a second interposer connection pad120may be provided on a lower surface of the circuit board160. Therefore, the interposer170interposed between the space transformer ST and the circuit board160may be joined to the first interposer connection pad110and the second interposer connection pad120to form an electrical connection between the space transformer ST and the circuit board160. The space transformer ST may be made of the anodic oxide film101. The anodic oxide film101is a film formed by anodizing a metal that is a base material, and pores101aare pores formed in the anodic oxide film101during the process of anodizing the metal to form the anodic oxide film101. For example, in a case where the metal as the base material is aluminum (Al) or an aluminum alloy, the anodization of the base material forms the anodic oxide film101consisting of anodized aluminum (Al2O3) on a surface SF of the base material. The anodic oxide film101foiled as described above is divided into a barrier layer BL in which no pores101aare formed and a porous layer PL in which pores101aare formed. The barrier layer BL is positioned on the base material, and the porous layer PL is positioned on the barrier layer BL. In a state in which the anodic oxide film101having the barrier layer BL and the porous layer PL is formed on the surface SF of the base material, when the base material is removed, only the anodic oxide film101consisting of anodized aluminum (Al2O3) remains. The resulting anodic oxide film101has the pores101athat have a uniform diameter, are formed in a vertical shape, and have a regular arrangement. In this case, when the barrier layer BL is removed, a structure in which the pores101avertically pass through the anodic oxide film101from top to bottom is formed. The anodic oxide film101has a coefficient of thermal expansion of 2 to 3 ppm/° C. This may result in less deformation due to temperature. According to the present disclosure, by configuring the space transformer ST using the anodic oxide film101, the space transformer ST having less thermal deformation under a high temperature environment may be implemented. The probe head1is provided below the space transformer ST. The probe head1may include: a guide plate GP including first and second plates10and20and upper, intermediate, and lower guide plates40,50, and60; and a reinforcing plate RP including the plurality of probes80and upper, intermediate, and lower reinforcing plates710,720, and730. The probe head1may be manufactured by means of bolt fastening as an example. However, since this is a coupling means described as an example, the coupling means is omitted in the drawings. The probe head1supports the probes80and may have a structure in which the second plate20is provided under the first plate10. Specifically, the first plate10may include an upper seating region15for having the upper guide plate40and the upper reinforcing plate710therein, and the second plate20may include a lower seating region25for having the lower guide plate50and the lower reinforcing plate720therein, and an intermediate seating region26for having the intermediate guide plate60and the intermediate reinforcing plate730therein. The probes80may sequentially pass through the upper guide plate40, the lower guide plate50, and the intermediate guide plate60to be provided toward the wafer W. Hereinafter, the configuration of the probe head1according to the present disclosure will be described in detail with reference toFIGS.2and3. FIG.2is a top view of the probe head constituting the present disclosure, andFIG.3is a perspective view when viewed from a surface cut along line A-A′ ofFIG.2. As illustrated inFIGS.2and3, the first plate10and the second plate20may be provided in corresponding shapes, and the second plate20may be provided under the first plate10. The first plate10may be provided with an upper coupling hole12and a first guide pin insertion hole13. In addition, the second plate20may be provided with a lower coupling hole (not illustrated) and a second guide pin insertion hole (not illustrated) respectively corresponding to the sizes of the upper coupling hole12and the first guide pin insertion hole13at positions respectively corresponding to the upper coupling hole12and the first guide pin insertion hole13. A coupling means may be provided in the upper coupling hole12and the lower coupling hole, and a guide pin may be provided in the first guide pin insertion hole13and the second guide pin insertion hole. In this case, the coupling means denotes a means for coupling the first plate10and the second plate20, and the guide pin denotes an auxiliary means for aligning the first plate10and the second plate20. The coupling means may be configured as a bolt as an example. Specifically, the guide pin may sequentially pass through the first guide pin insertion hole13and the second guide pin insertion hole to align the first plate10and the second plate20, and then the coupling means may sequentially pass through the upper coupling hole12and the lower coupling hole to couple the first plate10and the second plate20. In this case, the guide pin may be removed before the first and second plates10and20are coupled to each other by means of the bolt through the upper coupling hole12and the lower coupling hole. The positions, shapes, and numbers of the upper coupling hole12and the first guide pin insertion hole13of the first plate10illustrated inFIGS.2and3are illustrated as an example, and thus the positions, shapes, and numbers thereof are not limited thereto. The upper seating region15is formed on the first plate10, and the lower seating region25and the intermediate seating region26are formed on the second plate20. In this case, the upper seating region15may be formed on an upper side of the first plate10, the intermediate seating region26may be formed on an upper side of the second plate20, and the lower seating region25may be formed on a lower side of the second plate20. In addition, the upper seating region15, the lower seating region25, and the intermediate seating region26may have the same size and shape. After the first plate10and the second plate20are coupled to each other, the lower seating region25and the intermediate seating region26may be located on the same vertical line, but the upper seating region15may be located on a vertical line that is not the same as that of the lower seating region25and the intermediate seating region26. The first plate10and the second plate20are provided with a plurality of guide plates GP and reinforcing plates RP. Specifically, the guide plates GP and the reinforcing plates RP may be provided in the upper seating region15, the lower seating region25, and the intermediate seating region26. Therefore, each of the guide plates GP and the reinforcing plates RP may be formed to have a size smaller than that of the upper seating region15, the lower seating region25, and the intermediate seating region26. The guide plates GP include the upper guide plate40, the lower guide plate50, and the intermediate guide plate60. The upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may be formed in shapes corresponding to each other, and may include the same configuration (e.g., a guide hole GH for allowing insertion of each of the plurality of probes80). With this structure, handling of the probe head1may be facilitated. Specifically, when ends of the plurality of probes80first inserted through the respective guide holes GH are front ends of the probes80, the upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may serve to guide the front ends of the probes80. In other words, the upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may define probing areas of the probe card100. Therefore, on the first and second plates10and20defining the entire area of the probe head1, the areas50, occupied by the upper guide plate,40, the lower guide plate and the intermediate guide plate60may be the probing areas. Since the upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may have smaller areas than the first and second plates10and20, a problem in which the probing areas are broken or damaged may be minimized. Therefore, handling of the probe card100may be facilitated. Unlike the probe head1constituting the present disclosure, when the upper guide plate40, the lower guide plate50, and the intermediate guide plate60defining the probing areas define the entire area of the probe head1, an unnecessary area other than the probing areas in which the plurality of probes80are provided and performing a practical probing process may be included in the probing areas thereby defining the entire area of the probe head1. This structure may cause a problem in that handling is difficult because even if a portion of the probe head1is damaged, this means that the probing areas are damaged. However, in the probe head1constituting the present disclosure, since the upper guide plate40, the lower guide plate50, and the intermediate guide plate60defining the probing areas may have smaller areas than the first and second plates10and20defining the entire area of the probe head1, the risk of damage may be lowered and handling may be facilitated. In addition, in the probe head1constituting the present disclosure, since the upper guide plate40, the lower guide plate50, and the intermediate guide plate60defining the probing areas may have smaller areas than the first and second plates10and20defining the entire area of the probe head1, a relatively uniform flatness may be achieved compared to a structure in which the upper guide plate40, the lower guide plate50, and the intermediate guide plate60define the entire area of the probe head1. When the upper guide plate40, the lower guide plate50, and the intermediate guide plate60define the entire area of the probe head1, it is difficult to achieve uniform flatness due to a large area thereof. When the flatness of the upper guide plate40, the lower guide plate50, and the intermediate guide plate60each having the guide hole GH into which the probes80are inserted is not uniform, the positions of the probes80may be changed, resulting in an error in wafer pattern test. However, in the probe head1constituting the present disclosure, since the upper guide plate40, the lower guide plate50, and the intermediate guide plate60in which the probes80are inserted may have smaller areas than the probe head1, it may be advantageous to achieve uniform flatness thereof. The first plate10constituting the probe head1may serve to support, on an upper surface thereof, the upper guide plate40that serves to guide the front ends of the probes80. The first plate10may have a larger area than the upper guide plate40and may support the upper guide plate40in at least a partial area of the upper surface thereof. The first plate10may include the upper seating region15for seating the upper guide plate40therein. The upper seating region15may be configured as a concave recess in the upper surface of the first plate10. However, since the concave recess shape of the upper seating region15is illustrated as an example, the shape thereof is not limited thereto. Therefore, the upper seating region15may be formed in a suitable shape to allow the upper guide plate40to be provided on the upper surface of the first plate10more stably. The first plate10may include a first through-hole11. The first through-hole11may be provided to allow the plurality of probes80inserted through an upper guide hole43of the upper guide plate40to be positioned therein. Therefore, the first through-hole11may be formed at a position corresponding to a position where the upper guide hole43of the upper guide plate40is formed, to allow the plurality of probes80to be positioned therein, and in consideration of the elastic deformation of the plurality of probes80, may have an inner diameter capable of receiving the elastic deformation. The second plate20may be coupled to a lower portion of the first plate10. The second plate20may serve to support, on a lower surface thereof, the lower guide plate50and the intermediate guide plate60that serve to guide the front ends of the probes80. Specifically, the second plate20may serve to support the intermediate guide plate60on an upper surface thereof and support the lower guide plate50on the lower surface thereof. In this case, the second plate20may have an area corresponding to the first plate10. Therefore, the second plate20may support the lower guide plate50and the intermediate guide plate60in at least a portion of the upper surface thereof and at least a portion of the lower surface thereof. The lower seating region25for seating the lower guide plate50may be provided on the lower surface of the second plate20, and the intermediate seating region26for seating the intermediate guide plate60may be provided on the upper surface thereof. The lower guide plate50may be provided in the lower seating region25provided on the lower surface of the second plate20, and the intermediate guide plate60may be provided in the intermediate seating region26provided on the upper surface of the second plate20. In this case, the lower seating region25and the intermediate seating region26may be configured as concave recesses in the upper and lower surfaces of the second plate20. However, since this is illustrated as an example, the shapes of the lower seating region25and the intermediate seating region26are not limited thereto. The lower seating region25and the intermediate seating region26may be provided at positions that are inverted from each other with respect to the center of the second plate20. Therefore, the lower guide plate50and the intermediate guide plate60may also be provided at positions that are inverted from each other with respect to the center of the second plate20. However, since the inverted shapes of the lower seating region25and the intermediate seating region26are illustrated as an example, the shapes of the lower seating region25and the intermediate seating region26are not limited thereto. The second plate20may include a second through-hole21corresponding to the first through-hole11of the first plate10. This allows the probes80positioned in the first through-hole11to be also positioned in the second through-hole21. The second through-hole21may be formed to have the same inner diameter as the first through-hole11. However, the sizes of the inner diameters of the first through-hole11and the second through-hole21are not limited. For example, the second through-hole21may be formed at a position corresponding to the first through-hole11to have an inner diameter smaller than that of the first through-hole11and capable of securing a free space that allows, when the plurality of probes80positioned in the first through-hole11is elastically deformed, the elastic deformation to be received therein. Alternatively, the second through-hole21may be formed at a position corresponding to the first through-hole11to have an inner diameter larger than that of the first through-hole11. The plurality of probes80may be inserted into and through the upper guide hole43of the upper guide plate40and inserted into and through a lower guide hole53of the lower guide plate50through an intermediate guide hole63, so that the plurality of probes80may be positioned inside the first and second through-holes11and12. Therefore, the probe head1may have a structure with the first through-hole11formed in the first plate10and the second through-hole11formed in the second plate20correspondingly to the first through-hole11, so that the plurality of probes80is positioned inside the first and second through-holes11and21. At least one of the upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may be made of an anodic oxide film101. Therefore, the space transformer ST constituting the present disclosure, and the upper guide plate40the lower guide plate50, and the intermediate guide plate60defining substantial probing areas by having the guide holes GH into which the plurality of probes80are inserted may be made of the same anodic oxide film101. The probe card100may perform an electronic die sorting (EDS) process for testing electrical characteristics of each chip constituting a wafer. The EDS process may be performed under a high temperature environment. Therefore, as the overall temperature of the probe card100increases, the upper guide plate40, the lower guide plate50, and the intermediate guide plate60may be thermally expanded. In this case, when at least one of the upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may be made of the anodic oxide film101, such deformation may not easily occur. Each of the upper guide plate,40, the lower guide plate50, and the intermediate guide plate60may be made of a transparent anodic oxide film101, so that a problem of reducing positional accuracy of the upper guide hole43, the lower guide hole53, and the intermediate guide hole63may be prevented. The upper guide plate40, the lower guide plate50, and the intermediate guide plate60made of the anodic oxide film101may undergo an etching process to form the upper guide hole43, the lower guide hole53, and the intermediate guide hole63. In the case of the anodic oxide film101, the upper guide hole43, the lower guide hole53, and the intermediate guide hole63may be vertically formed by the etching process. This may make it possible to implement a fine size and fine pitch of the upper guide hole43, the lower guide hole53, and the intermediate guide hole63. In the probe card100according to the present disclosure, since the space transformer ST having the respective probe connection pads130, and the upper guide plate40, the lower guide plate50, and the intermediate guide plate60provided below the space transformer ST and having the probes80therein may be made of the same anodic oxide film101, a problem in which the probe connection pads130and the probes80coming into contact with the probe connection pads130are misaligned due to thermal deformation may be prevented. As a result, probing reliability of the vertical type probe card100, which is overdriven to test electrical characteristics of a wafer, may be increased. The reinforcing plate70and RP may be provided on at least one surface of each of the upper guide plate40, the lower guide plate50, and the intermediate guide plate60. In the present disclosure, as illustrated inFIGS.1to3, as an example, the reinforcing plate RP may be provided on each of lower surfaces of the upper guide plate40and the intermediate guide plate60and an upper surface of the lower guide plate50. This may increase mechanical strength of the upper guide plate40, the lower guide plate50, and the intermediate guide plate60. When the reinforcing plate RP is provided on at least the surface of each of the upper guide plate40, the lower guide plate50, and the intermediate guide plate60, the reinforcing plate RP may be composed of the upper reinforcing plate710coupled to the surface of the upper guide plate40, the lower reinforcing plate720coupled to the surface of the lower guide plate50, and the intermediate reinforcing plate730coupled to the surface of the intermediate guide plate60. Therefore, the upper guide plate40may include an upper guide pin insertion hole41for allowing alignment of the upper guide plate40with the upper reinforcing plate710provided on the surface of the upper guide plate40, by means of a guide pin. In addition, the upper guide plate40may include an upper main bolt fastening hole42for allowing insertion of a coupling means for coupling the upper reinforcing plate710and the first plate10. Since the lower guide plate50and the intermediate guide plate60may have a shape corresponding to the upper guide plate40, each of the lower guide plate50and the intermediate guide plate60may have a guide pin insertion hole and a main bolt fastening hole that perform the same function in the same shape at the same position as those of the upper guide plate40. The reinforcing plate70and RP may serve to support the upper guide plate40, the lower guide plate50, and the intermediate guide plate60, and thus may be made of a material having high mechanical strength. Specifically, for example, the reinforcing plate RP may be made of a Si3N4material. In another example, the reinforcing plate RP may be made of a ceramic material. The reinforcing plate RP, the upper guide plate40, the lower guide plate50, and the intermediate guide plate60may be coupled to each other by bonding or molding. With such a structure, the present disclosure may have an advantage in terms of mechanical strength while implementing a fine size and fine pitch of the upper guide hole43, the lower guide hole53, and the intermediate guide hole63in the upper guide plate40, the lower guide plate50, and the intermediate guide plate60each made of the anodic oxide film101. The anodic oxide film101having a small thickness may be more efficient in forming the upper guide hole43, the lower guide hole53, and the intermediate guide hole63vertically. In addition, the anodic oxide film101may be a material suitable for implementing a fine size and fine pitch of the upper guide hole43, the lower guide hole53, and the intermediate guide hole63. In the probe head1, by forming a structure in which at least one of the upper guide plate40, the lower guide plate50, and the intermediate guide plate60is made of the anodic oxide film101, and the reinforcing plates710,720, and730are coupled to the surfaces of the upper guide plate40, the lower guide plate50, and the intermediate guide plate60, it may be possible to provide fine probes80with a fine pitch arrangement. At the same time, the probe card100may have excellent durability in which warpage deformation is prevented. FIGS.4A and4Bare views illustrating the probe head ofFIG.1. Specifically,FIG.4Ais a view illustrating the probes80before undergoing elastic deformation, andFIG.4Bis a view illustrating the probes80after undergoing elastic deformation. Referring toFIGS.4A and4B, the probes80may vertically pass through the upper guide plate40, the lower guide plate50, and the intermediate guide plate60. In this case, the probes80may be provided in a vertical shape without deformation. Specifically, the probes80may pass through the upper guide hole43of the upper guide plate40, then pass through the intermediate guide hole63of the intermediate guide plate60, and finally pass through the lower guide hole53of the lower guide plate50. Therefore, the front ends of the probes80may be provided below the second plate20. Each of the upper guide plate40and the intermediate guide plate60may be made of a transparent material. Specifically, the probes80may sequentially pass through the upper guide plate40, the intermediate guide plate60, and the lower guide plate50. In this case, a user may more accurately identify the intermediate guide hole63and the lower guide hole53through the transparent upper guide plate40and intermediate guide plate60. In other words, an effect of facilitating the insertion of the probes80may be obtained. After the probes80sequentially pass through the upper guide plate40, the lower guide plate50, and the intermediate guide plate60, the first plate10is relatively moved horizontally. As illustrated inFIG.4A, when the first plate10and the second plate20are aligned, the upper guide plate40, the lower guide plate50, and the intermediate guide plate60may be located on the same vertical line. Therefore, the upper guide hole43, the lower guide hole53, and the intermediate guide hole63may be located on the same vertical line, and the probes80may vertically pass through the upper guide hole43, the intermediate guide hole63, and the lower guide hole53. When the insertion of the probes80is completed, as illustrated inFIG.4B, the first plate10may be moved horizontally (in the direction of the arrow). In this case, the first plate10may be moved after the guide pin is removed. When the first plate10is moved to one side, the position of the upper guide hole43may be also changed, and the probes80may be elastically deformed according to the positional movement of the upper guide hole43. In other words, upper sides of the probes80that have passed through the upper guide hole43may be deformed in the moving direction of the first plate10, and intermediate and lower portions of the probes80that have passed through the intermediate guide hole63and the lower guide hole53may be maintained in a vertical state. Therefore, when the first plate10is moved, as illustrated inFIG.4B, an elastically deformable structure of the probes80may be implemented. FIGS.5A,5B,5C, and5Dare views illustrating a method of manufacturing an intermediate guide plate ofFIG.1. A guide plate GP illustrated inFIGS.5A,5B,5C, and5Dmay be at least one of an upper guide plate40, a lower guide plate50, and an intermediate guide plate60, and hereinafter will be described as being the intermediate guide plate60as an example. InFIGS.5A,5B,5C, and5D, for convenience of explanation, a portion of the intermediate guide plate60having an intermediate guide hole63is enlarged and schematically illustrated. As illustrated inFIG.5A, an anodic oxide film101including pores101amay be provided. Then, as illustrated inFIG.5B, a film5may be provided under the anodic oxide film101. In this case, the anodic oxide film101may be provided in a state in which a barrier layer BL is not removed, and the barrier layer BL may be provided on an upper surface180of the anodic oxide film101on which the film5is not provided. In other words, a porous layer PL may be provided between the barrier layer BL and the film5. Since the upper surface180of the intermediate guide plate60may be composed of the barrier layer BL, a problem in which particles flow into the intermediate guide plate60through the pores101amay be prevented. In addition, inner walls of openings of the guide plate GP, into which front ends of the probes80are first inserted during insertion of the probes80, may be composed of the barrier layer BL having high density, so that high durability may be ensured. This may prevent abrasion of the inner walls of the openings of guide holes GH that may occur simultaneously with the insertion of the probes80. As a result, a particle generation problem due to abrasion of the inner walls of the openings of the guide holes GH may be minimized. The film5may be a photosensitive material, and preferably, the film5is a photosensitive film capable of lithography. In addition, the film5may be a material capable of adhesion, and thus, the anodic oxide film101and the film5may be bonded without use of a separate adhesive means. As illustrated inFIG.5C, at least a portion of the film5may be patterned by a photo process. Therefore, a plurality of film holes5amay be formed in the film5. As illustrated inFIG.5D, the anodic oxide film101may be etched through the film holes5a, which are areas removed by patterning. Therefore, by such etching, a plurality of intermediate guide holes63corresponding to the film holes5amay be formed in the anodic oxide film101. In other words, the intermediate guide holes63may be holes having the same size as the film holes5a. The intermediate guide plate60in which the intermediate guide holes63are formed may be provided on a second plate20after the film5is removed. However, the intermediate guide plate60is not limited thereto, and may be provided on the second plate20with the film5provided thereon. In a conventional guide plate, insertion holes for probes are formed by mechanical processing such as laser or drilling processing. Therefore, a residual stress is generated when mechanically processing the insertion holes in the guide plate, resulting in a problem of deteriorating durability during use of the probe card. In addition, since the holes formed by laser or drilling processing are not vertical, it is difficult to insert the probes into the holes. On the contrary, in the intermediate guide plate60according to the present disclosure, since the intermediate guide holes63are formed by etching, the problems caused by mechanical processing may be prevented, and the intermediate guide holes63in which inner walls thereof are vertical in a straight line may be formed. Therefore, the insertion of the probes80may be facilitated. In the present disclosure, only the method of manufacturing the intermediate guide plate60has been described, but when each of the upper guide plate40and the lower guide plate50is made of the anodic oxide film101, a plurality of upper guide holes43and a plurality of lower guide holes53may be formed through the same process. FIG.6is a view illustrating a probe head of a probe card according to a second embodiment of the present disclosure, andFIGS.7A and7Bare views each illustrating a modified state ofFIG.6. Compared to the first embodiment, the second embodiment has a difference in the shape of a guide plate. Therefore, the difference will be mainly described, and the description and reference numerals of the first embodiment will be used for the same parts. A guide plate GP each illustrated inFIGS.6,7A, and7Bmay be at least one of an upper guide plate40′, a lower guide plate50′, and an intermediate guide plate60′, and hereinafter will be described as being an intermediate guide plate60′ as an example. InFIGS.6,7A, and7B, for convenience of explanation, a portion of the intermediate guide plate60′ having an intermediate guide hole63′ is enlarged and schematically illustrated. First,FIG.6is a view illustrating a stacked structure when the intermediate guide plate60′ is composed of two unit anodic oxide film sheets200′. Specifically, in the intermediate guide plate60′, a first unit anodic oxide film sheet201′ and a second unit anodic oxide film sheet202′ may be sequentially stacked. In the intermediate guide plate60′, the first unit anodic oxide film sheet201′ forming a lower surface190′ may be made of an anodic oxide film101′ having a porous layer PL and a barrier layer BL under the porous layer PL. In addition, in the intermediate guide plate60′, the second unit anodic oxide film sheet202′ forming an upper surface180′ may be made of an anodic oxide film101′ having a porous layer PL and a barrier layer BL on the porous layer PL. In this case, a film5′ may be provided on one side of each of the first unit anodic oxide film sheet201′ and the second unit anodic oxide film sheet202′. Specifically, the film5′ may be provided at a position not overlapping with the barrier layer BL, and thus, the respective films5′ may be provided on the first unit anodic oxide film sheet201′ and under the second unit anodic oxide film sheet202′. Therefore, when the first unit anodic oxide film sheet201′ and the second unit anodic oxide film sheet202′ are stacked, the films5′ may be provided between the first unit anodic oxide film sheet201′ and the second unit anodic oxide film sheet202′. The first unit anodic oxide film sheet201′ and the second unit anodic oxide film sheet202′ may be bonded to each other by the films5′. Therefore, the first unit anodic oxide film sheet201′ and the second unit anodic oxide film sheet202′ may be stacked without use of a separate adhesive means. As illustrated inFIG.6, the intermediate guide plate60′ is configured so that a surface SF is composed of the respective barrier layers BL having a symmetrical structure, so that upper and lower surfaces of the intermediate guide plate may have a uniform density. Specifically, there may be a density difference between the barrier layer BL in which no pores exist and the porous layer PL in which orderly arranged pores exist. Therefore, when the intermediate guide plate60′ is configured with only one unit anodic oxide film sheet200′ having an asymmetric structure, warpage deformation may occur under a high temperature environment. In addition, a plurality of intermediate guide hole63′ for allowing insertion of the probes80may be formed in the intermediate guide plate60′. However, when the surface SF of the intermediate guide plate60′ is composed of the respective porous layers PL including the pores, there may be a problem in which fine particles are collected therein, and then discharged when the probes80are inserted through the guide holes GH. Therefore, in the present disclosure, at least one of the upper guide plate40′, the lower guide plate50′, and the intermediate guide plate60′ each having the guide holes GH for the insertion of the probes80may be configured by stacking a plurality of unit anodic oxide film sheets200′, and the surface SF may be composed of the barrier layers BL having a symmetrical structure. This may ensure a uniform density of the upper and lower surfaces180′ and190′ of each of the upper guide plate40′, the lower guide plate50′, and the intermediate guide plate60′, thereby preventing warpage deformation. FIGS.7A and7Bare views illustrating a stacked structure when an intermediate guide plate60′ is composed of three unit anodic oxide film sheets200′. Specifically, as illustrated inFIGS.7A and7B, in the intermediate guide plate60′, a first unit anodic oxide film sheet201′ forming a lower surface190′ may be made of an anodic oxide film101′ having a porous layer PL and a barrier layer BL under the porous layer PL. In addition, a third unit anodic oxide film sheet203′ forming an upper surface180′ may be made of an anodic oxide film101′ having a porous layer PL and a barrier layer BL on the porous layer PL. Then, a second unit anodic oxide film sheet202′ including a barrier layer BL as illustrated inFIG.7Aor a second unit anodic oxide film sheet202′ including a porous layer PL with a barrier layer BL removed as illustrated inFIG.7Bmay be provided between the first unit anodic oxide film sheet201′ and the third unit anodic oxide film sheet203′. In the present embodiment, the intermediate guide plate60′ has been described as having a two- or three-layered stacked structure, but the structure of the intermediate guide plate60′ is not limited thereto. As an example, the intermediate guide plate60′ may be formed a stacked structure of four or more lavers. Since the intermediate guide plate60′ may have a stacked structure as described above, strength of the intermediate guide plate60′ may be increased. In other words, the intermediate guide plate60′ may effectively support probes80. While particular embodiments of the probe head and the probe card having the same according to the present disclosure have been described, it is merely illustrative and is not intended to limit the scope of the present disclosure and should be construed as having widest range based on the spirit of present disclosure. Those of ordinary skill in the art may combine and substitute the disclosed embodiments to perform a particular pattern of shape that has not been noted, but it is also within the scope of the present disclosure. It will be apparent to those of ordinary skill in the art that various changes and modifications may be readily made without departing from the spirit and scope of the present disclosure. | 40,408 |
11860193 | DESCRIPTION OF EMBODIMENTS Embodiments of the present disclosure are elaborated below with reference to the accompanying drawings. 1. Embodiment 1 Anisotropic Conductive Sheet FIG.1Ais a plan view illustrating anisotropic conductive sheet10according to Embodiment 1, andFIG.1Bis a sectional view taken along line1B-1B ofFIG.1A.FIG.2Ais an enlarged view ofFIG.1B, andFIG.2Bis a perspective view of anisotropic conductive sheet10as viewed through it in plan from first surface12aside inFIG.2A. As illustrated inFIGS.1A and1B, anisotropic conductive sheet10includes a plurality of conduction paths11, and insulation layer12disposed to fill the gap therebetween and including first surface12aand second surface12b(seeFIG.1B). In the present embodiment, preferably, an inspection object is disposed at first surface12a. Conduction Path11 Conduction path11extends in the thickness direction of insulation layer12, and includes first end portion11aon the first surface12aside and second end portion11bon the second surface side12b(seeFIG.1B). To be more specific, preferably, conduction path11extends through insulation layer12in the thickness direction, with first end portion11aexposed to first surface12aside and second end portion11bexposed to second surface12bside. To be more specific, the configuration in which conduction path11extends in the thickness direction of insulation layer12means that the direction connecting first end portion11aand second end portion11bof conduction path11is approximately parallel to the thickness direction of insulation layer12. The “approximately parallel” means that the angle to the thickness direction of insulation layer12is ±10° or smaller. In the case where first end portion11aof conduction path11is exposed to first surface12aside of insulation layer12, first end portion11aof conduction path11may be flush with first surface12aof insulation layer12, or may be protruded from first surface12aof insulation layer12. Likewise, in the case where second end portion11bof conduction path11is exposed to second surface12bside of insulation layer12, second end portion11bof conduction path11may be flush with second surface12bof insulation layer12, or may be protruded from second surface12bof insulation layer12. Center-to-center distance (pitch) p of first end portions11aof the plurality of conduction paths11is not limited, and may be appropriately set in accordance with the pitch of the terminal of the inspection object (seeFIG.1B). The pitch of the terminal of a high bandwidth memory (HBM) as an inspection object is 55 μm, and the pitch of the terminal of a package on package (PoP) is 400 to 650 μm, and accordingly, center-to-center distance p of the plurality of conduction paths11may be 5 to 650 μm, for example. Or more preferably, center-to-center distance p of the plurality of conduction paths11is 5 to 55 μm from the viewpoint of eliminating the need for alignment (alignment free) of the terminal of the inspection object. Center-to-center distance p of the plurality of conduction paths11is the smallest center-to-center distance of the center-to-center distances of first end portions11aof the plurality of conduction paths11. The center of first end portion11aof conduction path11is the center of gravity of first end portion11a. Center-to-center distance p of first end portions11aof the plurality of conduction paths11and the center-to-center distance of second end portion11bmay be the same or different from each other. In the present embodiment, center-to-center distance p of first end portions11aof the plurality of conduction paths11and the center-to-center distance of second end portion11bare the same, and are also referred to as a center-to-center distance of the plurality of conduction paths11. It suffices that the circle equivalent diameter of first end portion11aof conduction path11is a value with which center-to-center distance p of first end portions11aof the plurality of conduction paths11can be adjusted to the above-mentioned range, and the continuity with the terminal of the inspection object can be ensured. To be more specific, preferably, the circle equivalent diameter of first end portion11aof conduction path11is 2 to 20 μm, for example. The circle equivalent diameter of first end portion11aof conduction path11means the circle equivalent diameter of first end portion11aof conduction path11as viewed along the thickness direction of insulation layer12. The circle equivalent diameters of first end portion11aand second end portion11bof conduction path11may be the same or different from each other. In the present embodiment, the circle equivalent diameters of first end portion11aand second end portion11bof conduction path11are the same, and they are also referred to as the circle equivalent diameter of conduction path11. When anisotropic conductive sheet10is viewed through it in such a manner that the center of first end portion11aand the center of second end portion11boverlap each other, at least a part between first end portion11aand second end portion11bin conduction path11(that is, at least a part of conduction path11inside anisotropic conductive sheet10) is disposed in such a manner as not to overlap first end portion11aand second end portion11b(seeFIGS.2A and2B). The configuration in which at least a part of conduction path11does not overlap first end portion11aand second end portion11bas viewed through it in the above-mentioned manner means that at least a part of conduction path11is away from first end portion11aand second end portion11bas viewed through it in the above-mentioned manner (seeFIG.2B). To be more specific, when anisotropic conductive sheet10is viewed along virtual straight line A-A′ connecting the center of first end portion11aand the center of second end portion11b(seeFIG.2A), at least a part between first end portion11aand second end portion11bin conduction path11is not located on virtual straight line A-A′, and is located outside virtual straight line A-A′ (the dotted line ofFIG.2B). That is, in a cross-section along the thickness direction of insulation layer12, at least a part between first end portion11aand second end portion11bin conduction path11includes non-linear part11c(seeFIG.2A). To be more specific, in a cross-section along the thickness direction of insulation layer12, non-linear part11cis a portion (bent portion) where conduction path11does not have a straight-line shape. The shape of non-linear part11cis not limited as long as non-linear part11chas a spring-like elasticity in the thickness direction of insulation layer12, and examples of such a shape include a wavy shape, a zigzag shape, and an arch shape. In the present embodiment, non-linear part11chas a zigzag shape. Conduction path11including non-linear part11cwith such a shape can have a spring-like elasticity in the thickness direction of insulation layer12. In this manner, the impact of placing the inspection object on the surface of anisotropic conductive sheet10and the pressing force from upper side of the inspection object for electrical connection are absorbed and dispersed, and thus the damage of the terminal of the inspection object due to a contact with first end portion11aof conduction path11of anisotropic conductive sheet10can be suppressed. Non-linear part11cis disposed at least in a part of conduction path11. Preferably, non-linear part11cis disposed at least in a part of conduction path11located on first end portion11aside than the midpoint between first end portion11aand second end portion11b(that is, it is disposed on first surface12aside). One reason for this is to facilitate the absorption of the impact of placing the inspection object on the surface of anisotropic conductive sheet10so as to suppress the damage of the terminal of the inspection object due to a contact with first end portion11aof conduction path11of anisotropic conductive sheet10. In the present embodiment, non-linear part11cis disposed in the entirety of a center portion between first end portion11aand second end portion11bin conduction path11(seeFIGS.1B and2A). Distance d (the distance between peaks adjacent to each other) and height h of the zigzag shape of non-linear part11cof conduction path11in a cross-section along the thickness direction of insulation layer12is not limited as long as a spring elasticity that can absorb the impact of placing the inspection object on the surface of anisotropic conductive sheet10. For example, distance d of the zigzag shape (the distance between the vertexes of peaks adjacent to each other) of non-linear part11cof conduction path11in a cross-section along the thickness direction of insulation layer12may be approximately 5 to 50% of the thickness of insulation layer12(seeFIG.2A). In addition, height h of the zigzag shape of non-linear part11cof conduction path11in a cross-section along the thickness direction of insulation layer12may be approximately 2 to 20% of the thickness of insulation layer12. Height h of the zigzag shape is the distance between the straight line connecting the vertexes of two peaks adjacent to each other, and the straight line connecting the bottom point of the valley formed between the two peaks and the bottom points of two valleys formed on both sides of that valley (seeFIG.2A). The material of conduction path11is not limited as long as the material has conductivity. Preferably, the volume resistivity of the material of conduction path11is 1.0×10×10−4Ω-cm or smaller, more preferably, 1.0×10×10−6to 1.0×10−9Ω-cm, for example. It suffices that the volume resistivity of the material of conduction path11satisfies the above-mentioned range, and examples of the material of conduction path11include metal materials such as copper, gold, nickel, tin, iron or alloys of them, and carbon materials such as carbon black. Among them, the material of conduction path11is preferably a metal material. Specifically, it is preferable that conduction path11be a metal line composed of a metal material. The metal line may be composed of a single layer, or a plurality of layers. For example, the metal line may have a multilayer structure with a copper alloy layer as the core material, a nickel or nickel alloy layer as the intermediate coating material, and a gold layer as the top surface coating material. For example, with an intermediate coating material, internal diffusion of the top surface coating material to the core material can be prevented. Insulation Layer12 Insulation layer12is disposed to fill the gap between the plurality of conduction paths11, and insulates the plurality of conduction paths11from each other. Such an insulation layer12includes first surface12aas one surface of anisotropic conductive sheet10, and second surface12bas the other surface. Insulation layer12may be composed of a first resin composition. Preferably, the glass transition temperature of the first resin composition is −40° C. or below, more preferably −50° C. or below. The glass transition temperature of the first resin composition can be measured by complying with JIS K 7095:2012. Preferably, the storage modulus of the first resin composition at 25° C. 1.0×107Pa or smaller, more preferably 1.0×105to 9.0×106Pa. In particular, the boundary surface between conduction path11and insulation layer12is prone to peeling due to repeated pressurization and depressurization, and in such a case, it is particularly effective to provide bonding layer14. The storage modulus of the first resin composition can be measured by complying with JIS K 7244-1:1998/ISO6721-1:1994. The glass transition temperature and the storage modulus of the first resin composition are adjusted by the amount of filler added and the type of elastomer contained in the resin composition. In addition, the storage modulus of the first resin composition can also be adjusted by the morphology of the resin composition (e.g., whether it is porous or not). The first resin composition can be anything that provides insulation, and there are no particular restrictions, but from the viewpoint of making it easier to meet the glass transition temperature or storage modulus described above, it is preferable to be a cross-linked product of a composition containing an elastomer (base polymer) and a cross-linking agent (hereinafter referred to as the “first elastomer composition”). Preferably, examples of elastomer include elastomers such as silicone rubber, urethane rubber (urethane polymer), acrylic rubber (acrylic polymer), ethylene-propylene-diene copolymer (EPDM), chloroprene rubber, styrene-butadiene copolymer, acrylic nitrile-butadiene copolymer, poly butadiene rubber, natural rubber, polyester thermoplastic elastomer, and olefin thermoplastic elastomer. Among them, silicone rubber is preferable. The cross-linking agent can be selected according to the type of elastomer. Examples of cross-linking agents for silicone rubber include organic peroxides such as benzoyl peroxide, bis-2,4-dichlorobenzoyl peroxide, dicumyl peroxide, and di-t-butyl peroxide. Examples of cross-linking agents for acrylic rubbers (acrylic polymers) include epoxy compounds, melamine compounds, and isocyanate compounds. The first elastomer composition may also further contain other components such as adhesion-imparting agents, silane coupling agents, and fillers as necessary, for example, from the viewpoint of facilitating adjustment of adhesion and storage modulus to the above ranges. The first elastomer composition may be porous, for example, from the perspective of facilitating adjustment of the storage modulus to the above range. In other words, porous silicone can be used. Thickness The thickness of insulation layer12may be, but not limited as long as it can ensure insulation, 20 to 100 μm, for example. The thickness of insulation layer12can be measured by complying with ASTM D6988. Other Layers Anisotropic conductive sheet10according to the present embodiment may further include layers other than the above-mentioned layers as necessary. For example, it is possible to further include electrolyte layer13(seeFIG.8described later) disposed on the surface of first surface12aside (or on first end portion11aof conduction path11) of anisotropic conductive sheet10, and/or a plurality of bonding layers14(seeFIGS.9A and9Bdescribed later) disposed between the plurality of conduction paths11and insulation layer12. Operations Operations of anisotropic conductive sheet10according to the present embodiment are described through comparison with anisotropic conductive sheet1for comparison.FIG.3Ais a partially enlarged sectional view of anisotropic conductive sheet1for comparison, andFIG.3Bis a plan view ofFIG.3A. As illustrated inFIGS.3A and3B, in anisotropic conductive sheet1for comparison, conduction path2is linearly formed in a cross-section along the thickness direction of insulation layer3. That is, conduction path2does not have the non-linear part. As such, it is difficult to disperse the pressing force when an inspection object is disposed and pressed on the surface of anisotropic conductive sheet1. Therefore, the terminal of the inspection object may be damaged due to the contact with first end portion2aof conduction path2of anisotropic conductive sheet1. In contrast, in anisotropic conductive sheet10according to the present embodiment, conduction path11includes non-linear part11c(seeFIGS.2A and2B). Non-linear part11chas an elasticity such that it expands and contracts in the thickness direction of insulation layer12(like a spring). In this manner, the force of pressing the inspection object disposed on the surface of anisotropic conductive sheet10can be dispersed at the portion of non-linear part11c. In this manner, the damage of the terminal of the inspection object due to a contact with first end portion11aof conduction path11of anisotropic conductive sheet10can be suppressed. In addition, in the portion of non-linear part11c, the contact area between conduction path11and insulation layer12increases, and therefore the adhesiveness between conduction path11and insulation layer12can also be increased. In this manner, the peeling of conduction path11from insulation layer12can be suppressed. Manufacturing Method of Anisotropic Conductive Sheet FIGS.4A to4Care schematic views illustrating a manufacturing process of anisotropic conductive sheet10according to the present embodiment. As illustrated inFIGS.4A to4C, for example, anisotropic conductive sheet10according to the present embodiment can be obtained through: 1) a step of preparing a plurality of composite sheets20in which a plurality of conduction lines22is disposed at the surface of insulating sheet21(seeFIG.4A); 2) a step of obtaining laminated body23by stacking and sequentially integrating the plurality of composite sheets20(seeFIGS.4A and4B); and 3) a step of obtaining anisotropic conductive sheet10by cutting the obtained laminated body23at a predetermined interval (seeFIGS.4B and4C). Step 1) The plurality of composite sheets20in which the plurality of conduction lines22is disposed is prepared on the surface of insulating sheet21. Insulating sheet21may be a resin sheet (such as a silicone sheet) composed of the first resin composition that constitutes insulation layer12. The plurality of conduction lines22is disposed on the surface of insulating sheet21with a predetermined distance therebetween. Conduction line22can be disposed on the surface of insulating sheet21by any methods. For example, in the case where conduction line22is composed of a metal line, the metal line may be disposed as it is, a metal paste may be formed through drawing with a dispenser and the like, or a metal ink may be printed by an ink-jet manner. Step 2) The obtained composite sheets20are stacked and sequentially integrated (FIGS.4A and4B). Normally, the method of the integration may be, but not limited thereto, a thermo-compression bonding, pressure bonding, or the like. Block-shaped laminated body23is obtained by sequentially repeating the lamination and integration of composite sheets20(seeFIG.4B). Step of 3) Obtained laminated body23is cut in the direction intersecting (preferably, orthogonal to) the extending direction of conduction line22along the lamination direction at a predetermined interval (t) (the broken line ofFIG.4B). In this manner, anisotropic conductive sheet10with a predetermined thickness of the (t) can be obtained (FIG.4C). That is, the plurality of conduction paths11in anisotropic conductive sheet10results from the plurality of conduction lines22, and insulation layer12results from insulating sheet21. Other Steps 4) The manufacturing method of anisotropic conductive sheet10according to the present embodiment may further include steps other than the above-mentioned steps 1) to 3) in accordance with the configuration of anisotropic conductive sheet10. For example, it is possible to further include a step of forming electrolyte layer13on the surface of the obtained anisotropic conductive sheet10(seeFIG.8described later). Anisotropic conductive sheet10according to the present embodiment can be used for electrical testing. Electrical Testing Apparatus and Electrical Testing Method Electrical Testing Apparatus FIG.5is a sectional view illustrating electrical testing apparatus100according to the present embodiment. Electrical testing apparatus100, which uses anisotropic conductive sheet10illustrated inFIG.1B, is an apparatus for inspecting electrical characteristics such as the continuity between terminals131of inspection object130(between measurement points), for example. It is to be noted that in this drawing, inspection object130is also illustrated from the viewpoint of describing the electrical testing method. As illustrated inFIG.5, electrical testing apparatus100includes holding container (socket)110, inspection substrate120, and anisotropic conductive sheet10. Holding container (socket)110is a container that holds inspection substrate120, anisotropic conductive sheet10and the like. Inspection substrate120is disposed in holding container110, and provided with a plurality of electrodes121that faces the measurement points of inspection object130on the surface that faces inspection object130. Anisotropic conductive sheet10is disposed on the surface of inspection substrate120on which electrode121is disposed, such that the electrode121and electrolyte layer13on second surface12bside in anisotropic conductive sheet10are in contact with each other. Inspection object130is not limited, but is, for example, various semiconductor devices (semiconductor packages) such as HBM and PoP, electronic components, printed boards and the like. In the case where inspection object130is a semiconductor package, the measurement point may be a bump (terminal). In addition, in the case where inspection object130is a printed board, the measurement point may be a component mounting land or a measurement land provided in the conductive pattern. Electrical Testing Method An electrical testing method using electrical testing apparatus100illustrated inFIG.5is described below. As illustrated inFIG.5, the electrical testing method according to the present embodiment includes a step of electrically connecting electrode121of inspection substrate120and terminal131of inspection object130through anisotropic conductive sheet10by stacking inspection substrate120including electrode121and inspection object130with anisotropic conductive sheet10therebetween. When performing the above-mentioned step, inspection object130may be pressurized (seeFIG.5), or may be brought into contact under a heated atmosphere as necessary from the viewpoint of facilitating sufficient conduction between electrode121of inspection substrate120and terminal131of inspection object130through anisotropic conductive sheet10. In the above-mentioned step, the surface (first surface12a) of anisotropic conductive sheet10makes contact with terminal131of inspection object130. In anisotropic conductive sheet10according to the present embodiment, conduction path11includes non-linear part11c. Non-linear part11ccan have a spring-like elasticity for moving upward and downward in the thickness direction of insulation layer12. In this manner, in comparison with a known anisotropic conductive sheet with a metal pin that is linearly formed and does not include non-linear part11c, the force of pressing the inspection object disposed on the surface of anisotropic conductive sheet10can be dispersed at the portion of non-linear part11c. In this manner, the damage of the terminal of the inspection object due to a contact with first end portion11aof conduction path11of anisotropic conductive sheet10can be suppressed. Modification While virtual line A-A′ connecting first end portion11aand second end portion11bof conduction path11is parallel to the thickness direction of insulation layer12(seeFIG.2A) in anisotropic conductive sheet10of the present embodiment described above, this is not limitative. FIG.6Ais a partial sectional view illustrating anisotropic conductive sheet10according to a modification, andFIG.6Bis a perspective view of conduction path11as viewed through it in such a manner that the center of first end portion11aand the center of second end portion11boverlap each other inFIG.6A. As illustrated inFIG.6A, virtual line A-A′ connecting between first end portion11aand second end portion11bof conduction path11may not be completely parallel to the thickness direction of insulation layer12. In addition, while non-linear part11cof conduction path11has a zigzag shape in anisotropic conductive sheet10in the present embodiment, this is not limitative. FIGS.7A to7Gare partial sectional views illustrating anisotropic conductive sheet10according to modifications. As illustrated inFIGS.7A to7G, the shape of non-linear part11cof conduction path11may have a wavy shape (seeFIG.7A), an arch shape (seeFIG.7B), or a v shape (seeFIG.7C). In addition, the shape may be a combination of a linear part and a wavy shape, a zigzag shape, an arch shape or a v shape (seeFIGS.7A to7G). Distance d and height h of these non-linear parts11care defined as described above. In addition, in the present embodiment, anisotropic conductive sheet10may further include layers other than the above-mentioned layers, such as electrolyte layer13and bonding layer14as described above. Electrolyte Layer13 FIG.8is a sectional view of anisotropic conductive sheet10according to a modification. As illustrated inFIG.8, anisotropic conductive sheet10further includes electrolyte layer13disposed on the surface on first surface12aside. Electrolyte layer13is a coating including lubricant, and can be disposed on first end portion11aof conduction path11, for example. In this manner, when placing the inspection object on first surface12a, deformation of the terminal of the inspection object can be suppressed, and adhesion of the electrode material of the inspection object to the surface of first end portion11aof conduction path11can be suppressed without impairing the electrical connection with the terminal of the inspection object. Examples of lubricants in the electrolyte layer13include fluoropolymer-based lubricants; lubricants based on inorganic materials such as boron nitride, silica, zirconia, silicon carbide, and graphite; hydrocarbon-based mold-releasing agents such as paraffin waxes, metallic soaps, natural and synthetic paraffins, polyethylene waxes, and fluorocarbons; fatty acid-based mold-releasing agents such as stearic acid, hydroxystearic acid, and other high-grade fatty acids and oxyfatty acids; fatty acid amide release agents such as stearic acid amides, fatty acid amides such as ethylene bis-stearoamide, and alkylene bis-fatty acid amides; alcohol-based release agents such as aliphatic alcohols such as stearyl alcohol and cetyl alcohol, polyhydric alcohols, polyglycols, and polyglycerols; fatty acid ester-based release agents such as aliphatic acid lower alcohol esters such as butyl stearate and pentaerythritol tetrastearate, fatty acid polyhydric alcohol esters, and fatty acid polyglycol esters; silicone based release agents such as silicone oils; and alkyl sulfonate metal salts. Among them, alkylsulfonic acid metal salts are preferred from the viewpoint that they have fewer adverse effects such as contaminating the electrodes of the inspection object, especially when used at high temperatures. Metal salts of alkylsulfonic acids are preferably alkali metal salts of alkylsulfonic acids. Examples of alkali metal salts of alkylsulfonic acids include sodium 1-decanesulfonate, sodium 1-undecanesulfonate, sodium 1-dodecanesulfonate, sodium 1-tridecane sulfonate, sodium 1-tetradecane sulfonate, sodium 1-pentadecane sulfonate, sodium 1-hexadecane sulfonate, sodium 1-heptadecane sulfonate, sodium octadecane sulfonate, sodium nonadecane sulfonate, sodium eicosanedecane sulfonate, potassium 1-decane sulfonate, potassium 1-undecane sulfonate, potassium 1-dodecane sulfonate, potassium 1-tridecane sulfonate, potassium 1-tetradecane sulfonate, potassium 1-pentadecane sulfonate potassium, potassium 1-hexadecane sulfonate, potassium 1-heptadecane sulfonate, potassium 1-octadecane sulfonate, potassium 1-nonadecane sulfonate, potassium 1-eicosanedecasulfonate, lithium 1-decane sulfonate, lithium 1-undecane sulfonate, lithium 1-dodecane sulfonate, lithium 1-tridecane sulfonate, lithium 1-tetradecane sulfonate, lithium 1-pentadecane sulfonate, lithium 1-hexadecane sulfonate, lithium 1-heptadecanesulfonate, lithium 1-octadecanesulfonate, lithium 1-nonadecanesulfonate, lithium 1-eicosanedecasulfonate, and their isomers. Among them, the sodium salt of alkylsulfonic acid is particularly preferred because of its excellent heat resistance. One type of these may be used alone, and two or more complexes may be used in combination. It is to be noted that the conductivity of conduction path11on the surface on first surface12aside can be ensured by making the thickness of electrolyte layer13extremely thin. Electrolyte layer13can be formed by any method, such as a method of applying the solution of electrolyte layer13, for example. The solution of electrolyte layer13can be applied by known methods such as spraying, brushing, dropping of the solution of electrolyte layer13, and dipping of anisotropic conductive sheet10into that solution. In the applying methods, it is possible to appropriately use a method in which the material of the electrolyte layer13is diluted with a solvent such as alcohol, and the diluted solution (solution of the electrolyte layer13) is applied to the surface of the anisotropic conductive sheet10(conduction path11), and then the solvent is evaporated. Thus, the electrolyte layer13can be uniformly formed on the surface of the anisotropic conductive sheet10(on the conduction path11of the sheet). In addition, in the case where the material of the electrolyte layer13that is in solid powder state at room temperature is used, it is possible to use a method in which an appropriate amount of it is put on the surface of anisotropic conductive sheet10, and then it is applied by melting the material by heating anisotropic conductive sheet10to a high temperature. Bonding Layer14 FIG.9Ais a partially enlarged view of a horizontal cross-section of anisotropic conductive sheet10according to a modification (a partial sectional view along the direction orthogonal to thickness direction),FIG.9Bis a partially enlarged view of a vertical cross section of anisotropic conductive sheet10ofFIG.9A(a partial sectional view along the thickness direction). As illustrated inFIGS.9A and9B, anisotropic conductive sheet10may further include a plurality of bonding layers14disposed between the plurality of conduction paths11and insulation layer12. Bonding layer14is disposed at least a part between conduction path11and insulation layer12(at least a part of the side surface of conduction path11). In the present embodiment, bonding layer14is disposed to surround the side surface of conduction path11(FIG.9B). Bonding layer14increases the adhesiveness between conduction path11and insulation layer12to make it difficult to peel off at the boundary surface. That is, bonding layer14functions as a junction layer that increases the adhesiveness between conduction path11and insulation layer12. The material of bonding layer14is not limited as long as conduction path11and insulation layer12can be sufficiently bonded with each other without impairing the function of conduction path11. The material of bonding layer14may be an organic-inorganic composite composition containing polycondensation products of alkoxysilane or its oligomers, or it may be a second resin composition. Organic-Inorganic Composite Composition The organic-inorganic composite composition contains polycondensation products of alkoxysilane or its oligomers. Alkoxysilane is an alkoxysilane compound in which two to four alkoxy groups are bonded to silicon. That is, an alkoxysilane can be a bifunctional alkoxysilane, a trifunctional alkoxysilane, a tetrafunctional alkoxysilane, or a mixture of one or more of these. Among them, from the viewpoint of forming three-dimensional cross-links and facilitating sufficient adhesion, it is preferable that the alkoxysilane contains a trifunctional or tetrafunctional alkoxysilane, and it is more preferable that it contains a tetrafunctional alkoxysilane (tetraalkoxysilane). Oligomers of alkoxysilanes can be partially hydrolyzed and polycondensed alkoxysilanes. Specifically, the alkoxysilane or its oligomer should preferably include, for example, the compound shown in Formula (1) below. In Formula (1), R is independently an alkyl group and n is an integer from 0 to 20. Examples of alkoxysilanes represented by Formula (1) include tetramethoxysilane, tetraethoxysilane, and tetrabutoxysilane. The alkoxysilane or its oligomer may be commercially available. Examples of commercially available oligomers of alkoxysilane include Colcoat N-103X and Colcoat PX manufactured by Colcoat. The organic-inorganic composite composition may further contain other components, such as conductive materials, silane coupling agents, and surfactants, as needed. Second Resin Composition The glass transition temperature of the second resin composition constituting bonding layer14is not limited, but it is preferably higher than the glass transition temperature of the first resin composition constituting insulation layer12. For example, preferably the glass transition temperature of the second resin composition is 150° C. or above, more preferably 160 to 600° C. The glass transition temperature of the second resin composition can be measured by the same method as the above-mentioned method. The second resin composition constituting bonding layer14is not limited, but from the viewpoint of facilitating the development of adhesiveness, the second resin composition may be a cross-linked product of a composition containing an elastomer and a cross-linking agent (hereinafter also referred to as the “second elastomer composition”), or a resin composition containing a non-elastomeric resin or it may be a cured product of a resin composition containing a curable resin that is not an elastomer and a curing agent. The elastomers included in the second elastomer composition can be the same as those listed as elastomers included in the first elastomer composition. The type of elastomer included in the second elastomer composition may be the same as or different from the type of elastomer included in the first elastomer composition. For example, from the viewpoint of facilitating increased affinity and adhesion between insulation layer12and adhesive layer14, the type of elastomer contained in the second elastomer composition may be the same as the type of elastomer contained in the first elastomer composition. The weight average molecular weight of the elastomer contained in the second elastomer composition is not limited, but from the viewpoint of making it easier to meet the glass transition temperature described above, it is preferable that it is higher than the weight average molecular weight of the elastomer contained in the first elastomer composition. The weight average molecular weight of the elastomer can be measured in polystyrene equivalent by gel permeation chromatography (GPC). The crosslinking agent in the second elastomer composition can be selected as appropriate according to the type of elastomer, and the same crosslinking agent as listed for the crosslinking agent in the first elastomer composition can be used. The content of the crosslinking agent in the second elastomer composition is not particularly limited, but from the viewpoint of facilitating the satisfaction of the glass transition temperature described above, it is preferable to have a higher content than that of the crosslinking agent in the first elastomer composition. In addition, the degree of crosslinking (gel fraction) of the crosslinked material of the second elastomer composition should be higher than that of the crosslinked material of the first elastomer composition. The same non-elastomeric resins (including curable resins) and curing agents included in the second resin composition can be used as the non-elastomeric resins and curing agents included in the first resin composition, respectively. The non-elastomeric resins included in the second resin composition are preferably polyimide, polyamide-imide, acrylic resin, and epoxy resin. Among them, it is preferable that the second resin composition is a resin composition containing a non-elastomeric resin or a cured product of a resin composition containing a curable resin that is not an elastomer and a curing agent, from the viewpoint of making it easier to meet the glass transition temperature described above. Thickness The thickness of bonding layer14is not limited as long as it can achieve sufficient bonding between conduction path11and insulation layer12without impairing the function of conduction path11. Normally, it is preferable that the thickness of bonding layer14be smaller than the circle equivalent diameter of conduction path11. To be more specific, preferably, the thickness of bonding layer14is 1 μm or smaller, more preferably 0.5 μm or smaller. In addition, bonding layer14may further be disposed in a region other than the region between conduction path11and insulation layer12. InFIG.9A, bonding layer14is additionally disposed in such a manner as to connect bonding layer14on the side surface of one conduction path11of two conduction paths11adjacent to each other and bonding layer14on the side surface of the other conduction path11. In this manner, not only the adhesiveness between conduction path11and insulation layer12, but also the adhesiveness between insulating sheets21described later that constitute insulation layer12can be increased (seeFIGS.10A and10Bdescribed later). In this manner, even when pressurization and depressurization are repeated in electrical testing, peeling less occurs at the boundary surface between conduction path11and insulation layer12of anisotropic conductive sheet10, and in addition, at the interface between insulating sheets21that constitute insulation layer12. In addition, also at the step of cutting in the above-mentioned step 3), the peeling at the boundary surface between conduction path11and insulation layer12, and the peeling at the interface between insulating sheets21that constitute insulation layer12can be made less occur. Anisotropic conductive sheet10including bonding layer14can be manufactured by the following method. FIGS.10A to10Care schematic views illustrating a manufacturing process of anisotropic conductive sheet10according to a modification. It is to be noted that inFIG.10C, the illustration of conduction path11in first surface11ais omitted. It can be manufactured in the same manner as the method described above except that at the step of the above-mentioned1), composite sheet20including insulating sheet21, the plurality of conduction lines22, and bonding layer24that covers at least a part of the side surface thereof in this order is prepared (seeFIG.10A), and, at the step of the above-mentioned2), the plurality of composite sheets20are stacked such that bonding layer24of one composite sheet20and insulating sheet21of the other composite sheet20are brought into contact with each other while sequentially integrating them (seeFIGS.10A to10C). At the step 1), composite sheet20can be obtained by any method. For example, the plurality of conduction lines22covered with bonding layer24may be disposed with a predetermined distance therebetween on the surface of insulating sheet21; or after the plurality of conduction lines22is disposed with a predetermined distance therebetween on the surface of insulating sheet21, bonding layer24may be formed in such a manner as to cover the plurality of conduction line22. Bonding layer24may be formed by applying a solution containing the aforementioned alkoxysilane or its oligomer, or the aforementioned elastomer composition, or by laminating sheets thereof. In the present embodiment, composite sheet20can be obtained by further applying the above-mentioned solution or composition after the plurality of conduction lines22is disposed with a predetermined distance therebetween on the surface of insulating sheet21(seeFIG.10A). The step 2) and the step 3) can be performed in the same manner as the steps described above. In this manner, anisotropic conductive sheet10with a predetermined thickness (t) can be obtained (seeFIG.10C). That is, in anisotropic conductive sheet10, conduction path11results from conduction line22, insulation layer12results from the integrated member of the plurality of insulating sheets21, and bonding layer14results from bonding layer24. 2. Embodiment 2 Anisotropic Conductive Composite Sheet An anisotropic conductive composite sheet according to the present embodiment includes a first anisotropic conductive sheet (first anisotropic conductivity layer), and a second anisotropic conductive sheet (second anisotropic conductivity layer) stacked (fixed) on the first anisotropic conductive sheet. The anisotropic conductive composite sheet may be used for electrical testing, and it is preferable that the sheet be disposed such that the first anisotropic conductive sheet is on the inspection substrate side and the second anisotropic conductive sheet is on the inspection object side. At least one of the first anisotropic conductive sheet and the second anisotropic conductive sheet may be the above-described anisotropic conductive sheet (anisotropic conductive sheet10according to Embodiment 1). In particular, anisotropic conductive sheet10according to Embodiment 1, which can easily achieve an elasticity of expanding and contracting in the thickness direction and can make the terminals of the object to be inspected less susceptible to damage, is preferably used as second anisotropic conductive sheet50. FIG.11Ais a plan view illustrating anisotropic conductive composite sheet30according to the present embodiment, andFIG.11Bis a sectional view taken along line11B-11B ofFIG.11A. As illustrated inFIG.11B, anisotropic conductive composite sheet30includes the first anisotropic conductive sheet (first anisotropic conductivity layer)40, and second anisotropic conductive sheet (second anisotropic conductivity layer)50stacked on it. Second anisotropic conductive sheet50is the above-described anisotropic conductive sheet (anisotropic conductive sheet10according to Embodiment 1). First Anisotropic Conductive Sheet First anisotropic conductive sheet40includes a plurality of conduction paths41(first conduction paths) extending through it in the thickness direction, and insulation layer42(first insulation layer) that insulates them from each other and includes third surface42aand fourth surface42b(seeFIG.11B). Conduction path41includes third end portion41aextending through it in the thickness direction of first anisotropic conductive sheet40and exposed to third surface42aside, and fourth end portion41bexposed to fourth surface42bside (seeFIG.11B). It is to be noted that as described above, the “thickness direction” means that the angle to the thickness direction of insulation layer42is ±10° or smaller. Preferably, center-to-center distance p1(pitch) of third end portions41aof the plurality of conduction paths41is greater than center-to-center distance p2(pitch) of fifth end portions51aof a plurality of conduction paths51of second anisotropic conductive sheet50from the viewpoint of easily ensuring the conductivity in the thickness direction (seeFIG.11B). To be more specific, although it depends on center-to-center distance p2of the plurality of conduction paths51of fifth end portion51aof second anisotropic conductive sheet50, center-to-center distance p1of third end portions41aof the plurality of conduction paths41of first anisotropic conductive sheet40may be 55 to 650 μm, for example. It is to be noted that as in Embodiment 1, center-to-center distance p1of third end portions41aof the plurality of conduction paths41and the center-to-center distance of fourth end portions41b(or center-to-center distance p2of fifth end portions51aof the plurality of conduction paths51and the center-to-center distance of sixth end portions51b) may be the same or different from each other. In the present embodiment, center-to-center distance p1of third end portions41aof the plurality of conduction paths41(or center-to-center distance p2of fifth end portions51aof the plurality of conduction paths51) and the center-to-center distance of fourth end portions41b(or the center-to-center distance of sixth end portions51b) are the same, and they are referred to also as the center-to-center distance of the plurality of conduction paths41(or the center-to-center distance of the plurality of conduction paths51). Conduction path41is exposed to both surfaces of first anisotropic conductive sheet40. In the present embodiment, from the viewpoint of increasing the electrical contact with second anisotropic conductive sheet50, it is preferable that conduction path41of first anisotropic conductive sheet40protrude than insulation layer42in the thickness direction at the surface of first anisotropic conductive sheet40on which second anisotropic conductive sheet50is stacked (seeFIG.11B). For example, the protruding height of conduction path41may be, but not limited thereto, 10 to 40 μm, or more preferably 15 to 30 μm. The circle equivalent diameter of conduction path41is not limited as long as the continuity can be ensured, and may be approximately 20 to 200 μm, for example. The material of conduction path41may be the same as the above-mentioned materials for conduction path11, or preferably metal materials. Insulation layer42is disposed to fill the gap between the plurality of conduction paths41and insulates the plurality of conduction paths41from each other (seeFIG.11B). Examples of the material of insulation layer42include the above-mentioned material for insulation layer12, or preferably the cross-linked product of the first elastomer composition. The rockwell hardness of the surface of first anisotropic conductive sheet40is not limited, and normally, may be substantially the same as the rockwell hardness of conduction path41(e.g., 90 to 100% of the rockwell hardness of conduction path41). The reason for this may be that since the circle equivalent diameter and center-to-center distance p1of third end portion41aof conduction path41are relatively large, the percentage of the surface area of insulation layer42that makes contact with a measurement indenter is small (when the measurement indenter is applied to the center of the cross-section of conduction path41), and that conduction path41protrudes than insulation layer42at the surface (third surface42aof insulation layer42) of first anisotropic conductive sheet40on which second anisotropic conductive sheet50is stacked. The rockwell hardness of the surface of first anisotropic conductive sheet40can be measured using a hardness meter in accordance with ASTM D785 as described later. Second Anisotropic Conductive Sheet Second anisotropic conductive sheet50includes the plurality of conduction paths51(second conduction paths) formed along the thickness direction, and insulation layer52(second insulation layer) filling the gaps therebetween and including fifth surface52aand sixth surface52b(seeFIGS.11A and11B). To be more specific, they are stacked such that third surface42aof first insulation layer42of first anisotropic conductive sheet40and sixth surface52bof second insulation layer52of second anisotropic conductive sheet50face each other (seeFIG.11B). Then, second anisotropic conductive sheet50may be anisotropic conductive sheet10according to Embodiment 1. That is, in second anisotropic conductive sheet50, conduction path51(second conduction path) corresponds to conduction path11in the above-mentioned anisotropic conductive sheet10, and insulation layer52(second insulation layer) corresponds to insulation layer12in the above-mentioned anisotropic conductive sheet10. Likewise, fifth end portion51aand sixth end portion51bof conduction path51correspond to first end portion11aand second end portion11bof conduction path11, respectively, and fifth surface52aand sixth surface52bof insulation layer52correspond to first surface12aand second surface12bof insulation layer12, respectively. Center-to-center distance p2(pitch) of fifth end portions51aof the plurality of conduction paths51in second anisotropic conductive sheet50is smaller than center-to-center distance p1(pitch) of third end portions41aof the plurality of conduction paths41in first anisotropic conductive sheet40. To be more specific, preferably, center-to-center distance p2of fifth end portions51aof the plurality of conduction paths51is 18 to 31% of the center-to-center distance p1of third end portions41aof the plurality of conduction paths41. With center-to-center distance p2of fifth end portions51aof the plurality of conduction paths51sufficiently smaller than center-to-center distance p1of third end portions41aof the plurality of conduction paths41, the need for alignment of inspection objects can be eliminated. Center-to-center distance p2of fifth end portions51aof the plurality of conduction paths51may be 10 to 200 μm, for example. In addition, normally, the circle equivalent diameter of fifth end portion41aof conduction path51is smaller than the circle equivalent diameter of third end portion41aof conduction path41. Insulation layer52is disposed to fill the gap between the plurality of conduction paths51and insulates them from each other. As the material of insulation layer52, the same material as that of insulation layer42may be used except that it is selected such that the rockwell hardness of the surface of second anisotropic conductive sheet50satisfies the range described later. For example, from the viewpoint of easily integrating second anisotropic conductive sheet50and first anisotropic conductive sheet40, the material of insulation layer52and the material of insulation layer42may be the same. Preferably, the rockwell hardness of the surface (preferably fifth surface52a) of second anisotropic conductive sheet50is lower than the rockwell hardness of the surface (preferably third surface42a) of first anisotropic conductive sheet40. As described above, in first anisotropic conductive sheet40, the circle equivalent diameter and center-to-center distance p1of third end portion41aof conduction path41are relatively large, and therefore the percentage of the surface area of the insulating material that makes contact with the indenter is small; whereas in second anisotropic conductive sheet50, the circle equivalent diameter and center-to-center distance p2of fifth end portion51aof conduction path51are relatively small, and therefore the percentage of the surface area of the insulating material that makes contact with the indenter tends to be large. As a result, conceivably, the rockwell hardness of the surface of second anisotropic conductive sheet50is substantially the same as the rockwell hardness between the metal line and the insulating material (preferably an insulating material), and is lower than the surface the rockwell hardness of first anisotropic conductive sheet40that is substantially the same as the rockwell hardness of conduction path41. To be more specific, preferably, the rockwell hardness of the surface of second anisotropic conductive sheet50is M120 or smaller. Such a second anisotropic conductive sheet50has a suitable flexibility, and can be less damaged by the terminal of the inspection object conduction path51and the like in comparison with the case where first anisotropic conductive sheet40is in direct contact with the inspection object. The rockwell hardness of the surface of second anisotropic conductive sheet50can be measured by complying with ASTM D785. To be more specific, after second anisotropic conductive sheet50is cut into a predetermined size, the rockwell hardness of the M scale of the obtained test specimen is measured using a hardness meter in accordance with ASTM D785. The rockwell hardness of the surface of second anisotropic conductive sheet50can be adjusted by the percentage of the area of the insulating material (insulation layer52) with respect to the surface area of second anisotropic conductive sheet50. To set the rockwell hardness of the surface of second anisotropic conductive sheet50to M120 or smaller, it is preferable to 1) increase the percentage of the surface area of the insulating material (insulation layer52) with respect to the surface area of second anisotropic conductive sheet50, for example than that of first anisotropic conductive sheet40, for example. To be more specific, it is preferable to set the percentage of the surface area of insulation layer52with respect to the surface area of second anisotropic conductive sheet50to 75% or greater. The percentage of the surface area of insulation layer52can be calculated from the two-dimensional information obtained through observation of the surface of second anisotropic conductive sheet50using a scanning electron microscope. Percentage (%) of Surface Area of Insulation Layer 52=Surface Area of Insulation Layer 52/Surface Area of Second Anisotropic Conductive Sheet 50×100 (Formula (1)) In the case where conduction path51is a metal line, the percentage of the surface area of the insulating material (insulation layer52) may be adjusted by the center-to-center distance p2and the circle equivalent diameter of the metal line, for example. To increase the percentage of the surface area of the insulating material (insulation layer52), it is preferable to set the circle equivalent diameter of the metal line to a small value, and set center-to-center distance p2to a large value (within a range smaller than center-to-center distance p1), for example. In addition, to set the rockwell hardness of the surface of second anisotropic conductive sheet50to M120 or smaller, it is preferable to 2) set the protruding height of the metal line from the surface of second anisotropic conductive sheet50to a small value, for example, than that of first anisotropic conductive sheet40, in the case where conduction path51is a metal line. To be more specific, it is preferable that conduction path51do not protrude from the surface of second anisotropic conductive sheet50. The reason for this is that conduction path51protruded from the surface of second anisotropic conductive sheet50likely to affect the rockwell hardness. It is preferable that the thickness of second insulation layer52of second anisotropic conductive sheet50be smaller than the thickness of first insulation layer42of first anisotropic conductive sheet40. That is, normally, second anisotropic conductive sheet50has a larger conduction resistance value than that of first anisotropic conductive sheet40. Therefore, when the ratio of the thickness of second anisotropic conductive sheet50is suitably small, the conduction resistance value of the entire anisotropic conductive composite sheet30does not excessively increase, and thus the inspection accuracy is less impaired. Preferably, the thickness of second insulation layer52of second anisotropic conductive sheet50is 20 to 100 μm, for example, from the viewpoint of not excessively increasing the conduction resistance value of the entire anisotropic conductive composite sheet30and the like. The thicknesses of second insulation layer52and first insulation layer42may be measured by the same method as the above-described method. The ratio T2/T1of thickness T2of second insulation layer52of second anisotropic conductive sheet50and thickness T1of first insulation layer42of first anisotropic conductive sheet40may be set to approximately 1/4 to 1/10. Layer Configuration Second anisotropic conductive sheet50may be stacked only on one surface of first anisotropic conductive sheet40, or on both surfaces. In the present embodiment, second anisotropic conductive sheet50is stacked on one surface (the side that makes contact with the terminal of the inspection object) of first anisotropic conductive sheet40. The inspection object is disposed on the surface (the surface opposite to the side of first anisotropic conductive sheet40) of second anisotropic conductive sheet50. Operation With anisotropic conductive composite sheet30according to the present embodiment, the following effect can be provided in addition to the effect described in the above-mentioned Embodiment 1. Specifically, a known common anisotropic conductive sheet has a favorable conductivity in the thickness direction, but the metal pin (referred to as a metal line) that is a conduction path is exposed or protruded at the surface of anisotropic conductive sheet. As such, when aligning the terminal of the inspection object and performing electrical testing on the anisotropic conductive sheet, the terminal of the inspection object is easily damaged by making contact with the exposed or protruded metal line. In addition, as the wiring density of inspection objects becomes higher, i.e., the pitch between terminals becomes finer, it is difficult to align (alignment) the terminals of inspection objects with high accuracy. In contrast, in the present embodiment, anisotropic conductive sheet10according to Embodiment 1 (second anisotropic conductive sheet50) is provided on the known anisotropic conductive sheet (first anisotropic conductive sheet40) for example. As described above, anisotropic conductive sheet10according to Embodiment 1 can easily provide the elasticity of expanding and contracting in the thickness direction, and can make the terminal of the inspection object less susceptible to damage, and it is thus possible to suppress the damage of the inspection object due to the contact with the metal line. In addition, by setting a small center-to-center distance p2of first end portion11aof conduction path11of anisotropic conductive sheet10according to Embodiment 1 (second anisotropic conductive sheet50), it is possible to eliminate the need for the positioning (alignment). Manufacturing Method of Anisotropic Conductive Composite Sheet Anisotropic conductive composite sheet30of the present disclosure can be manufactured by any method. For example, anisotropic conductive composite sheet30can be obtained through: 1) a step of preparing first anisotropic conductive sheet40and second anisotropic conductive sheet50; and 2) a step of stacking first anisotropic conductive sheet40and second anisotropic conductive sheet50and then integrating them by thermo-compression bonding and the like. Step 1) First anisotropic conductive sheet40can be manufactured by any method. For example, first anisotropic conductive sheet40as illustrated inFIGS.11A and11Bcan be obtained by obtaining a laminated body in which a layer in which a plurality of long metal lines is arranged at a predetermined pitch such that they do not make contact with each other (a layer composed of a metal line) and an insulating sheet are alternately stacked, and then cutting the obtained laminated body in the direction parallel to the lamination direction (or in the direction perpendicular to the metal line) in a predetermined thickness. In the case where conduction path51of second anisotropic conductive sheet50is a metal line (seeFIG.11B), it can be manufactured by the same method as that described above. Step 2) First anisotropic conductive sheet40and second anisotropic conductive sheet50can be integrated with each other by any method such as thermo-compression bonding, for example. It is to be noted that while first anisotropic conductive sheet40and second anisotropic conductive sheet50are integrated with each other (composite sheet) inFIGS.11A and11B, this is not limitative, and first anisotropic conductive sheet40and second anisotropic conductive sheet50may not be integrated with each other, and may be stacked when they are used. Anisotropic Conductive Sheet Set FIG.12is a sectional view illustrating anisotropic conductive sheet set60according to the present embodiment. Anisotropic conductive sheet set60includes first anisotropic conductive sheet40, and second anisotropic conductive sheet50configured to be stacked on at least one surface of it. To be more specific, anisotropic conductive sheet set60is used with third surface42aof first insulation layer42of anisotropic conductive sheet40and sixth surface52bof second insulation layer52of second anisotropic conductive sheet50are stacked to face each other (seeFIG.12). First anisotropic conductive sheet40and second anisotropic conductive sheet50are configured in the same manner as the above-described first anisotropic conductive sheet40and second anisotropic conductive sheet50, respectively. From the viewpoint of reducing the interface resistance between first anisotropic conductive sheet40and second anisotropic conductive sheet50and the like, the surface of second anisotropic conductive sheet50that makes contact with first anisotropic conductive sheet40may be provided with a surface shape (e.g., convex or concave) that mates with the surface shape of first anisotropic conductive sheet40. In addition, as described above, it is preferable that an inspection object is disposed at the surface of second anisotropic conductive sheet50(the surface on the side opposite to first anisotropic conductive sheet40). As described above, since first anisotropic conductive sheet40and second anisotropic conductive sheet50are not integrated with each other, the configuration of the anisotropic conductive sheet can be freely changed in accordance with the type of the inspection object. In addition, even when they are not integrated with each other, electrical connection can be sufficiently performed by exerting a pressure at the time of contact with the terminal of the inspection object. As described above, anisotropic conductive composite sheet30and anisotropic conductive sheet set60can be favorably used for electrical testing of inspection objects such as semiconductor devices. Electrical Testing Apparatus and Electrical Testing Method FIG.13is a sectional view illustrating electrical testing apparatus100according to the present embodiment. As illustrated inFIG.13, electrical testing apparatus100has the same configuration as that of the above-mentioned Embodiment 1 except that anisotropic conductive composite sheet30or a laminated of anisotropic conductive sheet set60is disposed on the surface of inspection substrate120on which electrode121is disposed such that the electrode121and metal line11make contact with each other. Then, the surface of second anisotropic conductive sheet20that constitutes anisotropic conductive composite sheet30or the laminated of anisotropic conductive sheet set60is disposed in contact with the terminal of inspection object130. Then, in the present embodiment, the rockwell hardness of the surface of second anisotropic conductive sheet50that makes contact with terminal131of inspection object130is, preferably, as low as M120 to have a suitable flexibility. In this manner, even when it is pressed with terminal131of inspection object130put thereon, damage of the terminal131of inspection object130due to second anisotropic conductive sheet50can be suppressed. In addition, second center-to-center distance p2of fifth end portions51aof the plurality of conduction paths51of anisotropic conductive sheet50is significantly smaller than that of first anisotropic conductive sheet40, it is possible to eliminate the need for the positioning (alignment) of terminal131of inspection object130. Modification While second anisotropic conductive sheet50is anisotropic conductive sheet10according to Embodiment 1 in the present embodiment, this is not limitative, and first anisotropic conductive sheet40may be anisotropic conductive sheet10according to Embodiment 1. FIG.14Ais a sectional view illustrating anisotropic conductive composite sheet30according to a modification, andFIG.14Bis a sectional view illustrating anisotropic conductive sheet set60according to a modification.FIG.15is a sectional view illustrating anisotropic conductive composite sheet30according to the modification.FIG.15Aillustrates a case where second anisotropic conductive sheet50is a dispersion type anisotropic conductive sheet inFIG.14A,FIG.15Bis an enlarged view of broken line part15B ofFIG.15A, andFIG.15Cillustrates a case where second anisotropic conductive sheet50is an anisotropic conductive sheet of an uneven type inFIG.14A. As described above, first anisotropic conductive sheet40may be anisotropic conductive sheet10according to Embodiment 1 (seeFIGS.14A and14B, andFIGS.15A to15C). In addition, conduction path51of second anisotropic conductive sheet50may be composed of a plurality of conductive particles dispersed in an insulating material and configured to form a conduction path in the thickness direction in a pressed state or non-pressed state (seeFIGS.15A to15C). To be more specific, as illustrated inFIGS.15A and15B, second anisotropic conductive sheet50may include an insulating material, and a plurality of conductive particles dispersed therein. From the viewpoint of obtaining sufficient continuity without the need for alignment, it is preferable that the plurality of conductive particles be aligned in the thickness direction of the sheet. The number of conductive particles aligned in the thickness direction may be one or more. The conductive particles aligned in the thickness direction may be dispersed in the entirety of the sheet (dispersed type, seeFIGS.15A and15B), or regularly unevenly distributed (uneven type, seeFIG.15C). It suffices that conduction path51of second anisotropic conductive sheet50is a member that conducts in the thickness direction in a non-pressed state or a pressed state (seeFIGS.15A to15C). From the viewpoint of eliminating the need for alignment, the volume resistivity with conduction of conduction path51satisfies the above-described range, or preferably 1.0×10×10−4to 1.0×10×10−5Ω-cm. Second anisotropic conductive sheet50includes a plurality of conductive particles P aligned in the thickness direction as conduction path51(seeFIGS.15A to15C). The plurality of conductive particles P aligned in the thickness direction is dispersed over the entire sheet. Preferably, conductive particles P are, but not limited thereto, conductive magnetic substance particles from the viewpoint of alignment in the thickness direction and the like, for example. Examples of conductive magnetic particles include particles made of magnetic metals such as iron, nickel, and cobalt or their alloys, or those plated with conductive metals such as gold, silver, copper, tin, palladium, and rhodium. The median diameter (d50) of the conductive particles P is, but not limited thereto, 5 to 100 μm, preferably 10 to 50 μm, for example. The median diameter of the conductive particles P can be measured by a light scattering method, for example, a laser analysis and scattering particle size analyzer. In the case where conduction path51is composed of the plurality of conductive particles P aligned in the thickness direction, center-to-center distance p2of the plurality of conduction paths51is specified as follows. Specifically, in the case of the dispersion type illustrated inFIGS.15A and15B, the distance between the center lines (lines each connecting the centers of the plurality of conductive particles P aligned in the thickness direction) of the plurality of conductive particles P aligned in the thickness direction is set as center-to-center distance p2of the plurality of conduction paths51(seeFIG.15B). On the other hand, in the case of the uneven type illustrated inFIG.15C, the uneven part is regarded as one conduction path51, and the distance between each center line of the uneven part is set as center-to-center distance p2of the plurality of conduction paths51(seeFIG.15C). Second anisotropic conductive sheet50in which conduction path51is composed of conductive particles P (seeFIGS.15A to15C) can be manufactured in the following procedure.i) First, a conductive elastomer composition including an insulating material and magnetic conductive particles P is prepared. Then, a conductive elastomer composition layer is formed by applying the conductive elastomer composition on a mold-releasing support plate.ii) Next, a magnetic field is applied in the thickness direction of the conductive elastomer composition layer, and thus conductive particles P dispersed in the conductive elastomer composition layer are aligned in the thickness direction. Then, while continuously applying the magnetic field to conductive elastomer composition layer, or after the application of the magnetic field is stopped, the conductive elastomer composition layer is cured to obtain a conductive elastomer layer in which conductive particles P are aligned in the thickness direction.iii) Then, second anisotropic conductive sheet50composed of the conductive elastomer layer is obtained by peeling off the mold-releasing support plate. As the mold-releasing support plate used in the step i), a metal plate, a ceramic plate, a resin plate and their combined materials and the like may be used. The application of the conductive elastomer composition can be performed by a printing method as screen printing, a roll application method, or a blade application method. The thickness of conductive elastomer composition layer is set in accordance with the length of the conduction path to be formed. The means for applying a magnetic field to conductive elastomer composition layer may be an electromagnet, a permanent magnet and the like. Preferably, in the step of ii), the strength of the magnetic field applied to the conductive elastomer composition layer is 0.2 to 2.5 Tesla. Normally, the conductive elastomer composition layer is cured through a heat treatment. The specific heating temperature and heating time are appropriately set in consideration of the type of the elastomer composition that constitutes the conductive elastomer composition layer, the time required for the movement of conductive particles P and the like. 3. Other Notes It is to be noted that while each anisotropic conductive sheet including the conduction path including non-linear part11cis used in the above-mentioned embodiments, an anisotropic conductive sheet including a conduction path that does not include non-linear part11cmay be used in accordance with the purpose (seeFIGS.16and17). FIGS.16A to16Cillustrate an anisotropic conductive sheet according to a modification.FIG.16Ais a perspective view,FIG.16Bis a partially enlarged view of a horizontal cross section, andFIG.16Cis a partially enlarged view of a vertical cross section. For example, peeling between conduction path11and insulation layer12tends to occur at conduction path11that does not include non-linear part11c, and therefore bonding layer14may be disposed between insulation layer12and conduction path11that does not include non-linear part11c(seeFIGS.16A to16C). FIG.17Ais a partially enlarged sectional view illustrating an anisotropic conductive composite sheet according to a modification, andFIG.17Bis a partially enlarged sectional view illustrating an anisotropic conductive sheet set according to a modification. For example, an anisotropic conductive sheet in which conduction path11does not include non-linear part11cis less deflected in the thickness direction, and may damage the terminal of the inspection object, but may be used as an anisotropic conductive composite sheet and an anisotropic conductive sheet set (seeFIGS.17A and17B). In addition, while electrical testing is performed by pressing inspection object130to inspection substrate120where anisotropic conductive sheet10is disposed in the present embodiment, this is not limitative, and electrical testing may be performed by pressing inspection substrate120where anisotropic conductive sheet10is disposed to inspection object130. In addition, while the anisotropic conductive sheet is used for electrical testing in the present embodiment, this is not limitative, and it may be used for an electrical connection between two electronic members, such as an electrical connection between a glass substrate and a flexible printed board and an electrical connection between a substrate and an electronic component mounted on it. This application is entitled to and claims the benefit of Japanese Patent Application No. 2018-218281 filed on Nov. 21, 2018, Japanese Patent Application No. 2019-54538 filed on Mar. 22, 2019, and Japanese Patent Application No. 2019-98814 filed on May 27, 2019 the disclosure each of which including the specification, drawings and abstract is incorporated herein by reference in its entirety. INDUSTRIAL APPLICABILITY According to the present disclosure, an anisotropic conductive sheet, an anisotropic conductive composite sheet, an anisotropic conductive sheet set, an electrical testing apparatus and an electrical testing method that can suppress damage of inspection objects can be provided. REFERENCE SIGNS LIST 10Anisotropic conductive sheet11,41,51Conduction path11aFirst end portion11bSecond end portion11cNon-linear part12,42,52Insulation layer12aFirst surface12bSecond surface13Electrolyte layer14,24Bonding layer20Composite sheet21Insulating sheet22Conduction line23Laminated body30Anisotropic conductive composite sheet40First anisotropic conductive sheet41aThird end portion41bFourth end portion42aThird surface42bFourth surface50Second anisotropic conductive sheet51aFifth end portion51bSixth end portion52aFifth surface52bSixth surface60Anisotropic conductive sheet set100Electrical testing apparatus110Holding container120Inspection substrate121Electrode130Inspection object131Terminal (of inspection object) | 73,266 |
11860194 | SUMMARY OF THE INVENTION This Summary is provided to introduce a brief selection of disclosed concepts in a simplified form that are further described below in the Detailed Description including the drawings provided. This Summary is not intended to limit the claimed subject matter's scope. One embodiment is a device, which comprises a meter portion having one or more blades, the blades configured to reside within both a premises-side opening and a utility-side opening within a socket, a supply unit connected to the meter portion, a metering unit connected to the supply unit and configured to measure an amount of electricity that passes through the meter portion, and a sensor connected to the meter portion, wherein when the meter portion is placed in the socket, the sensor collects data associated with the placing of the meter portion in the socket. Another embodiment is a method comprising enabling a computing module and a storage module within a meter portion, receiving placement of the meter portion into a socket, the meter portion having one or more blades, the blades configured to reside within both a premises-side opening and a utility-side opening within the socket, using a sensor connected to the meter portion to collect data associated with the placing of the meter portion in the socket, and storing the data in the storage module. In another embodiment, a method includes providing a procedure for a technician to place a meter portion into a socket, the meter portion having one or more blades, the blades configured to reside within both a premises-side opening and a utility-side opening within the socket, using a sensor connected to the meter portion to collect data associated with the placing of the meter portion in the socket, sending the data to a head-end system, analyzing the data at the head-end system, and sending an alert to the technician, based on the data sent to the head-end system. DETAILED DESCRIPTION OF THE INVENTION Disclosed aspects are described with reference to the attached FIGs, wherein like reference numerals are used throughout the FIGs to designate similar or equivalent elements. The FIGs are not drawn to scale and they are provided merely to illustrate certain disclosed aspects. Several disclosed aspects are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the disclosed aspects. FIG.1is a block diagram of a device140that includes an electric meter102having a meter shell103. An accelerometer195can be positioned on a PCB of the meter portion106, where the electric meter102can be configured to implement a socket protection module199at a premise. As shown, the electric meter102comprises a meter portion106generally implemented on the PCB including measurement circuitry104including a voltage measurement circuit104A and a current measurement circuit104B for measuring the amount of electricity that is being consumed. The electric meter102also includes a meter processor105such as a microprocessor or other computing device. The meter processor105has an associated memory112which can be accessed to run algorithms stored in the memory112and can be used to store functions for a controller to control the overall operation of the electric meter102. The memory112is generally also for storing program instructions. Although not shown, a display is generally also included for displaying at least the meter data, status information, an alert, and/or information associated with the current installation. As will be further described below, a display module196in the socket protection module199can be used to this end, in one example. The electric meter102besides the meter portion106comprises a communications unit108that can be a wireless communications unit, generally comprising a transceiver that can be coupled to an antenna, for wirelessly transmitting and receiving data to/from other electric meters that may be equivalent to electric meter102, and/or to a head-end system130. The wireless communication unit108may comprise, for example, a CC1110Fx/CC1111fX semiconductor device available from Texas Instruments Incorporated® which comprises a Low-Power SoC (System-on-Chip) with MCU, memory, Sub-1 GHz RF Transceiver, and a USB controller. Within the utility box100is also a meter socket114which is coupled between the other components of the electric meter102and the power lines shown as190. The power lines190electrically connect to the meter socket114to supply power to a premise from the utility company. The electrical power received from the power lines190may be routed through the meter socket114to allow the blades of the electric meter102(see blades303inFIG.2described below) to be positioned in the openings of the meter socket114to monitor the power levels consumed within the premises. The blades are generally sized to be positioned within openings of the meter socket114. Positioning the blades of the electric meter102within the meter socket114electrically connects the electric meter102to the meter socket114. The meter socket114may include springs or other features to provide a tension force on the blades to maintain the position of the blades within the opening of the meter socket114. The meter socket114and blades may each include one or more surfaces made out of an electrically conductive material to allow electricity to flow between the meter socket114and the blades. The measurement circuitry104using its voltage sense circuit104A and current sense circuit104B measures the power consumed by the premises from the electrical signals supplied to the premises through the power lines190. The voltage sense circuit104A may be connected to electric load terminals in the electric meter102, which are connected to the meter socket114using the blades of the electric meter102. The voltage sense circuit104A generally includes amplifiers, resistors, or other electrical devices to generate a voltage sense signal corresponding to an instantaneous voltage from the power lines190. The current sense circuit104B may include, for example transformers, inductors, or other coils connected to the meter socket114via the blades of the meter system102to generate a current sense signal corresponding to an instantaneous current flowing from the power lines190through the meter socket114. Voltage sense signals and current sense signals generated by the voltage sense circuit104A and the current sense circuit104B can be routed to the meter processor105for monitoring the signals and determining the power consumed by the premises. Based on one or more of the voltage sense signals and current sense signals, measurements may be generated by the meter processor105. The meter processor105may comprise one or more processors communicatively coupled to the memory device112. The meter processor105is configured to execute instructions stored by the memory device112, and for example includes a logical processing unit, a microprocessor, a digital signal processor, or another processing for processing the signals received from the voltage sense circuit104A and the current sense circuit104B. The memory device112device can include includes volatile or non-volatile, random-access memory (RAM), electrically erasable programmable read-only memory (EEPROM), or other readable and writeable memory devices. For example, the memory device112may include a non-volatile memory that stores data representing the measurements received from the voltage sense circuit104A and the current sense circuit104B. The meter portion106also optionally includes disconnect circuitry122which is a conventional feature provided by most commercially available smart electric meters. During operation, a system, such as a central system or head-end130, responsive to the alert signal sent by the electric meter will send a signal to the electric meter commanding the disconnect circuitry122to implement a disconnection. In some aspects, the disconnect circuitry122includes a switching device or other circuitry for disconnecting the power supplied from the power lines190to the premises through the meter socket114. Disconnecting the power may include interrupting electrical signals that are transmitted between the meter socket114and the electric meter102. The disconnect circuitry122may also include an actuator126coupled to the switching device. The actuator126may cause the switching device of the disconnect circuitry122to transition from a first position that allows power to flow into the premises to a second position that prevents the power from flowing into the premises, and from the second position to the first position. The actuator126may be communicatively coupled to the meter processor105, which can transmit control signals to the disconnect circuitry122to allow the meter processor105to operate the switching device. The communications unit108through its associated antenna107is shown communicatively coupled to a central system130, such as a central system associated with an operator of the power utility. In some disclosed aspects, the communication unit108may transmit a signal associated with the utility box100. For example, one aspect uses a socket protection module199during an installation procedure. In this case, a technician will remove an existing meter portion and swap it with a new one. This could be in the case of replacing an older meter with a smart meter, a new installation, a new meter portion where the old one experienced a hot socket, and the like. When a technician performs one of the procedures described above, the socket protection module199operates to ensure that the installation procedure is compliant with a process that ensures a proper installation (e.g., proper clamping force from the jaws on the blades). Thus, the socket protection module199operates to ensure that the installation is less likely to result in a hot socket in the future, and hence, is least likely to cause catastrophic damage. To this end, the socket protection module199has a wake-up module194, which could be coupled to a physical button on the meter, or other suitable input mechanism, to indicate to the meter that the installation process is beginning, and it should enter a more active mode of processing that can include activating one or more processing units and/or memory areas, such as meter processor105and memory112. The wake-up module194can be powered by a battery or capacitor, for example. The socket protection module199can also include a display module196for preparing information for the technician, so it can be displayed on a display to assist the technician in achieving a compliant installation. When the meter portion106is being installed, the accelerometer199can collect data, for example including the acceleration of the meter portion106into the socket114, the deceleration of the meter portion106into the socket114, and/or the amount of force the technician is using to push the meter portion106into the socket114. The meter processor105will process the data and store it in the memory112. In another example, the blades are formed in a plurality of rows and the technician performs a “rocking” motion with the meter portion106. By using a rocking motion, a first row of blades is pushed in before a second row of blades. In this manner, the data can include an installation profile that includes the data from the accelerometer195each time the technician performed the rocking motion and pushed in a row of blades. An analysis module192is included in the socket protection module199. The analysis module192connects to the head-end system130via the communication unit108. The analysis module192is capable of providing the data from the accelerometer195to the head-end system130, from the installation of the meter portion106. The head-end system130can use the data from the analysis module192to gather data from a plurality of installations at a plurality of meter. The head-end system130is capable of using artificial intelligence (AI) to better understand the parameters that lead to less likelihood of a hot socket. The head-end system130, in turn, can use the data to revise the procedures, that can later be used by the display module196to update a technician with better information and a safer procedure. Alternatively, an alert can be sent from the head-end system130to the display module196, to prepare an alert for the display of the meter, in order to alert the operator that there is a problem with the installation. In the case where the electric meter102comprises a smart meter, this enables two-way communication between the electric meter102and the head-end system130. Communications from the electric meter to the network as noted above may be wireless, or via a fixed, wired connections. Wireless communication options include cellular communications, Wi-Fi, wireless ad hoc networks over Wi-Fi, wireless mesh networks, low power long-range wireless (LoRa), ZigBee (low power, low data rate wireless), and Wi-SUN (Smart Utility Networks). FIG.2shows an electric meter302having blades303and399configured for insertion into openings shown as utility-side openings305A and premises-side openings305B of a meter socket314.FIG.3shows the electric meter302installed on a wall380A of a premise380. The utility box is shown as300. The openings305A and305B are sized and configured so that the blades303(not visible inFIG.3) may be positioned therein. The electric meter302may be fitted into the meter socket314, as indicated by the dotted lines shown inFIG.2, such that the blades303(visible inFIG.2) are positioned in the openings305A,305B. Positioning the blades303within the openings305A,305B electrically connects the electrical meter302to the meter socket314. In one aspect a first row of blades, shown as the bottom row ofFIG.2, which includes a blade399, is positioned below the second row of blades303. In one installation procedure, blade399is positioned into the socket314alternatively from blades303in a rocking motion, wherein each row of blades is pushed into the socket314individually until all the rows are completely inserted. The blades303and399and the utility-side openings305A can be configured such that electrical signals are transmitted between a utility-side of the meter socket314and the electrical meter302, and the premises-side openings305B are configured such that electrical signals are coupled between the electric meter302and a premises-side of the meter socket314. The electric meter302can be configured such that disconnect circuitry (such as disconnect circuitry122described above) allows the electrical signals to be transmitted between the utility-side and the premises-side. For example, the disconnect circuitry122may be configured such that triggering the actuator126may interrupt the transmission of the electrical signals between the utility-side and the premises-side by interrupting the transmission of electrical signals between the blades303on the utility-side and the blades303on the premises-side of the electric meter302. FIG.4is a flowchart showing an implementation of the socket-jaw protection module according to one embodiment. At step400, a processor and memory are awakened after a device receives a wake-up command. This can occur, for example, during an installation procedure of an electric meter. The meter can be awakened using a battery, a super-capacitor, or other internal power source. At step410, a meter portion is placed into a socket, for example by a technician, and the meter portion is received into the socket by the device. In one embodiment, there are a plurality of rows of blades and the technician performs a rocking motion, wherein each of the rows of blades is pushed in at different times, although this is not required. There may also be a checklist the technician uses and/or an installation profile for the meter which the technician uses. At step420, sensor data is collected during the placement of the meter body. This includes, for example, an accelerometer and the readings it takes (3-axis data, for example) as the meter is installed into the socket. At step,430the data is stored in a memory. FIG.5is a flowchart showing an implementation of the socket-jaw protection module according to another embodiment. At step500, it is determined whether a wake-up command has been received. For example, there may a dedicated button or buttons on the meter, or a combination of button actions that could be used to wake up the meter. The device waits until the command is received and wakes up a CPU and memory in response at step510. A battery or super-capacitor can be used, for example, and the memory can be a form of resilient storage capable of operating without the power supply. At step520, the system determines whether a meter installation is occurring. If not, the process ends. If so, the at step530, a sensor collects one or more readings associated with the installation. In one example, this includes the acceleration of the meter into the socket, the deceleration of the meter into the socket, and/or an amount of force the technician is using to push the meter into the socket. Other sensor readings can be used as well, and in one example one or more 3-axis accelerometers are used. At step540, information associated with the installation is displayed to the technician. This can include the sensor readings themselves, information about whether the sensor readings indicate a compliant installation procedure that will minimize the chance of a hot socket, warning signals, information associated with an installation profile and whether the installation matches the profile, and others. In another example, a socket jaw protection module (such as the socket-jaw protection module199ofFIG.1) can use a display module and/or a coupling to a head-end to determine the information to display at step540. At step550, the system determines whether there is an error in the install. For example, the acceleration of the meter body into the socket might be too high, or the force might be too low, or the procedure didn't follow the installation profile. If not, the process ends, and the meter is successfully installed. If there is an error, the display is updated at step560, based at least in part on the sensor readings, to indicate to the technician that the installation was erroneous. FIG.6is a flowchart showing an implementation of the socket-jaw protection module according to another embodiment. At step600, it is determined whether a wake-up command has been received. For example, there may a dedicated button or buttons on the meter, or a combination of button actions that could be used to wake up the meter. The device waits until the command is received and wakes up a CPU and memory in response, as well as providing an installation procedure at step610. In one example, the installation procedure can include a checklist for the technician. In another example, an installation profile is loaded into the memory and is used in real-time during this process. At step620, the system determines whether a meter installation is occurring. If not, the process ends. If so, the at step630, a sensor collects one or more readings associated with the installation, for example with an accelerometer. At step640, the data is sent to a head-end system. This can include, for example, sending a plurality of 3-axis data from one or more accelerometers to the head-end. The head-end uses this data at step650. For example, the head-end can apply one or more algorithms to the data in order to perform an analysis. In another example, mathematical, statistical, and/or analytical modeling is used on the data and the head-end can estimate an amount of insertion force that was used during the installation. In another example, an artificial intelligence (AI) process can be used by the head-end. The AI process can train, classify, and/or otherwise use the data and additional data from other installations, for example, to tailor a response, if needed, that uses more information than is available merely from the sensor of the current meter. At step660, the head-end determines whether an error has occurred in the installation (e.g., one that may increase the risk of a hot socket). If not, the head-end returns information to the display at step670. The information can indicate, for example, the amount of insertion force used during the installation. Otherwise, at step680, an alert is provided on the display. This can be used, for example, to indicate to the technician that the error occurred. After step670or680the process ends. Although the subject matter has been described in language specific to structural features and/or methodological acts, it is to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described above. Rather, the specific features and acts described above are disclosed as example forms of implementing the claims. | 21,150 |
11860195 | DETAILED DESCRIPTION This application discloses a non-contact electric potential meter system suitable for obtaining a determination of a voltage between a reference potential and an energized or “hot” conductor of an alternating current electrical circuit, without direct electrical contact to the first hot conductor and without reference to any other AC voltage signal. The technical advancements of the non-contact voltage sensing apparatus disclosed herein allow it to overcome the limitations of prior power meters and offer significant advantages over prior art electric voltage measuring devices. For example, the disclosed voltage sensing apparatus uses a non-contact capacitive coupling system and technique to measure the voltage signal in an AC conductor. This allows accurate voltage sensing—measurement of a properly shaped and scaled waveform, not just peak detection—without requiring physical contact to the hot conductor wire. The disclosed technology obtains an AC waveform in a target energized conductor via capacitive coupling between the conductor and a sense plate that is situated near the conductor. An electronic circuit samples a waveform representing a filtered version of the AC voltage between the energized conductor and a reference potential. Another circuit determines the coupling capacitance between the conductor and the sense plate. Together, these allow analog or digital signal processing circuitry to recover the shape or frequency spectrum of the line voltage and to correctly scale the recovered waveform. Thus, the non-contact voltage sensing apparatus disclosed herein accurately determines the AC line voltage. In addition, the disclosed technology allows a split-core current transformer (CT) to provide current measurements of the target conductor, and provides a multiplexing circuit to repurpose the current transformer (when it is not actively measuring current) as an energy harvester to supply runtime power for the non-contact voltage sensing apparatus. In some embodiments, a CT functions as a current sensor and energy harvester without multiplexing. For example, by estimation or calculation of energy harvester output voltage, current, or power, both energy harvesting and current sensing functions may be operated at the same time. Harvesting energy to power the non-contact voltage sensing apparatus and simultaneously measuring the current flowing in the target conductor can increase energy efficiency. For another example, components (such as energy harvesting electronic circuitry) may be turned off when current sensing circuitry is active. Reference is now made in detail to the description of the embodiments as illustrated in the drawings. While embodiments are described in connection with the drawings and related descriptions, there is no intent to limit the scope to the embodiments disclosed herein. On the contrary, the intent is to cover all alternatives, modifications, and equivalents. In alternate embodiments, additional sensing devices, or combinations of illustrated devices, may be added to, or combined, without limiting the scope to the embodiments disclosed herein. Each of the Figures discussed below may include many more or fewer components than those shown and described. Moreover, not all of the described components may be required to practice various embodiments, and variations in the arrangements and types of the components may be made. However, the components shown are sufficient to disclose various illustrative embodiments for practicing the disclosed technology. The embodiments set forth below are primarily described in the context of measuring electric circuits such as residential, commercial, industrial, or utility-level wiring (including, e.g., power transmission and distribution networks). However, the embodiments described herein are illustrative examples and in no way limit the disclosed technology to any particular size, construction, or application of conductor. The phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment. The terms “comprising,” “having,” and “including” are synonymous, unless the context dictates otherwise. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. For example, “a scaling factor” generally includes multiple scaling factors. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. The terms “sense”/“sensing,” “meter”/“metering,” “detect”/“detecting,” and “measure”/“measuring” are generally synonymous, unless the context dictates otherwise. For example, “detecting” an AC waveform generally refers to obtaining measurement of a continuously varying alternating current, and not only obtaining a Boolean value representing whether an AC waveform is present. A “detector” likewise should be interpreted as a device to obtain measurements and not only to detect presence. The terms “electric” and “electrical” are generally synonymous. The terms “voltage” and “potential” or “electric(al) potential” are also generally synonymous. Similarly, the terms “amperage” and “current” or “electric(al) current” are generally synonymous. Thus, the terms “voltage sensing apparatus” and “electric potential meter system” are used synonymously. The disclosed non-contact voltage sensing apparatus can take a variety of form factors.FIGS.1through12illustrate a variety of different arrangements, designs, and subsystem possibilities. The illustrated sensing systems and methods are not an exhaustive list; in other embodiments, a measurement region for receiving a conductor or circuitry for powering and/or controlling a non-contact voltage sensing apparatus could be formed in different arrangements. However, it is not necessary to exhaustively show such optional implementation details to describe illustrative embodiments. FIG.1is a block diagram illustrating operational components of an example non-contact voltage sensing apparatus100in accordance with one embodiment. The apparatus100typically includes a housing110. The apparatus100includes a measurement region120that is configured to receive a conductor, e.g., a “hot” AC conductor (or “line”) such as an insulated copper wire. The non-contact voltage sensing apparatus100is configured so that when the measurement region120receives the conductor, the conductor is not interrupted. In various embodiments, the measurement region120in operation receives a single conductor, and is configured to exclude other conductors (including, e.g., any additional “hot” wire). In various embodiments, the housing110provides, defines, or indicates the measurement region120. In various embodiments, the measurement region120is configured such that the conductor passes along, into, or through the measurement region120. Thus, the measurement region120may be arranged inside and/or outside the housing110. The non-contact voltage sensing apparatus100further includes a shield125. The shield125may be constructed of material having high conductivity, such as a metallic foil or mesh. In various embodiments, the shield125may form a Faraday cage around a portion or all of the components of the non-contact voltage sensing apparatus100, such as around the measurement circuitry and conductive sense plate described below with respect to thisFIG.1. In some embodiments, the shield125may extend only partly around a portion or all of the components of the non-contact voltage sensing apparatus100, and/or may include one or more apertures, e.g., to accommodate a conductor in the measurement region120. In some embodiments, the shield125does not surround the measurement region120. In some embodiments, the shield125is connected to a ground potential. The illustrated non-contact voltage sensing apparatus100includes a capacitive AC voltage sensing mechanism140. The voltage sensing mechanism140includes components configured to obtain a determination of an AC voltage in a conductor in the measurement region120, such as by sensing an AC line voltage waveform, determining a scaling factor based on coupling capacitance, and processing the waveform and the scaling factor as further described in this disclosure. The capacitive AC voltage sensing mechanism140includes a conductive sense plate130configured so that the conductive sense plate130forms a capacitive coupling with a conductor in the measurement region120. The non-contact voltage sensing apparatus100further includes an electrical connection to a reference potential145, used in determining a voltage between the conductor and the reference potential. The electrical connection to a reference potential145may include a resistive or capacitive connection to a ground potential. In various embodiments, the non-contact voltage sensing apparatus100is configured so that the conductive sense plate130has a determined or otherwise determinable geometric relationship to the conductor when the measurement region120receives the conductor. The geometric relationship between the conductor and the conductive sense plate130, and, e.g., the size or other physical characteristics of the conductor (such as a wire gauge and/or insulation jacket thickness) may be known, fixed, or pre-set; or they may be measurable or similarly determinable. In some embodiments, the non-contact voltage sensing apparatus100includes conductor measurement means131to fix and/or measure one or more aspects of the conductor at the measurement region120. An example conductor measurement means131and digital caliper electronic circuitry133is described in further detail with reference toFIG.11below. In some embodiments, the non-contact voltage sensing apparatus100includes a humidity sensor135, so that the apparatus100can identify and compensate for changes in the dielectric constant of air associated with changes in humidity. The capacitive AC voltage sensing mechanism140includes waveform-sensing electronic circuitry142configured to sense an AC line voltage waveform. The waveform-sensing electronic circuitry142may obtain the AC waveform by measuring a current induced via capacitive coupling between the sense plate130and an energized conductor in the measurement region120. The waveform-sensing electronic circuitry142may include an operational amplifier (“op-amp”) circuit, such as described in further detail with reference toFIG.7below. The capacitive AC voltage sensing mechanism140further includes capacitance-determining electronic circuitry144(also referred to herein as coupling capacitance tracking electronic circuitry) configured to sense a coupling capacitance between the conductive sense plate130and the conductor in the measurement region120. In some embodiments, the capacitance-determining electronic circuitry144includes elements having an output frequency that depends in part on the coupling capacitance, such as described in further detail with reference toFIG.8below. The capacitive AC voltage sensing mechanism140further includes signal processing electronic circuitry146. The signal processing electronic circuitry146processes the AC line voltage waveform representation obtained by the waveform-sensing electronic circuitry142to recover a shape or frequency spectrum of the line voltage waveform. The signal processing electronic circuitry146may process the coupling capacitance determination obtained by the capacitance-determining electronic circuitry144to obtain a scaling factor. The signal processing electronic circuitry146may also use the determination of one or more physical characteristics of the conductor obtained by the conductor measurement means131(e.g., via the digital caliper electronic circuitry133) to obtain a scaling factor. The signal processing electronic circuitry146scales the recovered shape or frequency spectrum of the line voltage waveform according to the scaling factor(s) to obtain an accurate measurement of the true AC line voltage in the conductor in the measurement region120. In various embodiments, the signal processing electronic circuitry146may include analog and/or digital signal processing components. In some embodiments, the non-contact voltage sensing apparatus100includes a current sensing mechanism150. The current sensing mechanism150may include a current transformer (“CT”)151, a current sensor152, and current processing electronic circuitry155. The current sensor152may be a split-core current transformer, a solid-core current transformer, a Rogowski coil, an anisotropic magnetoresistance (AMR) sensor, a giant magnetoresistance (GMR) sensor, a Hall effect sensor, a current-sensing resistor, an inductor, etc. In some embodiments, the signal processing electronic circuitry146can process and time-synchronize a current waveform and a voltage waveform to obtain a determination of a power factor based on the detected AC electric current and voltage. In various embodiments, the non-contact voltage sensing apparatus100includes power supply means160configured to power the electronic circuitry of the non-contact voltage sensing apparatus100. For example, the power supply means160may include a stored energy system such as a capacitor or battery164; an external power supply such as a direct current (“DC”) voltage source166; or means for obtaining energy from an AC conductor, such as energy harvesting electronic circuitry162. For example, the power supply means160may be configured to obtain power from the conductor via the measurement region120, e.g., via the current transformer151of the current sensing mechanism150. In various embodiments, the non-contact voltage sensing apparatus100includes control circuitry180. The control circuitry180may include multiplexing circuitry185configured to share or switch components among or between circuits. For example, the control circuitry180may multiplex185the conductive sense plate130between the waveform-sensing electronic circuitry142and the capacitance-determining electronic circuitry144. As another example, the control circuitry180may multiplex185the current transformer151between the current sensing mechanism150and the energy harvesting electronic circuitry162. The multiplexing circuitry185may reconfigure subcircuits to include or exclude components such that they can perform multiple functions without interfering with one another. The multiplexing circuitry185may operate according to various algorithms based on time (e.g., at even, uneven, or irregular intervals or according to a schedule), need (e.g., based on one or more signals received by the control circuitry180, such as a battery164level), sensed voltage and/or current values, or other factors. In addition, the control circuitry180may be configured to combine properly scaled sampled waveforms measured by the voltage sensing mechanism140(e.g., by the waveform-sensing electronic circuitry142, scaled according to the capacitance-determining electronic circuitry144) with current measurements obtained by the current sensing mechanism150, allowing the non-contact voltage sensing apparatus100to calculate a power dissipation, power delivery, or power factor, among other possible calculations including real, reactive, and apparent power. In various embodiments, a data bus connects the various internal systems and logical components of the non-contact voltage sensing apparatus100. For example, the control circuitry180may include circuitry to cause measurements to be recorded to memory190and/or transmitted via input/output (“I/O”) components170such as a radio175transceiver for transmitting and/or receiving radio frequency (“RF”) signals (e.g., via low-power wide-area network (“LPWAN”) [e.g., LoRa, Sigfox, LTE-M, NB-IoT, etc.], Bluetooth, Wi-Fi, ZigBee, cellular network connection, NFC, RFID, etc.) or other interface (e.g., a wired communication port such as USB, UART, etc.). The I/O components170may allow data (including, e.g., recorded voltage measurements) to be sent from the non-contact voltage sensing apparatus100to an external device or destination. The I/O components170may also allow instructions to be transmitted to the control circuitry180or other components of the non-contact voltage sensing apparatus100such as the memory190. The I/O components170may interface with, e.g., specialized meter reading devices, mobile phones, desktop computers, laptops, tablets, wearable computers, or other computing devices that are configured to connect to the non-contact voltage sensing apparatus100. The memory190can include a combination of temporary and/or permanent storage, and both read-only memory (“ROM”) and writable memory (e.g., random access memory (“RAM”), processor registers, and on-chip cache memories), writable non-volatile memory such as flash memory or other solid-state memory, hard drives, removable media, magnetically or optically readable discs and/or tapes, nanotechnology memory, synthetic biological memory, and so forth. A memory is not a propagating signal divorced from underlying hardware; thus, a memory and a computer-readable storage medium do not refer to a transitory propagating signal per se. The memory190includes data storage that contains programs, software, and/or information, such as an operating system (e.g., an embedded real-time operating system), application programs or functional routines, and data (e.g., data structures, database entries, waveform representations, measurement records, calculation results, etc.). The non-contact voltage sensing apparatus100may include a subset or superset of the components described above. Additional components may include, e.g., a display screen (such as an LCD, LED, or OLED display screen or an e-ink display), a speaker for playing audio signals, a haptic feedback device for tactile output such as vibration, etc., an environmental sensor such as a temperature sensor, power managing or regulating systems, etc. In various embodiments, additional infrastructure as well as additional devices may be present. Further, in some embodiments, the functions described as being provided by some or all of the non-contact voltage sensing apparatus100may be implemented via various combinations of physical and/or logical devices, e.g., one or more replicated and/or distributed physical or logical devices. For example, in some embodiments, the non-contact voltage sensing apparatus100includes a sensor configured to capture and wirelessly transmit voltage parameters (shape, frequency components and phases, etc.) to an external device, and may or may not include any current sensing or signal processing circuitry. Aspects of the non-contact voltage sensing apparatus100can be embodied in a specialized or special purpose computing device or data processor that is specifically programmed, configured, or constructed to perform one or more of the computer-executable instructions explained in detail herein. For example, the control circuitry180can be embodied in a microcontroller or an application-specific integrated circuit (“ASIC”). Various circuits or circuitry of the non-contact voltage sensing apparatus100may include or be embodied in a processing component that controls operation of the non-contact voltage sensing apparatus100in accordance with computer-readable instructions stored in memory190. A processing component may be any logic processing unit, such as one or more central processing units (“CPUs”), graphics processing units (“GPUs”), digital signal processors (“DSPs”), field-programmable gate arrays (“FPGAs”), ASICs, etc. A processing component may be a single processing unit or multiple processing units in an electronic device or distributed across multiple devices. Aspects of the disclosed systems and methods can also be practiced in distributed computing environments where tasks or modules are performed by remote processing devices that are linked through a communications network, such as a local area network (LAN), wide area network (WAN), or the Internet, e.g., computing resources provisioned from a “cloud computing” provider. In a distributed computing environment, modules can be located in both local and remote memory storage devices. For example, in some embodiments, the non-contact voltage sensing apparatus100includes a sensor configured to capture voltage parameters at a first location, and signal processing circuitry at a second location remote from the first location. Such implementations allow remote voltage sensing around an electrical network at low cost with centralized computational resources. Alternative implementations of the systems disclosed herein can employ systems having blocks arranged in different ways; and some blocks can be deleted, moved, added, subdivided, combined, and/or modified to provide alternative or sub combinations. Each of these blocks can be implemented in a variety of different ways. However, it is not necessary to show such infrastructure and implementation details or variations inFIG.1in order to describe an illustrative embodiment. FIG.2illustrates an exploded isometric view of components of an example non-contact voltage sensing apparatus200in accordance with one embodiment. The example non-contact voltage sensing apparatus200includes a housing in two parts: an upper housing210and a lower housing215. The upper housing210includes an upper hinge part214, and the lower housing215includes a lower hinge part216. Together, the upper hinge part214and the lower hinge part216form a hinge that allows the upper housing210and the lower housing215to hinge open and remain connected, providing an advantageous way to place the non-contact voltage sensing apparatus200around a conductor. When the hinges214,216are engaged and the upper housing210and lower housing215are closed, a hook212on the upper housing210engages with a bar218on the lower housing215to secure the non-contact voltage sensing apparatus200around a conductor. The illustrated hinge and closure mechanisms are examples; alternative approaches could include any of a wide variety of mechanisms such as a friction fit or snap fit between the upper housing210and lower housing215, a screw-on attachment, a magnetic attachment, a locking pin, a fastener securement, a hook-and-loop fabric securement, another style of mechanical latch, etc. Implementations without a split-core current transformer (e.g., with a one-piece transformer core or no current transformer) may have a housing in one part and include no closure mechanism at all. When the non-contact voltage sensing apparatus200is closed, it provides a measurement region220for a conductor. In the illustrated embodiment, the measurement region220passes through the non-contact voltage sensing apparatus200. In other embodiments, the measurement region may be arranged in other ways, such as along a non-contact voltage sensing apparatus, so that the apparatus can measure voltage in a conductor located at or against the apparatus without requiring the conductor to pass through the apparatus. Adjacent to the measurement region220is a conductive sense plate230. The conductive sense plate230is electrically connected to circuitry or electronics240by a connection235. The non-contact voltage sensing apparatus200includes a current transformer (e.g., a ferro-magnetic core, such as a ferrite or nanocrystalline core) divided into two parts: an upper core251within the upper housing210, and a lower core252within the lower housing215. The upper core251and the lower core252come together to form a complete loop around the measurement region220. In some embodiments, the current transformer (CT) is one piece rather than a split core, which may only partially surround a conductor or may require a conductor to be threaded through the current transformer core. When a conductor is energized in the measurement region220and the non-contact voltage sensing apparatus200is closed around the conductor, windings253around the lower core252and connected to the electronics240enable the current transformer to sense current through the conductor and/or harvest energy from the conductor. The present disclosure encompasses various arrangements and shapes of voltage sensing systems, and is not limited to the embodiment described via this illustrative example. FIG.3illustrates a different exploded breakout view of components of an example non-contact voltage sensing apparatus300in accordance with one embodiment. This illustration of an example non-contact voltage sensing apparatus300includes elements of the non-contact voltage sensing apparatus200ofFIG.2. For example, it includes the components of the housing: the upper housing210and the lower housing215, the upper hinge part214and the lower hinge part216, the hook212on the upper housing210that engages with the bar218on the lower housing215. The example non-contact voltage sensing apparatus300also includes the measurement region220, around which are situated the upper core251and the lower core252that form the core of the current transformer (the windings253are not shown in this illustration). In this embodiment, resilient foam pieces310are configured to fit within the upper housing210, on opposite sides of the current transformer upper core251. The resilient foam pieces310provide a mechanical spring force to press a conductor in the measurement region220toward the conductive sense plate230when the non-contact voltage sensing apparatus300is assembled and closed around a conductor. In various embodiments, other mechanical means may be employed to fix or locate a conductor within the non-contact voltage sensing apparatus300, e.g., a spring or a spring-loaded plate to provide a pushing force on a conductor, or a strap or clip to pull a conductor into a position. The lower core252and an aligner350are shown, for illustrative purposes, out of the order in which they would be assembled in this example non-contact voltage sensing apparatus300. In operation, the aligner350and lower core252would be located below the carrier330for the conductive sense plate230. Similarly, for visibility the electronics240are shown broken out outside the lower housing215. In this embodiment, a separator320separates the measurement region220from the conductive sense plate230, and in operation below it the carrier330, the aligner350, the current transformer lower core252, and the electronics240. The separator320is illustrated as a plastic piece having a thickness of approximately 1 mm In various embodiments, the separator320can be formed of various materials, be of various thicknesses, be of implicit construction (so that a space is provided between the measurement region220and the conductive sense plate230) or be omitted (so that no space is provided between the measurement region220and the conductive sense plate230). In the illustrated embodiment, the conductive sense plate230is a conductive metal layer on a flexible printed circuit board (“PCB”) with an insulating layer, e.g. polyamide. The connection235is made of the same material. The conductive sense plate230is shown configured with a curve or the flexibility to curve around a conductor in the measurement region220to improve capacitive coupling, e.g., by providing a reduced average distance between the conductive sense plate230and the conductor. A conductive sense plate230may also be flat (e.g., for simplicity of manufacture) or have a different shape. In this example embodiment, the electronics240include three PCBs342,344,346. In an implementation, each PCB may include circuitry to perform a discrete function or set of functions. For example, the electronics240may include a voltage sensing circuitry PCB342, an energy harvesting and current sensing circuitry PCB344, and a microcontroller and RF communication circuitry PCB346. Another embodiment could integrate some or all of these functions onto a single PCB. Yet another embodiment could integrate some or all of these functions into an ASIC. FIG.4illustrates a perspective view of an assembled example non-contact voltage sensing apparatus400in accordance with one embodiment. The upper housing210and the lower housing215ofFIG.2are closed, and the hook212on the upper housing210is engaged with the bar218on the lower housing215. This illustrates an elegant closure by which a person can simply press on the hook212to release it from the bar218and open the non-contact voltage sensing apparatus400(via the hinge, which is not visible) for placing the apparatus400onto, or removing it from, a conductor420. The example non-contact voltage sensing apparatus400shows, by dashed line, a conductor420located in the measurement region of the apparatus400, pressed into a position relative to the conductive sense plate by the mechanical spring force from the resilient foam pieces310. FIG.5illustrates an operational routine500of a non-contact voltage sensing system in accordance with one embodiment. In various embodiments, the operational routine500is performed by one or more non-contact voltage sensing apparatuses such as those illustrated above with reference toFIGS.1-4. Portions of the operational routine500may be performed by circuitry such as illustrated below with reference toFIGS.6-9. As those having ordinary skill in the art will recognize, not all events of an operational routine are illustrated inFIG.5. Rather, for clarity, only those aspects reasonably relevant to describing the non-contact sensing of an AC voltage are shown and described. Those having ordinary skill in the art will also recognize that the presented embodiment is merely one example embodiment and that variations on the presented embodiment may be made without departing from the scope of the broader inventive concept set forth in the description herein and the claims below. The operational routine500begins in starting block501. In block515, the operational routine500receives a single AC conductor in or at a measurement region. As described above with reference toFIGS.1and2, a measurement region for a conductor may be arranged so that the conductor passes along, into, or through the measurement region provided by the housing. The operational routine500receives a single AC conductor and excludes other conductors. In block525, the operational routine500shields some or all of the measurement region and/or the measurement circuitry or electronics. This allows the operational routine500to reduce interference from other conductors that may be nearby, and thus to improve the quality (e.g., precision and accuracy) of voltage measurement relative to a reference potential. For example, in a multiphase (e.g., three-phase) electrical system, conductors of alternate phases may produce unwanted capacitances to a conductive sense plate, as described below with reference toFIG.12. In such an environment, arranging shielding to help isolate capacitance to the target conductor can enable the operational routine500to provide improved results. In block535, the operational routine500obtains an AC waveform by measuring a current induced via capacitive coupling between the energized conductor in the measurement region and a conductive sense plate of the non-contact voltage sensing apparatus. Example waveform-sensing electronic circuitry is described in further detail with reference toFIG.7below. The operational routine500may sample an AC waveform, though the sampled waveform may be a filtered and/or distorted representation of a voltage between the energized conductor and a reference potential. The illustrated operational routine500branches to show two alternative approaches to determine a coupling capacitance and/or a scaling factor based on coupling capacitance. One branch includes blocks540,550, and560; the other branch includes blocks545and555. Various implementations of a non-contact voltage sensing system may utilize either approach, or combinations or permutations thereof. Turning to the approach illustrated in blocks540,550, and560: in some implementations, the routine500uses capacitance-determining electronic circuitry to measure a coupling capacitance between the energized conductor in the measurement region and the conductive sense plate of the non-contact voltage sensing apparatus. In block540, the routine500calibrates the capacitance-determining electronic circuitry. Calibration may be performed under controlled conditions at the time of manufacture. Calibration may also be performed during operation, e.g., to correct for effects of parasitic capacitance. Example calibration circuitry is described in further detail with reference toFIG.9below. In block550, the operational routine500senses the coupling capacitance between the energized conductor and the conductive sense plate. For example, the routine500may determine the coupling capacitance using the capacitance-determining electronic circuitry calibrated in block540. In some embodiments, the operational routine500produces a value for the coupling capacitance; in other embodiments, the operational routine500obtains an indication that does not directly provide a value for the coupling capacitance but may be used to produce a scaling factor for signal processing. In block560, the operational routine500determines a scaling factor based at least in part on the coupling capacitance between the conductor and the conductive sense plate of the non-contact voltage sensing apparatus. Example scaling factor- or capacitance-determining electronic circuitry is described in further detail with reference toFIG.8below. Turning to the approach of blocks545and555: in some implementations, the routine500measures a dimension of a conductive wire located in or at the measurement region. In block545, the operational routine500fixes (or determines) the location of the conductor received in block515and determines a size of the conductor. To facilitate both physical measurement of the conductor and consistent electric measurement of voltage in the conductor, the routine500provides a mechanism for holding the conductor in a place or position. Some example mechanisms are described above with reference toFIG.3and below with reference toFIG.11. For example, a digital caliper may determine a width or diameter of a conductor including its insulating jacket. In other embodiments, an optical measuring system may determine a size of the conductor. In other embodiments, a measurement device may, e.g., obtain a circumferential measurement of a conductor, or use a shaped aperture (e.g., a vee shape or stepped opening) to locate a conductor according to its size. In some embodiments, the routine500determines a wire gauge or cross-sectional area of the conductor based on a measurement of the conductor. For example, the routine500may determine that a range of diameters of any conductive wire plus its insulating jacket corresponds to a particular gauge of wire. Thus, even though different brands of conductors may have different thicknesses of insulation and therefore overall diameters, the operational routine500can accurately determine a size of the conductive wire to provide an improved contactless determination of a voltage in the conductor. In block555, the operational routine500determines a scaling factor or coupling capacitance between the conductor and the conductive sense plate of the non-contact voltage sensing apparatus. In this approach, capacitance-determining electronic circuitry is configured to produce a scaling factor or estimated capacitance from a combination of fixed or known factors (e.g., the size of the conductive sense plate and distance from the conductive sense plate to the measurement region) and measured variables (e.g., the location and/or size of the target hot conductor). The operational routine500may determine the scaling factor or coupling capacitance based in part on the determination of the size of the conductor from block545, so that the routine500accounts for how a gauge of the conductive wire affects the coupling capacitance. For example, based on the geometry of the measurement space and location of the conductor relative to the conductive sense plate, together with the size of the conductor determined in block545, the routine500can perform a calculation or use a lookup table to obtain a computed capacitance or scaling factor. In block565, the operational routine500performs signal processing with respect to the AC waveform obtained in block535and the scaling factor or coupling capacitance determined in block560or block555. As described above with reference toFIG.1, the routine500processes the obtained waveform representation and may recover a shape or frequency spectrum of the line voltage waveform. The operational routine500processes the coupling capacitance and/or the size of the conductor and may obtain a scaling factor. This may include multiple scaling factors, e.g., scaling factors that are frequency dependent and/or scaling factors that account for different influences on the obtained waveform representation. The operational routine500performs signal processing that scales the obtained waveform representation (e.g., the recovered shape or frequency spectrum of the line voltage waveform) based on the coupling capacitance (e.g., according to the scaling factor(s)). In some implementations, the routine500applies one or more scaling factors to account for, e.g., nonlinearities in a circuit, attenuation at particular frequencies, or complex impedances, to separate out multiple correction factors that may affect measurement. In some implementations, calibration of a circuit may provide an additional scaling factor to account for parasitic capacitance. In block575, the operational routine500determines, based on the signal processing in block565, an AC voltage of the conductor relative to a reference potential. The operational routine500is thus able to determine conductor voltage without interrupting the conductor, without contact to the conductor wire, and without reference to any other AC signal. The operational routine500ends in ending block599. Alternative implementations of the operational routine500can perform routines having processes in a different order, and some processes or blocks can be deleted, moved, added, subdivided, combined, and/or modified to provide alternative or sub combinations. Each of these processes or blocks can be implemented in a variety of different ways. While some processes or blocks may be shown as being performed in series, they may instead be performed or implemented in parallel, or can be performed at different times. FIG.6is a wiring diagram schematically illustrating a multiplexing circuit600of electrical components configured to measure a waveform and a capacitance in accordance with one embodiment. A hot conductor620carries an AC line current and voltage to be measured by a non-contact voltage sensing apparatus. The line voltage625in conductor620is labeled “Vline” inFIGS.6-9. The hot conductor620is part of an electrical circuit that typically includes a load (not shown), a neutral615(e.g., a line, conductor, bus, or node), and a connection to earth610. The hot conductor620is capacitively coupled with a conductive sense plate630. The capacitance635between the conductive sense plate630and the hot conductor620is labeled “Csense” inFIGS.6-9and12. In various embodiments, the conductive sense plate630may comprise one or more components of a capacitor such as the conductive sense plate130described above with reference toFIG.1or the conductive sense plate230described above with reference toFIGS.2-3. The conductive sense plate630may be switchably connectable to a waveform detector650(e.g., waveform-sensing electronic circuitry such as described in further detail with reference toFIG.7below) via switch S0640, and/or to a capacitance detector660(e.g., capacitance-determining electronic circuitry such as described in further detail with reference toFIG.8below) via switch S1645. In some embodiments, switches S0640and S1645are physically connected or logically controlled so that when switch S0640is closed, switch S1645is open, and/or vice versa. For example, switch S0640and switch S1645can be implemented as one single-pole double-throw (“SPDT”) switch. In such embodiments, the conductive sense plate630is switched between the waveform detector650and the capacitance detector660so that the conductive sense plate630is connected to either the waveform detector650or the capacitance detector660but not to both at the same time. In some embodiments, switches S0640and S1645can be switched independently, e.g., allowing both to be in an open state so that the conductive sense plate630is connected to neither the waveform detector650nor the capacitance detector660. In some embodiments, switches S0640and S1645can be switched such that both are in a closed state so that the conductive sense plate630is connected to both the waveform detector650and the capacitance detector660at the same time. In some embodiments, the switches S0640and S1645are connected to separate conductive sense plates630, such that the waveform detector650is switchably connected to one conductive sense plate630and the capacitance detector660is switchably connected to another conductive sense plate630. In an embodiment in which the conductive sense plate630is configured to be switchable between the waveform detector650and the capacitance detector660, the switches S0640and S1645may be operated in two or more phases. For example, in one phase, switch S0640may be closed, and switch S1645may be open, so that the waveform detector650can amplify and sample a filtered Vlinewaveform “Vsense”655of voltage in the hot conductor620. In another phase, switch S0640may be open, and switch S1645may be closed, so that the capacitance detector660can measure capacitance Csense635of the conductive sense plate630to obtain a scaling factor665for scaling the filtered Vlinewaveform “Vsense”655. Accordingly, by controlling the switches S0640and/or S1645, a multiplexing circuit600of a non-contact voltage sensing apparatus according to the present disclosure can selectively couple a conductive sense plate630to a waveform detector650and/or a capacitance detector660. By multiplexing the conductive sense plate630between the waveform detector650and the capacitance detector660, the same conductive sense plate630used by the waveform detector650can be shared with the capacitance detector660for accurate measurement and signal processing. In addition, the multiplexing allows duplication of components to be minimized. FIG.7is a wiring diagram schematically illustrating electrical components of a waveform detector circuit700in accordance with one embodiment. The waveform detector circuit700includes the hot conductor620with voltage Vline625, earth610(reference voltage), conductive sense plate630with capacitance Csense635, and switch S0640in a closed position, as described in further detail above with reference toFIG.6. The illustrated waveform detector circuit700is configured to convert an induced current “i1”735into an amplified voltage signal proportional to the hot conductor620voltage Vline625. The time-varying AC voltage625of the hot conductor620induces, through capacitive coupling, the induced current i1735, so that the waveform detector circuit700produces a filtered or distorted Vlinewaveform output “Vsense”655. In the illustrated embodiment, the waveform detector circuit700is implemented as a transimpedance amplifier circuit. Those having ordinary skill in the art will recognize that alternative implementations, e.g., other means of current measurement that may include different current-to-voltage circuitry or other current-sensing circuitry, may equivalently be used to sense the induced current i1735. The illustrated waveform detector circuit700includes an amplifier750, e.g., an op-amp. At one input of the amplifier750, a constant DC reference voltage “Vref”745may be used to properly bias the waveform detector circuit700. At another input of the amplifier750, the induced current i1735is combined with a feedback loop connected to the output Vsense655. The feedback loop resistance “Rf”755may be chosen to provide a gain for the amplifier750. The amplifier750is also connected to a DC supply voltage VCC752and a sensor ground751. The illustrated waveform detector circuit700produces an output signal Vsense655that scales linearly with the coupling capacitance Csense635and with the voltage Vline625in the hot conductor620. For example, the illustrated circuit may determine the output Vsense655as follows, based on the time-varying voltage Vline625in the hot conductor620, the coupling capacitance Csense635between the conductive sense plate630and the hot conductor620, the induced current i1735, and the feedback loop resistance Rf755: i1(t)=Csensedvline(t)dtvsense(t)=-i1(t)RFvsense(t)=-RFCsensedvline(t)dtvline(t)=-1RFCsense∫vsense(t)dt Integrating the determined output Vsense655, the waveform detector circuit700or signal processing circuitry produces a response proportional to the time-varying AC line voltage Vline625. Thus, if the coupling capacitance Csense635can be known, the output Vsense655of the waveform detector circuit700can be used by a non-contact voltage sensing apparatus according to the present disclosure to measure the AC line voltage Vline625in the hot conductor620. FIG.8is a wiring diagram schematically illustrating electrical components of a capacitance detector circuit800(or capacitance-determining electronic circuitry) in accordance with one embodiment. The capacitance detector circuit800includes the hot conductor620with voltage Vline625, earth610(reference voltage), and conductive sense plate630with capacitance Csense635, as described in further detail above with reference toFIG.6. The illustrated capacitance detector circuit800includes node A825and node B830to illustrate the formation of capacitance Csense635in the conductive sense plate630. In the illustrated embodiment, the metallic wire of the energized hot conductor820forms node A, and a conductive sense plate (e.g., the conductive sense plate130described above with reference toFIG.1or the conductive sense plate230described above with reference toFIGS.2-3) forms node B. The capacitance Csense635is dependent on physical characteristics of node A825and node B830, such as, e.g., a size (e.g., wire gauge) of the node A825hot conductor820and an area and/or shape of the node B830conductive sense plate. The capacitance Csense635is also dependent on a geometry of a relationship between node A825and node B830, such as, e.g., alignment and distance between node A825and node B830. Such factors may differ with each installation or application of a non-contact voltage sensing apparatus according to the present disclosure. Therefore, a capacitance detector circuit800allows more accurate determination of the voltage Vline625in the hot conductor620. The illustrated capacitance detector circuit800includes a relaxation oscillator configured to generate a signal having a frequency proportional to the capacitance Csense635between the hot conductor620and the conductive sense plate630. The conductive sense plate630is discharged by current flow in the following loop: node B830, sensor ground851, earth610, neutral, node A825. The conductive sense plate630is charged by current flow in the following loop: node B830, VCC852, sensor ground851, earth610, neutral, node A825. The capacitance detector circuit800produces a voltage output “Vout”860that switches at a switching frequency fvout865. The switching frequency fvout865of the capacitance detector circuit800output Vout860can provide a scaling factor665for scaling the filtered Vlinewaveform output Vsense655from the waveform detector circuit700. In the illustrated embodiment, the capacitance detector circuit800is implemented as an astable multivibrator circuit. Those having ordinary skill in the art will recognize that alternative implementations, e.g., other means of capacitance measurement that may include different relaxation oscillator or capacitance-to-frequency circuitry or other capacitance-sensing circuitry, may equivalently be used to sense the capacitance Csense635. The illustrated relaxation oscillator circuit allows a non-contact voltage sensing apparatus in accordance with this disclosure to synthesize a transfer function to easily recover the Vlinewaveform from the filtered Vsense655, at a low power expenditure. The illustrated capacitance detector circuit800is astable and continuously switches its output Vout860between VCC852and the sensor ground851potential (e.g., connected to earth610or another reference potential). The astable multivibrator circuit includes an op-amp850, a feedback resistance Rf840, a constant DC reference voltage Vref842, and resistances R1841and R2842. Its output Vout860switching frequency fvout865depends on a time constant set by feedback resistance Rf840and the coupling capacitance Csense635formed between the hot conductor620and the conductive sense plate630. For example, the switching frequency fvout865of the illustrated circuit800is inversely proportional to Csense635. Accordingly, the capacitance detector circuit800may determine the switching frequency fvout865as follows: β=R2R1+R2TVout2RFCsenseln(1+β1-β)fVout=1TVout Signal processing electronic circuitry may be configured to use the output frequency fvout865of the coupling capacitance-determining electronic circuitry800to obtain a determination of the capacitance Csense635and apply that determination to scale the filtered Vlinewaveform output Vsense655produced by the waveform detector circuit700, which depends on the capacitance Csense635. FIG.9is a wiring diagram schematically illustrating electrical components of a calibration circuit900configured to calibrate a capacitance detector circuit in accordance with one embodiment. In the illustrated embodiment, the calibration circuit900includes a capacitance detector950that can be calibrated to more accurately measure a capacitance, e.g., the capacitance Csense635. For example, the capacitance detector950may include a portion or all of the capacitance detector circuit800described above with reference toFIG.8(e.g., a relaxation oscillator). It also includes two capacitive elements: a fixed, known, or background (e.g., parasitic [possibly unknown and/or changing]) capacitance Cpar935between a ground or reference potential and the capacitance detector950, and the possibly variable or unknown coupling capacitance Csense635formed between the hot conductor620and the conductive sense plate630. The calibration circuit900also includes a calibration phase switch “S1”940. The illustrated calibration circuit900is configured to operate in two phases to calibrate the capacitance detector950. In Phase A, the calibration phase switch S1940is open, and the capacitance detector950is only influenced by the capacitance Cpar935. In Phase A, the capacitance detector950is isolated from the coupling capacitance Csense635. Accordingly, the output (e.g., frequency) of the capacitance detector950in Phase A is proportional to the capacitance Cpar935. In Phase B, the calibration phase switch “S1”940is closed, so that the coupling capacitance Csense635is also connected into the circuit including the capacitance Cpar935and the capacitance detector950. Accordingly, the output (e.g., frequency) of the capacitance detector950in Phase B is proportional to both the capacitance Cpar935and the coupling capacitance Csense635. In the illustrated embodiment, the frequency of the capacitance detector950in Phase B is proportional to the sum of capacitance Cpar935and coupling capacitance Csense635. By comparing the output of the capacitance detector950in Phase A with the output of the capacitance detector950in Phase B, the calibration circuit900(or, e.g., signal processing circuitry and/or control circuitry) can obtain a more accurate determination of the coupling capacitance Csense635. For example, in the illustrated embodiment, calibration may include taking a difference between two frequencies: Csenseα (fB-fA). The calibration circuit900provides a way to dynamically correct for the effect of any parasitic capacitance Cpar935, and/or to calibrate a capacitance detector950. In other implementations, such a correction may be performed by a different type of calibration circuit, e.g., calibration via controlled conditions at manufacturing. FIGS.10A-10Bare graphs illustrating a sensitivity of sensed capacitance to conductor size and distance from a conductive sense plate, for a given dielectric constant. FIG.10Aillustrates a graph1000showing a modeled relationship between distance and capacitance Csensefor wires of different gauges. The x-axis of graph1000displays a distance “d_wire_plate”1020between a conductive sense plate and a conductive wire, measured in millimeters (ranging linearly from approximately 1 mm to 20 mm in divisions of 2.5 mm) The y-axis of graph1000displays a capacitance “C_sense”1030, measured in Farads (ranging from approximately 10−14to 10−11Farads, in a logarithmic or other non-linear scale). Capacitance-versus-distance curves are plotted for four wires of different sizes: 6-gauge (AWG6) wire1006, 8-gauge (AWG8) wire1008, 10-gauge (AWG10) wire1010, and 12-gauge (AWG12) wire1012. For a given wire, capacitance is greater for a smaller distance. For a given distance between the sense plate and wire, capacitance is greater for larger wire. FIG.10Billustrates a graph1050showing a modeled relationship between wire positioning error and percent absolute error of measuring voltage Vsensein wires of different gauges. The x-axis of graph1050displays a distance “d_wire_plate”1070between a conductive sense plate and a conductive wire, measured in millimeters (ranging linearly from approximately 9 mm to 11 mm in divisions of 0.25 mm) The y-axis of graph1050displays a “Percent Error Amplitude (V_sense)”1080, measured in percentages (ranging linearly from 0 to 20 percent). Error-versus-distance curves are plotted for four wires of different sizes: 6-gauge (AWG6) wire1056, 8-gauge (AWG8) wire1058, 10-gauge (AWG10) wire1060, and 12-gauge (AWG12) wire1062. The curves are all calibrated for a target 10-millimeter distance from the conductive sense plate to the center of the wire, so all of the curves show no error where d_wire_plate is exactly 10 mm. For a given wire, as actual distance to the sense plate diverges in either direction from the calibrated distance, the absolute value of the error percentage grows larger. For a given distance between the sense plate and wire, percentage error is greater for larger wire. FIG.11illustrates physical components of an example conductor fixing and measurement system1100including a digital caliper in accordance with one embodiment. A conductor fixing and measurement system1100may improve measurement of a capacitance Csenseand reduce error in determining an AC voltage Vsense(e.g., as illustrated above with reference toFIGS.10A-10B), for example by enabling a non-contact voltage sensing apparatus to determine and/or account for a location of the conductor to be measured and a size of the conductor to be measured. In some embodiments, the non-contact voltage sensing apparatus of the present disclosure includes conductor measurement means to fix and/or measure one or more aspects of a conductor at a measurement region. For example, the apparatus may include one or more features configured to guide a conductor to the measurement region (e.g., to a location and/or into an orientation at the measurement region) and/or to hold the conductor at or in the measurement region. In some embodiments, the apparatus is configured to fix at least a portion of a conductor in or against a known position. The apparatus may include one or more features to obtain a determination of at least one physical dimension of the conductor. For example, the apparatus may be configured to determine a diameter of a conductor (e.g., a wire including an outer insulating jacket/layer), a circumference of the conductor (e.g., a partial circumference), a wire gauge of the conductor, and/or a cross-sectional area of the conductor. In the illustrated example, a conductor fixing and measurement system1100includes a digital caliper. The system1100includes an upper housing1110and a lower housing1115connected by a hinge1116. The upper housing1110and lower housing1115close around a measurement region1120configured to receive a conductor1122. The conductor1122is pressed between a fixed caliper jaw1130within the lower housing1115and a movable caliper jaw1140(e.g., slidable) within the upper housing1110. The movable caliper jaw1140is connected to a pair of guide rails1145that can move within rail receivers1140in the upper housing1110, allowing the movable caliper jaw1140to adjust to accommodate different sizes of conductor1122. A tension leaf spring1160or other resilient element provides spring force to move or press the movable caliper jaw1140against the conductor1122. In operation, the conductor fixing and measurement system1100includes a sliding measure1170(e.g., attached to or part of a guide rail1145) that indicates, by the location of the movable caliper jaw1140, the size (e.g., diameter) of the conductor1122. For example, the sliding measure1170may include conductive, capacitive, resistive, magnetic, and/or optical elements readable by an electrical contact, magnetic head, or optical reader1180. For example, the sliding measure1170may include a PCB with metallic fingers at a periodic spacing, and the reader1180may include a conductive sense electrode and/or capacitance detector circuit (e.g., on another PCB). When the measure1170PCB moves with respect to the reader1180, the capacitance changes periodically as the conductive fingers (e.g., triangles) of the sliding measure1170slide past the reader1180. A microcontroller can count the periodic changes in capacitance corresponding to the changing positioning of the sliding measure1170attached to the movable caliper jaw1140. Thus, based on the known finger spacing and the count of capacitance changes, the microcontroller may determine an absolute positioning of the movable caliper jaw1140with respect to the reader1180. This allows precise measurement of the diameter of the conductor1122. In some embodiments, the conductor fixing and measurement system1100includes an absolute positioning identification system (e.g., a binary code reader) that incorporates mechanical, electro-magnetic, and/or optical position coding. From the total diameter of the conductor1122(including approximately twice the thickness of its insulating jacket), the microcontroller can calculate a probable wire gauge and probable insulation thickness. For example, known insulation and/or wire gauge standards may enable the conductor fixing and measurement system1100to categorize the conductor into one of a set of discrete categories, e.g., if a thickest 6-gauge wire is smaller than a thinnest 4-gauge wire. Based on those calculations, the conductor fixing and measurement system1100can estimate a distance between the center of the wire and the conductive sense plate. A non-contact voltage sensing apparatus may include a conductor fixing and measurement system1100or a functional equivalent to improve the accuracy of determining a coupling capacitance between a target conductor and a conductive sense plate, so that the apparatus provides a more accurate determination of an AC voltage in the target conductor. FIG.12is a wiring diagram1200schematically illustrating multiphase coupling capacitances. Conventionally, shielding (such as shield125described above with reference toFIG.1) has not been considered necessary in measuring an AC circuit. The inventors have discovered, however, that shielding can be surprisingly important for non-contact voltage sensing in multiphase (e.g., three-phase) environments. For example, without shielding, a current can undesirably be injected into a waveform detector650from an energized non-target AC voltage phase. The wiring diagram1200schematically illustrates an example multiphase environment in which the waveform detector650is intended to measure the voltage on a first conductor VA1220with respect to a ground1210potential. The multiphase environment includes additional conductors VB1230and VC1240energized with AC voltages of different phases. As a result, the waveform measured by waveform detector650may be corrupted by capacitances1235,1245between the waveform detector650and conductors VB1230and VC1240, and potentially capacitances1237,1247between the sensor ground1215potential and conductors VB1230and VC1240. As a result of the currents injected from these non-target phases, the output Vsense655may be incorrect. Therefore, the disclosed non-contact voltage sensing apparatus is shielded to reduce the influence of undesired capacitances as described above. Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that alternate and/or equivalent implementations may be substituted for the specific embodiments shown and described without departing from the scope of the present disclosure. For example, although various embodiments are described above in terms of a housing that snaps around a conductor, in other embodiments various other form factors may be used. In addition, processing and/or output readings may be provided locally at the apparatus and/or performed or displayed remotely. The spirit and scope of this application is intended to cover any adaptations or variations of the embodiments discussed herein. Thus, although the subject matter has been described in language specific to structural features and/or methodological acts, it is also to be understood that the subject matter defined in the appended claims is not necessarily limited to the specific features or acts described. Rather, the specific features and acts are disclosed as example forms of implementing the claims. This application is intended to cover any adaptations or variations of the embodiments discussed herein. | 62,457 |
11860196 | In the drawings, reference numbers may be reused to identify similar and/or identical elements. DETAILED DESCRIPTION A detection system according to the present disclosure detects changes in capacitance values of one or more capacitors arranged at one or more DC inputs of one or more power inverters supplying vehicle loads such as a motor or other vehicle loads. While the following description relates to a power control system including a single DC capacitor and a single power inverter, the detection system can be used to detect changes in the total capacitance value of multiple capacitors and power inverters connected in parallel. The detection system monitors changes in the capacitance value to diagnose degradation of the capacitor and to estimate the lifetime of the capacitor. The detection system may be implemented in power control systems using a solid state relay or efuse or power control systems using traditional precharge circuits and mechanical relays. For example, pulse width modulation of a power switch in the solid-state relay or efuse is used to generate impulse currents that are output by the battery to the capacitor. Current measured by a current sensor in the solid-state relay or efuse is used to estimate a capacitance value of the capacitor and is used to detect early signs of failure of the capacitor and/or to alter one or more operating parameters of the inverter or vehicle in response to changes in the capacitance value. Referring now toFIG.1, a detection system10detects changes in a capacitance value of a capacitor C1that is connected across a DC input of a power inverter12. As noted above, the detection system can also be used to detect changes in the total capacitance value of multiple capacitors and power inverters connected in parallel. The power inverter12includes power switches T1, T2, T3, T4, T5and T6. First terminals of power switches T1, T3and T5are connected to a first terminal of the capacitor C1. Second terminals of the power switches T1, T3and T5are connected to first, second and third phases of a motor14, respectively, and to first terminals of the power switches T2, T4and T6, respectively. Second terminals of the power switches T2, T4and T6are connected to a second terminal of the capacitor C1. A battery system BATT1includes one or more battery packs that are connected in series and/or parallel. Each of the battery packs includes one or more battery modules that are connected in series and/or parallel. Each of the battery modules includes one or more battery cells that are connected in series and/or parallel. Power is supplied by the battery system BATT1to the capacitor C1and the power inverter12via a protection circuit16. In some examples, a solid state relay is used and includes a gate driver, a transistor, a current sensor and/or other circuits. In other examples, an electronic fuse is used and includes a gate driver, a transistor, a current sensor and/or other circuits. A voltage sensor24sense a voltage across the capacitor C1. During startup of the vehicle, a controller26selectively causes the protection circuit16to supply a current pulse for a predetermined period to the capacitor C1. In some examples, the power switch is turned on for a predetermined period in a range from 1 microseconds to 5 microseconds, although shorter or longer periods can be used. The pulse width is selected to keep the sensor error effects to minimum, perform quick capacitance check and minimize non-linearities. In some examples, prior to supplying the current pulse, the controller26measures the current offset using the current sensor in the protection circuit16. After sensing the current pulse, the controller26receives an output of the voltage sensor24and the current sensor in the protection circuit16and calculates the capacitance value of the capacitor C1. If the current offset is measured, compensation for the current is performed. The controller26calculates the capacitance value of the capacitor C1by integrating the measured current (I) over a predetermined period and dividing by the voltage V (or C=∫ldt/V). Alternately, the capacitance value can be calculated by taking current divided by the inverse of the change in voltage over a predetermined period (or C=I/(dV/dt)). The controller26compares the measured capacitance to one or more thresholds and selectively alters operation of the power control system. In some examples, the measured capacitance is compared to multiple thresholds TH1, TH2, TH3, etc. that are monotonically decreasing and the controller26takes different actions depending upon the particular threshold that is crossed. For example, when the measured capacitance falls below a first threshold, a first action can be taken to alter the operation of the vehicle. When the measured capacitance falls below a second threshold (less than the first threshold), a second action (or the first and second actions) can be taken to alter the operation of the vehicle. When the measured capacitance falls below a third threshold (less than the second threshold), a third action (or the first, second and third actions) can be taken to alter the operation of the vehicle. In other examples, a rate of change of the capacitance value is monitored over time and compared to a predetermined rate threshold. If the measured rate of change exceeds the predetermined rate threshold, another type of action can be taken to alter the operation of the vehicle. In still other examples, the lifetime of the capacitor is estimated based on the comparisons to the thresholds. For example, the controller26may de-rate by reducing the maximum voltage and/or current that is output to the motor14(via the power inverter12and the capacitor C1). Alternately, the controller26may use a higher maximum frequency for switching of the power switch in the inverter since lower frequency switching tends to stress the capacitor C1more than higher frequency switching via ripple. In other examples, the controller26may take other action such as illuminating a warning light, remotely contacting service via a telematics system34, shutting the vehicle down, or taking other remedial actions. Referring now toFIG.2, an example of a solid state relay48is shown to include a gate driver50. In some examples, the gate driver50provides isolation and includes a light emitting diode (LED)52and a photodiode56, although other types of isolating gate drivers can be used. The solid state relay48further includes a power switch62that includes a gate that is driven by the gate driver50. In this example, the power switch62is driven by the output of the photodiode56. A first terminal of the power switch62is connected to the battery system BATT1. A second terminal of the power switch62is connected to capacitor via a current sensor66. In some examples, the current sensor66may include a Hall Effect sensor, although other types of current sensors such as a shunt resistor and voltage sensor or other type of current sensor can be used. Referring now toFIGS.3A to3D, graphs of simulated current, voltage, capacitor value, and turn on pulse are shown as a function of time in a circuit with a solid state relay and a snubber circuit. InFIG.3D, a turn-on pulse is generated by the controller. The turn-on pulse generates current into the capacitor C1(FIG.3A) and voltage across the capacitor C1(FIG.3B). The controller26calculates the capacitance value of the capacitor C1(FIG.3C) based thereon and selectively alters operation of the power inverter12based on the comparison with one or more thresholds as described above and below. Referring now toFIG.4, a detection system100includes mechanical relays SW1and SW2(instead of the solid-state relay or efuse that are shown above). The detection system100is configured to detect degradation of the capacitor C1of the power inverter12. A mechanical relay SW1includes a first terminal connected to the battery system BATT1and a second terminal connected to the power inverter12. A precharge resistor R2has a first terminal connected to the battery system BATT1and the first terminal of the mechanical relay SW1, a second terminal connecter to a first terminal of a mechanical relay SW2. A second terminal of the mechanical relay SW2is connected to the power inverter12and the second terminal of the mechanical relay SW1. A voltage sensor110senses a voltage VR2across the precharge resistor R2. Since the resistance value of the resistor R2is known, the current can be calculated. In some examples, the pulse is a first pulse of a group pre-charging pulses to precharge the capacitor C1. In use, the voltage sensor110optionally determines the sensor offset current before the mechanical relay SW2is closed. Then, the controller26closes the mechanical relay SW2to output the current pulse and calculates the capacitance value of the capacitor C1as described above. Referring now toFIG.5, a method200is shown for detecting changes in the capacitance value. At210, the method determines whether the electric vehicle is being started. If210is true, the method optionally measures current offset. At218, the method generates a current pulse having a predetermined period. At222, the method measures the voltage and current. At226, the method calculates the capacitance. In some examples, the method optionally adjusts for current offset. At230, the method compares the measured capacitance to a capacitance threshold. In some examples, one or more thresholds are used as described above. If230is true, the method adjusts an operating parameter of the electric vehicle as described above. The foregoing description is merely illustrative in nature and is in no way intended to limit the disclosure, its application, or uses. The broad teachings of the disclosure can be implemented in a variety of forms. Therefore, while this disclosure includes particular examples, the true scope of the disclosure should not be so limited since other modifications will become apparent upon a study of the drawings, the specification, and the following claims. It should be understood that one or more steps within a method may be executed in different order (or concurrently) without altering the principles of the present disclosure. Further, although each of the embodiments is described above as having certain features, any one or more of those features described with respect to any embodiment of the disclosure can be implemented in and/or combined with features of any of the other embodiments, even if that combination is not explicitly described. In other words, the described embodiments are not mutually exclusive, and permutations of one or more embodiments with one another remain within the scope of this disclosure. Spatial and functional relationships between elements (for example, between modules, circuit elements, semiconductor layers, etc.) are described using various terms, including “connected,” “engaged,” “coupled,” “adjacent,” “next to,” “on top of,” “above,” “below,” and “disposed.” Unless explicitly described as being “direct,” when a relationship between first and second elements is described in the above disclosure, that relationship can be a direct relationship where no other intervening elements are present between the first and second elements, but can also be an indirect relationship where one or more intervening elements are present (either spatially or functionally) between the first and second elements. As used herein, the phrase at least one of A, B, and C should be construed to mean a logical (A OR B OR C), using a non-exclusive logical OR, and should not be construed to mean “at least one of A, at least one of B, and at least one of C.” In the figures, the direction of an arrow, as indicated by the arrowhead, generally demonstrates the flow of information (such as data or instructions) that is of interest to the illustration. For example, when element A and element B exchange a variety of information but information transmitted from element A to element B is relevant to the illustration, the arrow may point from element A to element B. This unidirectional arrow does not imply that no other information is transmitted from element B to element A. Further, for information sent from element A to element B, element B may send requests for, or receipt acknowledgements of, the information to element A. In this application, including the definitions below, the term “module” or the term “controller” may be replaced with the term “circuit.” The term “module” may refer to, be part of, or include: an Application Specific Integrated Circuit (ASIC); a digital, analog, or mixed analog/digital discrete circuit; a digital, analog, or mixed analog/digital integrated circuit; a combinational logic circuit; a field programmable gate array (FPGA); a processor circuit (shared, dedicated, or group) that executes code; a memory circuit (shared, dedicated, or group) that stores code executed by the processor circuit; other suitable hardware components that provide the described functionality; or a combination of some or all of the above, such as in a system-on-chip. The module may include one or more interface circuits. In some examples, the interface circuits may include wired or wireless interfaces that are connected to a local area network (LAN), the Internet, a wide area network (WAN), or combinations thereof. The functionality of any given module of the present disclosure may be distributed among multiple modules that are connected via interface circuits. For example, multiple modules may allow load balancing. In a further example, a server (also known as remote, or cloud) module may accomplish some functionality on behalf of a client module. The term code, as used above, may include software, firmware, and/or microcode, and may refer to programs, routines, functions, classes, data structures, and/or objects. The term shared processor circuit encompasses a single processor circuit that executes some or all code from multiple modules. The term group processor circuit encompasses a processor circuit that, in combination with additional processor circuits, executes some or all code from one or more modules. References to multiple processor circuits encompass multiple processor circuits on discrete dies, multiple processor circuits on a single die, multiple cores of a single processor circuit, multiple threads of a single processor circuit, or a combination of the above. The term shared memory circuit encompasses a single memory circuit that stores some or all code from multiple modules. The term group memory circuit encompasses a memory circuit that, in combination with additional memories, stores some or all code from one or more modules. The term memory circuit is a subset of the term computer-readable medium. The term computer-readable medium, as used herein, does not encompass transitory electrical or electromagnetic signals propagating through a medium (such as on a carrier wave); the term computer-readable medium may therefore be considered tangible and non-transitory. Non-limiting examples of a non-transitory, tangible computer-readable medium are nonvolatile memory circuits (such as a flash memory circuit, an erasable programmable read-only memory circuit, or a mask read-only memory circuit), volatile memory circuits (such as a static random access memory circuit or a dynamic random access memory circuit), magnetic storage media (such as an analog or digital magnetic tape or a hard disk drive), and optical storage media (such as a CD, a DVD, or a Blu-ray Disc). The apparatuses and methods described in this application may be partially or fully implemented by a special purpose computer created by configuring a general purpose computer to execute one or more particular functions embodied in computer programs. The functional blocks, flowchart components, and other elements described above serve as software specifications, which can be translated into the computer programs by the routine work of a skilled technician or programmer. The computer programs include processor-executable instructions that are stored on at least one non-transitory, tangible computer-readable medium. The computer programs may also include or rely on stored data. The computer programs may encompass a basic input/output system (BIOS) that interacts with hardware of the special purpose computer, device drivers that interact with particular devices of the special purpose computer, one or more operating systems, user applications, background services, background applications, etc. The computer programs may include: (i) descriptive text to be parsed, such as HTML (hypertext markup language), XML (extensible markup language), or JSON (JavaScript Object Notation) (ii) assembly code, (iii) object code generated from source code by a compiler, (iv) source code for execution by an interpreter, (v) source code for compilation and execution by a just-in-time compiler, etc. As examples only, source code may be written using syntax from languages including C, C++, C#, Objective-C, Swift, Haskell, Go, SQL, R, Lisp, Java®, Fortran, Perl, Pascal, Curl, OCaml, Javascript®, HTML5 (Hypertext Markup Language 5th revision), Ada, ASP (Active Server Pages), PHP (PHP: Hypertext Preprocessor), Scala, Eiffel, Smalltalk, Erlang, Ruby, Flash®, Visual Basic®, Lua, MATLAB, SIMULINK, and Python®. | 17,416 |
11860197 | DETAILED DESCRIPTION Referring toFIG.1A, a patient102is undergoing or about to undergo medical treatment by medical equipment122. In an exemplary embodiment, medical equipment122is a blood treatment device, such that patient102is connected to the blood treatment device by one or more hollow fluid lines14that can convey blood and/or other fluids between the patient102and the blood treatment device. Although only a single line is illustrated, it is understood that the illustration represents one or more such lines. In various embodiments, medical equipment122may be a hemodialysis treatment device, a hemofiltration treatment device, and any other device that conveys blood and/or other fluids between the patient and the medical equipment122. In some embodiments, medical equipment122is a peritoneal dialysis treatment device that is configured to pump dialysate into the patient's peritoneal cavity and to withdraw spent dialysate from the patient's peritoneal cavity and certain times and/or intervals. It can be appreciated that the fluid line14, when filled with a conductive fluid such as blood or dialysate, creates a conductive connection between the patient102and the medical equipment122. This conductive connection creates a possibility of a leakage current18and/or19to flow between the patient102and ground124, as shown inFIG.1A. Leakage current18could flow from the patient102through the medical equipment122and to ground124via a ground connection between the medical equipment122and the ground124, such as a ground connection as part of an electrical power connection. Alternatively, or additionally, leakage current19could flow from the patient to the medical equipment122and to ground124through another fluid connection of the medical equipment122, such as a drain line125. In some embodiments, the medical equipment122generates waste (e.g., spent dialysate fluid) that is discarded into a drain126. The drain126may be itself at ground potential. For example, some drain plumbing is made of copper, which is highly conductive and is eventually in physical contact with earth ground. Thus, when a conductive fluid flows through drain line125, there is a possibility of forming a conductive connection to ground124through drain126. In some embodiments, drain line125is a hollow tube formed from an insulating material (e.g., PVC, rubber, plastic, etc.) and the floor127of the medical facility where the medical equipment122is used is made of metal or other conductive material. In this situation, the conductive fluid in drain line125could become capacitively coupled to the floor127, which is at ground potential, thus creating yet another conductive path for leakage current19. Turning next toFIG.1B, an example of the leakage current reduction system100is described. Embodiments of system100reduce current leakage from the patient (e.g., electrified patient) to the medical device by selectively injecting or inducing AC (alternating current) into the conductive fluid (e.g., blood lines) causing a voltage drop from the blood line entering the medical device. The AC is induced by transducer116. In some embodiments, transducer116is contactless, while in other embodiments the transducer116may be a contact transducer. A contactless transducer does not come into direct contact with the conductive fluid into which current is induced. Instead, the transducer generates a magnetic field, which in turn induces current in the fluid. Exemplary embodiments of such a transducer include a toroid that surrounds the fluid line14and/or125conveying conductive fluid. The toroid has wire windings on one or more sides thereof, and when current passes through the wire windings, a magnetic field is generated in the toroid. The magnetic field may be oriented circularly around the tube with conductive fluid, and it may induce an electrical current in the fluid. A contact transducer is in direct contact with the conductive fluid, so that an electrical current can be injected into the fluid directly from the transducer. In embodiments, the contact transducer includes a conductive tube that is fluidly coupled to the fluid line (14and/or125) conveying conductive fluid. The fluid coupling can be achieved via a luer connector, or another similar coupling device. In this configuration, the conductive tube can be conductively connected to, and driven by, a controller to inject a specified current into the conductive fluid passing through the conductive tube. If the current which is induced in or injected into the conductive fluid is substantially equal to or a threshold less than the leakage current (18,19), the leakage current can be reduced by the degree of the injected or induced current. Other embodiments can selectively inject or induce any other suitable amount of current to reduce the current leakage from the patient to the medical device. Referring still toFIG.1B, patient102is illustrated as being connected to AC source104to represent a voltage of the patient. The patient102is further connected by a fluid line14to medical equipment122. The leakage current reduction system100is illustrated as installed on fluid line14, between the patient102and the medical equipment122. However, system100can also be installed on drain line125in addition to, or instead of, on the fluid line14. The system100includes a proximal current sensor108and a distal current sensor118, as shown inFIG.1B. Both of the current sensors detect electrical current flowing through fluid line14(i.e., in the conductive fluid that flows through the fluid line14). System100also includes a transducer116which is operatively coupled to transducer controller112. The transducer controller112may include signal conditioners110and120, as shown. The signal conditioners may amplify and/or filter the signal output from sensors108and118. The transducer controller112is powered by a power supply114. In embodiments, only a single current sensor is used (not shown). In other embodiments, the distal current sensor118measures electrical current in fluid line14. In embodiments, the distal current sensor118is a contactless sensor, similar to the transducer116. For example, sensor118may have a generally toroidal shape with one or more wire windings, and be placed around the fluid line14. In some embodiments, the toroid of sensor118may be a single piece, such that fluid line14will need to be inserted through the opening in the toroid. In other embodiments, the toroid may have an air gap which allows the toroid to open and close around fluid line14. Further examples of embodiments of sensors108and118are described below. Referring toFIG.1C, an example of an embodiment of contactless current sensor108,118is described. The sensor has a body170which has a toroidal shape, such that an opening in the center is surrounded by a material. The body170may be round, square, rectangular, oval, and may have rounded corners. An example of a square with rounded corners is illustrated. The body170can be made from a laminated material, such as Carpenter High Permeability 49 alloy (“Carpenter 49”) which is a 48% nickel-iron alloy that has high saturation flux density, high magnetic permeability and low core loss. Fluid line14is shown passing through the central opening of the toroidal shape, but it is understood that the sensor can be used on any fluid line (e.g., drain line125) in addition or instead of fluid line14. In some embodiments, multiple fluid lines may pass through the central opening at the same time (e.g., a venous blood line and an arterial blood line of a hemodialysis machine). A wire with a first winding173and a second winding174has ends171and172. The two windings can be connected in series, as shown. In embodiments, the windings may be connected in parallel (not shown). When electrical current, such as alternating current is present in fluid line14, it generates a magnetic field in the body170, which in turn induces an electrical current in the wire of the two windings. Thus, a signal representative of the electrical current in the fluid line14can be output from ends171and172, and supplied to the controller112. In embodiments, the body170is split into two halves by an air gap. An example of one half of the body170is shown inFIG.21. It will be understood that the transducer116may have a similar or same design as the sensor108. In embodiments, transducer116has four windings connected in series, each on one side of the body170(not shown). In embodiments, the sensor108,118is a contact sensor, such that it is in direct contact with the conductive fluid flowing through fluid line14. It will be understood that sensor108can be the same as sensor118, but does not need to be. In embodiments, one or both of the sensors108and118will be a contactless sensor. In embodiments, one or both of the sensors108and118will be a contact sensor. It will be further understood that contact sensors and contact free sensors can be combined with contact transducers and contactless transducers in all possible combinations. In embodiments, the distal sensor118is used to drive the transducer116, while the proximal sensor108is used as a safety measure to monitor the leakage current from patient102and thus verify the operation and status of system100. In embodiments, the transducer116may have the same design as sensors108and118. In some embodiments, one of the sensors108and118may be omitted. Embodiments of system100can reduce the amount of leakage current when a patient is electrified (e.g., by AC mains). For example, a fault condition mitigated by embodiments is when patient102is accidentally connected to AC source104(e.g., AC mains). An issue can arise when electrical current flows from patient102to a low potential, such as earth ground124. The current can flow from patient102to electrically coupled medical device122(e.g., a kidney dialysis machine) through a conductive fluid (e.g., fluid line14) and out of medical device122to a drain. In this illustrative example, there are multiple current leakage paths to earth ground124. Some of the leakage paths are in the medical device, another leakage path might be through the drain line to a conductive floor, and yet another leakage path might be the drain line emptying into a copper drain pipe. Because of the potential fault and the multiple potential leakage current paths, various current mitigation techniques are disclosed. Embodiments utilize the fluid resistance (e.g., patient blood resistance) to assist in limiting the leakage current. A reduction to the voltage potential drop across the conductive fluid electrical resistance can achieve this objective. Referring back toFIG.1C, if the patient voltage VP2in fluid line14measured at location150and the voltage measured at location160are nearly the same voltage, then the current through the blood line is nearly zero. This can be achieved by measuring the current (and/or voltage) by sensors108and/or118, and inducing an appropriate current in the fluid line14by transducer116. Embodiments inject current into the fluid line14(e.g., magnetically induce an alternating current via transducer116) in phase with leakage current IPLC measured in the fluid line14. The induced current can replace the leakage current into the machine and force VP2to a voltage closer to VP1measured at location140, thus reducing leakage current IPLC measured at location155. Because embodiments of the design have reactive elements, capacitors and inductors, the phasing of the reducing current is non-trivial. Therefore, leakage current IPLC130is measured before and after transducer116by leakage current sensors108and118. By using the before and after current signals, transducer controller112can adjust the phase to be in phase with the IPLC130current signal using power supply114. For example, using the current sensed by leakage current sensors108and118, sensor signal conditioners110and120can determine input leakage current voltage VCI132and output leakage current voltage VCO136, and provide these voltages to transducer controller112such that an induced current IC134can be determined. In some embodiments, the current sensed by leakage current sensor108can be controlled at or near a predefined threshold or range, such as 10 μA or 20 μA via transducer controller112. The induced current IC134is injected into the fluid stream and summed with the patient leakage current IPLC130. The resultant current is equal to the current that would have passed through the patient if the canceling transducer was not functional. Another embodiment of a sensor/transducer200that may be used in connection with a leakage current-management system as described above is illustrated inFIGS.2A-C,3A-E,4A-B,5,6A-C,7, and8A-B. Advantageously, identical sensor/transducers200may be used as 1) a leakage current-cancelling transducer116or as 2) a proximal current sensor108located upstream of the current-cancelling transducer116and/or a distal current sensor118located downstream of the current-cancelling transducer116, which sense or senses the flow of current within the fluid lines. In the description below, reference is made to fluid line224, but it should be appreciated that the sensor/transducer200can be used with an insulated wire wrapped in the manner of fluid line224, without deviating from the concepts further described below, resulting in a high sensitivity current sensor. In general, the embodiment of a sensor/transducer200may include an E-shaped core202, which has outer arms204aand204band a middle arm206all extending from a base portion208of the E-shaped core202as shown inFIGS.2C,7, and8A-B in particular. For uniformity of performance of the sensor/transducer200regardless of how the sensor/transducer200is attached to a fluid line (as described further below), each of the longitudinal centerlines of the outer arms204aand204bmay be the same distance from the longitudinal centerline of the middle arm206, or the arms may be spaced apart from each other (204ato206,204bto206) by distances that are not the same. Additionally, the outer arms204a,204band the middle arm206may all extend the same distance from the base portion208to facilitate assembly of the sensor transducer200, or they may extend by different distances. As further illustrated inFIGS.2C,7, and8A-B, the embodiment of a sensor/transducer200may include a cross-bar210, which extends across the width of the E-shaped core202from the outermost side of one of the outer arms204a,204bto the outermost side of the other of the outer arms204a,204b. Notably, the cross-bar210may be arranged to make contact with each of the free ends212a,212bof the outer arms204a,204band the free end214of the middle arm206. When brought together in this manner, given that the base portion208of the E-shaped core202and the cross-bar210may be generally parallel to each other with the outer arms204a,204band the middle arm206extending between them like rungs of a ladder, the composite assembly of the E-shaped core202and the cross-bar210may be referred to as an ladder-shaped core. Furthermore, in this manner, two separate sub-circuits216aand216b, which each function as a magnetic circuit as addressed more fully below, can be formed with the middle branch206(also referred to as middle arm) of the E-shaped core202common to both sub-circuits216aand216b, as illustrated inFIGS.8A and8B. Alternatively, magnetic sub-circuits can be formed even with a slight gap present between the cross-bar210and the free ends212a,212bof the outer arms204a,204band the free end214of the middle arm206. Although not illustrated, the cross-bar210may be embedded in a carrier medium such as a plastic or soft vinyl bar, with cutouts formed at locations along one side of the cross-bar210corresponding to locations of the outer arms204a,204band the middle arm206of the E-shaped core202. The carrier member would be thick enough in a direction extending toward the base portion208of the E-shaped core202, and the cutouts would be sized to receive the free ends212a,212b, and214of the outer arms204a,204band middle arm206, respectively, with a friction fit, such that the cross-bar210could be joined to the E-shaped core202generally like a cap, thereby forming the composite ladder-shaped core and establishing the magnetic sub-circuits216aand216b. Other arrangements to facilitate connection between the cross-bar210and the free ends212a,212b, and214of the outer arms204a,204band middle arm206will, of course, occur to those having skill in the art. As alluded to above, the E-shaped core202and the cross-bar210cooperate to form the magnetic sub-circuits216aand216b. Therefore, the E-shaped core202and the cross-bar210may be fabricated from highly magnetically conductive materials, e.g., as laminates formed from material such as Carpenter High Permeability 49 alloy (“Carpenter 49”) which is a 48% nickel-iron alloy that has high saturation flux density, high magnetic permeability and low core loss. It will be appreciated that other materials may also be used. As further illustrated inFIGS.2A,2B, and2C, the sensor/transducer200may include a fluid line cassette220, which slides onto and fits around the middle arm206of the E-shaped core202, and a pair of coil-winding bobbins222aand222b, which slide onto and fit around the outer arms204a,204b, respectively, of the E-shaped core202. The fluid line cassette220facilitates wrapping a fluid line around the middle arm206of the E-shaped core202to form a coil, e.g., with on the order of nine or ten loops around the middle arm206of the E-shaped core202, in some embodiments. Suitably, the fluid line cassette220may be configured to facilitate double-wrapping of the fluid line224around the middle arm206of the E-shaped core202, as illustrated inFIG.5and indicated schematically inFIG.7and as explained more fully below. Increasing the number of loops of the fluid line224enhances sensitivity and resolution of the sensor/transducer200. Similarly, as indicated schematically inFIG.7, the coil-winding bobbins222aand222bmay each support a large number loops of a conductor such as insulated wire with a conductive core, which is wound into coils226aand226bsupported on the outer arms204a,204b, respectively, of the E-shaped core202. In embodiments, the core may be made from copper or a copper alloy, and the wire may be of a predetermined wire gauge. As is the case with the fluid line224, increasing the number of loops of the conductor in each of the coils226a,226benhances sensitivity and resolution of the sensor/transducer200. The coils226aand226bcan be connected in series within a sensing circuit (other components of which are not shown), or they can be connected in parallel within the sensing circuit, with the direction of winding (i.e., in the sense of right-hand winding or left-hand winding) being selected accordingly as addressed further below. Components that can form the fluid line cassette220are illustrated in greater detail inFIGS.3A-E,4A-B, and5. These components can include a fluid line spool230, which is illustrated inFIGS.3A-Eand5. The fluid line spool230can be formed, e.g., from a polymer, such as nylon, polyethylene, or similar, as a generally cylindrical spool tube232, with a circular flange234located at one end of the spool tube232and a groove235extending around circumferentially around the inner surface of the spool tube232at its opposite end. The spool tube may be a part of a fluid line kit, such as a disposable fluid circuit used for dialysis with a length of tubing wrapped around it. In the present context, generally cylindrical includes shapes that do not have a circular cross section, but may have other cross-sectional shapes, such as oval, polygonal, or polygons with rounded vertices. The spool tube232is open at both ends to allow the fluid line cassette220to fit over the middle arm206of the E-shaped core202, and the internal diameter of the spool tube232may be sized for a friction fit—or even a slight interference fit—with the middle arm206of the E-shaped core202. An external helical thread236may be formed on the outer surface of the spool tube232, extending almost all the way from near the circular flange234to the opposite end of the spool tube232. The “pitch” of the external helical thread236may be selected such that the external thread236passes around the spool tube232on the order of four or five times in total, with sufficient space between successive thread crests for a segment of fluid line224to fit between each of a pair of successive thread crests as illustrated inFIG.3B. Furthermore, a circumferentially oriented, “double-width” pass-through slot240may be formed in the circular flange234. As illustrated, the pass-through slot240is shaped generally like two narrow, circumferentially extending ovals placed side-by-side in the radial direction, with the ovals offset relative to each other in the circumferential direction. As further illustrated, the ends of the ovals may each have a slanted surface, e.g., with the radially inner oval having a slanted surface242with a surface normal facing upwardly at one end of the oval and the radially outer oval having a slanted surface244with a surface normal facing upwardly at the circumferentially opposite end of the oval. On the other hand, for each of the inner and outer ovals, the respective opposite end of the oval has a slanted surface with a surface normal facing downward, e.g., slanted surface246for the radially inner oval and slanted surface248for the radially outer oval, as shown inFIG.3D. As illustrated inFIGS.3B and3Ein particular, this configuration makes it possible for a length of fluid line224to be inserted through the slot240from below (relative to the circular flange234) via the radially inner oval and helically wound around the length of the spool tube232from the flange end of the spool tube232to the opposite end of the spool tube232, with the fluid line224located between successive segments of the helical thread236. The fluid line224can then be helically wound “back down” the length of the spool tube232and passed back through the pass-through slot240via the radially outer oval, with the second “wrap” of the fluid line224(not illustrated) overlying the first “wrap” of the fluid line224, which is the “layer” illustrated inFIG.3B. Furthermore, the slanted surfaces242,244,246, and248allow the fluid line224to lie relatively flush against the upper and lower surfaces of the circular flange234, with the fluid line224passing through the circular flange at a relatively shallow angle, i.e., on the order of 15° or less. As a result, the overall “course” a given fluid line follows between a patient and a medical device, or from one medical device to another medical device, remains essentially unchanged by installation of sensor/transducer200onto the fluid line. To help keep the fluid line224wrapped securely around the spool tube232, which improves sensing and transducing performance (addressed more fully below) by holding the fluid line224uniformly close to the spool tube232, the fluid line cassette220can also include a spool cover250, as illustrated inFIGS.2C,4A-B, and5. The spool cover250may be formed, e.g., from plastic or hard nylon, as a relatively thin-walled cylindrical outer wall252, which is open at its lower end and which has an outer diameter that matches the diameter of the circular flange234of the fluid line spool230. A washer-shaped end wall254is located at the upper end of the spool cover250, with a short length of a cylindrical inner wall256surrounding the opening258in the middle of the end wall254. The outer diameter of the cylindrical inner wall256matches the diameter of the groove235extending around circumferentially around the inner surface of the spool tube232, and the height of the cylindrical inner wall256(in the axial direction of the spool cover250) matches the height of the groove235(in the axial direction of the spool tube232). The thickness of the cylindrical inner wall256can match the depth of the groove235(in the radial direction of the spool tube232). With this configuration, the spool cover250can be joined to the fluid line spool230, with the lower end of the spool cover's outer wall252bearing against the circular flange234of the fluid line spool230and the cylindrical inner wall256fitting neatly within the groove235, as illustrated inFIG.5. In this manner, the fluid line224can be held in a coiled arrangement surrounding the spool tube232—and surrounding the middle arm206of the E-shaped core202when the fluid line cassette220is installed onto it—with the fluid line224residing in an annular chamber260formed between the spool tube230and the spool cover250. In an embodiment, the spool230may have a length of tubing bonded to it (e.g. by heat welding or glue, etc.) and the cover250may be attached to the spool, such that ends of the bonded tubing protrude out of the combined structure, and have connectors compatible with various medical tubing. In this way, a coiled fluid pathway can be provided for easy use during a medical treatment, without requiring the coiling of tubing around the spool at the time of treatment, but instead at an earlier time, making the treatment itself faster. In other embodiments, a monolithic structure that mimics the shape of a tube coiled around spool230can be made by 3-D printing or molding. This monolithic structure has a coiled fluid channel formed in a material, and has two fluid line connectors (e.g., luer type connectors) for easy attachment to a fluid line. As illustrated inFIGS.6A-C, the coil-winding bobbins222a,222bmay be formed, e.g., from plastic or hard nylon, with a fairly simple columnar configuration that is open at upper and lower ends. To prevent the coil-winding bobbins222a,222bfrom rotating on the arms of the E-shaped core202to which they are mounted, which could cause the electrical conductors that are coiled around the coil-winding bobbins to unspool or perhaps even break, the coil-winding bobbins222a,222bmay have a non-circular cross-section—e.g. rectangular—that matches the cross-sectional shape of the arms of the E-shaped core to which they are mounted. Flanges262aand262bare formed at the upper and lower ends of the coil-winding bobbins222a,222bto prevent the conductor coils226aand226bfrom sliding off of the coil-winding bobbins. In general, the sensor/transducer200operates in accordance with the same principles. Current flowing within the coils of the fluid line224, carried by the conductive fluid being transported by the fluid line, establishes a magnetic field that extends locally along the middle arm206of the core. The direction in which the magnetic field extends along the middle arm206of the core depends on the direction in which the current is flowing within the fluid line coils, in accordance with a right-hand rule, and the strength of the magnetic field will be proportional to the number of coils that are wrapped around the middle arm206of the core. Magnetic flux will, in turn, extend along the magnetic sub-circuits216aand216b, as illustrated inFIGS.8A and8B, with the direction of circulation likewise depending on the direction in which the electrical current is flowing relative to the middle arm206of the core. For direct current (DC) flowing within the fluid line coils, the magnetic fields will be constant, and there will be no effect on the conductor coils226aand226blocated on the outer arms204a,204bof the core. On the other hand, if alternating current (AC) flows within the fluid line coils, the magnitude and direction of the magnetic field established by the current and extending along the middle arm206of the core will vary sinusoidally with the alternating current, as will the magnitude and direction of the magnetic flux extending along the magnetic sub-circuits216aand216bas illustrated schematically inFIGS.8A and8B. Furthermore, as the magnetic flux passing through the conductor coils226aand226bvaries in magnitude and direction, voltages (emf) will be induced across the conductor coils in accordance with Faraday's law of induction. The magnitude of the induced voltage in each of the conductor coils226a,226bwill be proportional to the time rate of change in magnetic flux through the given conductor coil as well as the number of loops in the given coil. Additionally, the ratio of the voltage induced across a conductor coil to the voltage drop across the fluid line coils (associated with current flowing along the coiled length of the fluid line) will be the same as the ratio of the number of loops in a conductor coil to the number of loops in the fluid line coil. Furthermore, the induced voltage will act in a direction that causes induced current to flow along the conductor coil in a direction such that the magnetic field associated with the induced current opposes the time-varying nature of the magnetic flux through the coil, in accordance with Lenz's law. Thus, by measuring the induced voltage across the conductor coils226a,226b, knowing the ratio of the number of conductor coil turns to the number of fluid line loops in the fluid line coil, and using a predetermined value of resistance through fluid along the coiled length of the fluid line, the amount of current flowing along the coiled length of the fluid line can be ascertained based on the formula i=V/R. Alternatively, the sensor/transducer200can be used to counteract leakage current flowing within the fluid line, as alluded to above, by applying appropriate voltage to the conductor coils226a,226b. In particular, by applying a voltage across the conductor coils226a,226b, the electromagnetic principles explained above (Faraday's law and Lenz's law) will operate “in the reverse direction” to induce a voltage potential across the coiled length of fluid line, with attendant induced current in the fluid line. Thus, if the amount and direction of leakage current in the fluid line is detected (e.g., by measuring it using a proximal current sensor108located upstream of the current-cancelling transducer116and/or a distal current sensor118located downstream of the current-cancelling transducer116) so that the amount of induced current that needs to be injected into the fluid line is known, then the amount of voltage to be applied to the conductor coils226a,226bcan be determined using the same principles as those described immediately above. As noted above, the conductor coils226aand226bcan be connected in series within a sensing circuit or they can be connected in parallel within the sensing circuit, and the direction of winding should be selected accordingly. In particular, as illustrated inFIGS.8A and8B, the magnetic flux in each of the outer arms204aand204bflows in the same direction relative to the base portion208of the E-shaped core202. Therefore, if the conductor coils206aand206bare connected in series, they can be installed onto the outer arms204aand204binverted relative to each other so that the direction of advance of the loops of one of the coils (e.g., using a right-hand rule) is, for example, from the base portion208toward the cross-bar210and the direction of advance of the loops of the other coil is from the cross-bar210to the base portion208. With this arrangement, the magnetic fields associated with current flowing along the conductor coils206aand206bin series will be aligned in the same direction as each other (base portion208to cross-bar210or vice-versa), which prevents counter-acting or cancelling flows of magnetic flux along the middle arm206of the core. On the other hand, if the conductor coils206aand206bare connected in parallel, they can be installed onto the outer arms204aand204bwith the direction of advance of their respective being the same, i.e., both extending from the base portion208of the E-shaped core toward the cross-bar210or vice-versa. With this arrangement, current flowing simultaneously through the conductor coils206a,206b(i.e., in parallel) will generate associated magnetic fields that are co-aligned so as not to produce counter-acting or cancelling flows of magnetic flux along the middle arm206of the core. According to a first further embodiment, there is provided a device for detecting an electrical current flowing through a fluid line, comprising a magnetically conductive core with a centrally located support member configured to receive a length of coiled conductor, and at least two electrically conducting coils located at positions that are spaced from the centrally located support member on opposite sides thereof. The magnetically conductive core comprises a magnetically conductive central branch on which the centrally located support member is disposed in surrounding relationship and a pair of magnetically conductive outer branches with at least one of the electrically conducting coils disposed in surrounding relationship to each of the outer branches. According to a second further embodiment, there is provided the device of the first further embodiment, wherein the magnetically conductive core forms two magnetic sub-circuits with the magnetically conductive central branch forming a common portion of each of the two magnetic sub-circuits and with each of the magnetically conductive outer branches forming a portion of one of the two magnetic sub-circuits. According to a third further embodiment, there is provided the device of the first further embodiment, wherein the conductor is a hollow tube filled with an electrically conductive fluid. According to a fourth further embodiment, there is provided the device of the third further embodiment, wherein the hollow tube is made of an electrically insulating material. According to a fifth further embodiment, there is provided the device of the fourth further embodiment, wherein the electrically insulating material includes a polymer. According to a sixth further embodiment, there is provided the device of the third further embodiment, wherein the hollow tube is made of a semi-conductive material. According to a seventh further embodiment, there is provided the device of the sixth further embodiment, wherein the semi-conductive material includes carbon impregnated polymer. According to an eighth further embodiment, there is provided the device of the seventh further embodiment, wherein the carbon impregnated polymer includes polyvinylchloride. According to a ninth further embodiment, there is provided the device of the first further embodiment, wherein the magnetically conductive core comprises a ladder-shaped core. According to a tenth further embodiment, there is provided the device of the ninth further embodiment, wherein the ladder-shaped core comprises a magnetically conductive E-shaped core member and a magnetically conductive cross-bar. According to an eleventh further embodiment, there is provided the device of the tenth further embodiment, wherein the magnetically conductive E-shaped core member forms the magnetically conductive central branch and the magnetically conductive outer branches and wherein the magnetically conductive cross-bar is removably connectable to free ends of the magnetically conductive central branch and the magnetically conductive outer branches. According to a twelfth further embodiment, there is provided the device of the first further embodiment, wherein the centrally located support member comprises a spool which surrounds the magnetically conductive central branch. According to a thirteenth further embodiment, there is provided the device of the twelfth further embodiment, wherein the spool has an external helical thread, and the thread has a size that accommodates the tube conveying conductive fluid, such that the tube fits tightly into the treads. According to a fourteenth further embodiment, there is provided the device of the twelfth further embodiment, wherein the spool comprises an externally helically threaded central spool tube and a circular flange located at an end thereof. According to a fifteenth further embodiment, there is provided the device of the fourteenth further embodiment, wherein the circular flange has a circumferentially oriented pass-through slot that is generally adjacent to the central spool tube. According to a sixteenth further embodiment, there is provided the device of the fifteenth further embodiment, wherein the pass-through slot is formed as a pair of circumferentially extending ovals that are positioned side-by-side and that are circumferentially shifted relative to each other. According to a seventeenth further embodiment, there is provided the device of the sixteenth further embodiment, wherein ends of the circumferentially extending ovals are slanted to facilitate passage of tubing through the pass-through slot at an angle of 15° or less relative to a plane in which the circular flange lies. According to an eighteenth further embodiment, there is provided the device of the fourteenth further embodiment, wherein the centrally located support member comprises a cassette comprising the spool and a spool cover configured to mate with the spool, defining an annular chamber surrounding the externally helically threaded central spool tube. According to a nineteenth further embodiment, there is provided the device of the fourteenth further embodiment, wherein the electrically conducting coils are supported by respective bobbins. According to a twentieth further embodiment, there is provided a system for sensing and counteracting leakage current from a patient fluidly connected to a medical device by tubing filled with a conductive fluid. The system comprises at least a pair of sensor/transducers, each of the sensor/transducers including a magnetically conductive core with a centrally located support member configured to receive a length of tubing filled with the conductive fluid, and at least two electrically conducting coils located at positions that are spaced from the centrally located support member on opposite sides thereof. The magnetically conductive core comprises a magnetically conductive central branch on which the centrally located support member is disposed in surrounding relationship and a pair of magnetically conductive outer branches with at least one of the electrically conducting coils disposed in surrounding relationship to each of the outer branches. The magnetically conductive core forms two magnetic sub-circuits with the magnetically conductive central branch forming a common portion of each of the two magnetic sub-circuits and with each of the magnetically conductive outer branches forming a portion of one of the two magnetic sub-circuits. The at least a pair of sensor/transducers are disposed on the tubing with the tubing supported by the centrally located support member and coiled around the magnetically conductive central branch of each sensor/transducer, with a first one of the sensor/transducers arranged to detect leakage current flowing within the conductive fluid and a second one of the sensor/transducers arranged to induce current within the conductive fluid to counteract the leakage current when voltage is applied to the electrically conducting coils of the second sensor/transducer. It is, thus, apparent that there is provided, in accordance with the present disclosure, system and method for reducing current flowing in a conductive fluid. Many alternatives, modifications, and variations are enabled by the present disclosure. Features of the disclosed embodiments can be combined, rearranged, omitted, etc., within the scope of the invention to produce additional embodiments. Furthermore, certain features may sometimes be used to advantage without a corresponding use of other features. Accordingly, Applicants intend to embrace all such alternatives, modifications, equivalents, and variations that are within the spirit and scope of the present disclosure. | 39,936 |
11860198 | DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS The present specification is concerned with A.C. current sensing using mutual inductance current sensors. Mutual inductance current sensors have voltage outputs proportional to the rate of change of current, particularly but not exclusively when used in electrical power measurement applications. One example of a mutual inductance current sensor is a Rogowski Coil type sensor. However, the methods of the present specification are not limited by the type of mutual inductance sensor used, and are applicable to any current sensors having an output proportional to the rate of change of a monitored current. As discussed in the background section, in traditional power distribution systems, currents which are drawn may be predominantly sinusoidal. However, modern power distribution systems are increasingly supplying loads which draw significantly non-sinusoidal currents. Currents drawn by such loads may contain harmonic components with significant amplitudes, in some cases exceeding even the amplitude of the fundamental (or base) frequency of the power supply. As noted in the background section, the frequency response of a mutual inductance current sensor increases at 20 dB/decade, so that signals associated with these harmonic components can be very large in a signal from the mutual inductance current sensor. For measurements employing digital integration (or other digital processing methods), the signal from the mutual inductance current sensor must be digitised. In such systems, harmonic components having amplitudes exceeding or significantly exceeding the fundamental frequency of a current may present a problem for providing adequate performance of an analogue-to-digital converter (ADC). An N-bit ADC digitises an analogue signal into 2N−1 signal levels (or binary words). For an N-bit ADC having minimum Vminand maximum Vmaxinput voltages, the input range is ΔV=Vmax−Vmin, the quantisation step is approximately δV=ΔV/(2N−1) and the dynamic range is ˜ΔV/δV. The input range ΔV should be large enough to accommodate the peak-to-peak amplitude of the harmonic components without signal clipping. However, when the fundamental frequency and/or lower harmonic components have significantly lower amplitude than the higher harmonics, the quantisation step δV may have a poor dynamic range for measuring the fundamental frequency and/or lower harmonic components. For example, if the fundamental frequency has amplitude ˜V0in the signal from the current sensor, the dynamic range for the fundamental frequency is ˜V0/δV. Extending this, for the nthharmonic having amplitude Vn(n an integer ≥2), the measurement has a dynamic range of ˜Vn/δV. Ideally, a measurement system should have adequate dynamic range across the fundamental frequency and all harmonics of interest. This may be achieved by simply increasing the number N of bits for the ADC. However, this will require the use of high performance ADCs, which are more complex and expensive. Furthermore, even with a high performance ADC, the lower amplitude signal components will still experience reduced dynamic range for digitisation. The present specification addresses this problem by using low-pass filtering in the analogue domain to attenuate the higher harmonics. The low-pass filtering has a second transfer function (a first transfer function corresponds to the frequency and phase response of the mutual inductance current sensor itself). In systems where the higher harmonics have greater amplitude than a fundamental frequency and/or lower harmonic components, this permits the input range ΔV to be set lower, improving dynamic range across all signal components of the low-pass filtered signal. Following digitisation by the ADC, the digitised signal is processed using a digital processing chain including one or more digital filters. A first digital filter stage has a third transfer function configured to compensate for the frequency and phase responses of the current sensor. In other words, the first digital filter stage is configured to invert (as much as is practicable) the first transfer function. The first digital filter stage may correspond to a filter in the form of a numerical integration. The first digital filter stage is a typical feature for processing signals from a mutual inductance current sensor. The present invention concerns the combination of the analogue domain low-pass filter having the second transfer function with a digital processing chain including a second digital filter stage having a third transfer function configured to compensate for the frequency and phase responses of the low-pass filter. In other words, the second digital filter stage is configured to invert (as much as is practicable) the second transfer function. The digital processing chain may include one or more further filter stages configured to compensate for frequency and phase effects of any other circuitry present in the analogue domain signal path in addition to the current sensor and low-pass filter. The amplitude of the reconstructed harmonic components should be within a few %, and at least within 10%, of the amplitude of the same components in the originally measured current (before the effects of measurement by the mutual inductance current sensor and attenuation by the low-pass filtering. In some examples, the order of the first and second digital filter stages may be reversed. In other examples, the transfer functions of the first and second digital filter stages may be multiplied together and the product applied as a single stage. Power Management System Referring toFIG.1, an apparatus1including a mutual inductance current sensor11is shown. The apparatus1may take the form of an electricity meter or other device for monitoring and/or measuring an alternating current (A.C.) electrical system. The current sensor11may be connected between a power line2(FIG.2) and a load3(FIG.2) via respective sets of terminals4,5(FIG.2). The apparatus1can measure current only, or current and voltage. The apparatus1includes a current sensing section6, an optional voltage sensing section7, a controller8in the form of a microcontroller, a wired and/or wireless network interface(s)9for connecting the apparatus1to external device(s) (not shown), such as meter reader, and/or remote devices(s) or system(s) (not shown), such as a computer server (not shown), for example, via the Internet, and an optional display10. The current sensing section6includes a mutual inductance current sensor11, such as a Rogowski coil or other suitable coil arrangement for measuring current using mutual inductance. The mutual inductance current sensor11corresponds to a first transfer function Gsens. As will be explained in more detail hereinafter, the current sensing section6includes a low-pass filter12interposed between the current sensor11and an analogue-to-digital converter (ADC)13. The low-pass filter12corresponds to a second transfer function Gfilt. The voltage sensing section7includes a voltage sensor14and an ADC15. The controller8includes at least one processor16and memory17. Application code18and code19for correcting for the current sensor11and low-pass filter12(“correction code”) are stored in non-volatile memory (not shown) and loaded into memory17for execution by the at least one processor16. The current sensor11measures a signal S(t). The signal S(t) is actually related (by first transfer function Gsens) to the time derivative of an actual current I(t) though an inductively coupled conductor21(FIG.2), i.e.S(t)∝ddt(I(t)). The low-pass filter12receives the signal S(t) from the current sensor11. The low-pass filter12has a frequency response (or second transfer function) Gfiltwhich is configured to attenuate one or more harmonic components of the signal S(t) received from the current sensor11. The combination of the first transfer function Gsensof the current sensor11and the second transfer function Gfiltof the low-pass filter12may be viewed as having an overall analogue domain transfer function G, which describes the changes (as a function of frequency) in amplitude and phase of a filtered signal Sfilt(t) output from the low-pass filter12, when compared to the original current I(t). The analogue-to-digital converter13receives and digitises the filtered signal Sfilt(t) output from the low-pass filter12. The controller8is configured to process a digitised signal SADCfrom the analogue-to-digital converter using a digital processing chain having an overall digital domain transfer function H (the digital processing chain may also be referred to as H herein). The digital processing chain H may typically be a multi-stage filter which includes a first digital filter stage which has a third transfer function Hsensconfigured to at least partially invert the first transfer function (e.g. a stage which implements an integrator to recover a current signal from the dI/dt signal SADC). The digital processing chain H also includes a second digital filter stage having a fourth transfer function Hfilt, which is configured to at least partially invert the frequency response of the low-pass filter12. The first digital filter stage may also be referred to as Hsensherein and the second digital filter section may also be referred to as Hfiltherein. The correction code19implements the digital processing chain H (including first and second digital filter stages Hsens, Hfilt) when executed by the processor16. The overall digital domain transfer function H is configured to compensate for the frequency and phase response of the overall analogue domain transfer function G. In other words, the overall digital domain transfer function H is configured to reconstruct the actual current I(t) as accurately as possible. The digitised signal SADCprocessed using the first and second digital filter stages Hsens, Hfiltmay correspond to the direct output of the ADC13. However, the digital processing chain H may also include further stages, for example one or more additional filtering and/or signal conditioning stages (not shown), which may be arrange before, after and/or between the first and second digital filter stages Hsens, Hfilt. The low-pass filter12may take the form of a first order filter, a second order filter, or a higher order filter. In some examples the low-pass filter12may take the form of a cascaded filter. The low-pass filter12may take the form of any analogue components disposed on a signal path between the mutual inductance current sensor11and the ADC13, and having an overall second transfer function Gfiltwhich has the effect of attenuating one or more harmonic components in the signal S(t) received from the current sensor11. In some examples, the low-pass filter12may include at least one series resistance R and at least one parallel capacitance C. In particularly simple examples, the low-pass filter12may take the form of a simple, single stage RC filter. Herein, a series resistance R is located directly on a signal path from the mutual inductance current sensor11to the ADC13. A parallel capacitance connects between a ground or reference potential and the signal path from the mutual inductance current sensor11to the ADC13. As mentioned hereinbefore, the digital processing chain H includes first and second digital filter stages Hsens, Hfilt, and may also include one or more further filters (or stages), for example in the form of a cascaded filter. The digital processing chain H may include one or more infinite impulse response, IIR, filters and/or one or more finite impulse response, FIR, filters to provide the individual digital filter stages. The controller8may also be configured to calculate one or more parameters of the actual current I(t) based on the output of the digital processing chain H. One or more parameters of the actual current I(t) may include a root mean square current, the amplitudes and phases of the current harmonics, and when combined with a voltage waveform in a power or electricity meter or a power analyser, the active, reactive and apparent powers, either total, fundamental-only or per-harmonic, and so forth. The overall digital domain transfer function H may be an inverse of the overall analogue domain transfer function G across at least part of a bandwidth of the ADC13. To this end, the third transfer function Hsensmay be substantially inverse to the first transfer function Gsensacross at least part of a bandwidth of the ADC13, and the fourth transfer function Hfiltmay be substantially inverse to the second transfer function Gfiltacross at least part of a bandwidth of the ADC13. Compensating for the frequency and phase response of the first and second transfer functions Gsens, Gfiltmay correspond to reducing or removing changes in phase and/or amplitude introduced by the current sensor11and low-pass filter12. In general, for an individual apparatus1, the digital processing chain (overall digital domain transfer function) H stored by the correction code19of that apparatuses1controller8should be calibrated specifically to the current sensor11and low-pass filter12of that apparatus1(and their corresponding first and second transfer functions Gsens, Gfilt, or equivalently the overall analogue domain transfer function G). The variability is expected to be dominated by the low-pass filter12(and corresponding second transfer function Gfilt), in particular the precise values of passive components such as resistors and capacitors. In other words, once each apparatus1has been assembled/fabricated, the first transfer function Gsenscorresponding to the current sensor11and the second transfer function Gfiltcorresponding to the low-pass filter12(plus any transfer function(s) corresponding to other components in the analogue signal path) are measured, and the measurements are used to fine-tune the overall digital domain transfer function H (fine-tuning may be dominated by, or consist entirely of, adjustments to the fourth transfer function Hfilt). In this way, the apparatus1may take account of tolerances/variations of the parameters of components used to provide the apparatus1, and in particular the low-pass filter12. In some examples, the fourth transfer function Hfiltmay be calibrated independently of the current sensor11, for example, the second transfer function Gfiltmay be measured without making measurements of the current sensor11transfer function Gsens, or even before the current sensor11is added to the apparatus1. In some examples, the third transfer function Hsensmay not require calibration and only the fourth transfer function Hfiltmay be calibrated. The apparatus1may be intended for measuring a signal S(t) corresponding to a current I(t) having a fundamental frequency f0(sometimes also referred to as the “first” harmonic) and wherein the apparatus is configured to measure up to a predetermined harmonic of the fundamental frequency f0. For example, the apparatus may be intended to measure up to a Kthharmonic having frequency Kf0, with K an integer ≥2 (though in practice it is likely that harmonics up to at least K=10 would be desired, if not higher e.g. K=50. The second transfer function Gfiltcorresponding to the low-pass filter has a −3 dB corner frequency which lies between the fundamental frequency f0and the predetermined harmonic Kf0. In some examples, the −3 dB corner frequency of the second transfer function Gfiltmay be as little as five times the fundamental frequency f0. In general, the −3 dB corner frequency of the second transfer function Gfiltmay be configured in dependence on the specific application. For example, the −3 dB frequency response of the second transfer function Gfiltmay be less than 90%, less than 80%, less than 50%, less than 20%, or less than 10% of the predetermined harmonic Kf0. For specific applications relating to metering and/or monitoring of A.C. power distribution systems, the −3 dB frequency response of the second transfer function Gfiltmay be less than 100 Hz, less than 150 Hz, less than 200 Hz, less than 300 Hz, less than 400 Hz, less than 500 Hz, less than 600 Hz, less than 800 Hz, or less than 1000 Hz, depending on the fundamental frequency f0of the A.C. power distribution system and the desired accuracy. In some examples, one or more apparatuses1may be built into, or connected to, an electrical meter (not shown). Each mutual inductance current sensor11is inductively coupled to a conductor, for example a first line21(FIG.2), which supplies power from a supply line2(FIG.2) to a load3(FIG.2). An electricity meter (not shown) may be used to measure energy or power supplied to the load3(FIG.2). The conductor(s), for example the first line21(FIG.2), may be part of the electrical meter (not shown) or separate from the electrical meter (not shown). The electrical meter (not shown) may satisfy non-sinusoidal accuracy requirements of one or more standard including, for example, American National Standards Institute, ANSI, C12.20:2015. Using the apparatus1, a dynamic range of measurements obtained using the apparatus1may be improved compared to a second electricity meter which is identical to the electricity meter except that the second electricity meter does not include the low-pass filter. For example, a dynamic range of current measurements obtained using the apparatus1may be greater than or equal to two times, four times, six times, eight times, or ten times larger than a dynamic range of current measurements obtained using an apparatus (not shown) which is identical to the apparatus1except that the low-pass filter is omitted. The comparison of dynamic ranges may refer to the signal component having minimum dynamic range in the apparatus (not shown) which is identical to the apparatus1except that the low-pass filter is omitted, also assuming that the input range ΔV of the ADC is increased to avoid any clipping of higher frequency harmonics. Although shown as a separate component to the controller8, the ADC13may instead be integrated as part of the controller8. In other words, the controller8may be a controller which includes one or more ADC input ports (not shown), and one of these ADC input ports (not shown) may provide the ADC13. Similarly, in some examples the low-pass filter12may be integrated as a single unit with the current sensor11. Referring toFIG.2, an example of an analogue front end20of the apparatus1and the ADCs13,15is shown. The current sensor11includes a coil L2inductively coupled to a first line21between a first input terminal4and a first load terminal5. The coil L2is connected to the low-pass filter12which includes first and second resistors R1, R2and a capacitor C1. The first resistor R1is arranged in series in a first dI/dt signal path23between a first end of the coil L2and a first input in+ of the first ADC13. The second resistor R2is arranged in series in a second dI/dt signal path25between a second end of the coil L2and a second input in− of the first ADC13. The capacitor C1is arranged in parallel with the coil L2between the first and second dI/dt current paths23,25. The voltage sensor14includes a voltage divider between the first and second line-load paths21,22having a tap27connected via a first voltage signal path28to a first input in+ of the second ADC15. The second line-load path22is connected via a second voltage signal path29to the second input in− of the second ADC15. Limiting the Analogue Bandwidth and Dynamic Range For the purposes of understanding the benefits of the invention, it is useful to consider a waveform with a sharp current step, such as might be seen when a triac (not shown) supplying a resistive load (not shown) is turned on. Referring toFIG.3, an example of a waveform with a sharp current step is shown which is in the form of a waveform for a 100-ohm load switched with a triac at 135° on a 50 Hz supply. The waveform shown inFIG.3is only one example of an A.C. current I(t) which may be measured using the apparatus1. A mutual inductance sensor (not shown) measuring the resulting current step current will produce a voltage whose peak amplitude is proportional to the bandwidth of the apparatus1prior to analog-to-digital conversion, including the low-pass filter and any other filtering or signal conditioning components (not shown) prior to the ADC13of the current sensing section6. Referring also toFIG.4, to limit the bandwidth, a simple passive RC network acting as a low-pass filter12can be placed before an analogue-to-digital converter13reducing the response bandwidth down to typically between 100 Hz and 1,000 Hz. In this type of power application, full amplitude current steps are typically limited by supply reactance, usually to no more than 2 kHz, so these bandwidth limits give reductions in the required dynamic range of an ADC of between 2 and 20 times. Phase Shift When considering performance of such a bandwidth-limited mutual inductance sensor in a power measurement application, a key parameter of concern is the phase shift at the fundamental line frequency, which is typically 50 or 60 Hz. Just 0.1° of phase shift can produce 0.3% error in power measurement at a power factor of 0.5, which is a typical measurement point in an energy metering standard. The 60 Hz phase lag from a simple RC low-pass filter at 600 Hz is about 6°, and if the RC value is in error by 1%, the phase will change by 0.06°, which will cause a change of ˜0.2% in the power calculated at a power factor of 0.5. As the RC filter −3 dB corner frequency is lowered, this effect becomes proportionately larger. Another way of stating this is that if the bandwidth is ten times the line frequency, a 1% shift in bandwidth will give a 1 mrad (milli-radian) shift in line frequency phase and if it is five times the line frequency, then it will be 2 mrad. Digital Correction An RC low-pass filter at, for example, five- to ten-times the fundamental frequency f0, if applied to a current sensor in a power metering application, would by itself lead to an unacceptably poor system response, both in terms of frequency and phase response (resulting from the corresponding overall analogue domain transfer function G). One way to compensate for a small phase mismatch between current and voltage channels in a power measurement system is simply to delay one signal relative to the other. Whilst it is technically feasible to apply a delay to correct for 6° of phase lag in the example hereinbefore described (namely by 28 μs), this does not correct for the frequency dependence of the amplitude response, either when the fundamental frequency varies, or to accurately measure the effects of harmonic current. In the example given, there would be 3 dB attenuation at a 10thharmonic, which would be unacceptable. A solution to this is to create a second digital filter stage Hfiltwhich includes the inverse amplitude and phase characteristics of the RC filter, so that both the phase and amplitude effects from the low-pass filter12are exactly or substantially reversed. In an ideal case, the fourth transfer function Hfiltwould be the inverse of the second transfer function Gfilt. Exact reversal/inversion of the second transfer function Gfiltwill be difficult in practice, so that substantially reversed refers to reversed (inverted) as much as is practical. For example, substantially reversed may corresponds to the phase and amplitude effects of the RC filter being reduced so that at the harmonics of interest, i.e. up to a predetermined Kthharmonic, the amplitude of the kthharmonic component (2≤k≤K) in the digitally filtered signal is within a few percent (10%, 5%, 2%) of the kthharmonic component prior to low-pass filtering. This processing by the controller8will mean that the phase is correct at the fundamental frequency f0, changes in fundamental frequency f0do not cause amplitude errors, and harmonic amplitudes are correct. The second digital filter stage (having fourth transfer function) Hfiltmay be implemented as an Infinite Impulse Response (IIR) filter, derived from the inverse response of the low-pass filter12, for example an RC filter, effectively a zero compensating for the pole of the RC filter (filters may often defined in terms of a number of zeroes and a number of poles). For stability near the Nyquist frequency, a higher frequency pole may also be needed, so a 3-tap IIR can be used. The digital processing chain H will typically also include one or more further stages, for example the first digital filter stage Hsensproviding compensation for the first transfer function Gsensof the current sensor11response, and/or additional signal conditioning stages. In some examples, one or more digital filter stages (for example having third or fourth transfer functions Hsens, Hfilt) may each be implemented as one or more difference equations having coefficients set in dependence on the desired transfer functions Hsens, Hfilt. Coefficients of the one or more difference equations may be iteratively determined based on comparing an overall transfer function of the difference equation(s) to the desired transfer function (inverse response) Hsens, Hfilt, and adjusting the coefficients of the difference equation(s) until a close enough match is obtained. In some examples, closed form methods for determining the coefficients of one or more difference equations providing digital filter stages Hsens, Hfiltof the processing chain H may be available or may be derived. A worked example of a digital processing chain H for a 50 Hz current I(t) and corresponding signal S(t) and a relatively low order difference equation is described hereinafter (in relation toFIGS.10to15). The coefficients of the difference equation(s) are preferably set, or adjusted, based on calibrations performed after assembly/fabrication of an apparatus1. The controller8may also be configured to numerically integrate the output of the difference equation (third transfer function Hsensfollowing the fourth transfer function Hfilt), or alternatively the difference equation may be configured to integrate the digitised signal SADCprior to, or at the same time as, compensating for the frequency and phase response of the second transfer function Gfilt(third transfer function Hsensbefore, or merged with, the third transfer function Hfilt). FIG.5shows the amplitude response (related to the second transfer function Gfilt) of a typical RC filter such as might be used to provide the low-pass filter12for an apparatus intended for a 60 Hz power measurement application.FIG.6shows the corresponding amplitude response of a matching inverse IIR filter (related to the fourth transfer function Hfilt), andFIG.7shows the resulting combined amplitude response. In one example which is most favourable when the low-pass filter12takes the form of a single-pole low-pass filter, processing the digitised signal SADCusing the digital processing chain H may include numerically integrating the digitised signal SADCto generate an integrated output signal ∫SADC, then adding a fraction of the digitised signal SADCto the integrated output signal ∫SADC. The fraction of the digitised signal SADCadded to the integrated output signal should preferably be calibrated for the specific low-pass filter12of a particular apparatus1, in order to maximise the degree of compensation for the frequency and phase response of the low-pass filter12. Numerical integration of the digitised signal SADCmay be based on the most recently sampled value of the digitised signal SADC, any number of previously sampled values of the digitised signal SADCand/or any previously calculated values of the integrated output signal ∫SADC. As mentioned hereinbefore, numerical integration may be implemented as one stage of a multi-part digital processing chain H. Referring again toFIG.4, an example of implementing the compensation by adding a fraction of the digitised signal SADCto the integrated output signal ∫SADCis to consider that the error introduced by the RC filter is just the voltage that appears across the resistor R in the RC network. By calculating this voltage, it can be added back onto a digitised signal SADCin the form of a voltage Vadcsampled by the ADC13to create the voltage on the other side of the resistor, i.e., the unfiltered voltage. The voltage across the resistor R equals the current multiplied by the resistance R. The current is equal to the current through the capacitor C, which is equal to the capacitance C multiplied by the rate of change of voltage. Because the capacitor is effectively connected across the ADC input, the voltage is just the ADC value. Hence, for a current sensor11with mutual inductance M: MdIdt=Vadc+RCdVadcdt(1) Consider that a subsequent signal processing step for a mutual inductance sensor is typically to integrate to recover the originally measured current signal: I=1M∫[Vadc+RCdVadcdt]dt(2) Integrating the second term, the correction term becomes RCVadc, which can simply be added to the output of the integrator to reconstruct the current signal: I=1M[∫Vadcdt+RCVadc](3) Hence the RC filter response may simply be corrected by adding in a proportion of the ADC signal to the integrated signal. In practical terms, care is needed to match the group delay between the ADC signal and the integrated signal. For example, when implementing a digital integrator, it may typically have a group delay of e.g.1sample (or more depending on the specific implementation). Consequently, when the fraction of the unintegrated signal is added, it should be delayed by the same group delay (see also the worked example described in relation toFIGS.10to15). This arrangement becomes particularly advantageous as the system bandwidth extends to higher harmonics. At frequencies near the fundamental f0, the majority of the reconstructed current signal is produced directly by the digital integrator, as the relative contribution of the raw ADC signal is small, yielding all the advantages noted above for carrying out the signal processing in the digital domain. Any variations of time and temperature in the RC filter constant have a minimal effect on the measurement performance at the fundamental, much less than would be seen in an analogue integrator with the same component stability. At frequencies above the RC filter −3 dB point, where typically the requirements on absolute accuracy are less demanding, the majority of the reconstructed current signal comes from the raw ADC signal, as the relative contribution of the digitally integrated part becomes smaller. The RC filter is simply providing most of the integration function at the higher frequencies in this case, whilst at the same time limiting the dynamic range that would otherwise be required of the ADC. Phase Response As noted hereinbefore, it can be difficult to construct stable analogue filters with low corner frequencies, particularly passive filters that ideally should be placed between a mutual inductance sensor11and an ADC13input, where high series impedances typically cannot be tolerated. Low-cost, large-value (e.g., 100 nF) NPo ceramic capacitors recently became available. These capacitors have nominally zero temperature coefficient and excellent long-term stability relative to film capacitors (which used to be the only choice for stable capacitors at 100 nF and above). Combined with low temperature coefficient resistors, this enables a sufficiently stable low frequency low-impedance RC filter to be incorporated into a commercial electric meter between a mutual inductance sensor and an ADC input at a reasonable cost. However, although these capacitors are extremely stable, they are not made to tight tolerance (5% or 10% tolerances are typically), so the RC time constant (and hence filter bandwidth and line frequency phase shift) will vary from part to part. Hence, it is not possible simply to fix the filter coefficients in the digital reconstruction filter described above, or to fix the fraction of the ADC value added back, because these fixed characteristics will not exactly cancel out the variation of effects of the RC filter with tolerance, leading to large phase and amplitude errors. Calibration of Phase Response Considering a power measurement system comprising one or more mutual inductance current sensors (for example current sensors11of apparatus1) and one or more voltage sensors, such as might form the basis of a power or electricity meter, it is generally necessary to find a way to match the current and voltage phase responses at line frequency, and to set the current and voltage (or at least power) gain so that the system measures power accurately over the requisite range of power factors. Commonly this is achieved by a calibration process. Whilst there are many variants, this is usually implemented either by applying precisely known currents and voltages with a precise phase angle relationship such that the expected power is known and can be compared against the measured power, or by comparing the response of the device under test against a precise reference power meter whilst applying approximately known, but not precise, currents and voltages. Depending on the information calculated or reported by the devices, this may require more than one set of conditions to determine all the required coefficients. Verification measurements may also need to be done with different currents and phase angles depending on jurisdiction. Using a low-pass filter in the form of a low-frequency RC filter between a mutual inductance current sensor (e.g. current sensor11) and the ADC (e.g. ADC13), nearly all the phase variation between current and voltage channels to be eliminated through calibration will result from variations in the RC time constant as a result of the tolerance of the capacitance C. The calibration process may make best use of this fact, and phase variations measured at the line frequency may be trimmed out by adjusting the nominal RC value in the compensation described above, instead of following the traditional methods of varying the delay between the current and voltage inputs. Having determined the phase error by a suitable measurement, then in the case where an IIR filter is used to reconstruct the current signal, a calculation is carried out to update the IIR coefficients to modify the digital processing chain H response (in particular the fourth transfer function Hfiltof the second digital filter stage) to change the phase response to correct the phase error, and these updated IIR filter coefficient are then substituted for the original values. In the case where the reconstruction is achieved by adding the Vadcto the integrator output, the phase adjustment is achieved simply by varying the coefficient (fraction) used to multiply Vadc, i.e., by varying RC in the RCVadccorrection term. Other system phase errors may still exist, for example, from higher frequency anti-alias bandwidth limits in the current and voltage channels, or from sampling skew between current and voltage channels. These can be correctly compensated for at line frequency, but lack of matching will lead to phase errors in the harmonic and sub harmonic response of the meter, so good design practice should still be taken to minimise these effects and to match the channels. Referring toFIG.8, examples of the size of these effects are shown. Referring toFIG.9, the processor16of the controller8(FIG.1) corrects for the effect of the low-pass filter12(FIG.1). The controller8receives the low-pass filtered dI/dt signal S(t) (step S1), reconstructs the current I(t) compensating for the effect of the current sensor11and low-pass filter12as herein described (step S2) and outputs a reconstructed current signal (step S3). The correction process may be implemented in software, using correction code19(FIG.1) to apply the digital processing chain (having overall digital domain transfer function H). Characteristics of Systems Covered Embodiments of the invention can be used in situations where the frequency response of a mutual inductance sensor is attenuated in the analogue domain at a frequency significantly below the highest harmonic required to be measured by the application, significantly below the Nyquist frequency of the subsequent sampling system, but generally above the fundamental, such that the only way that the application is able to measure the highest harmonic sufficiently accurately is by compensating to some extent for the attenuation and phase shift caused by the frequency attenuation. For example, in a 50 Hz or 60 Hz power measurement system, this might be by using an RC filter with a −3 dB frequency of 500 Hz or 600 Hz respectively, ten times the fundamental frequency, or lower. To determine whether a particular apparatus is applying the teachings of the present specification, the frequency response (or second transfer function Gfilt) of the analogue signal components (e.g. low-pass filter12) connecting between a mutual inductance current sensor and an analogue-to-digital converter of such apparatus may be measured. For example using a frequency sweep and monitoring using an oscilloscope. Alternatively, the frequency response (or second transfer function Gfilt) of the analogue signal components (e.g. low-pass filter12) connecting between a mutual inductance current sensor and an analogue-to-digital converter of such apparatus may be simulated based on a schematic and/or inspection of the circuit. The measured frequency response (or second transfer function Gfilt) may be compared to the measurement requirements of said apparatus. If, for example, the apparatus is required to measure up to the Kthharmonic and/or outputs data corresponding to harmonics up to the Kthharmonic, yet the measured frequency response (or second transfer function Gfilt) of the low-pass filter has a −3 dB point at a frequency below the Kthharmonic, then digital compensation using a fourth transfer function Hfilt(i.e. an approximate inverse to the low-pass filter frequency response) must logically be applied by said apparatus in the digital domain. For example, if an apparatus is required to and/or outputs data up to the 50thharmonic, yet has a low-pass filter with a measured analogue frequency response (or second transfer function Hfilt) with −3 dB point around the 10thharmonic, then it can be concluded that some digital domain compensation is being employed (or else any measurements provided by such an apparatus would be inaccurate). Analogue Response Compensation The methods and approach hereinbefore described are not limited to RC filters, nor to single pole filters, nor to low-pass filters. In any signal processing application where digital filtering can be applied, the digital filters can be used to correct for non-ideality in any analogue signal processing. For example, if a signal chain contains a second-order cascaded RC filter, for example a higher frequency anti-alias filter, then a similar technique can be used to compensate for the frequency and phase response of this filter too, for example by calculating the inverse response function of this second filter and implementing as an IIR filter (e.g. a further stage of the digital processing chain H). More generally, it is possible to calculate the inverse response of all the input filter components in a more complex analogue input network, and to implement this as an IIR filter so that the frequency and phase response more closely matches the response required by the application. Compensation in Frequency Domain Many sophisticated power measurement devices measure the harmonic content by carrying out a Fast Fourier Transform (FFT) on the current and voltage waveforms. The frequency and phase response of the input filter network is known from pre-calibration experiments using known signals (e.g. the second transfer function Gfilt), allowing a transfer function inverse to that low-pass filter to be derived (e.g. the fourth transfer function Hfilt). The inverse transfer function is applied in the frequency domain, i.e., on the output of the FFT, and similarly to carry out the power calculations in the frequency rather than the time domain (multiplying each frequency component by its complex conjugate). Digital integration (which typically compensates for the current sensor11response Gsens) is just another filter, and it too may be applied in the frequency domain. Alternatively, after applying the fourth transfer function Hfilt(substantially) inverse to the second transfer function Gfiltof the low-pass filter, the signal may be transformed back into the time domain using an inverse Fast Fourier Transform (iFFT), prior to integration and determination of the power (or other properties of the current I(t)). The controller8may be configured to calculate one or more parameters of the original current signal I(t), for example RMS current, the amplitudes and phases of the current harmonics, and when combined with a voltage waveform in a power or electricity meter or a power analyser, the active, reactive and apparent powers, either total, fundamental-only or per-harmonic, and so forth, based on the filtered frequency domain signal. Although FFT methods are usually preferred, any other methods for obtaining the discrete Fourier transform may be used instead. Alternatively, signals may be transformed between time and frequency domains using Laplace transform methods. Applications Outside Power and Energy Measurement The application of this combination of reduced analogue bandwidth and digital reconstruction with a mutual inductance sensor is not limited to the power measurement applications hereinbefore described. Any applications where a wide bandwidth A.C. current measurement with a mutual inductance sensor is needed can make use of this technique, with the dynamic range advantage increasing proportionately to the ratio between the highest and lowest frequencies of interest. Particular examples outside the fields of electrical metering or monitoring of A.C. power systems (e.g. measurements of RMS current) may include, but are not limited to, are fault detection, measurements of conducted emissions for electromagnetic compatibility (EMC) compliance, combination wave surge current measurement, and so forth. Worked Example An example of signal processing appropriate to a 50 Hz power system will now be described. This worked example is not intended to limit the teachings of this specification described hereinbefore. Features and/or principles of the description hereinbefore should be assumed to be applicable to modifying the worked example, unless explicitly indicated otherwise or where such features and/or principles would be self-evidently incompatible. Referring also toFIG.10, a block diagram of an exemplary signal processing chain30is shown, including a digital processing chain34,35,36,37,38. The input31from a dI/dt sensor (e.g. current sensor11) is passed through a low-pass filter12in the form of a first-order low-pass analogue filter32, with a −3 dB frequency of 600 Hz. This signal is then fed into an Analogue to Digital Converter (ADC)13, creating a digital representation of the input signal at a word rate of 6.4 kHz. (Preferably the ADC is a sigma-delta type, with a modulator frequency in excess of 1 MHz, to avoid the need for further anti-aliasing filters). The first transfer function Gsensin this example corresponds to the amplitude and phase effects of the dI/dt sensor, and the second transfer function Gfiltcorresponds to the amplitude and phase effects of the first-order low-pass analogue filter32. In the controller8, the signal is passed through a digital processing chain including a digital high pass filter34, a digital integrator block35, a matching delay block36, an adder37and a gain trimmer block38. These elements34,35,36,37,38collectively correspond to the digital domain transfer function H for this example. The signal is first passed through the high-pass filter34, which is of conventional design, with a −3 dB frequency of 1 Hz, substantially below the power system frequency of 50 Hz. The purpose of the high-pass filter34is to remove any D.C. components of the signal before integration, as D.C. components would integrate to give a continuously rising or falling signal. The next stage is the digital integrator block35. One simple form can be described by the difference equation: yi[n]=(1-k)×y[n-1]+Δt[x[n]+x[n-1]2](4) where yi[n] denotes the output samples, x[n] denotes the input samples, k<<1 in order to ensure that the integrator is stable, and Δt is the time between successive samples of the ADC13. This integrator has a group delay of ˜half a sample period, so an unintegrated signal path with matching group delay can be created by the matching delay block36using the difference equation: yu[n]=w×x[n]+x[n-1]2(5) where w is a weighting factor chosen such that at the calibration frequency (50 Hz in this example), the amplitudes of the integrated and unintegrated signal paths are the same. The weighting means that the integrated and unintegrated waveforms at 50 Hz are equal amplitude signals with 900 phase difference, which makes it easy to alter the phase without affecting the amplitude during calibration. The final corrected output39is created by the adder37and gain trim block38using a combination of the integrated and unintegrated samples: y[n]=gain×(√{square root over (1−p2)}×yi[n]+p×yu[n]) (6) The calibration process is used to set the values of p (to achieve the phase trim) and gain as follows: Initially set p=0 and gain=1. Measure a sinusoidal current waveform at the calibration frequency of, for example, 50 Hz. The expected output is a sinusoidal current waveform exactly in phase with and the same amplitude as the input. First determine the phase error, theta. Calculate: t=tan theta (7) Then calculate the updated value of the phase trim p as: p=t21+t2(8) If necessary, re-measure the output and iteratively fine tune the value of p until the phase error theta is zero. Second, measure the output amplitude, and adjust the value of gain such the output amplitude matches the input signal level. The principle of operation is illustrated with reference also toFIG.11to15. InFIG.11, two different current waveforms of approximately equal amplitude are shown. The dotted trace40shows a sinusoidal 50 Hz current waveform such as might be seen with a resistive load, or such as might be used to calibrate the gain and phase. The solid trace41shows a phase-fired triac waveform at approximately 135° such as might be seen when using a triac dimmer circuit on a load such as a switch-mode power supply for an LED light. This second waveform41represents a relatively extreme waveform in terms of the ratio of the fundamental to the harmonics. The sinusoidal 50 Hz current waveform40and the phase-fired triac waveform41are examples of currents I(t) which an apparatus1may be used to measure/monitor/meter etc. FIG.12shows the outputs40b,41bof a dI/dt sensor measuring these two waveforms. Although the original currents had substantially the same amplitude, the amplitude of the dI/dt signal41bfor the phase-fired waveform41bis substantially higher (about 16 times in this example) than that of the sinusoidal waveform40b. Note also the 90 degree phase shift of the sinusoidal waveform40b(these effects on amplitude and phase correspond to the contributions of the current sensor11to the first transfer function Gsens). The sinusoidal waveform40band the phase-fired waveform41bare examples of signals S(t). FIG.13shows the signals40c,41cfromFIG.12after a 600 Hz low-pass first order analogue filter12,32has been applied. The dI/dt signal41cis now 7 times larger amplitude than the sinusoidal signal40c, rather than 16 times. This is the signal which is digitised by the subsequent analogue to digital converter (ADC)13,33. Clearly, if a lower corner frequency such as 300 Hz were to be chosen, a larger degree of attenuation could be achieved. The benefit of the attenuation is to reduce the dynamic range requirements for the ADC13,33and to minimise the chance of clipping. The sinusoidal waveform40cand the phase-fired waveform41care examples of filtered signals Sfilt(t) which would be sampled to obtain digitised signals SADC. The sinusoidal waveform40cand the phase-fired waveform41cinclude the effects of the first and second transfer functions Gsens, Gfiltwith respect to the original current I(t) signals40,41). FIG.14shows the result of conventional digital signal processing of the low-pass filtered and digitised signals40d,41dshown inFIG.13. It is clear that the amplitude of the phase-fired current waveform41dhas been significantly attenuated—it is now 63% of the amplitude of the sinusoidal signal40d. This is because the phase-fired current waveform41dcontains significant harmonics above 600 Hz which have been attenuated by the analogue low-pass filter12,32. FIG.15shows the results40e,41eof the new digital signal processing of the low-pass filtered and digitised signals shown inFIG.13. The reconstructed phase-fired waveform41eis restored to the same amplitude as the sinusoidal waveform40eas a result of the signal processing to compensate for the analogue low-pass filter12,32. Modifications It will be appreciated that various modifications may be made to the embodiments hereinbefore described. Such modifications may involve equivalent and other features which are already known in the design, manufacture and use of A.C. current measurement using a mutual inductance sensor and component parts thereof and which may be used instead of or in addition to features already described herein. Features of one embodiment may be replaced or supplemented by features of another embodiment. Although claims have been formulated in this application to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel features or any novel combination of features disclosed herein either explicitly or implicitly or any generalization thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. The applicants hereby give notice that new claims may be formulated to such features and/or combinations of such features during the prosecution of the present application or of any further application derived therefrom. | 50,674 |
11860199 | DESCRIPTION FIG.1is a schematic of an apparatus100comprising a current sensor102in accordance with a first embodiment of the present disclosure. The current sensor102is for a switching converter104comprising an inductor106and a switch108. The switch108is arranged to selectively couple the inductor106to a voltage V1. By “selectively couple” it is meant that the switch108acts to couple the inductor106to the voltage V1or to decouple the inductor106from the voltage V1based on a control signal received by the switch108during operation of the switching converter. The switch108may comprise a transistor, for example a p-type or an n-type transistor, with the control signal being received at a gate of the transistor. The voltage V1may, for example, be referred to as a supply voltage, an input voltage or ground depending on the application. The switching converter104may be a DC-DC converter, for example a buck converter, a boost converter or a buck-boost converter. The current sensor102is configured to generate an output current Iout1that is dependent on an inductor current IL flowing through the inductor106, and to at least partially compensate for an error arising due to the switch108in the generation of the output current Iout1. The error may arise due to an impedance of the switch108. This may, for example, be an on-resistance of the transistor, when the switch108comprises the transistor. The error may, for example, arise due to a temperature dependency of the impedance of the switch108, and/or an offset in a physical value of the impedance of the switch108from an intended value of the impedance. The intended value may be the value of the impedance resulting from the switching converter104specification with the true value of the impedance of the physical implementation of the switch108varying from the intended value, for example, due to process variations and/or imperfections. The sensed current (in the present example Iout1) can be used for current monitoring or as a loop control mechanism. FIG.2is a schematic of the apparatus100, showing a specific implementation of the current sensor102, and according to a second embodiment of the present disclosure. The current sensor102comprises a first stage200configured to generate a current I1that is dependent on the inductor current IL. The current I1comprises a current error. The current error results from the error arising due to the switch108. The current sensor102further comprises a second stage202configured to receive the current I1and to generate the output current Iout1by at least partially compensating for the current error of the current I1. Common reference numerals and variables between Figures denote common features. FIG.3Ais a schematic of the apparatus100, showing a specific implementation of the first stage200in accordance with a third embodiment of the present disclosure. In the present example the switching converter104further comprises a switch300, a capacitor302and is coupled to a load304. The switches108,300may comprise, for example, be PMOS or NMOS transistors. In the present example, the switches108,300comprise high voltage NMOS transistors that function as pass devices. The first stage200comprises a differential amplifier306a resistive element R1, and a resistive element R2. The resistive element R2is coupled in parallel to the switch108. The differential amplifier306is configured to generate the current I1, which is dependent on the impedances of the resistive element R1, R2and the switch108. The resistive elements may, for example, comprise one or more of a transistor and/or a resistor. An output of the differential amplifier306may be coupled to a gate G1of a transistor308. A negative input of the differential amplifier306may be coupled to a first terminal T1of the transistor308. In the present example the switch108comprises a high side switch of the switching converter104. The high side switch comprises a terminal N1coupled to the voltage V1and a terminal N2coupled to the inductor106at an inductor node LX. The resistive element R1may be coupled to the negative input of the differential amplifier306, and a parallel combination of the resistive element R2and the high side switch (provided by the switch108) may be coupled to a positive input of the differential amplifier306. Also shown is a voltage V2which is less than the voltage V1in the present example. In the present example V2is a ground. FIG.3Bis a schematic of the apparatus100, showing a specific implementation of the first stage200in accordance with a fourth embodiment of the present disclosure. In the present embodiment, the switch108comprises a low side switch. The low side switch comprises the terminal N1coupled to the voltage V1and the terminal N2coupled to the inductor106at the inductor node LX. Also shown is a voltage V2which is greater than the voltage V1in the present example. In the present example V1is a ground. In the present embodiment, the resistive element R1is coupled to the positive input of the differential amplifier306, and the parallel combination of the resistive element R2and the low side switch (provided by the switch108) is coupled to the negative input of the differential amplifier306. It should be noted that this differs from the configuration shown inFIG.3A. FIG.3Cis a schematic of a further embodiment of the apparatus100ofFIG.3A, where the current sensor102further comprises a safety switch310having a terminal S1coupled to the resistive element R2and a terminal S2coupled to the inductor106. The switch310may comprise a transistor, for example a field effect transistor (FET), such as a MOSFET. In operation, the switch310may be controlled by the same control signal of the switch108, so they are opened and closed simultaneously. The switch310is arranged to disconnect an input of the first stage200from the LX node. When the high side is OFF (the switch108is open) and the low side is ON (the switch300is closed). Without the switch310, there would be a path from the voltage V1to ground which can compromise the state of the circuit components, causing current consumption and changing the operating point of the first stage200every time that switch108changes between ON and OFF. Therefore, the switch310can mitigate or resolve these issues. FIG.3Dis a schematic of a further embodiment of the apparatus100ofFIG.3Bincluding the safety switch310. FIG.4Ais a schematic of the apparatus100, showing a specific implementation of the second stage202in accordance with a fifth embodiment of the present disclosure. In the present example, the switch108is a high side switch of the switching converter104. The second stage202comprises a differential amplifier400, a resistive element R3, a resistive element R4, and a resistive element R5. The resistive element R5is coupled in parallel with the resistive element R4, thereby forming a parallel combination. The differential amplifier400is configured to generate the output current Iout1that is dependent on the current I1and the impendences of the resistive elements R3, R4, R5. The differential amplifiers306,400may be implemented as common gate amplifiers. Each of the resistive elements R3, R4, R5may comprise one or more of a transistor and/or a resistor. In the present embodiment the resistive element comprises a transistor having its gate coupled to a supply voltage VCC. An output of the differential amplifier400may be coupled to a gate G2of a transistor402. A negative input of the differential amplifier400may be coupled to a terminal TA of the transistor402. The resistive element R3may be coupled to a positive input of the differential amplifier400and a parallel combination of the resistive elements R4, R5may be coupled to the negative input of the differential amplifier400. In operation, the resistive element R3may receive a first portion of the current I1and the parallel combination of the resistive elements R4, R5may receive a second portion of the current I1. Preferably, first and second current portions are each approximately equal to half of the current I1. In a further embodiment, the second stage202may comprise a current divider404configured to divide the current I1into the first portion and the second portion. In the present embodiment, the current divider comprises the transistor308and a transistor405. The current divider404may be configured to provide the first portion of the current I1to the resistive element R3, and to provide the second portion of the current I1to the parallel combination of the resistive elements R4, R5. In the present embodiment, the apparatus further comprises a sense resistor Rsense coupled to the supply voltage VCC which is used to generate a sense voltage Vsense that is representative the output current Iout1. The sense voltage Vsense, may, for example, be measured at the location as shown on the Figure. In operation, the first stage200senses a voltage across the high side switch108and generates the current I1that is proportional to the inductor current IL. The current I1also contains information of the temperature variation, as the resistive elements R1, R2vary in temperature differently from the switch108. In operation the current I1of the first stage200is split in two and it is pushed into two impedances (provided by the resistive elements R3and the parallel combination of R4, R5). The impedances of the second stage202provided by the resistive elements R3, R4, R5are of the same type of the impedance of the first stage200(as provided by the resistive elements R1, R2) but connected in opposite way to the differential amplifier. Specifically, R1is of the same type as R3but they are coupled to different differential amplifier input types. Furthermore, R2is of the same type as R4and R5is of the same type as the switch108. In this way, the gain function provided by the differential amplifier400of the second stage202has an opposite temperature characteristic compared to the gain function of the differential amplifier306of the first stage200, thereby performing the temperature compensation. Furthermore, as the resistive elements R1, R2, R3, R4, R5are all subject to the same process variations, any variations due to process are also compensated for. Therefore in operation, the output current Iout1is compensated in respect of these errors. In summary, in the present example, the two operations of sensing the inductor current IL and compensating errors are split in the two different gain stages provided by the first stage200and the second stage202. FIG.4Bis a schematic of the apparatus100, showing a specific implementation of the second stage202in accordance with a sixth embodiment of the present disclosure. In the present example, the switch108is a low side switch of the switching converter104. In the present embodiment, the current sensor102comprises a current mirror transistor407configured to receive the current I1, and to mirror the current I1to the second stage202. In the present example, the current divider404comprises a transistor406and a transistor408. All of the observations made for the high side, as described inFIG.4Aare valid for the low side sense as described inFIG.4B. There are however some minor differences:The current mirror is used for practical reason such as:Generating an output current from the second stage202of the same type of the high side second stage202, so they can be connected to the same node.It is easier to drive the transistor of the R5device because its source is connected to ground and its gate can be connected to the existing voltage rail VCC. FIG.5Ais a schematic of an alternative embodiment of the apparatus100presented inFIG.4A. In the present embodiment, the first stage200further comprises an offset resistive element Roffset, for example a resistor. The parallel combination of the resistive element R2and the switch108are coupled to the positive input of the differential amplifier306via the offset resistive element Roffset. FIG.5Bis a schematic of an alternative embodiment of the apparatus100presented inFIG.4B. In the present embodiment, the first stage200further comprises the offset resistive element Roffset, for example a resistor. The parallel combination of the resistive element R2and the switch108are coupled to the negative input of the differential amplifier306via the offset resistive element Roffset. The offset resistive element Roffset may be a resistor used to adjust an offset current of the first stage200, in order to improve accuracy at low current and dynamic response. The LX node is connected between R2and Roffset because, the current in the low side pass device (the switch108) flows from source to drain (opposite direction of the high side) and it generates a negative drain source voltage Vds. Since the LX node is negative, it is connected to the negative input terminal of the differential pair of the differential amplifier306. FIG.6is a schematic of an apparatus600comprising two current sensors602,604in accordance with a seventh embodiment of the present disclosure. The current sensors602,604may be provided by any of the current sensors as described herein and in accordance with the understanding of the skilled person. The current sensor602is applied to a high side switch of the switching converter104and the current sensor604is applied to a low side switch of the switching converter104. For clarity of labelling, the reference numerals as previously defined in relation to single current sensors will be followed by “a” when described in relation to the current sensor602, and will be followed by “b” when described in relation to the current sensor604. For example, the switch108ais the high side switch that the current sensor602is coupled to, whereas the switch108bis the low side switch, that the current sensor604is coupled to. In operation, there is generated a total output current Itotal, that comprises the output currents Iout1aand Iout1bas generated by the current sensor602and the current sensor604, respectively. The total output current Itotal represents a reproduction of the inductor current IL as it includes the combination of the output currents Iout1a,Iout1b. FIG.7is a schematic of an apparatus700in accordance with an eighth embodiment. The apparatus700comprises current sensors702,704, where the current sensor702is coupled to the high side switch108aand the current sensor704is coupled to the low side switch108b.In the present example, the second stage202is shared between both of the current sensors702,704. The apparatus600functions otherwise as described for the apparatus700as will be clear to the skilled person. In the present example, the second stage202performs the same gain function in the high side current sense (the current sensor702) and the low side current sense (the current sensor704). FIG.8is a schematic of the apparatus100for a specific embodiment of the stages200,202for use with a high side switch108. In the present embodiment, the differential amplifier306comprises transistors800,802and current sources804,806, where the current source804has a current la and the current source806has a current Ib. The differential amplifier400comprises transistors808,810. FIG.9AandFIG.9Bare schematics illustrating current flows through the first stage200of the apparatus ofFIG.8. FIG.10AandFIG.10Bare schematics illustrating current flows through the second stage202of the apparatus ofFIG.8. The following is provided as analysis for the first stage200for high side switch current sensing. The feedback loop forces node “A” equal to node “B”, assuming that the resistance of the switch310is negligible, compared with R2. VA=(Ib+I1)·R1 (1) VB=VC+Ia·Roffset (2) VC=Ia·Rtotal(R2, R108)+IL·Rtotal(R2, R108) (3) Rtotal(R2, R108) is the total resistance of the parallel combination of the resistive element R2and the switch108, where R108is the resistance of the switch108. Imposing VA=VByields the following relation: (Ib+I1)·R1=Ia·Rtotal(R2, R108)+IL·Rtotal(R2, R108)+Ia·Roffset (4) The output current of the first stage200is then as follows: I1=IL·Rtotal(R2,R108)R1+Ia·Rtotal(R2,R108)+RoffsetR1-Ib(5) The following is provided as analysis for the second stage202for high side switch current sensing. The feedback loop forces node “D” equal to node “E”. VD=(Iout1+I12)·Rtotal(R4,R5)(6) Rtotal(R4, R5) is the total resistance of the parallel combination of the resistive elements R4, R5. VE=I12·R3(7) Imposing VD=VE: (Iout1+I12)·Rtotal(R4,R5)=I12·R3(8) The output current Iout1of the second stage202is: Iout1=I12·(R3Rtotal(R4,R5)-1)=I12·(R3-Rtotal(R4,R5)Rtotal(R4,R5))(9) We may then consider the combinations of the gain functions provided by the first and second stages200,202. The output current I1of the first stage200is provided by equation (5). The output current Iout1of the second stage202is provided by equation (9). Combining these equations yields the following: Iout1=12[(IL·Rtotal(R2,R108)R1)·(R3Rtotal(R4,R5)-1)+(Ia·Rtotal(R3,R108)+RoffsetR1-Ib)·(R3Rtotal(R4,R5)-1)](10) Equation (10) includes an offset term: Ioffset=12·(Ia·Rtotal(R3,R108)+RoffsetR1-Ib)·(R3Rtotal(R4,R5)-1)](11) and a gain term: Igain=12·(IL·Rtotal(R2,R108)R1)·(R3Rtotal(R4,R5)-1)(12) In order to cancel the offset Ioffset, which is not compensated in process and temperature, it is possible to impose: Ia·Rtotal(R2,R108)+RoffsetR1=Ib(13) Since the term Rtotal(R2, R108) is negligible compared with R2(for example, R2≈1 kOhm and R108≈50 mOhm), then it is possible to cancel any offset by imposing: Ia·RoffsetR1=Ib(14) Igain may be written as follows: Igain=12·(IL·Rtotal(R2,R108)R1)·(R3Rtotal(R4,R5)-1)=12·IL·(Rtotal(R2,R108)R1·R3Rtotal(R4,R5)-Rtotal(R2,R108)R1(15) Igain includes the following two terms: 12·IL·(Rtotal(R2,R108)R1·R3Rtotal(R4,R5))(16)12·IL·(Rtotal(R2,R108)R1)(17) Term (16) is compensated, as R3/R1varies with process and temperature in the same way as Rtotal(R3, R108)/Rtotal(R4, R5). Term (17) is not compensated but, since the term Rtotal(R2, R108) is negligible if R2and R1are in the order of kOhm and R108is in the order of mOhms, the overall number is in the order of mOhm/kOhm. FIG.11is a schematic of the apparatus100for a specific embodiment of the stages200,202for use with a low side switch108. FIG.12AandFIG.12Bare schematics illustrating current flows through the first stage200of the apparatus ofFIG.11. FIG.13AandFIG.13Bare schematics illustrating current flows through the second stage202of the apparatus ofFIG.11. The following is provided as analysis for the first stage200for low side switch current sensing. The feedback loop forces node “A” equal to node “B”, assuming that the resistance of the switch310is negligible compared with R2. VA=(Ib+I1)·Roffset+VC(18) VB=Ia·R1 (19) VC=(Ib+I1)·Rtotal(R2, R108)−IL·Rtotal(R2, R108) (20) Rtotal(R2, R108) is the total resistance of the parallel combination of the resistive element R2and the switch108, where R108is the resistance of the switch108. Imposing VA=VByields the following relation: (Ib+I1)·Roffset+(Ib+I1)·Rtotal(R2, R108)−IL·Rtotal(R2, R108)=Ia·R1 (21) The output current of the first stage200is: I1=IL·Rtotal(R2,R108)Roffset+Rtotal(R2,R108)+Ia·R1Roffset+Rtotal(R2,R108)-1b(22) The following is provided as analysis for the second stage202for low side switch current sensing. The feedback loop forces node “D” equal to node “E”. VD=(Iout1+I12)·Rtotal(R4,R5)(23) Rtotal(R4, R5) is the total resistance of the parallel combination of the resistive elements R4, R5. VE=I12·R3(24) Imposing VD=VE: The output current Iout1of the second stage202is: Iout1=I12·(R3Rtotal(R4,R5)-1)=I12·(R3-Rtotal(R4,R5)Rtotal(R4,R5))(25) We may then consider the combinations of the gain functions provided by the first and second stages200,202. The output current I1of the first stage200is provided by equation (22). The output current Iout1of the second stage202is provided by equation (25). Combining these equations yields the following: Iout1=12·[IL·Rtotal(R2,R108)Roffset+Rtotal(R2,R108)·(R3Rtotal(R4,R5)-1)+(IaR1(Roffset+Rtotal(R2,R108)-Ib)·(R3Rtotal(R4,R5)-1)](26) Equation (26) includes an offset term: Ioffset=12·(Ia·R1Roffset+Rtotal(R2,R108)-Ib)·(R3Rtotal(R4,R5)-1)](27) and a gain term: Igain=12·(IL·Rtotal(R2,R108)Roffset+Rtotal(R2,R108))·(R3Rtotal(R4,R5)-1)(28) In order to cancel the offset Ioffset, which is not compensated in process and temperature, it is possible to impose: Ia·R1(Roffset+Rtotal(R2,R108))=Ib(29) Since the term Rtotal(R2, R108) is negligible compared with Roffset (for example, Roffset≈1 kOhm and R108≈50 mOhm), then it is possible to cancel any offset by imposing: Ia·R1Roffset=Ib(30) Igain may be written as follows: Igain=12·(IL·Rtotal(R2,R108)Roffset+Rtotal(R2,R108))·(R3Rtotal(R4,R5)-1)=12·IL·(Rtotal(R2,R108)(Roffset+Rtotal(R2,R108)·R3Roffset(R4,R5)-Rtotal(R2,R108)(Roffset+Rtotal(R2,R108))(31) Igain includes the following two terms: 12·IL·(Rtotal(R2,R108)Roffset+Rtotal(R2,R108)·R3Roffset(R4,R5))(32)12·IL·(Rtotal(R2,R108)(Roffset+Rtotal(R2,R108))(33) By considering the approximation that: Roffset>>Rtotal(R2, R108)≈R108 (34) (for example, Roffset in the order of kOhm and R108in the order of mOhm), the term (32) can be written as follows: 12·IL·Rtotal(R2,R108)Roffset·R3Rtotal(R4,R5)(35) With this approximation, we can assume that R3and Roffset have the same variation with process and temperature, as well as Rtotal(R2, R108) and Rtotal(R4, R5) and the term is therefore compensated with process and temperature variation. Considering the approximation given by (34), the term (33) may be written as: 12·IL·Rtotal(R2,R108)Roffset(36) This term is not compensated but, since the term Rtotal(R2, R108) is negligible if Roffset is in the order of kOhm and R108is in the order of mOhms, the term is negligible. In summary, the output current Iout1is composed of two terms: one is proportional to the inductor current IL and the other is an offset. The offset term is partially compensated in temperature and can be nulled with the appropriate choice of component values. The gain term is also partially compensated against process and temperature; the residual part not compensated can be negligible with the appropriate choice of component values. DC sweep test simulations were performed for practical implementations of the apparatuses shown inFIGS.8and11whilst ILOAD varies from 0 to 5A. The following parameters were used for the simulation:NMOS switches for the high side and low side switches (108a,108b)V1afrom 4.5V to 42VVCC 5V+/−10%Current sense gain: 50 μA/AMaximum current, I_max=5A The DC sweep test was run for the following conditions:Temp=[−40:150]CACTIVE_STD=[ff ss fnsp_tbip snfp_tbip]ACTIVE_HV=[ff ss fnsp snfp]Passive_tsmc=[pass_lo pass_nom pass_hi] FIG.14is a graph showing the results for the DC sweep of the simulation of the circuit ofFIG.6. Shown on the graph are traces1500of the current I1a,traces1502of the current I1b,traces1504of the current Iout1aand traces1506of the current Iout1bacross the DC sweep of V1a. FIG.15Ashows the results of the simulation for the high side current sensor, including a schematic. FIG.15Bshows the results of the simulation for the low side current sensor, including a schematic. In the first stage200the current has a large variation, because it includes the temperature variation and the load information at the same time. The second stage204removes the temperature variation thereby compressing the spread of the output current Iout1a,Iout1bacross PVT. The table below shows the datapoints related to the DC sweep simulation across PVT. The error of measurement from the first stage is in the order of hundred percent, while the error in the second stage is compressed down to few unit of percentage. TABLE 1Slope/Intercept/RSQ across PVT-High SideMin (uA)Nom (uA)Max (uA)Min error (%)Max error (%)High SideHS_1st_stage_1A5.5416.2039.00−66%141%HS_1st_stage_2A10.9032.2078.10−66%143%HS_1st_stage_3A16.4048.30118.00−66%144%HS_1st_stage_4A21.8064.50157.00−66%143%HS_1st_stage_5A27.3080.70197.00−66%144%HS_2nd_stage_1A49.3051.5054.30−4%5%HS_2nd_stage_2A97.40102.00104.00−5%2%HS_2nd_stage_3A147.00152.00155.00−3%2%HS_2nd_stage_4A196.00203.00207.00−3%2%HS_2nd_stage_5A246.00253.00259.00−3%2%Low SideLS_1st_stage_1A11.4029.2074.00−61%153%LS_1st_stage_2A22.7058.50148.00−61%153%LS_1st_stage_3A34.0087.90222.00−61%153%LS_1st_stage_4A45.50117.00295.00−61%152%LS_1st_stage_5A56.90147.00363.00−61%147%LS_2nd_stage_1A49.0051.5056.10−5%9%LS_2nd_stage_2A98.90102.00107.00−3%5%LS_2nd_stage_3A148.00152.00158.00−3%4%LS_2nd_stage_4A196.00202.00210.00−3%4%LS_2nd_stage_5A240.00252.00261.00−5%4% It should be noted that optimisation for resistors values and replica device values has not been performed, so it will be possible to achieve an improvement on these results. Looking at the linearity of the output current Iout1a,Iout1b,it is possible to notice that either the first or second stage200,202output currents have very high RSQ coefficient. The slope in the first stage200has a considerable variation as the output current I1a,I1bis not compensated across process and temperature variation. The output current Iout1a,Iout1bof the second stage202, which implements the same uncompensated function but inverted, has a much more reduced slope variation. TABLE 2Slope/Intercept/RSQ across PVT-High SideMinNomMaxSlope 1st stage6.312515.826837.8875(uA/A)Intercept1st0.22680.33650.5446stage (uA)RSQ 1st stage1.00000.99990.9998Slope 2nd stage46.902549.590750.6850(uA/A)Intercept 2nd0.52251.81643.3936stage (uA)RSQ 2nd stage0.99990.99990.9997 TABLE 3Slope/Intercept/RSQ across PVT-Low SideMinNomMaxSlope 1st stage11.79729.74068.373(uA/A)Intercept1st27.358−1.166163.805stage (uA)RSQ 1st stage0.99900.99980.9998Slope 2nd stage44.92949.37750.697(uA/A)Intercept 2nd−0.338−0.9750.330stage (uA)RSQ 2nd stage0.99940.99990.9999 The data in the tables above, show the functioning of the current sensors of the present disclosure. Transient test simulations were performed for a practical implementation of the apparatus shown inFIG.6with the switching converter operating in PWM mode and the load varying from 0A to 4A with 1A of peak to peak current ripple. The following parameters were used for the simulation:DC load from 1 to 4 AI_ripple=1 Apk_pkfsw=1 MHzV1a=12VVCC=[4.5; 5.5]Temp=[−40; 150] The transient test was run for the following conditions:ACTIVE_STD =[ff ss fnsp_tbip snfp_tbip]ACTIVE_HV =[ff ss fnsp snfp]Passive_tsmc=[pass_lo pass_nom pass_hi] FIG.16is a graph showing the results for the transient test of the simulation of the circuit ofFIG.6for a low load. Shown on the graph are traces1800of the total output current Itotal, traces1802of the load current, traces1804of the output current Iout1band traces1806of the output current Iout1a. FIG.17is a graph showing the results for the transient test of the simulation of the circuit ofFIG.6for a medium load. Shown on the graph are traces1900of the total output current Itotal, traces1902of the load current, traces1904of the output current Iout1band traces1906of the output current Iout1a. FIG.18is a graph showing the results for the transient test of the simulation of the circuit ofFIG.6for a high load. Shown on the graph are traces2000of the total output current Itotal, traces2002of the load current, traces2004of the output current Iout1band traces2006of the output current Iout1a. Advantages of the current sensing methods described herein include:High voltage and low voltage operationSelf-compensated against process and temperatureNo trimming for gain is necessaryDesign suitable for PMOS/NMOS high side and low side switches The current sensing methods as described herein provide improved current measurement accuracy as they reduce errors arising, for example, due to temperature variations and/or process variations. The current measurements may, for example, be used as an output of the system for a user to determine whether the system is functioning correctly. Alternatively, or in addition to, providing the current measurement as an output to the user, the current measurement may be used internally by the system to control certain operations, to evaluate the functioning of the system and/or to take action in response to a specific current measurement, for example if it is indicative of a problem within the system. Various improvements and modifications may be made to the above without departing from the scope of the disclosure. | 28,849 |
11860200 | DETAILED DESCRIPTION Description will be made clearly and completely of technical solutions in the embodiments of the present disclosure in conjunction with accompanying drawings in the embodiments of the present disclosure. Obviously, the embodiments described here are only part of the embodiments of the present disclosure and are not all embodiments of the present disclosure. Based on the embodiments of the present disclosure, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present disclosure. Embodiment 1 As illustrated inFIG.1, the embodiment provides a zero crossing point signal output method. To achieve the above object, the technical solution of the present disclosure is specifically implemented as follows. At S101, zero crossing point square wave signals input by a zero-crossing detection circuit are continuously received, and each zero crossing point square wave signal is periodically sampled at a predetermined sampling frequency. In the embodiment, since a power system may typically provide a power frequency voltage of 50 HZ, that is, a power frequency voltage with a periodicity of 20 ms, for alternating current power, there are two moments in each alternating current periodicity at which the voltage value is zero, that is, two zero crossing points. However, the periodicity of the mains power supply generally has an error of about 1%-2%. Thus, when the zero crossing points are used for power line carrier communications, the periodicity and the zero crossing points cannot be determined directly by using the nominal mains frequency 50 HZ. Instead, the actual periodicity and zero crossing points need to be obtained again through detection and calculation, and then the actual periodicity and zero crossing points are used to conduct power line carrier communications. However, since the zero-crossing detection circuit generally includes analog devices, there is large error in detection of zero crossing points, and the zero crossing point square wave signals output by the zero-crossing detection circuit also have large errors. For example, for the zero crossing point square wave signals output by the zero-crossing detection circuit as illustrated inFIG.2, the periodicity of the zero crossing point square wave signals varies significantly. Hereinafter, the solution of the embodiment will be described by taking the received zero crossing point square wave signals being the zero crossing point square wave signals shown inFIG.2as an example. In the embodiment, each zero crossing point square wave signal is periodically sampled at a predetermined sampling frequency, that is, each zero crossing point square wave signal is sampled by using a high-speed clock. For example, the predetermined sampling frequency may be set to 2000 HZ. Taking the mains frequency being 50 HZ as an example, the sampling number of the zero crossing point square wave signal is usually about 40. By periodically sampling each zero crossing point square wave signal, the periodicity of each zero crossing point square wave signal can be obtained, and the next calculation is further executed. In an optional implementation of the embodiment, after the zero crossing point square wave signals input by the zero-crossing detection circuit are continuously received, filtering is performed on each of the received zero crossing point square wave signals to filter out high-frequency noise in each zero crossing point square wave signal, and then each zero crossing point square wave signal is periodically sampled at the predetermined sampling frequency. In the optional implementation, the zero crossing point square wave signals are periodically sampled after the high-frequency noise is filtered out, so that misjudgment of the zero crossing point square wave signals caused by the existence of the high-frequency noise and hence incorrect sampling results due to the misjudgment are avoided. At S102, a sampling number of each zero crossing point square wave signal in 1stto Mthzero crossing point square wave signals is acquired to obtain sampling numbers of M zero crossing point square wave signals, an average value of the sampling numbers of the M zero crossing point square wave signals is calculated to obtain an average sampling number S, and a first zero crossing point interval T1 is calculated based on the average sampling number S and the predetermined sampling frequency; and a zero crossing point signal output interval is set as the first zero crossing point interval T1, where M is a predetermined value and is a positive integer greater than or equal to 1. In the embodiment, partial sampling results are obtained by periodically sampling each zero crossing point square wave signal at S101. For example, supposing a predetermined value M=5, the sampling number of each zero crossing point square wave signal in the 1stto 5thzero crossing point square wave signals is obtained. The sampling numbers of the 1stto 5thzero crossing point square wave signals are 38, 39, 45, 36, and 37, respectively, and the average value of the sampling numbers of the five zero crossing point square wave signals is calculated to be 39, that is, the average sampling number S=39. In the embodiment, the first zero crossing point interval T1 is calculated based on the average sampling number S and a predetermined sampling frequency. That is, the average periodicity of the zero crossing point square wave signals is calculated based on the average sampling number S and the predetermined sampling frequency, and then the first zero crossing point interval T1 is obtained based on the average periodicity of the zero crossing point square wave signals. Alternatively, since in alternating current there are 2 zero crossing points in each periodicity of the zero crossing point square wave signals, the interval between zero crossing points should be one half of a periodicity. For example, when the average sampling number S is 39 and the predetermined sampling frequency is 2000 HZ, the average periodicity of the zero crossing point square wave signals is 0.0195 s, that is, the first zero crossing point interval T1 is 0.00975 s, and the zero crossing point signal output interval is set to 0.00975 s. By calculating the average value of the sampling numbers of the M zero crossing point square wave signals and obtaining the first zero crossing point interval by using the average sampling number, the error of the zero crossing point square wave signals output by the zero-crossing detection circuit can be reduced, and more accurate zero crossing point interval can be obtained. At S103, zero crossing point signals with an interval being the zero crossing point signal output interval are continuously output; a sampling number of each zero crossing point square wave signal in M+1thto M+Nthzero crossing point square wave signals is obtained to obtain sampling numbers of N zero crossing point square wave signals, a difference value between each of the sampling numbers of the N zero crossing point square wave signals and the average sampling number S is calculated to obtain N sampling number difference values, and the N sampling number difference values are summed to obtain an accumulated difference value Δs, where N is a predetermined value and is a positive integer greater than or equal to 1. In the embodiment, the zero crossing point signals may be output in various modes. For example, in a first mode, zero crossing point signals are output as high-frequency pulses. As illustrated inFIG.3, when the output interval of the zero crossing point signals is 0.00975 s, a high-frequency pulse is output every 0.00975 s, and the time when the high-frequency pulse occurs is the zero crossing point time. In a second mode, the zero crossing point signals are output as alternating high and low levels. As illustrated inFIG.4, when the output interval of the zero crossing point signals is 0.00975 s, a high-level signal with a duration of 0.00975 s and a low-level signal with a duration of 0.00975 s are alternately output, and the time when the level changes is the zero crossing point time. Zero crossing point signals with an interval being the zero crossing point signal output interval are output, the zero crossing point signals can be used to conduct zero crossing point power line communications, and the problem of inaccurate zero crossing point time and low communication efficiency due to a large error in the detection result of analog devices in the zero-crossing detection circuit can be avoided. In the embodiment, while using the zero crossing point signal output interval to output the zero crossing point signals, the zero crossing point signal output interval is calculated and corrected by further using a sum of difference values between sampling numbers of a predetermined number of the zero crossing point square wave signals and the average sampling number S. For example, as illustrated inFIG.2, supposing the predetermined value N=5, the sampling number of each zero crossing point square wave signal of the 6thto 10thzero crossing point square wave signals is obtained. The sampling numbers of the five zero crossing point square wave signals are 38, 36, 35, 32, and 40, respectively. Thus, the difference value between each of the sampling number 38, 36, 35, 32 and 40 and the average sampling number 39 is calculated to obtain five sampling number difference values as 1, 3, 4, 7, and −1 respectively, and the five sampling number difference values are summed to obtain the accumulated difference value Δs as 14. At S104, when Δs is not within a predetermined change range, a second zero crossing point interval T2 is calculated in accordance with a predetermined rule, and the zero crossing point signal output interval is set as the second zero crossing point interval T2; and when Δs is within the predetermined change range, the zero crossing point signal output interval is kept unchanged. In the embodiment, after the accumulated difference value Δs is calculated, the zero crossing point signal output interval is adjusted based on Δs. That is, by determining whether Δs is within the predetermined change range, it is determined whether the second zero crossing point interval T2 is calculated in accordance with the predetermined rule. When Δs is not within the predetermined change range, the second zero crossing point interval T2 is calculated in accordance with the predetermined rule, and the zero crossing point signal output interval is set as the second zero crossing point interval T2; and when Δs is within the predetermined change range, the zero crossing point signal output interval is still T1. By determining whether the accumulated difference value Δs is within the predetermined change range to adjust the zero crossing point signal output interval, the zero crossing point signal output interval can be more precise, and the communication efficiency is improved. For example, when the predetermined change range is [−5,5] and Δs=14 which is not within the predetermined change range, the current zero crossing point signal output interval is considered to be inaccurate and needs to be adjusted, and the second zero crossing point interval T2 is calculated in accordance with the predetermined rule. When Δs=3 which is within the predetermined change range, the current zero crossing point signal output interval is considered to be accurate without having to be adjusted. Thus, the zero crossing point signal output interval is still T1. In an optional implementation of the embodiment, T2=T1−Δt is calculated when Δs is smaller than a minimum value of the predetermined change range, and T2=T1+Δt is calculated when Δs is greater than a maximum value of the predetermined change range, wherein Δt is a predetermined correction value. For example, the predetermined change range is [−5,5], the predetermined correction value Δt is 0.00005 s. When Δs is =−7, that is, Δs is smaller than the minimum value of the predetermined change range, the second zero crossing point interval T2=T1−Δt=0.00975 s−0.00005 s=0.0097 s. When Δs=14, that is, Δs is greater than the maximum value of the predetermined change range, the second zero crossing point interval T2=T1+Δt=0.0098 s. In the optional implementation, the zero crossing point signal output interval is corrected by setting the predetermined correction value Δt instead of directly setting the zero crossing point signal output interval based on the sampling numbers of the zero crossing point square wave signals, thereby avoiding an inaccurate zero crossing point signal output interval, which results from great fluctuation and large error in the output zero crossing point square wave signals of the zero-crossing detection circuit due to its components being affected by environmental changes and the like. At S105, zero crossing point signals with an interval being the zero crossing point signal output interval are continuously output. In the embodiment, when the zero crossing point output interval is T1, zero crossing point signals having an interval of 0.00975 s are continuously output, and when the zero crossing point output interval is T2, zero crossing point signals having an interval of 0.0098 s are continuously output. The mode of outputting the zero crossing point signals may be the same as that at S103. By adjusting the zero crossing point signal output interval according to the sampling numbers of the input zero crossing point square wave signals, the accuracy of outputting the zero crossing point signals can be improved, and the communication efficiency can be improved. According to the technical solution of the present disclosure, the embodiment provides a zero crossing point signal output method. After receiving zero crossing point square wave signals input by a zero-crossing detection circuit, an average sampling number is obtained by calculating a sampling number of each zero crossing point square wave signal in 1stto Mthzero crossing point square wave signals, a zero crossing point signal output interval is obtained based on the average sampling number and a predetermined sampling frequency, zero crossing point signals are output based on the zero crossing point signal output interval, sampling numbers of subsequent zero crossing point square wave signals are continuously calculated, the zero crossing point signal output interval is adjusted based on the subsequent zero crossing point square wave signals, the zero crossing point signals are output based on the adjusted result. By using the zero crossing point signal output method to correct a detection result of the zero-crossing detection circuit and output the zero crossing point signals, the zero crossing point signals output according to the method have small errors, and when such zero crossing point signals are used to conduct power line communications, the problem of low zero crossing point communication efficiency due to a large error in the detection result of the zero-crossing detection circuit is avoided, and the communication efficiency is improved. Embodiment 2 The present embodiment provides a computer-readable storage medium including computer instructions. The computer instructions, when executed, implement the zero crossing point signal output method in Embodiment 1. For specific operations, reference may be made to Embodiment 1, which is not described herein again. Embodiment 3 As illustrated inFIG.5, the embodiment provides a power line data transmitting method, which includes the zero crossing point signal output method in Embodiment 1. Therefore, specific implementations and optional implementations of S201, S202, S204, and S205are the same as those of S101, S102, S104, and S105in Embodiment 1, and the same parts as those in Embodiment 1 are not repeated and only different parts are described in detail. At S201, zero crossing point square wave signals input by a zero-crossing detection circuit are continuously received, and each zero crossing point square wave signal is periodically sampled at a predetermined sampling frequency. At S202, a sampling number of each zero crossing point square wave signal in 1stto Mthzero crossing point square wave signals is acquired to obtain sampling numbers of M zero crossing point square wave signals, an average value of the sampling numbers of the M zero crossing point square wave signals is calculated to obtain an average sampling number S, and a first zero crossing point interval T1 is calculated based on the average sampling number S and the predetermined sampling frequency; and a zero crossing point signal output interval is set as the first zero crossing point interval T1, where M is a predetermined value and is a positive integer greater than or equal to 1. At S203, zero crossing point signals with an interval being the zero crossing point signal output interval are continuously output; a 1stzero crossing point time t is determined based on the zero crossing point signals, and a start time of transmitting a synchronization signal of a data packet to be transmitted is determined based on the 1stzero crossing point time, where the start time of transmitting the synchronization signal of the data packet to be transmitted is t+t1, t+t1is earlier than a 2ndzero crossing point time t+T, t+T is contained in a time period from a time point of transmitting a first synchronization bit signal to a time point of transmitting a last synchronization bit signal, and t1is a first predetermined fixed value; and data bit signals of the data packet to be transmitted are sequentially transmitted; a sampling number of each zero crossing point square wave signal in M+1thto M+Nthzero crossing point square wave signals is obtained to obtain sampling numbers of N zero crossing point square wave signals, a difference value between each of the sampling numbers of the N zero crossing point square wave signals and the average sampling number S is calculated to obtain N sampling number difference values, and the N sampling number difference values are summed to obtain an accumulated difference value Δs, where N is a predetermined value and is a positive integer greater than or equal to 1. In the embodiment, after continuously outputting zero crossing point signals with an interval being the zero crossing point signal output interval, the 1stzero crossing point time is determined based on the zero crossing point signals, and the start time of the synchronization signal of the data packet to be transmitted is determined based on the 1stzero crossing point time, where the start time is earlier than the 2ndzero crossing point time, so that the time period for transmitting the synchronization signal of the data packet to be transmitted contains the 2ndzero crossing point time. After the transmission of the synchronization signal is finished, the data bit signals of the data packet to be transmitted are sequentially transmitted. Since the time period for transmitting the synchronization signal of the data packet to be transmitted contains the zero crossing point time, near which there is less interference, the efficiency of signal transmission is higher. This also facilitates a signal receiving end to determine whether received signals are near a zero crossing point and determine whether the signals are the synchronization signal, so that information can be transmitted efficiently and completely. At S204, when Δs is not within a predetermined change range, a second zero crossing point interval T2 is calculated in accordance with a predetermined rule, and the zero crossing point signal output interval is set as the second zero crossing point interval T2; and when Δs is within the predetermined change range, the zero crossing point signal output interval is kept unchanged. At S205, zero crossing point signals with an interval being the zero crossing point signal output interval are continuously output. According to the technical solutions provided by the present disclosure, the present disclosure provides a power line data transmitting method. After receiving zero crossing point square wave signals input by the zero-crossing detection circuit, the average sampling number is obtained by calculating the sampling number of each zero crossing point square wave signal in 1stto Mthzero crossing point square wave signals, the zero crossing point signal output interval is obtained based on the average sampling number and the predetermined sampling frequency, the zero crossing point signals are output based on the zero crossing point signal output interval, a zero crossing point time is determined based on the zero crossing point signals, the start time of transmitting the synchronization signal of the data packet to be transmitted is determined, such that the zero crossing point time is contained in the time period during which the synchronization signal is transmitted, sampling numbers of subsequent zero crossing point square wave signals are continuously calculated, the zero crossing point signal output interval is adjusted based on the subsequent zero crossing point square wave signals, and the zero crossing point signals are output based on the adjusted result. By using the power line data transmitting method as described above to correct a detection result of the zero-crossing detection circuit and output the zero crossing point signals, when the zero crossing point signals output according to the method are used to conduct power line communications, the problem of low zero crossing point communication efficiency due to a large error in the detection result of the zero-crossing detection circuit is avoided, and the communication efficiency is improved. Meanwhile, the zero crossing point signal is contained in the time period for transmitting the synchronization signal of the data packet to be transmitted, so that a receiving end can determine whether received signals are near a zero crossing point and determine whether the signals are the synchronization signal, and thus information can be transmitted efficiently and completely. Embodiment 4 The present embodiment provides a computer-readable storage medium including computer instructions. The computer instructions, when executed, implement the power line data transmitting method in Embodiment 3. For specific operations, reference may be made to Embodiment 3, which is not described herein again. Embodiment 5 In the embodiment, a zero crossing point signal output apparatus is provided. The apparatus corresponds to the zero crossing point signal output method in Embodiment 1, and thus details thereof are not repeated herein and only a brief description is provided. In an optional implementation of the embodiment, for specific operations performed by each unit in the zero crossing point signal output apparatus, reference may be made to Embodiment 1. In the embodiment, the zero crossing point signal output apparatus may be included in any communication terminal in power line communications, for example, a camera, a Personal Computer (PC), a server, or the like, or may be an independent device. FIG.6illustrates an optional zero crossing point signal output apparatus300of the present embodiment, including: a receiving module301, a sampling module302, a calculating module303and an outputting module304. The receiving module301is configured to continuously receive zero crossing point square wave signals input by a zero-crossing detection circuit. The sampling module302is configured to periodically sample each zero crossing point square wave signal at a predetermined sampling frequency. The calculating module303is configured to acquire a sampling number of each zero crossing point square wave signal in 1stto Mthzero crossing point square wave signals to obtain sampling numbers of M zero crossing point square wave signals, calculate an average value of the sampling numbers of the M zero crossing point square wave signals to obtain an average sampling number S, and calculate a first zero crossing point interval T1 based on the average sampling number S and the predetermined sampling frequency; and set a zero crossing point signal output interval as the first zero crossing point interval T1, wherein M is a predetermined value and is a positive integer greater than or equal to 1. The outputting module304is configured to continuously output zero crossing point signals with an interval being the zero crossing point signal output interval. The calculating module303is further configured to obtain a sampling number of each zero crossing point square wave signal in M+1thto M+Nthzero crossing point square wave signals to obtain sampling numbers of N zero crossing point square wave signals, calculate a difference value between each of the sampling numbers of the N zero crossing point square wave signals and the average sampling number S to obtain N sampling number difference values, and sum the N sampling number difference values to obtain an accumulated difference value Δs, where N is a predetermined value and is a positive integer greater than or equal to 1; calculate, when Δs is not within a predetermined change range, a second zero crossing point interval T2 in accordance with a predetermined rule, and set the zero crossing point signal output interval as the second zero crossing point interval T2; and when Δs is within the predetermined change range, keep the zero crossing point signal output interval unchanged. As a preferred implementation of the embodiment, the zero crossing point signal output apparatus300of the embodiment further includes a filtering module (not shown). The filtering module is configured to, subsequent to the receiving module continuously receiving the zero crossing point square wave signals input by the zero-crossing detection circuit, perform filtering on each of the received zero crossing point square wave signals to filter out high-frequency noise in each zero crossing point square wave signal. In the optional implementation, the zero crossing point square wave signals are periodically sampled after using the filtering module to filter the high-frequency noise out, so that misjudgment of the zero crossing point square wave signals caused by the existence of the high-frequency noise and hence incorrect sampling results due to the misjudgment are avoided. As a preferred implementation of the embodiment, the calculating module303being configured to calculate, when Δs is not within the predetermined change range, the second zero crossing point interval T2 in accordance with the predetermined rule includes: the calculating module303being configured to calculate T2=T1−Δt when Δs is smaller than a minimum value of the predetermined change range, and calculate T2=T1+Δt when Δs is greater than a maximum value of the predetermined change range, wherein Δt is a predetermined correction value. For example, the predetermined change range is [−5,5], the predetermined correction value Δt is 0.00005 s; when Δs=−7, that is, Δs is smaller than the minimum value of the predetermined change range, the second zero crossing point interval T2=T1−Δt=0.00975 s−0.00005 s=0.0097 s; and when Δs=14, that is, Δs is greater than the maximum value of the predetermined change range, the second zero crossing point interval T2=T1+Δt=0.0098 s. In the optional implementation, the zero crossing point signal output interval is corrected by setting the predetermined correction value Δt instead of directly setting the zero crossing point signal output interval based on the sampling numbers of the zero crossing point square wave signals, thereby avoiding an inaccurate zero crossing point signal output interval, which results from great fluctuation and large error in the output zero crossing point square wave signals of the zero-crossing detection circuit due to its components being affected by environmental changes and the like. According to the technical solutions provided by the present disclosure, the present embodiment provides a zero crossing point signal output apparatus300. After the receiving module301receives the zero crossing point square wave signals input by the zero-crossing detection circuit, the sampling module302periodically samples the zero crossing point square wave signals, the calculating module303obtains an average sampling number by calculating the sampling number of each zero crossing point square wave signal in the 1stto Mthzero crossing point square wave signals, and obtains a zero crossing point signal output interval based on the average sampling number and a predetermined sampling frequency, the outputting module304outputs zero crossing point signals based on the zero crossing point signal output interval, the calculating module303continuously calculates sampling numbers of subsequent zero crossing point square wave signals, the zero crossing point signal output interval is adjusted based on the subsequent zero crossing point square wave signals, and the outputting module304outputs the zero crossing point signals based on the adjusted result. By using the apparatus to correct the detection result of the zero-crossing detection circuit and output the zero crossing point signals, the zero crossing point signals output by the apparatus have small errors, and when such zero crossing point signals are used for conducting power line communications, the problem of low zero crossing point communication efficiency caused by a large error in the detection result of the zero-crossing detection circuit is avoided, and the communication efficiency is improved. Embodiment 6 The embodiment provides a power line data transmitting device, which includes the zero crossing point signal output apparatus300in Embodiment 5. The device corresponds to the power line data transmitting method in Embodiment 3, and thus details of the same parts are not repeated herein and only a brief description is provided. In an optional implementation of the embodiment, for the specific operations performed by each unit in the power line data transmitting device, reference may be made to Embodiments 3 and 5. In the embodiment, the power line data transmitting device may be any communication terminal in power line communications, for example, a camera, a PC, a server, etc., and may also be included in another terminal device. FIG.7is an optional power line data transmitting device400of the embodiment, which includes the zero crossing point signal output apparatus300disclosed in Embodiment 5, and a data outputting module401. The data outputting module401is configured to, subsequent to receiving the zero crossing point signals with the interval being the zero crossing point signal output interval which are continuously output by the outputting module304, determine a 1st zero crossing point time t based on the zero crossing point signals, and determine a start time of transmitting a synchronization signal of a data packet to be transmitted based on the 1st zero crossing point time, wherein the start time of transmitting the synchronization signal of the data packet to be transmitted is t+t1, t+t1is earlier than a 2nd zero crossing point time t+T, t+T is included in a time period from a time point of transmitting a first synchronization bit signal to a time point of transmitting a last synchronization bit signal, and t1 is a first predetermined fixed value; and sequentially transmit data bit signals of the data packet to be transmitted. According to the above technical solutions provided by the present disclosure, the embodiment provides a power line data transmitting device400, which includes the zero crossing point signal output apparatus300in Embodiment 5. After the receiving module301receives zero crossing point square wave signals input by the zero-crossing detection circuit, the sampling module302periodically samples the zero crossing point square wave signals, the calculating module303obtains an average sampling number by calculating the sampling number of each zero crossing point square wave signal from the 1stto the Mthzero crossing point square wave signal, and obtains a zero crossing point signal output interval based on the average sampling number and a predetermined sampling frequency, the outputting module304outputs zero crossing point signals based on the zero crossing point signal output interval, the data outputting module401determines a zero crossing point time based on the zero crossing point signals, and determines a start time of transmitting a synchronization signal of a data packet to be transmitted, so that the zero crossing point time is contained in a time period for transmitting the synchronization signal, the calculating module303continuously calculates the sampling numbers of the subsequent zero crossing point square wave signals, and adjusts the zero crossing point signal output interval based on the subsequent zero crossing point square wave signals, and the outputting module304outputs the zero crossing point signals based on the adjusted result. By using the device to correct the detection result of the zero-crossing detection circuit and output the zero crossing point signals, when the zero crossing point signals output by the device are used to conduct power line communications, the problem of low zero crossing point communication efficiency caused by a large error in the detection result of the zero-crossing detection circuit is avoided, and the communication efficiency is improved. Meanwhile, the zero crossing point signal is contained in the time period for transmitting the synchronization signal of the data packet to be transmitted, so that a receiving end can determine whether received signals are near a zero crossing point and determine whether the signals are the synchronization signal, and thus information can be transmitted efficiently and completely. Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made to the embodiments within the scope of the present disclosure by those skilled in the art, without departing from the principle and purpose of the present disclosure. The scope of the present disclosure is defined by the appended claims and equivalents thereof. | 34,582 |
11860201 | DESCRIPTION Various examples are now described more thoroughly with reference to the accompanying figures, which depict a few examples. The thicknesses of lines, layers and/or regions in the figures may be exaggerated for clarification purposes. While further examples are suitable for various modifications and alternative forms, some specific examples thereof are accordingly shown in the figures and are described thoroughly below. However, this detailed description does not restrict further examples to the specific forms described. Further examples can cover all modifications, counterparts and alternatives that fall within the scope of the disclosure. Throughout the description of the figures, identical or similar reference signs refer to identical or similar elements which can be implemented identically or in modified form in comparison with one another, while they provide the same or a similar function. It goes without saying that if one element is referred to as “connected” or “coupled” to another element, the elements can be connected or coupled directly or via one or more intermediate elements. If two elements A and B are combined using an “or”, this should be understood such that all possible combinations are disclosed, e.g. only A, only B, and A and B, unless explicitly or implicitly defined otherwise. An alternative wording for the same combinations is “at least one of A and B” or “A and/or B”. The same applies, mutatis mutandis, to combinations of more than two elements. The terminology used here to describe specific examples is not intended to have a limiting effect for further examples. When a singular form, e.g. “a, an” and “the”, is used, and the use of only a single element is defined neither explicitly nor implicitly as obligatory, further examples can also use plural elements in order to implement the same function. If a function is described below as implemented using multiple elements, further examples can implement the same function using a single element or a single processing entity. Furthermore, it goes without saying that the terms “comprises”, “comprising”, “has” and/or “having” in their usage specify the presence of the indicated features, integers, steps, operations, processes, elements, components and/or a group thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, processes, elements, components and/or a group thereof. Unless defined otherwise, all terms (including technical and scientific terms) are used here in their customary meaning in the field with which examples are associated. FIG.1Ashows a conventional system comprising a current sensor chip10and a microcontroller30. The current sensor chip10measures a current in a line11and provides an analog measurement signal on a unidirectional analog signal interface12(AOUT). A unidirectional overcurrent interface13(OCD: OverCurrent Detection) of the current sensor chip10can be used to indicate an overcurrent signal to the microcontroller30, which signal signals whether the measured current is above a threshold value. The overcurrent signal may be a digital or binary signal (e.g. “high”=current too high, “low”=current not too high). The analog signal path between the analog signal interface12of the current sensor chip10and an ADC31of the microcontroller30has an analog circuit40for signal conditioning. In order to provide a functionally safe sensor system, the implementation shown inFIG.1Ahas the entire signal chain from the sensor chip10to the output of the ADC31monitored. A possible implementation of the current sensor chip10is shown inFIG.1B. The current through the line11is measured using a differential Hall sensor14as primary sensor. A resultant analog raw measurement signal15is supplied to an analog signal conditioning circuit16, an analog amplifier circuit17and an analog filter circuit18before the resultant filtered analog measurement signal19is provided on the analog signal interface12(AOUT). An amplified measurement signal at the filter input is additionally supplied to one or more overcurrent detection circuits20in order to compare it with one or more threshold values21. The overcurrent detection circuits20provide (digital) overcurrent signals on one or more unidirectional overcurrent interfaces13. The (programmable) threshold values21can be stored in a nonvolatile memory22of the current sensor chip10. Further, the current sensor chip10shown here additionally contains a secondary temperature sensor23and a secondary mechanical stress sensor24, the measurement signals of which influence a gain of the analog amplifier circuit17and can therefore be used for temperature and stress compensation. In conventional implementations, the integrity of sensor reading can be checked by adding redundancy in the system. In some implementations, this is accomplished by doubling the number of sensors (for example for battery management systems), as shown inFIG.1C(left). In electrical drive applications (seeFIG.1C, right), it is possible to employ current sensors in all of the phases. Since the sum of all currents must be zero (provided that there is no leakage current), the integrity of the sensor system (consisting of the sensors for all of the phases) can be checked. However, it is not possible to identify a single sensor that causes an incorrect output value, which is a problem particularly in high-availability systems, which must continue to operate despite a single error, for example a faulty sensor. In conventional sensor systems, there is thus merely a unidirectional flow of information from the sensor chip10to the microcontroller30via the signal interfaces12and13. This is shown schematically inFIG.1Dfor n sensors10-1, . . . ,10-non a microcontroller (μC)30. Diagnosis modes for the sensors10-1, . . . ,10-nare difficult to initiate and there is no (dynamic) change of sensor settings. Furthermore, nonvolatile memories22in the current sensor chips10-1, . . . ,10-nare programmed in an EoL programming. There is furthermore also no transmission channel for additional sensor information (for example functional safety or diagnosis). In order to overcome at least some of these disadvantages, a contrastingly improved sensor system200is proposed, which is depicted schematically inFIG.2A. The sensor system200comprises a microcontroller230and at least one sensor chip210. In the example implementation shown, there are n sensor chips210-1, . . . ,210-npresent (n=1, 2, 3, . . . ). Each of the sensor chips210-1, . . . ,210-nis designed to measure at least one physical quantity, such as for example a magnetic field or an electric current. The microcontroller230and the sensor chips210-1, . . . ,210-nare coupled via n unidirectional analog signal interfaces for conveying analog measurement data from the respective sensor chip210-1, . . . ,210-nto the microcontroller230. From each sensor chip210-1, . . . ,210-n, at least one analog line212-1, . . . ,212-nis thus routed to the microcontroller230. From each sensor chip210-1, . . . ,210-n, it is optionally also possible for a unidirectional digital overcurrent interface213-1, . . . ,213-nto be routed to the microcontroller230. The microcontroller230and the sensor chips210-1, . . . ,210-nare additionally coupled to one another via a bidirectional digital signal interface250for conveying digital secondary information between the sensor chips210-1, . . . ,210-nand the microcontroller230. The digital secondary information is not a unidirectional 1-bit overcurrent indicator. According to some example implementations, the digital secondary information is also not measured values of the physical quantity that is primarily measured (for example magnetic field, current, angle, etc.), since these are conveyed in analog fashion via the lines212-1, . . . ,212-n, of course. Instead, it may be contrasting digital sensor control data (commands), sensor diagnosis data, configuration and/or calibration data, sensor parameters, etc. The digital secondary information via the bidirectional digital signal interface250, such as for example control commands, addresses, data, can have far more than just one bit. In order to increase functional safety of the sensor system200, there may be provision in some implementations, however, for digital measured values of the physical quantity that are to be measured to also be transmitted from at least some of the sensor chips210-1, . . . ,210-nto the microcontroller230via the bidirectional digital signal interface250in addition to the analog measured values via the analog lines212-1, . . . ,212-n. In order both to solve the diagnosis problem and to allow transmission of extended diagnosis information between a sensor chip210-1, . . . ,210-nand the microcontroller230, a bidirectional, digital control and diagnosis interface250(DCDI) is thus proposed. The bidirectional digital signal interface250can then allow the following functions: a) Download of configuration and calibration data during startup. The applicable information can be stored in a nonvolatile memory of the microcontroller230and loaded into a sensor chip210when the sensor chip is switched on. b) Dynamic adaptation of the sensor parameters. Critical parameters such as bandwidth, gain and offset of individual sensor chips210-1, . . . ,210-ncan be adjusted during normal operation of the sensor chip. c) Upload of extended diagnosis information. Available diagnosis data inside a sensor chip210can be monitored via the diagnosis interface250. The sensor chip210can deliver e.g. realtime information about the supply voltage monitoring, a busbar temperature, etc. d) Controlled activation of test sequences. In order to activate a test sequence, the microcontroller230can perform timely activation of a test sequence. As an example, a sensor chip210can be forced to imitate a defined current pattern so as not only to monitor the integrity of the sensor but also to check the integrity of the entire signal processing path from the sensor chip210to the μC processing core. FIG.2Bschematically shows a microcontroller230coupled to a plurality of n sensor chips210-1, . . . ,210-nvia analog and digital interfaces. Each of the sensor chips210-1, . . . ,210-nhas a respective unidirectional analog signal output via lines212-1, . . . ,212-n(AOUT) for a current measurement signal. The microcontroller230provides appropriate analog signal inputs232-1, . . . ,232-ntherefor. Each of the sensor chips210-1, . . . ,210-nhas a respective unidirectional digital overcurrent output213-1, . . . ,213-n(OCD), which is coupled by the microcontroller230to the overcurrent interface233thereof. Each of the sensor chips210-1, . . . ,210-nhas a respective bidirectional digital control and diagnosis interface250-1, . . . ,250-n(DCDI), which are coupled by the microcontroller230to the bidirectional digital control and diagnosis interface250thereof. Optionally, the sensor chips210-1, . . . ,210-neach also have an analog input260-1, . . . ,260nfor a setting voltage (VREF) for setting an operating point of the respective analog amplifier circuit17. The microcontroller230provides appropriate analog signal outputs234-1, . . . ,234ntherefor. The unidirectional analog signal outputs on lines212-1, . . . ,212nand the bidirectional digital signal interface250can be used to convey analog and digital signals between the sensor chips210-1, . . . ,210nand the microcontroller230in parallel. While analog measurement signals (for example current measurement signals) go to the microcontroller230via the signal outputs on lines212-1, . . . ,212nof the sensor chips210-1, . . . ,210n, the digital signal interface250can be used to interchange other, digital data between the microcontroller230and the sensor chips210-1, . . . ,210n. As indicated inFIG.2B, the bidirectional digital signal interface250between the sensor chips210-1, . . . ,210nand the microcontroller230may be in the form of a bidirectional 1-line interface and act as a single-master (microcontroller230) multi-slave (sensor chips210-1, . . . ,210n) bus system. The microcontroller230thus gives commands and makes requests; the sensor chips210-1, . . . ,210nmerely react. The bus master controls the flow of communication by sending master request frames to the sensor bus. Depending on the master request frames, information is transmitted to the sensor (write frame) or received from the sensor (read frame). It is therefore easily possible for numerous sensors to be interfaced with the microcontroller230. The digital control and diagnosis interface250may be in the form of a single-wire UART interface (UART=Universal Asynchronous Receiver Transmitter), for example. Digital secondary information can be transmitted between the microcontroller230and the sensor chips210-1, . . . ,210nvia the digital control and diagnosis interface250as a serial digital data stream having fixed frames. As depicted in the example inFIG.3A, a frame310can comprise a start bit311, some data bits312, an optional parity bit for detecting transmission errors and a stop bit313. The transmitter does not need to notify the receiver of the transmission clock via a separate control line. Instead, the receiver can calculate the clock of the transmitter from the clock of the data line and synchronize itself thereto using the start and stop bits. The frames310can be encoded as 8-bit UART frames, for example. Information can be transmitted on a physical layer comprising a single wire. For example, there may be provision for a baud rate, programmable via the microcontroller230, of e.g. 38.4 kbit/s to 115.2 kbit/s (for example 4 speeds). By way of example, the line code used for transmitting information can be a Manchester code or a Non-Return-to-Zero (NRZ) or Non-Return-to-Zero-Inverted (NRZI) coding. Different types of communication can take place between the microcontroller230and the sensor chips210-1, . . . ,210n. A first possible communication is shown inFIG.3B(top). FIG.3B(top) shows a possible data communication relating to a request for sensor data—such as e.g. sensor state data or diagnosis data—by the microcontroller230. The microcontroller230first uses a command frame320to request specific sensor state data or diagnosis data via the digital signal interface250. Depending on the number of possible commands, a corresponding number of bits can be used for encoding them. The microcontroller230uses a subsequent address frame330via the digital signal interface250to indicate which of the sensor chips210-1, . . . ,210nit would like the sensor state data or diagnosis data from. Depending on the number of possible addresses, a corresponding number of bits can be used for encoding them. On the basis of the sensor ID contained in the address frame330, the addressed sensor chip conveys the requested sensor state data or diagnosis data to the microcontroller230in one or more data frames340, followed by a safety data frame350. It will be obvious to a person skilled in the art that an order for the depicted command, address and data frames could also be chosen differently. FIG.3B(bottom) further shows a possible data communication relating to a sending of data—such as e.g. configuration or control data—from the microcontroller230to a sensor chip210. The microcontroller230first uses a command conveyed in a command frame320to announce the configuration or control data via the digital signal interface250. The microcontroller230uses an address frame330to indicate via the digital signal interface250which of the sensor chips210-1, . . . ,210nit would like to convey the configuration or control data to. The microcontroller230subsequently conveys the announced configuration or control data to the previously addressed sensor chip210in one or more data frames345via the digital signal interface250. The sensor chip210confirms receipt of the data with a safety data frame350via the digital signal interface250. It will be obvious to a person skilled in the art that in this case too an order for the depicted command, address and data frames could also be chosen differently. Bus collisions can be prevented by the master-slave control principle. The communication is controlled by the (single) bus master (microcontroller230) by transmitting a command frame or address frame. Each frame transmitted by the bus master230contains address information that is used to address the sensors210-1, . . . ,210nindividually. Only following successful receipt of the address frame and concordant address information does the addressed slave sensor210-1, . . . ,210ntransmit its information to the digital signal interface250. For this addressing principle to work, each bus slave of slave sensors210-1, . . . ,210nhas an allocated biunique address. This allocation can be accomplished in a wide variety of ways. First, the bus address could be provided by hardware solutions, such as e.g. address-specific circuitry for the individual sensors210-1, . . . ,210n. Alternatively, it would also be possible to write the biunique addresses to a nonvolatile memory of the sensors210-1, . . . ,210ninvolved using a programming step before the sensor network is started up. Both the hardware circuitry and the programming of the individual sensors210-1, . . . ,210nhave the disadvantage that either an additional programming step is used during manufacture or multiple different components (sensor modules) are to be kept in accordance with the number of bus subscribers. It is therefore advantageous to introduce an auto-addressing mechanism that can be used to allocate biunique addresses to the individual bus subscribers of sensors210-1, . . . ,210ndynamically, that is to say during system initialization. While various auto-addressing methods are already known (e.g. daisychain method), the presence of one or more analog, biunique sensor-to-microcontroller connections is advantageously used in the present case. For the auto-addressing, the bus master230can use a first command transmitted via the digital signal interface250to reset the sensor addresses of all of the bus slaves of slave sensors210-1, . . . ,210ninvolved. The bus master230can then use a second command to stimulate the generation of random addresses in the individual slave sensors210-1, . . . ,210n. These are then transmitted to the bus master230by using an analog connection and/or the digital signal interface250. Those sensors210-1, . . . ,210nthat already have a biunique address available are allocated, by the bus master230, an address that is used in the application. For those sensors210-1, . . . ,210nthat have not yet generated a biunique address using the random generation, the bus master230is used to initiate a new random cycle with a further rating loop. The very low probability of identical random addresses in combination with multiple iterations thus allows the address allocation to be performed very efficiently. According to one possible example implementation, after receiving the command to generate a random address, each sensor210-1, . . . ,210ncan generate its address and output it to the digital signal interface250as a digital bit stream. This can involve an asymmetric output stage being used, which means that a logic level is output to the digital interface250much more weakly than the opposite level. In the event of a bus conflict, the stronger output stage overrides the respective weaker one (dominant as opposed to recessive level). The sensors210-1, . . . ,210ncontinuously monitor the data stream that is output. In the event of a bus conflict with a recessive output level, the affected sensor210-1, . . . ,210ndetects the conflict and resolves it by switching the output stage of the digital signal interface250to high impedance. After the sensor210-1, . . . ,210nhas output its random address, the success or failure (detected bus conflict) is indicated using a respective defined level on the analog output line212-1, . . . ,212n. This allows the bus master230to tell which sensor has successfully completed the cycle with the address received from it. In this case, the successful sensor can be allocated an address and a next arbitration cycle can be initiated. During an initial bus setup phase (address allocation), it is thus possible for bus collisions (multiple slaves transmitting data to the bus simultaneously) to occur. The bus setup phase can be initiated using a dedicated frame (arbitration frame) to allocate unique addresses to individual sensors. The frame contains an addressing command that is used to initiate the address allocation. For the bus setup phase, the microcontroller230may thus be designed to use the digital signal interface250to provide an addressing command to the sensor chips210-1, . . . ,210n. Each of the sensor chips210-1, . . . ,210nmay be designed to respond to the addressing command by using its respective analog signal line212-1, . . . ,212nor its respective digital signal interface250-1, . . . ,250-nto convey a random output signal to the microcontroller230. Each of the sensor chips210-1, . . . ,210nmay be designed to receive, from the microcontroller230, a unique address in response to its random output signal. According to some example implementations, the microcontroller is designed to allocate each of the sensor chips a unique address on the basis of the random output signals. The microcontroller230can use the digital signal interface to allocate each of the sensor chips its unique address. The sensor chips210-1, . . . ,210ncan therefore be allocated and notified of addresses for the further communication between the microcontroller and the sensor chips in an initial addressing phase using an unsophisticated addressing protocol. FIG.4Adepicts an example implementation of a method400for address allocation from the point of view of the microcontroller230. A command “Auto_Reset.all” via the digital signal interface250is first used by the microcontroller230at411to instruct all sensor chips210-1, . . . ,210nto set their analog output interfaces212-1, . . . ,212nto “high” (H). The microcontroller230subsequently provides a further command “Auto_Reset.test” via the digital signal interface250at412. In response to the command “Auto_Reset.test”, each sensor chip210-1, . . . ,210nuses its respective digital signal interface212-1, . . . ,212nto output a random digital output signal. After the sensor chip210-1, . . . ,210nhas output its random address, the success or failure (detected bus conflict) is indicated using a respective defined level on the analog output line212-1, . . . ,212n. The analog output signals can then be read at413via the analog signal inputs232-1, . . . ,232nof the microcontroller230and the ADCs thereof. It is therefore possible for the microcontroller230to tell which sensor chip210-1, . . . ,210nhas successfully completed the cycle with the address received from it. In this case a successful sensor chip210-1, . . . ,210ncan be allocated an address at415. If the analog output signal from a sensor thus indicates no bus conflict (success), this sensor can be allocated an address via the digital signal interface250at415. As a result, its analog output signal is set e.g. to “L” for the duration of the addressing method400. If the analog output signal from the sensor indicates a bus conflict (failure), step412is repeated until no further conflicts have been able to be detected. After the one sensor has been allocated an address at415, a test is performed at416to determine whether all of the sensors210-1, . . . ,210nhave already been allocated an address. If this is not the case, the command “Auto Reset (unassigned)” is used to set the analog output interfaces212of all the remaining sensors without an allocated address to “H” at417and then to put them back into the random mode at412. FIG.4Bdescribes the method400for address allocation from the point of view of a sensor chip210. First, a logical variable “Address_assigned” is set to “false” at421. This means that the sensor chip has not yet been allocated an address. At422and423the sensor chip210checks whether a command has been received from the microcontroller via the digital signal interface250, and the state of the logical variable “Address_assigned”. If the logical variable “Address_assigned”=“false”, the sensor checks at424whether the received command corresponds to “Auto_Reset.test”. If this is the case, the sensor chip210uses its digital signal interface250to output a random digital output signal. The digital output signal can then be subjected to a collision test at426. In the event of a collision with other sensor chips, the analog output signal is set to “L” at427, the output stage of the digital signal interface250is switched to high impedance and the method is continued at422by waiting for a new command. If a collision with other sensor chips is not detected, the method is likewise continued at422by waiting for a new command “Auto_Reset.Assign”. If this command was received at428, a check is performed at429to determine whether the analog output interface212of the sensor chip210is at “high” (H). If this is the case, the sensor is allocated an address via the digital signal interface250at430. The logical variable “Address_assigned” is subsequently set to “true” at431and the analog output signal is set to “L” for the duration of the addressing method400. The method is then continued at422by waiting for a new command. In an alternative implementation, the initialization command (e.g. “Auto_Reset.all”) can be followed by a second command (e.g. “Auto_Reset.test”) again being used to generate random addresses. In this implementation the addresses are output on one of the analog lines212or260as a representative voltage via a digital-to-analog converter. By reading in the analog voltages, the bus master230detects uniqueness of the addresses or possible overlaps. The sensors210-1, . . . ,210nthat have generated a unique address are allocated a productive address via the digital signal interface250. The sensors with the same address undertake a new cycle to generate a random address. After all of the sensor chips have been allocated an address using the method, digital data can be interchanged between the microcontroller230and the sensor chips210via the digital single-master multi-slave bus line (digital signal interface250) in parallel with the analog measurement signals. In this regard,FIG.5shows a schematic communication method500between the microcontroller230and at least one sensor chip210. The method500comprises conveying502analog measurement data between the sensor chip210and the microcontroller230via at least one analog signal interface212and, in parallel therewith, conveying504digital secondary information between the sensor chip210and the microcontroller230via the bidirectional digital signal interface250. The essence of example implementations of the present disclosure is to continue to use an analog signal path for the wideband realtime current information, but to introduce a digital interface that allows control and diagnosis of both the current sensor and the system to which the current sensor is connected. The bus capability of the proposed digital interface allows the additional complexity and number of wires (single wire!) to be reduced to an absolute minimum. The proposed digital interface allows simple and inexpensive sensor systems having high safety and availability demands and reduces the complexity of the end-of-line calibration. The aspects and features that have been described together with one or more of the examples and figures detailed above can also be combined with one or more of the other examples in order to replace an identical feature of the other example or in order to introduce the feature into the other example additionally. Examples can furthermore be or relate to a computer program having a program code for carrying out one or more of the methods above when the computer program is executed on a computer or processor. Steps, operations or processes of different methods described above can be carried out by programmed computers or processors. Examples can also cover program storage apparatuses, e.g. digital data storage media, which are machine-, processor- or computer-readable and code machine-executable, processor-executable or computer-executable programs of instructions. The instructions carry out some or all of the steps of the methods described above or cause them to be carried out. The program storage apparatuses can comprise or be e.g. digital memories, magnetic storage media such as, for example, magnetic disks and magnetic tapes, hard disk drives or optically readable digital data storage media. Further examples can also cover computers, processors or control units programmed to carry out the steps of the methods described above, or (field) programmable logic arrays ((F)PLAs) or (field) programmable gate arrays ((F)PGAs) programmed to carry out the steps of the methods described above. The description and drawings present only the principles of the disclosure. Furthermore, all examples mentioned here are intended to be used expressly only for illustrative purposes, in principle, in order to assist the reader in understanding the principles of the disclosure and the concepts contributed by the inventor(s) for further development of the art. All statements herein regarding principles, aspects and examples of the disclosure and also concrete examples thereof encompass the counterparts thereof. A function block designated as “means for . . . ” carrying out a specific function can relate to a circuit designed for carrying out a specific function. Consequently, a “means for something” can be implemented as a “means designed for or suitable for something”, e.g. a component or a circuit designed for or suitable for the respective task. Functions of different elements shown in the figures, including any function blocks referred to as “means”, “means for providing a signal”, “means for generating a signal”, etc., can be implemented in the form of dedicated hardware, e.g. “a signal provider”, “a signal processing unit”, “a processor”, “a controller”, etc., and as hardware capable of executing software in conjunction with associated software. When provided by a processor, the functions can be provided by a single dedicated processor, by a single jointly used processor or by a plurality of individual processors, some or all of which can be used jointly. However, the term “processor” or “controller” is far from being limited to hardware capable exclusively of executing software, but rather can encompass digital signal processor hardware (DSP hardware), network processor, application specific integrated circuit (ASIC), field programmable logic array (FPGA=Field Programmable Gate Array), read only memory (ROM) for storing software, random access memory (RAM) and nonvolatile memory apparatus (storage). Other hardware, conventional and/or customized, can also be included. A block diagram can depict for example a rough circuit diagram implementing the principles of the disclosure. In a similar manner, a flow diagram, a flowchart, a state transition diagram, a pseudo-code and the like can represent various processes, operations or steps represented for example substantially in a computer-readable medium and thus carried out by a computer or processor, regardless of whether such a computer or processor is explicitly shown. Methods disclosed in the description or in the patent claims can be implemented by a component having a means for carrying out each of the respective steps of the methods. It goes without saying that the disclosure of multiple steps, processes, operations or functions disclosed in the description or the claims should not be interpreted as being in the specific order, unless explicitly or implicitly indicated otherwise, e.g. for technical reasons. The disclosure of multiple steps or functions thus does not limit them to a specific order, unless the steps or functions are not interchangeable for technical reasons. Furthermore, in some examples, an individual step, function, process or operation can include multiple substeps, subfunctions, subprocesses or suboperations and/or be subdivided into them. Such substeps may be included and be part of the disclosure of the individual step, provided that they are not explicitly excluded. Furthermore, the claims that follow are hereby incorporated in the detailed description, where each claim may stand alone as a separate example. While each claim may stand alone as a separate example, it should be taken into consideration that—although a dependent claim can refer in the claims to a specific combination with one or more other claims—other examples can also encompass a combination of the dependent claim with the subject matter of any other dependent or independent claim. Such combinations are explicitly proposed here, provided that no indication is given that a specific combination is not intended. Furthermore, features of a claim are also intended to be included for any other independent claim, even if this claim is not made directly dependent on the independent claim. | 33,318 |
11860202 | DETAILED DESCRIPTION Embodiments of the present disclosure will be described herein below with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any configuration or design described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other configurations or designs. Herein, the phrase “coupled” is defined to mean directly connected to or indirectly connected with through one or more intermediate components. Such intermediate components may include both hardware and software based components. It is further noted that, unless indicated otherwise, all functions described herein may be performed in either hardware or software, or some combination thereof. In one embodiment, however, the functions are performed by at least one processor, such as a computer or an electronic data processor, digital signal processor or embedded micro-controller, in accordance with code, such as computer program code, software, and/or integrated circuits that are coded to perform such functions, unless indicated otherwise. It should be appreciated that the present disclosure can be implemented in numerous ways, including as a process, an apparatus, a system, a device, a method, or a computer readable medium such as a computer readable storage medium or a computer network where program instructions are sent over optical or electronic communication links. Embodiments of the present disclosure will be described herein below with reference to the accompanying drawings. As used herein, intelligent electronic devices (“IEDs”) can be any device that senses electrical parameters and computes data including, but not limited to, Programmable Logic Controllers (“PLC's”), Remote Terminal Units (“RTU's”), electric power meters, panel meters, protective relays, fault recorders, phase measurement units, serial switches, smart input/output devices and other devices which are coupled with power distribution networks to manage and control the distribution and consumption of electrical power. A meter is a device that records and measures power events, power quality, current waveforms, voltage waveforms, harmonics, transients and other power disturbances. Revenue accurate meters (“revenue meters”) relate to revenue accuracy electrical power metering devices with the ability to detect, monitor, report, quantify and communicate power quality information about the power that they are metering. FIG.1is a block diagram of an intelligent electronic device (IED)10for monitoring and determining power usage and power quality for any metered point within a power distribution system and for providing a data transfer system for faster and more accurate processing of revenue and waveform analysis. The IED100ofFIG.1includes a plurality of sensors112coupled to various phases A, B, C and neutral N of an electrical distribution system111, a plurality of analog-to-digital (A/D) converters114, including inputs coupled to the sensor112outputs, a power supply116, a volatile memory118, an non-volatile memory120, a multimedia user interface122, and a processing system that includes at least one central processing unit (CPU)150(or host processor) and/or one or more digital signal processors, two of which are shown, i.e., DSP1160and DSP2170. The IED100may also include a Field Programmable Gate Array180which performs a number of functions, including, but not limited to, acting as a communications gateway for routing data between the various processors150,160,170, receiving data from the A/D converters114, performing transient detection and capture and performing memory decoding for CPU150and/or the DSP processor160. In one embodiment, the FPGA80is internally comprised of two dual port memories to facilitate the various functions. It is to be appreciated that the various components shown inFIG.1are contained within a housing190. Exemplary housings will be described below in relation toFIGS.2A-2H. The plurality of sensors112sense electrical parameters, e.g., voltage and current, on incoming lines (i.e., phase A, phase B, phase C, neutral N) of an electrical power distribution system111, e.g., an electrical circuit, that are coupled to at least one load113that consumes the power provided. In one embodiment, the sensors112will include current transformers and potential transformers, wherein one current transformer and one voltage transformer will be coupled to each phase of the incoming power lines. A primary winding of each transformer will be coupled to the incoming power lines and a secondary winding of each transformer will output a voltage representative of the sensed voltage and current. The output of each transformer will be coupled to the A/D converters114configured to convert the analog output voltage from the transformer to a digital signal that can be processed by the CPU150, DSP1160, DSP2170, FPGA180or any combination thereof. A/D converters114are respectively configured to convert an analog voltage output to a digital signal that is transmitted to a gate array, such as Field Programmable Gate Array (FPGA)180. The digital signal is then transmitted from the FPGA180to the CPU150and/or one or more DSP processors160,170to be processed in a manner to be described below. The CPU150and/or DSP Processors160,170are configured to operatively receive digital signals from the A/D converters114(seeFIG.1) to perform calculations necessary to determine power usage and to control the overall operations of the IED100. In some embodiments, CPU150, DSP1160and DSP2170may be combined into a single processor, serving the functions of each component. In some embodiments, it is contemplated to use an Erasable Programmable Logic Device (EPLD) or a Complex Programmable Logic Device (CPLD) or any other programmable logic device in place of the FPGA180. In some embodiments, the digital samples, which are output from the A/D converters114are sent directly to the CPU150or DSP processors160,170, effectively bypassing the FPGA180as a communications gateway. The power supply116provides power to each component of the IED100. In one embodiment, the power supply116is a transformer with its primary windings coupled to the incoming power distribution lines and having windings to provide a nominal voltage, e.g., 5 VDC, +12 VDC and −12 VDC, at its secondary windings. In other embodiments, power may be supplied from an independent power source to the power supply116. For example, power may be supplied from a different electrical circuit or an uninterruptible power supply (UPS). In one embodiment, the power supply116can be a switch mode power supply in which the primary AC signal will be converted to a form of DC signal and then switched at high frequency, such as, for example, 100 Khz, and then brought through a transformer to step the primary voltage down to, for example, 5 Volts AC. A rectifier and a regulating circuit would then be used to regulate the voltage and provide a stable DC low voltage output. Other embodiments, such as, but not limited to, linear power supplies or capacitor dividing power supplies are also contemplated. The multimedia user interface122is shown coupled to the CPU150inFIG.1for interacting with a user and for communicating events, such as alarms and instructions to the user. The multimedia user interface122may include a display for providing visual indications to the user. The display may be embodied as a touch screen, a liquid crystal display (LCD), a plurality of LED number segments, individual light bulbs or any combination. The display may provide information to the user in the form of alpha-numeric lines, computer-generated graphics, videos, animations, etc. The multimedia user interface122further includes a speaker or audible output means for audibly producing instructions, alarms, data, etc. The speaker is coupled to the CPU150via a digital-to-analog converter (D/A) for converting digital audio files stored in a memory118or non-volatile memory120to analog signals playable by the speaker. An exemplary interface is disclosed and described in commonly owned U.S. Pat. No. 8,442,660, entitled “POWER METER HAVING AUDIBLE AND VISUAL INTERFACE”, which claims priority to expired U.S. Provisional Patent Appl. No. 60/731,006, filed Oct. 28, 2005, the contents of which are hereby incorporated by reference in their entireties. The IED100will support various file types including but not limited to Microsoft Windows Media Video files (.wmv), Microsoft Photo Story files (.asf), Microsoft Windows Media Audio files (.wma), MP3 audio files (.mp3), JPEG image files (.jpg, .jpeg, .jpe, .jfif), MPEG movie files (.mpeg, .mpg, .mpe, .m1v, .mp2v .mpeg2), Microsoft Recorded TV Show files (.dvr-ms), Microsoft Windows Video files (.avi) and Microsoft Windows Audio files (.wav). The IED100further comprises a volatile memory118and a non-volatile memory120. In addition to storing audio and/or video files, volatile memory118will store the sensed and generated data for further processing and for retrieval when called upon to be displayed at the IED100or from a remote location. The volatile memory118includes internal storage memory, e.g., random access memory (RAM), and the non-volatile memory120includes removable and/or non-removable memory such as magnetic storage memory; optical storage memory, e.g., the various types of CD and DVD media; solid-state storage memory, e.g., a CompactFlash card, a Memory Stick, SmartMedia card, MultiMediaCard (MMC), SD (Secure Digital) memory; or any other memory storage that exists currently or will exist in the future. By utilizing removable memory, an IED can be easily upgraded as needed. Such memory will be used for storing historical trends, waveform captures, event logs including time-stamps and stored digital samples for later downloading to a client application, web-server or PC application. In a further embodiment, the IED100will include a communication device124, also known as a network or communication interface, for enabling communications between the IED or meter, and a remote terminal unit, programmable logic controller and other computing devices, microprocessors, a desktop computer, laptop computer, other meter modules, etc. The communication device124may be a modem, network interface card (NIC), wireless transceiver, etc. The communication device124will perform its functionality by hardwired and/or wireless connectivity. The hardwire connection may include but is not limited to hard wire cabling, e.g., parallel or serial cables, RS232, RS485, USB cable, Firewire (1394 connectivity) cables, Ethernet, and the appropriate communication port configuration. The wireless connection may operate under any of the various wireless protocols including but not limited to Bluetooth™ interconnectivity, infrared connectivity, radio transmission connectivity including computer digital signal broadcasting and reception commonly referred to as Wi-Fi or 802.11.X (where x denotes the type of transmission), satellite transmission or any other type of communication protocols, communication architecture or systems currently existing or to be developed for wirelessly transmitting data including spread spectrum 900 MHz, or other frequencies, Zigbee, WiFi, or any mesh enabled wireless communication. The IED100may communicate to a server or other computing device via the communication device124. The IED100may be connected to a communications network, e.g., the Internet, by any means, for example, a hardwired or wireless connection, such as dial-up, hardwired, cable, DSL, satellite, cellular, PCS, wireless transmission (e.g., 802.11a/b/g), etc. It is to be appreciated that the network may be a local area network (LAN), wide area network (WAN), the Internet or any network that couples a plurality of computers to enable various modes of communication via network messages. Furthermore, the server will communicate using various protocols such as Transmission Control Protocol/Internet Protocol (TCP/IP), File Transfer Protocol (FTP), Hypertext Transfer Protocol (HTTP), etc. and secure protocols such as Hypertext Transfer Protocol Secure (HTTPS), Internet Protocol Security Protocol (IPSec), Point-to-Point Tunneling Protocol (PPTP), Secure Sockets Layer (SSL) Protocol, etc. The server may further include a storage medium for storing a data received from at least one IED or meter and/or storing data to be retrieved by the IED or meter. In an additional embodiment, the IED100may also have the capability of not only digitizing waveforms, but storing the waveform and transferring that data upstream to a central computer, e.g., a remote server, when an event occurs such as a voltage surge or sag or a current short circuit. This data may be triggered and captured on an event, stored to memory, e.g., non-volatile RAM, and additionally transferred to a host computer within the existing communication infrastructure either immediately in response to a request from a remote device or computer to receive said data or in response to a polled request. The digitized waveform will also allow the CPU150to compute other electrical parameters such as harmonics, magnitudes, symmetrical components and phasor analysis. Using the harmonics, the IED100will also calculate dangerous heating conditions and can provide harmonic transformer derating based on harmonics found in the current waveform. In a further embodiment, the IED100will execute an e-mail client and will send e-mails to the utility or to the customer direct on an occasion that a power quality event occurs. This allows utility companies to dispatch crews to repair the condition. The data generated by the meters are used to diagnose the cause of the condition. The data is transferred through the infrastructure created by the electrical power distribution system. The email client will utilize a POP3 or other standard mail protocol. A user will program the outgoing mail server and email address into the meter. An exemplary embodiment of said metering is described in U.S. Pat. No. 6,751,563, which all contents thereof are incorporated by reference herein. The techniques of the present disclosure can be used to automatically maintain program data and provide field wide updates upon which IED firmware and/or software can be upgraded. An event command can be issued by a user, on a schedule or by digital communication that will trigger the IED100to access a remote server and obtain the new program code. This will ensure that program data will also be maintained allowing the user to be assured that all information is displayed identically on all units. It is to be understood that the present disclosure may be implemented in various forms of hardware, software, firmware, special purpose processors, or a combination thereof. The IED10also includes an operating system and micro instruction code. The various processes and functions described herein may either be part of the micro instruction code or part of an application program (or a combination thereof) which is executed via the operating system. It is to be further understood that because some of the constituent system components and method steps depicted in the accompanying figures may be implemented in software, or firmware, the actual connections between the system components (or the process steps) may differ depending upon the manner in which the present disclosure is programmed. Given the teachings of the present disclosure provided herein, one of ordinary skill in the related art will be able to contemplate these and similar implementations or configurations of the present disclosure. Furthermore, it is to be appreciated that the components and devices of the IED10ofFIG.1may be disposed in various housings depending on the application or environment. For example, the IED100may be configured as a panel meter200as shown inFIG.2Aan2B. The panel meter200ofFIGS.2A and2Bis described in more detail in commonly owned U.S. Pat. No. 7,271,996, the contents of which are hereby incorporated by reference. As seen inFIGS.2A and2B, the IED200includes a housing202defining a front surface202a, a rear surface202b, a top surface202c, a bottom surface202d, a right side surface202e, and a left side surface (not shown). Electrical device200includes a face plate204operatively connected to front surface202aof housing202. Face plate204includes displays206, indicators208(e.g., LEDs and the like), buttons210, and the like providing a user with an interface for visualization and operation of electrical device200. For example, as seen inFIG.2A, face plate204of electrical device200includes analog and/or digital displays206capable of producing alphanumeric characters. Face plate204includes a plurality of indicators208which, when illuminated, indicate to the user the “type of reading”, the “% of load bar”, the “parameter designation” which indicates the reading which is being displayed on displays206, a “scale selector” (e.g., Kilo or Mega multiplier of Displayed Readings), etc. Face plate204includes a plurality of buttons210(e.g., a “menu” button, an “enter” button, a “down” button, a “right” button, etc.) for performing a plurality of functions, including and not limited to: viewing of meter information; enter display modes; enter configuring parameters; performing re-sets; performing LED checks; changing settings; viewing parameter values; scrolling parameter values; and viewing limit states. The housing202includes voltage connections or inputs212provided on rear surface202bthereof, and current inputs214provided along right side surface202ethereof. The IED200may include a first interface or communication port216for connection to a master and/or slave device. Desirably, first communication port216is situated in rear surface202bof housing202. IED200may also include a second interface or communication port218situated on face plate204. In other embodiment, the IED100may be configured as a socket meter220, also known as a S-base type meter or type S meter, as shown inFIG.2Can2D. An exemplary socket meter is described in more detail in commonly owned U.S. Pat. No. 8,717,007, the contents of which are hereby incorporated by reference. Referring toFIGS.2C and2D, the meter220includes a main housing222surrounded by a cover224. The cover224is preferably made of a clear material to expose a display226disposed on the main body222. An interface228, to access the display and interact with the IED, and a communication port230are also provided and accessible through the cover224. The meter220further includes a plurality of current terminals232and voltage terminals234disposed on backside of the meter extending through a base235. The terminals232,234are designed to mate with matching jaws of a detachable meter-mounting device, such as a revenue meter socket. The socket is hard wired to the electrical circuit and is not meant to be removed. To install an S-base meter, the utility need only plug in the meter into the socket. Once installed, a socket-sealing ring236is used as a seal between the meter220and/or cover224and the meter socket to prevent removal of the meter and to indicate tampering with the meter. In a further embodiment, the IED100ofFIG.1may be disposed in a switchboard or draw-out type housing240as shown inFIGS.2E and2F, whereFIG.2Eis a front view andFIG.2Fis a rear view. The switchboard enclosure242usually features a cover244with a transparent face246to allow the meter display248to be read and the user interface250to be interacted with by the user. The cover244also has a sealing mechanism (not shown) to prevent unauthorized access to the meter. A rear surface252of the switchboard enclosure242provides connections for voltage and current inputs254and for various communication interfaces256. Although not shown, the meter disposed in the switchboard enclosure242may be mounted on a draw-out chassis which is removable from the switchboard enclosure242. The draw-out chassis interconnects the meter electronics with the electrical circuit. The draw-out chassis contains electrical connections which mate with matching connectors254,256disposed on the rear surface252of the enclosure242when the chassis is slid into place. In yet another embodiment, the IED100ofFIG.1may be disposed in an A-base or type A housing as shown inFIGS.2G and2H. A-base meters260feature bottom connected terminals262on the bottom side of the meter housing264. These terminals262are typically screw terminals for receiving the conductors of the electric circuit (not shown). A-base meters260further include a meter cover266, meter body268, a display270and input/output means272. Further, the meter cover266includes an input/output interface274for interacting with the IED. The cover266encloses the meter electronics268and the display270. The cover266has a sealing mechanism (not shown) which prevents unauthorized tampering with the meter electronics. It is to be appreciated that other housings and mounting schemes, e.g., circuit breaker mounted, are contemplated to be within the scope of the present disclosure. FIG.3illustrates an exemplary environment300in which the present disclosure may be practiced. The environment300includes at least one IED310and at least one computing device390,391,392. The network302may be the Internet, a public or private intranet, an extranet, wide area network (WAN), local area network (LAN) or any other network configuration to enable transfer of data and commands. An example network configuration uses the Transport Control Protocol/Internet Protocol (“TCP/IP”) network protocol suite, however, other Internet Protocol based networks are contemplated by the present disclosure. Communications may also include IP tunneling protocols such as those that allow virtual private networks coupling multiple intranets or extranets together via the Internet. The network302may support existing or envisioned application protocols, such as, for example, telnet, POPS, Mime, HTTP, HTTPS, PPP, TCP/IP, SMTP, proprietary protocols, or any other network protocols. During operation, the IED310may communicate using the network302as will be hereinafter discussed. It is to be appreciated that are at least two basic types of networks, based on the communication patterns between the machines: client/server networks and peer-to-peer networks. On a client/server network, every computer, device or IED has a distinct role: that of either a client or a server. A server is designed to share its resources among the client computers on the network. A dedicated server computer often has faster processors, more memory, and more storage space than a client because it might have to service dozens or even hundreds of users at the same time. High-performance servers typically use from two to eight processors (and that's not counting multi-core CPUs), have many gigabytes of memory installed, and have one or more server-optimized network interface cards (NICs), RAID (Redundant Array of Independent Drives) storage consisting of multiple drives, and redundant power supplies. Servers often run a special network OS—such as Windows Server, Linux, or UNIX—that is designed solely to facilitate the sharing of its resources. These resources can reside on a single server or on a group of servers. When more than one server is used, each server can “specialize” in a particular task (file server, print server, fax server, email server, and so on) or provide redundancy (duplicate servers) in case of server failure. For demanding computing tasks, several servers can act as a single unit through the use of parallel processing. A client device typically communicates only with servers, not with other clients. A client system may be a standard PC that is running an OS such as Windows, Linux, etc. Current OSes contain client software that enables the client computers to access the resources that servers share. Older OSes, such as Windows 3.x and DOS, required add-on network client software to join a network. By contrast, on a peer-to-peer network, every computer or device is equal and can communicate with any other computer or device on the network to which it has been granted access rights. Essentially, every computer or device on a peer-to-peer network can function as both a server and a client; any computer or device on a peer-to-peer network is considered a server if it shares a printer, a folder, a drive, or some other resource with the rest of the network. Note that the actual networking hardware (interface cards, cables, and so on) is the same in client/server versus peer-to-peer networks, it is only the logical organization, management and control of the network that varies. A client may comprise any computing device, such as a server390, mainframe, workstation, personal computer391,392, hand held computer, laptop telephony device, network appliance, an IED310, Programmable Logic Controller, Power Meter, Protective Relay etc. The client may include system memory, which may be implemented in volatile and/or non-volatile devices, and one or more client applications which may execute in the system memory. Such client applications may include, for example, FTP client applications. File Transfer Protocol (FTP) is an application for transfer of files between computers attached to Transmission Control Protocol/Internet Protocol (TCP/IP) networks, including the Internet. FTP is a “client/server” application, such that a user runs a program on one computer system, the “client”, which communicates with a program running on another computer system, the “server”. In one embodiment, IED310includes at least an FTP server. While FTP file transfer comprises one embodiment for encapsulating files to improve a data transfer rate from an IED to external clients, the present disclosure contemplates the use of other file transfer protocols, such as the Ethernet protocol such as HTTP or TCP/IP for example. Of course, other Ethernet protocols are contemplated for use by the present disclosure. For example, for the purpose of security and firewall access, it may be preferable to utilize HTTP file encapsulation as opposed to sending the data via FTP. In other embodiments, data can be attached as an email and sent via SMTP, for example. Such a system is described in a co-owned U.S. Pat. No. 6,751,563, titled “Electronic Energy meter”, the contents of which are incorporated herein by reference. In the U.S. Pat. No. 6,751,563, at least one processor of the IED or meter is configured to collect the at least one parameter and generate data from the sampled at least one parameter, wherein the at least one processor is configured to act as a server for the IED or meter and is further configured for presenting the collected and generated data in the form of web pages, as will be described in relation toFIG.3. IED310includes a digital sampler320for digitally sampling the voltage and current of the power being supplied to a customer or monitored at the point of the series connection in the power grid. Digital sampler320digitally samples the voltage and current and performs substantially similar to the A/D converters114described above in relation toFIG.1. The digital samples are then forwarded to processor330for processing. It is to be appreciated that the processor may be a single processing unit or a processing assembly including at least one CPU150, DSP1160, DSP2170and FPGA180, or any combination thereof. Also connected to processor330is external device interface340for providing an interface for external devices350to connect to meter310. These external devices might include other power meters, sub-station control circuitry, on/off switches, etc. Processor330receives data packets from digital sampler320and external devices350, and processes the data packets according to user defined or predefined requirements. A memory360is connected to processor330for storing data packets and program algorithms, and to assist in processing functions of processor330. These processing functions include the power quality data and revenue calculations, as well as formatting data into different protocols which will be described later in detail. Processor330provides processed data to network302through network interface370, similar to the communication device124described above in relation toFIG.1In one embodiment, the network interface converts the data to an Ethernet TCP/IP format. The use of the Ethernet TCP/IP format allows multiple users to access the power meter simultaneously. In a like fashion, network interface370might be comprised of a modem, cable connection, or other devices that provide formatting functions. A web server program (web server) is contained in memory360, and accessed through network or communication interface370. The web server provides real time data through any known web server interface format. For example, popular web server interface formats consist of HTML and XML formats. The actual format of the programming language used is not essential to the present disclosure, in that any web server format can be incorporated herein. The web server provides a user-friendly interface for the user to interact with the meter310. The user can have various access levels to enter limits for e-mail alarms. Additionally, the user can be provided the data in a multiple of formats including raw data, bar graph, charts, etc. The currently used HTML or XML programming languages provide for easy programming and user-friendly user interfaces. The processor330formats the processed data into various network protocols and formats. The protocols and formats can, for example, consist of the web server HTML or XML formats, Modbus TCP, RS-485, FTP or e-mail. Dynamic Host Configuration Protocol (DHCP) can also be used to assign IP addresses. The network formatted data is now available to users at computers390-392through network302, that connects to meter310at the network interface370. In one embodiment, network interface370is an Ethernet interface that supports, for example, 100 base-T or 10 base-T communications. This type of network interface can send and receive data packets between WAN connections and/or LAN connections and the meter310. This type of network interface allows for situations, for example, where the web server may be accessed by one user while another user is communicating via the Modbus TCP, and a third user may be downloading a stored data file via FTP. The ability to provide access to the meter by multiple users, simultaneously, is a great advantage over the prior art. This can allow for a utility company's customer service personnel, a customer and maintenance personnel to simultaneously and interactively monitor and diagnose possible problems with the power service. FIG.4is a functional block diagram of processor330according to the embodiment of the present disclosure. Processor330is shown containing at least four main processing functions. The functions shown are illustrative and not meant to be inclusive of all possible functions performed by processor330. Power Quality and Revenue Metering functions (metering functions)410consists of a complete set of functions which are needed for power quality and revenue metering. Packet data collected by digital sampler320is transmitted to processor330. Processor330calculates, for example, power reactive power, apparent power, and power factor. The metering function410responds to commands via the network or other interfaces supported by the meter. External Device Routing Functions430handle the interfacing between the external device350and meter310. Raw data from external device350is fed into meter310. The external device350is assigned a particular address. If more than one external device is connected to meter310, each device will be assigned a unique particular address. The Network Protocol Functions450of meter310are executed by processor330which executes multiple networking tasks that are running concurrently. As shown inFIG.4, these include, but are not limited to, the following network tasks included in network protocol functions450: e-mail460, web server470, Modbus TCP480, FTP490, and DHCP492. The e-mail460network protocol function can be utilized to send e-mail messages via the network302to a user to, for example, notify the user of an emergency situation or if the power consumption reaches a user-set or pre-set high level threshold. As the processor receives packets of data it identifies the network processing necessary for the packet by the port number associated with the packet. The processor allocates the packet to a task as a function of the port number. Since each task is running independently the meter310can accept different types of requests concurrently and process them transparently from each other. For example, the web server may be accessed by one user while another user is communicating via Modbus TCP and at the same time a third user may download a log file via FTP. The Network to Meter Protocol Conversion Function440is used to format and protocol convert the different network protocol messages to a common format understood by the other functional sections of meter310. After the basic network processing of the packet of data, any “commands” or data which are to be passed to other functional sections of meter310are formatted and protocol converted to a common format for processing by the Network to Meter Protocol Conversion Function440. Similarly, commands or data coming from the meter for transfer over the network are pre-processed by this function into the proper format before being sent to the appropriate network task for transmission over the network. In addition, this function first protocol converts and then routes data and commands between the meter and external devices. Although the above described embodiments enable users outside of the network the IED or meter is residing on to access the internal memory or server of the IED or meter, IT departments commonly block this access through a firewall to avoid access by dangerous threats into corporate networks. A firewall is a system designed to prevent unauthorized access to or from a private network, e.g., an internal network of a building, a corporate network, etc. Firewalls can be implemented in both hardware and software, or a combination of both. Firewalls are frequently used to prevent unauthorized Internet users from accessing private networks connected to the Internet, especially intranets. All messages entering or leaving the intranet pass through the firewall, which examines each message and blocks those that do not meet the specified security criteria. A firewall may employ one or more of the following techniques to control the flow of traffic in and of the network it is protecting: 1) packet filtering: looks at each packet entering or leaving the network and accepts or rejects it based on user-defined rules; 2) Application gateway: applies security mechanisms to specific applications, such as FTP and Telnet servers; 3) Circuit-level gateway: applies security mechanisms when a TCP or UDP connection is established, once the connection has been made, packets can flow between the hosts without further checking; 4) Proxy server: intercepts all messages entering and leaving the network, effectively hides the true network addresses; and 5) Stateful inspection: doesn't examine the contents of each packet but instead compares certain key parts of the packet to a database of trusted information, if the comparison yields a reasonable match, the information is allowed through, otherwise it is discarded. Other techniques and to be developed techniques are contemplated to be within the scope of the present disclosure. In one embodiment, the present disclosure provides for overcoming the problem of not being allowed firewall access to an IED or meter installed within a facility, i.e., the meter is residing on a private network, by enabling an IED to initiate one-way communication through the firewall. In this embodiment, the IED or meter posts the monitored and generated data on an Internet site external to the corporate or private network, i.e., on the other side of a firewall. The benefit is that any user would be able to view the data on any computer or web enabled smart device without having to pierce or bypass the firewall. Additionally, there is a business opportunity to host this data on a web server and charge a user a monthly fee for hosting the data. The features of this embodiment can be incorporated into any telemetry application including vending, energy metering, telephone systems, medical devices and any application that requires remotely collecting data and posting it on to a public Internet web site. In one embodiment, the IED or metering device will communicate through the firewall using a protocol such as HTTP via a port that is open through the firewall. Referring toFIG.5, IEDs or meters510,512,514reside on an internal network516, e.g., an intranet, private network, corporate network, etc. The internal network516is coupled to an external network522, e.g., the Internet, via a router520or similar device over any known hardwire or wireless connection521. A firewall518is disposed between the internal network516and external network522to prevent unauthorized access from outside the internal network516to the IEDs or meters510,512,514. Although the firewall518is shown between the internal network516and the router520it is to be appreciated that other configurations are possible, for example, the firewall518being disposed between the router520and external network522. In other embodiments, the firewall518and router520may be configured as a single device. It is further to be appreciated that firewall518can be implemented in both hardware and software, or a combination of both. The communication device or network interface of the meter (as described above in relation toFIGS.1and4) may communicate through the firewall518and read a web site server524. It is to be appreciated that the one way communication from the IED through the firewall may be enabled by various techniques, for example, by enabling outbound traffic to the IP address or domain name of the server524or by using a protocol that has been configured, via the firewall settings, to pass through the firewall such as HTTP (Hyper Text Transfer Protocol), IP (Internet Protocol), TCP (Transmission Control Protocol), FTP (File Transfer Protocol), UDP (User Datagram Protocol), ICMP (Internet Control Message Protocol), SMTP (Simple Mail Transport Protocol), SNMP (Simple Network Management Protocol), Telnet, etc. Alternatively, the IED may have exclusive access to a particular port on the firewall, which is unknown to other users on either the internal or external network. Other methods or techniques are contemplated, for example, e-mail, HTTP tunneling, SNTP trap, MSN, messenger, IRQ, Twitter™, Bulletin Board System (BBS), forums, Universal Plug and Play (UPnP), User Datagram Protocol (UDP) broadcast, UDP unicast, Virtual Private Networks (VPN), etc. The server524will provide instructions in computer and/or human readable format to the IED or meter. For instance, the web server524might have XML tags that state in computer readable format to provide data for the last hour on energy consumption by 15 minute intervals. The meter510,512,514will then read those instructions on that web server524and then post that data up on the server524. In this manner, the IED or meter initiates communication in one direction, e.g., an outbound direction, to the server524. Another server (or can be in one server) will read the data that the meter510,512,514posts and will format the meter data into data that can be viewed for humans on a web site or a software application, i.e., UI Server526. Servers524,526can also store the data in a database or perform or execute various control commands on the data. Clients528may access the IED data stored or posted on servers524,526via a connection to the network522. Since the meters are only communicating in an outbound direction only, the meters510,512,514can read data or instructions from an external network application (e.g., server524), the external network application cannot request information directly from the meter. The server524posts the data or instructions on the web site and waits for the meter to check the site to see if there has been a new post, i.e., new instructions for the meter. The meter can be programmed at the user's discretion as to frequency for which the meter510,512,514exits out to the external network to view the postings. The meter instruction server524will post instructions in a directory programmed/located on the server or into XML or in any fashion that the meter is configured to understand and then the meter will post whatever data it is instructed to do. The meter can also be configured to accomplish control commands. In addition to the meter instruction server524, a user interface (UI) server526is provided that can be used to enable a user interface to the user. The user can provide input on the UI server526that might trigger the meter instruction server524to produce a message to control the energy next time the meter reads that server. In another embodiment, the IED or metering device will communicate through the firewall using a server (not shown) disposed on an internal network protected by a firewall. In this embodiment, the server aggregates data from the various IEDs510,512,514coupled to the internal or private network516. Since the server and the IEDs510,512,514are all on the same side of the firewall518, generally communications and data transfers among the server and the IEDs510,512,514is unrestricted. The server then communicates or transfers the data from the IEDs to server524on the external network on the other side of the firewall518. The communication between server on the internal network and server524may be accomplished by any one of the communication means or protocols described in the present disclosure. The server524then posts the data from the IEDs510,512,514making the data accessible to clients528on external networks, as described above. In a further embodiment, the server disposed on the internal network communicates or transfers the data from the IEDs to clients528on the external network on the other side of the firewall518, without the need to transfer or pass data to a server on the external network. In another embodiment, each IED510,512,514may be configured to act as a server to perform the functionality described above obviating the need for a separate server. Furthermore, in another embodiment, each IED510,512,514and each client device528may be configured as a server to create a peer-to-peer network, token ring or a combination of any such topology. The systems and methods of the present disclosure may utilize one or more protocols and/or communication techniques including, but not limited to, e-mail, File Transfer Protocol (FTP), HTTP tunneling, SNTP trap, MSN, messenger, IRQ, Twitter™, Bulletin Board System (BBS), forums, Universal Plug and Play (UPnP), User Datagram Protocol (UDP) broadcast, UDP unicast, Virtual Private Networks (VPN), etc. Common chat protocols, such as MSN, AIM, IRQ, IRC, and Skype, could be used to send a message, containing the meter's data, to a public chat server which could then route that message to any desired client. A public social server that supports a common web interface for posting information, such as Twitter™, Facebook™, BBS's, could be used to post a status, containing the meter's data, to a user on the public social server for that service, e.g., server440,540,640. This post could then be viewed by the clients to see the meter's data, or read by another server for further parsing and presentation. Hosted data services, such as a hosted database, cloud data storage, Drop-Box, or web service hosting, could be used as an external server to store the meter's data, called Hosting. Each of these Hosts, e.g., server540, could then be accessed by the clients to query the Hosted Data. In another embodiment, the IEDs can communicate to devices using Generic Object Oriented Substation Event (GOOSE) messages, as defined by the IEC-61850 standard, the content of which are herein incorporated by reference. A GOOSE message is a user-defined set of data that is “published” on detection of a change in any of the contained data items sensed or calculated by the IED. Any IED or device on the LAN or network that is interested in the published data can “subscribe” to the publisher's GOOSE message and subsequently use any of the data items in the message as desired. As such, GOOSE is known as a Publish-Subscribe message. With binary values, change detect is a False-to-True or True-to-False transition. With analog measurements, IEC61850 defines a “deadband” whereby if the analog value changes greater than the deadband value, a GOOSE message with the changed analog value is sent. In situation where changes of state are infrequent, a “keep alive” message is periodically sent by the publisher to detect a potential failure. In the keepalive message, there is a data item that indicates “The NEXT GOOSE will be sent in XX Seconds” (where XX is a userdefinable time). If the subscriber fails to receive a message in the specified time frame, it can set an alarm to indicate either a failure of the publisher or the communication network. The GOOSE message obtains high-performance by creating a mapping of the transmitted information directly onto an Ethernet data frame. There is no Internet Protocol (IP) address and no Transmission Control Protocol (TCP). For delivery of the GOOSE message, an Ethernet address known as a Multicast address is used. A Multicast address is normally delivered to all devices on a Local Area Network (LAN). Many times, the message is only meant for a few devices and doesn't need to be delivered to all devices on the LAN. To minimize Ethernet traffic, the concept of a “Virtual” LAN or VLAN is employed. To meet the reliability criteria of the IEC-61850, the GOOSE protocol automatically repeats messages several times without being asked. As such, if the first GOOSE message gets lost (corrupted), there is a very high probability that the next message or the next or the next will be properly received. In one embodiment, a client device, e.g., client computer528, may include a suite of software utilities or a module for verifying the setup of an IED or meter. The meter setup verification feature provides a user with a list of possible problems detected with meters and the system, so that the user may identify and correct faults quickly and easily. In one embodiment, a utility or module is provided for setup verifications. For example, the software utility or module may perform a wiring check, i.e., verifies the voltage and current hookups are in the correct order and that the current transformers (CT's) are not reversed. Referring toFIG.6, a screen shot600generated by the utility or module illustrates a meter list602for a plurality of meters, that may be on a single network or may be owned or associated to a single utility or organization over several networks. The meter list602includes at least two elements, to indicate to the user that a possible problem has been detected with one of the meters, or with the system. The meter list602displays a warning icon604next to the meter name when an issue with that meter is detected, and a status bar panel606includes a warnings item608to indicate a number of issues and/or warnings identified. The meter list warning icon604is displayed in a meter name column610when an issue with that meter is detected. Clicking the icon604jumps to a problems list panel, which will be described below in relation toFIG.7. Hovering over the icon604lists the known issues, e.g., Potential Current A CT Reversal. Additionally, the status bar warnings item608lists the number of issues detected by the system, including both meter and system issues. Clicking the warnings item608jumps to the problems list panel, as illustrated inFIG.7. The problems list panel700displays all of the registered issues detected by the utility or module and provides the user with the ability to search and filter the issues, and instruct the utility or module to retest each of the issues. In one embodiment, the problems list panel700is only shown when the system isn't scanning for problems; when the system is scanning for problems, the problems scan panel800(as shown inFIG.8) is shown instead. Once loaded, the user interface (UI) shown inFIG.7may be refreshed on a periodic interval, e.g., a user defined predetermined interval, or reloaded when a refresh button is pressed. Individual elements of problems list panel700are described below:Banner701—Displays the number of problems detected, or No Problems Detected if there are none registered or detected.Issue Type Filter702—Allows the user to filter by different issue types, e.g., wiring, offline, log retrieval, etc.Time Range Filter703—Limits the displayed issues to only those in the time range specified, based on the last detected date.Refresh List704—Upon selection, queries the issues list from the utility or module, and refreshes the issues list based on the filter options.Rescan705—Upon selection, instructs the utility or module to retest issues that it knows how to process.Issues List706—Displays the list of problems or issues, given the filters selected by the user. Note, there may be multiple rows for a single meter, if there are multiple issues detected. The Issues List706includes at least five columns of information as follows:Group—The group the meter is assigned to.Meter—The meter the problem is associated with.Issue—The category of problem detected, e.g., wiring, offline, log retrieval, etc.Detected—When the issue was last detected or confirmed.Description—Description of the problem, e.g., Potential Current A CT Reversal, unable to communicate to meter, unable to retrieve logs, etc.Issue Actions707—Right clicking on a problem in the issues list706brings up a menu of actions to perform on the problem. For example: Retest Issue—Instructs the utility or module to retest the specific issue, and update its status. Clear Issue—Removes the issue from the list of problems. If the problem is detected again, it will be reinserted to the list. Show Phasor—Displays a live diagram of the current state of the phasors being monitored by the meter. Exemplary phasor diagrams are described and illustrated below inFIGS.12and14. Corrective Measures—Provides executable instructions to the meter/IED to rectify the incorrect wiring, e.g., by reassigning actual connections to the meter/IED to the proper expected value.Report708—Upon selection, generates a CSV report of the issues list, that can be given to a technician for service. An exemplary report is shown below: MeterSerialConnectionIssueDescriptionOffice0123456789mn://172.20.166.98WiringPotential CurrentRKA CT ReversalW15.640123456788mn://172.20.166.99OfflineUnable tocommunicate tometer When the utility or module is scanning for problems, the problems list panel700displays the problem scan panel800instead, as shown inFIG.8. The problem scan panel800displays a list of each known problem, and the current status of its check. Once the scan has completed, the panel automatically reverts to the problems list panel700. Using a periodic status query, the current status and list are updated by a user adjustable period, e.g., every couple of seconds. Individual elements of problems scanning panel800are described below:Banner801—The banner changes to indicate that the utility or module is checking problems and how many issues/problems remain to be tested.Issue/Status802—Displays the current status of each problem being tested. For example,Possible Problem—The issue is still a problem after the test.Resolved—The issue was resolved, and will be removed from the problems list.Testing—The utility or module is currently testing the problem.Pending—The utility or module has not yet retested the problem.Results803—Displays the results of the test, or the current actions being performed for the test. It is to be appreciated that some issues, such as log retrieval and connection issues, are incidentally detected through the normal operation of the utility or module. When these issues are detected, they can be reported through various methods such as email, an API, etc. Some issues, such as the wiring check, may only ever need to be checked on demand, or periodically. An on-demand testing service can be run from a predefined script to perform this, and the issue retesting functionality. This service may be a thread that is run on demand via RPC (Remote Procedure Call), as opposed to a script. The client device may store the meter data generated in a storage device disposed in or coupled to the client device. In one exemplary embodiment, the stored data may have the following structure:Type—The type (or category) of the problem.WiringOfflineLog RetrievalMeter—The device key for the meter. Note, the display name or group is not stored, as that can change, and should be handled by the user interface (UI).Detected Date—The date the problem was last detected or updated.Description—A text description of the possible problem, to be displayed to the user, and providing more information than just the type. In one embodiment, the problems list may be stored as an XML structure. An exemplary problems list stored as an XML structure is illustrated below, which includes “issue type”, “meter”, “detected_date” and “desc” for description as described above: <root><header version=“1” last_updated=“2018/03/25 12:15:17”/><issues><issue type=“wiring” meter=“0091234567”detected_date=“2018/03/23 17:18:13”desc=“Potential Current A CT Reversal”/><issue type=“offline” meter=“0091234567”detected_date=“2018/03/24 23:57:01”desc=“Unable to communicate to meter”/><issue type=“log retrieval” meter=“0000000012345678”detected_date=“2018/03/1512:12:03” desc=“Unable to retrieve logs, security required but notconfigured”/></issues></root> A RPC may be employed to query issues, for example:issues.list This text is an example (and could be arbitrary), and other commands which execute similar code are envisioned. The RPC queries a list of all the issues detected by the meter setup verification utility or module. If the utility or module is currently retesting the problems list, this command will return that status. When tests are not running: <root><header version=“1” last_updated=“2018/03/25 12:15:17”status=“ready”/><issues><issue type=“wiring” meter=“0091234567”detected_date=“2018/03/23 17:18:13”desc=“Current CT's Reversed”/><issue type=“offline” meter=“0091234567”detected_date=“2018/03/24 23:57:01”desc=“Unable to communicate to meter”/><issue type=“log retrieval” meter=“0000000012345678”detected_date=“2018/03/1512:12:03” desc=“Unable to retrieve logs, security required but notconfigured”/></issues></root> When tests are running: <root><header version=“1” last_updated=“2018/03/25 12:15:17”/status=“testing”><issues><issue type=“wiring” meter=“0091234567”detected_date=“2018/03/23 17:18:13”status=“problem” desc=“Current CT's Reversed”/><issue type=“offline” meter=“0091234567”detected_date=“2018/03/24 23:57:01”status=“ok” desc=“Able to connect”/><issue type=“log retrieval” meter=“0000000012345678”detected_date=“2018/03/1512:12:03” status=“pending” desc=“Attempting to login...”/></issues></root> As described above, issues may be retested from the problems list panel700. Right clicking on a problem in the issues list706brings up a menu of actions to perform on the problem, i.e., issue actions707. Selecting “Retest Issue”, instructs the utility or module to retest a specific issue, or all the issues, according to the command below. issues.test [meter] [type]issues.test “meter=0091234567,type=wiring” Issuing this command will prevent querying the issues list until completed.[meter]—The meter to retest. If not specified, issues for all meters will be retested.[type]—The type of issues to retest. If not specified, all types of issues are retested (as available). Other command formats are envisioned, such as separate commands to test all meters, a list of meters, and a single meter. When a meter has been hooked up to the electrical power distribution system in a 3 Element Wye or 2 CT Delta configuration, the utility or module may use voltage and current phase angles as determined by the meter to determine if the meter has been wired incorrectly. Referring toFIG.16, an exemplary wiring diagram of an electronic power meter1602in a 3 Element Wye configuration is provided. The meter1602includes voltage inputs1612(e.g., Va, Vb, Vc, Vref) and current inputs1614(e.g., Ia, Ib, Ic). Each voltage input is coupled to a respective line1620of the electrical distribution system, e.g., input Va is coupled to line or phase A, input Vb is coupled to line or phase B, input Vc is coupled to line or phase C and input Vref is coupled to line or phase N (neutral). Each voltage input1612may be connected directly to a respective line or phase, or alternatively, each voltage input1612may be coupled to a respective line or phase via an optional potential transformer1622. Each current input1614is coupled to a respective line1620of the electrical distribution system via a current transformer (CT)1624,1626,1628. As can be seen inFIG.16, each current input for a respective line or phase includes a HI input and a LO input. Referring toFIG.17, an exemplary wiring diagram of an electronic power meter1702in a 2 CT Delta configuration is provided. The meter1702includes voltage inputs1712(e.g., Va, Vb, Vc, Vref) and current inputs1714(e.g., Ia, Ib, Ic). Each voltage input is coupled to a respective line1720of the electrical distribution system, e.g., input Va is coupled to line or phase A, input Vb is coupled to line or phase B, and input Vc is coupled to line or phase C. Each voltage input1612may be connected directly to a respective line or phase, or alternatively, the voltage inputs1612may be coupled to the three lines or phases via two optional potential transformers1722. The current inputs1714are coupled to three lines or phases1720of the electrical distribution system via two current transformers (CTs)1724and1726. As can be seen inFIG.17, each current input for a respective line or phase includes a HI input and a LO input. Since a result that the meter has been wired incorrectly will not change until rectified (e.g., a technician has rewired the meter, a user initiated reprogramming based on the actual wiring has been implemented, etc.), and will not become incorrect again after it has been rectified, this verification can be done on an as needed basis. For example, often a meter is first installed by a contractor or electrician, that may not have the ability or knowledge to verify that the voltage and current has been wired up correctly. This is particularly troublesome when phases are connected in the wrong order, as the raw voltage and current may look normal, but the energy accumulated and the phase angles reported, may be completely wrong. By checking and reporting the meter hookup issues, an administrator can quickly check the wiring of the meters in the associated system, and send technicians out with specific instructions to repair. Referring toFIG.9, a method for verifying a meter setup is provided. It is to be appreciated that the method ofFIG.9may be performed by a client device including the software utility or module of the present disclosure, such as client device528described above. In step902, the client device polls hookup or configuration settings for the meter or meters, e.g., 3 Element Wye or 2 CT Delta. It is to be appreciated that the hookup or configuration setting may be initially selected via a user interface on a display device of the meter (e.g., via display206and buttons210of meter200as inFIGS.2A and2B) or via a software program running on a client device coupled to the meter. The selected hookup or configuration setting may then be stored in a memory of the meter. Next, in step904, voltage and current RMS values are polled. The voltage and current phase angles are polled, in step906. In step908, the hookup settings, RMS values, and phase angles are normalized. Because each meter type may return phase angles and hookup or configuration settings in different formats, in one embodiment, the client device uses a DeviceLib class for each meter, i.e., a library module customized for each meter type, to individually translate the meters phase angle format to one useable by the wiring check utility or module. Referring toFIG.10, five meter types1002,1004,1006,1008,1010are in communication and polled for data to perform the wiring check. Each of the five meters1002,1004,1006,1008,1010have a different, predefined phase angle or data format, i.e., format 1, format 2, format 3, respectively. A poller1012in the client device employs a DeviceLib class for each meter to individually translate the phase angle format of each meter to a common format1014. Using the common phase angle format output by DeviceLib, the wiring check utility or module applies the various tests (as will be described below) to generate a result for each meter tested, in step910. These results are then stored in the problems list table, in step912. Before conducting a wiring check, the utility or module verifies at least three conditions. Initially, the utility or module determines if the wiring configuration setup programmed into the meter is a 3 Element Wye or a 2 CT Delta. It is to be appreciated that the wiring configuration setup may be selectable from a user interface coupled to the meter, e.g., a display device on the meter, via a software program executing on the client device, etc., and stored in memory of the meter. Depending on the wiring configuration, the utility or module performs different checks or tests to determine if the wiring setup is correct. Additionally, the utility or module determines if the RMS voltage is above 5 V secondary and RMS current is above 0.05 A secondary. Referring toFIG.11, a method1100for verifying the wiring setup of a meter in a 3 Element Wye configuration is provided. It is to be appreciated that the method ofFIG.11may be performed by a client device including the software utility or module of the present disclosure, such as client device528described above. It is to be appreciated that the tests or checks performed inFIG.11are the tests referred to in step910ofFIG.9. Initially, in step1102, if it is determined that the meter is configured in a 3 Element Wye configuration, the tests or checks for the 3 Element Wye configuration are retrieved, e.g., from a memory device. In step1104, RMS voltage and current (that were retrieved in step904ofFIG.9) are analyzed to determine if they are above a predetermined threshold, e.g., voltage is above 5 V secondary and current is above 0.05 A secondary. If the voltage and current are below the respective predetermined threshold, the test fails, in step1106, and method1100stops where no further tests or checks are performed. If the voltage and current are above the respective predetermined threshold, method1100proceeds to step1108. In step1108, the utility or module determines if the voltage phases are swapped, e.g., if all of the current phases are within ±45 degrees of a voltage, but two of the currents are associated with the wrong voltage. If the voltage phases are swapped, the test fails, in step1110, and method1100stops where no further tests or checks are performed; otherwise, method1100proceeds to step1112. Note, this test need not be performed if one of the current phase RMS values are below the threshold, as the relative phase angle between the current and voltage may be unreliable. In step1112, a voltage phase check is preformed, e.g., it is determined if the voltage phases are 120 degrees+/−5 degrees apart. If the voltage phases are not in compliance, the test fails, in step1114, and method1100stops where no further tests or checks are performed; otherwise, if all three voltage phases are 120 degrees+/−5 degrees apart, method1100proceeds to step1116. In step1116, a CT reversal check is preformed, e.g., it is determined that a current phase is 180 degrees+/−45 degrees from a corresponding voltage. If the currents are associated with the wrong voltage phases, the test fails, in step1118, and method1100stops where no further tests or checks are performed; otherwise, method1100proceeds to step1120. In step1120, a current to voltage check is preformed, e.g., it is determined if each current phase is +/−45 degree from a corresponding voltage. If a respective current phase is greater than +/−45 degree from a corresponding voltage phases (e.g., if Ia is greater than +/−45 degrees from Va), the test fails, in step1122, and method1100stops where no further tests or checks are performed; otherwise, method1100determines both voltage and current are wired correctly, in step1124. In one embodiment, if the voltages are above the predetermined voltage threshold, but the currents are below the predetermined current threshold in step1104, the voltage tests or checks, e.g., steps1108and1112, may still be performed without performing the current tests or checks, e.g., steps1116and1120. In another embodiment, if the voltages are above the predetermined voltage threshold, but only certain currents are above the predetermined current threshold (e.g., only the current for phase A is above the predetermined current threshold), the current tests or checks may be performed on those individual current phases that have current values above the predetermined current threshold, in addition to the voltage checks being performed. It is to be appreciated that in certain embodiments all steps, tests and/or checks, e.g., steps1104,1108,1112,1116,1120, may be performed even if one or more tests and/or checks have failed. In other embodiments, the steps, tests and/or checks may be performed in any order or simultaneously. For example, after each test, a flag may be set indicating if a particular test has passed or failed. After all test are completed, an indication may be presented indicating at least one test has failed and/or the indication may present a list of which tests have failed. Referring toFIG.12, exemplary results of the analysis performed in method1100for a 3 Element Wye configuration are illustrated in the various phasor diagrams. The results of the voltage phase check of step1112, where voltage phases must be 120 degrees±5 degrees apart, are illustrated in Examples 1 and 2 ofFIG.12. Example 1 shows the ideal (or passing) case where voltage phases Va, Vb and Vc are 120 degrees apart. Example 2 shows a failing case, where at least the voltage phase between Va and Vb is 180 degrees apart (i.e., more than 120 degree apart). The results of the current phase check of step1120, where current phase must be within ±45 degrees of its corresponding voltage phase, is illustrated in Examples 3 and 4 ofFIG.12. Example 3 shows the ideal or passing case where Ia is approximately 20 degrees from Va, and Example 4 shows a failing case where current Ia is more than 45 degrees from voltage Va, i.e., in this example, Ia is 90 degrees from Va. If the current phase is 180 degrees±45 degrees of the voltage phase as determined in step1116, this indicates a reversed CT. Example 5 ofFIG.12shows this failing case, where current Ia is approximately 170 degrees from Va. Step1108determines if the voltage phases are swapped by determining if all of the current phases are within ±45 degrees of a voltage, but two of the currents are associated with the wrong voltage. Example 6 ofFIG.12shows a case where Va and Vb are swapped, as shown by the fact that the Ib angle matches the Va angle, Ia matches the Vb angle, and Ic matches the Vc angle. Similarly, Example 7 ofFIG.12shows a case where Va and Vc are swapped. Referring toFIG.13, a method1300for verifying the wiring setup of a meter in a 2 CT Delta configuration is provided. It is to be appreciated that the method ofFIG.13may be performed by a client device including the software utility or module of the present disclosure, such as client device528described above. It is to be appreciated that the tests or checks performed inFIG.13are the tests referred to in step910ofFIG.9. Initially, in step1302, if it is determined that the meter is configured in a 2 CT Delta configuration, the tests or checks for the 2 CT Delta configuration are retrieved. In step1304, RMS voltage and current (that were retrieved in step904ofFIG.9) are analyzed to determine if they are above a predetermined threshold, e.g., voltage is above 5 V secondary and current is above 0.05 A secondary. If the voltage and current are below the respective predetermined threshold, the test fails, in step1306, and method1300stops where no further tests or checks are performed. If the voltage and current are above the respective predetermined threshold, method1300proceeds to step1308. In step1308, the utility or module determines if the voltage phases are swapped. If the voltage phases are swapped, the test fails, in step1310, and method1300stops where no further tests or checks are performed; otherwise, method1300proceeds to step1312. In step1312, a Vbc voltage phase check is preformed, e.g., it is determined if the Vcb voltage phase is 60 degrees+/−5 degrees from the Vab phase. If the voltage phases are not in compliance, the test fails, in step1314, and method1300stops where no further tests or checks are performed; otherwise, method1300proceeds to step1316. In step1316, a check is preformed to determine if Ia phase is within a predetermined threshold of Vab phase, e.g., it is determined if the Ia current phase is 30 degrees+/−45 degrees from the Vab phase. If the Ia current phase is not in compliance, the test fails, in step1318, and method1300stops where no further tests or checks are performed; otherwise, method1300proceeds to step1320. In step1320, a check is preformed to determine if Ic phase is within a predetermined threshold of Vcb phase, e.g., it is determined if the Ic current phase is 30 degrees+/−45 degrees from the Vcb phase. If the Ic current phase is not in compliance, the test fails, in step1322, and method1300stops where no further tests or checks are performed; otherwise, method1300proceeds to step1324. In step1324, a CT reversal check is preformed, e.g., it is determined that a current phase is 180 degrees+/−45 degrees from the ideal (where the current angles should be in a perfect system, i.e., seeFIG.14, Example 3). If the currents are reversed, the test fails, in step1326, and method1300stops where no further tests or checks are performed; otherwise, method1300determines the CTs are wired correctly, in step1324. In one embodiment, if the voltages are above the predetermined voltage threshold, but the currents are below the predetermined current threshold in step1304, the voltage tests or checks, e.g., steps1308and1312, may still be performed without performing the current tests or checks, e.g., steps1316,1320and1324. In another embodiment, if the voltages are above the predetermined voltage threshold, but only certain currents are above the predetermined current threshold (e.g., only the current for phase A is above the predetermined current threshold), the current tests or checks may be performed on those individual current phases that have current values above the predetermined current threshold, in addition to the voltage checks being performed. It is to be appreciated that in certain embodiments all steps, tests and/or checks, e.g., steps1304,1308,1312,1316,1320,1324, may be performed even if one or more tests and/or checks have failed. In other embodiments, the steps, tests and/or checks may be performed in any order or simultaneously. For example, after each test, a flag may be set indicating if a particular test has passed or failed. After all test are completed, an indication may be presented indicating at least one test has failed and/or the indication may present a list of which tests have failed. Referring toFIG.14, exemplary results of the analysis performed in method1300for a 2 CT Delta configuration are illustrated in the various phasor diagrams. The results of the Vbc voltage phase check of step1312, where Vcb must be 60 degrees±5 degrees from the Vab phase, are illustrated in Examples 1 and 2 ofFIG.14. Example 1 shows the ideal case, where Vab is 60 degrees+/−5 degrees from Vcb, and Example 2 shows a failing case, where Vab is 180 degrees from Vcb. The results of the checks performed in steps1316and1320, where Ic phase must be +30 degrees±45 degrees from Vcb, and Ia must be −30 degrees±45 degrees from Vab, are illustrated in Examples 3 and 4 ofFIG.13. Example 3 shows the ideal case where Ic is within 30 degrees of Vcb and Ia is within −30 degrees of Vab, and Example 4 shows a failing case where Ia is approximately 135 degrees from Vab. If the current phase is 180 degrees±45 degrees of the ideal (−30 degrees in the case of Ia), as determined in step1324, this indicates a reverse CT, as illustrated in Example 5. The results of the voltage swap check of step1308are illustrated in Examples 6 and 7 ofFIG.14. If Ic is where Ia should be, and Ia is −135 degrees±45 degrees from Vab, this indicates that Va and Vb are swapped, as shown in Example 6 ofFIG.14. If Vcb is −60 degrees±5 degrees from Vab, Ia is −30 degrees±45 degrees from Vcb, and Ic is 30 degrees±45 degrees from Vab, this indicates that Va and Vc are swapped, as shown in Example 7 ofFIG.14. Based on the results of the analysis above, the following messages or descriptions/meanings may be generated and displayed in the description section of the issues list706ofFIG.7: MessageMeaningGeneralOKBoth voltage and current are correctly wired.Not supportedSupports 3 Element Wye and 2 CT Delta,only.Unable to communicate with meterMeter is offline.Voltage Low - One or more of the voltageBelow 5 V secondaryphases has too lowof an RMS value.Current Low - One or more of the currentBelow 0.05 A secondaryphases has too lowof an RMS value.Phase A CT Reversal - A current phaseCurrent phase 180° ± 45 of the voltage phase.may be reversed.Phase B CT Reversal - B current phaseCurrent phase 180° ± 45 of the voltage phase.may be reversed.Phase C CT Reversal - C current phaseCurrent phase 180° ± 45 of the voltage phase.may be reversed.3 Element WyeVoltage Phases Va and Vb may beAll Current phases ±45° from a voltage, butswapped - Two of the voltage phases maytwo of the currents are associated with thebe swapped.wrong voltage.Voltage Phases Va and Vc may beAll Current phases ±45° from a voltage, butswapped - Two of the voltage phases maytwo of the currents are associated with thebe swapped.wrong voltage.Current Out of Phase - The current phasesCurrent phase ±45° from correspondingmay be incorrect.voltage.Voltage Out of Phase - The voltage phasesVoltage phase 120° ± 5 apart.may be incorrect.2 CT DeltaVoltage Phases A-C Swapped - Two of theVab 60° ± 5 from Vcbvoltage phases may be swapped.Voltage Phases B-C Swapped - Two of theVcb 60° ± 5 from Vabvoltage phases maybe swapped.Voltage Phase C may be incorrect.Vcb 60° ± 5 from VabCurrent Phase A Bad may be incorrect.Ia must be −30° ± 45° from VabCurrent Phase C Bad may be incorrect.Ic phase must be +30° ± 45° from Vcb. It is to be appreciated that based on the results of the wiring check, software utility or module (executed by at least one processor of the client device or by at least one processor of a meter) may trigger various events to occur. For example, the at least one processor may generate a notification indicating that the meter is wired incorrectly. In one embodiment, the notification may include a work order indicating the problem/issue and sent to the appropriate personnel to correct the issue, e.g., a field technician. The notification or work order may be sent via email, text message, computer-generated voice message, etc. without user intervention. The notification or work order may include information identifying the meter, its location, and/or corrective measures to rectify the incorrect wiring. In another embodiment, the wiring check utility or module may trigger an output on a respective meter having a wiring issue, for example, to trip a relay to shut off power being delivered to a load. Other outputs/triggers are contemplated to be within the scope of the present disclosure. In another embodiment, the incorrect wiring setup may be rectified by reprogramming the meter. In one embodiment, if the at least one processor determines that the at least one electronic power meter is wired incorrectly, the at least one processor generates executable instructions to rectify the determined incorrect wiring of the at least one electronic power meter and transmits the executable instructions to the at least one electronic power via the communication interface without user intervention. In another embodiment, if the at least one processor determines that the at least one electronic power meter is wired incorrectly, the at least one processor prompts a user via a user interface to initiate corrective measures and, if the user activates the corrective measures via the user interface, the at least one processor generates executable instructions to rectify the determined incorrect wiring of the at least one electronic power meter and transmits the executable instructions to the at least one electronic power via the communication interface. In one example, a CT may be reversed, i.e., the leads from the CT coupled to the HI and LO current inputs1614may be reversed. If it is determined that at least one CT is reversed, corrective measures may be initiated, either by the user or automatically by the software utility or module, and the meter's instructions (e.g., firmware, software, programmable settings, etc.) shifts the current phase by 180 degrees for that phase, rather than rewiring the meter. This will reverse the power direction, and all computations such as power factor, phase angle etc., which are derived after that will be affected. The option to rectify an incorrect wiring setup (or take corrective measures) may be presented to a user on a user interface of the client device, e.g., the problem list700shown inFIG.7. Where upon selection, e.g., selection of “Corrective Measures” on issue actions707, the corrective measures may be placed in effect. Alternatively, the corrective measures for a particular problem may be automatically implemented by at least one processor of the meter with user input or intervention. After the corrective measures have been implemented, a user may select “Retest Issue” on issue actions707to observe whether the corrective measures actually corrected the particular problem. In another example, it may be determined that the voltages are swapped, as determined in step1108of method1100and shown in Ex.6ofFIG.12where Va and Vb are swapped. Upon a user selecting corrective measures or the corrective measures being automatically initiated by the client device or meter, executable instructions will be implemented to now assigned the values as being read at voltage input Va as values for Vb and vice versa. After the corrective measures have been implemented, a user may select “Retest Issue” to observe whether the corrective measures actually corrected the particular problem (where, in this case, the problem is that Va and Vb were swapped). FIG.15is a block diagram illustrating physical components of a computing device1502, for example a client computing device, with which examples of the present disclosure may be practiced. Among other examples, computing device1502may be an exemplary computing device configured for execution of a wiring check module that is used to verify a meter wiring setup as described herein. In a basic configuration, the computing device1502may include at least one processing unit1504and a system memory1506. Depending on the configuration and type of computing device, the system memory1506may comprise, but is not limited to, volatile storage (e.g., random access memory), non-volatile storage (e.g., read-only memory), flash memory, or any combination of such memories. The system memory1506may include an operating system (OS)1507and one or more program modules1508suitable for running software programs/modules1520such as IO manager1524, other utility1526and application1528, for example, the wiring setup verification utility. As examples, system memory1506may store instructions for execution. Other examples of system memory1506may store data associated with applications. The operating system1507, for example, may be suitable for controlling the operation of the computing device1502. Furthermore, examples of the present disclosure may be practiced in conjunction with a graphics library, other operating systems, or any other application program and is not limited to any particular application or system. This basic configuration is illustrated inFIG.15by those components within a dashed line1522. The computing device1502may have additional features or functionality. For example, the computing device1502may also include additional data storage devices (removable and/or non-removable) such as, for example, magnetic disks, optical disks, or tape. Such additional storage is illustrated inFIG.15by a removable storage device1509and a non-removable storage device1510. As stated above, a number of program modules and data files may be stored in the system memory1506. While executing on the processing unit1504, program modules1508(e.g., Input/Output (I/O) manager1524, other utility1526and application1528) may perform processes including, but not limited to, one or more of the stages of the operations described throughout this disclosure, for example, the operation of verifying a meter wiring setup. Other program modules that may be used in accordance with examples of the present disclosure may include electronic mail and contacts applications, word processing applications, spreadsheet applications, database applications, slide presentation applications, drawing or computer-aided application programs, photo editing applications, authoring applications, etc. Furthermore, examples of the present disclosure may be practiced in an electrical circuit comprising discrete electronic elements, packaged or integrated electronic chips containing logic gates, a circuit utilizing a microprocessor, or on a single chip containing electronic elements or microprocessors. For example, examples of the meter setup verification of the present disclosure may be practiced via a system-on-a-chip (SOC) where each or many of the components illustrated inFIG.15may be integrated onto a single integrated circuit. Such an SOC device may include one or more processing units, graphics units, communications units, system virtualization units and various application functionality all of which are integrated (or “burned”) onto the chip substrate as a single integrated circuit. When operating via an SOC, the functionality described herein may be operated via application-specific logic integrated with other components of the computing device1502on the single integrated circuit (chip). Examples of the present disclosure may also be practiced using other technologies capable of performing logical operations such as, for example, AND, OR, and NOT, including but not limited to mechanical, optical, fluidic, and quantum technologies. In addition, examples of the present disclosure may be practiced within a general purpose computer or in any other circuits or systems. The computing device1502may also have one or more input device(s)1512such as a keyboard, a mouse, a pen, a sound input device, a device for voice input/recognition, a touch input device, etc. The output device(s)1514such as a display, speakers, a printer, etc. may also be included. The aforementioned devices are examples and others may be used. The computing device1504may include one or more communication connections or interfaces1516allowing communications with other computing devices1518and/or meters/IEDs1519. Examples of suitable communication connections or interfaces1516include, but are not limited to, a network interface card; RF transmitter, receiver, and/or transceiver circuitry; universal serial bus (USB), parallel, and/or serial ports. The term computer readable media as used herein may include computer storage media. Computer storage media may include volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information, such as computer readable instructions, data structures, or program modules. The system memory1506, the removable storage device1509, and the non-removable storage device1510are all computer storage media examples (i.e., memory storage.) Computer storage media may include RAM, ROM, electrically erasable read-only memory (EEPROM), flash memory or other memory technology, CD-ROM, digital versatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other article of manufacture which can be used to store information and which can be accessed by the computing device1502. Any such computer storage media may be part of the computing device1502. Computer storage media does not include a carrier wave or other propagated or modulated data signal. Communication media may be embodied by computer readable instructions, data structures, program modules, or other data in a modulated data signal, such as a carrier wave or other transport mechanism, and includes any information delivery media. The term “modulated data signal” may describe a signal that has one or more characteristics set or changed in such a manner as to encode information in the signal. By way of example, and not limitation, communication media may include wired media such as a wired network or direct-wired connection, and wireless media such as acoustic, radio frequency (RF), infrared, and other wireless media. It is to be appreciated that the computing device1520may, in certain embodiments, be a mobile computing device, for example, a mobile telephone, a smart phone, a personal data assistant, a tablet personal computer, a phablet, a slate, a laptop computer, and the like, with which examples of the present disclosure may be practiced. In another embodiment, the meter/IED may perform the meter setup verification. For example, a software utility or module disposed within a meter/IED may perform a wiring check, i.e., verifies voltage and current hookups are in the correct order and that the current transformers (CT's) are not reversed based on the meter wiring configuration and the voltage and current phase angles determined by the meter. In one embodiment, the meter/IED generates a notification indicating that it is wired incorrectly. The notification may be in the form of a pop-up display or screen display on a display device coupled to the meter/IED. In one aspect, the notification is at least one of an email, text message and/or voice message that may be transmitted to an end user or technician. In another aspect, the notification may include corrective measures to rectify the incorrect wiring. For example, the corrective measures may include instructions on how to rewire the meter/IED. In a further aspect, the corrective measures may include a selectable option, presented to the user via a user interface displayed on the display device, to enable executable instructions on the meter/IED to rectify the incorrect wiring, e.g., by reassigning actual connections to the meter/IED to the proper expected value. In yet another aspect, the executable instructions are initiated by the meter/IED automatically without user intervention. It is to be appreciated that the various features shown and described are interchangeable, that is a feature shown in one embodiment may be incorporated into another embodiment. While non-limiting embodiments are disclosed herein, many variations are possible which remain within the concept and scope of the present disclosure. Such variations would become clear to one of ordinary skill in the art after inspection of the specification, drawings and claims herein. The present disclosure therefore is not to be restricted except within the spirit and scope of the appended claims. Furthermore, although the foregoing text sets forth a detailed description of numerous embodiments, it should be understood that the legal scope of the present disclosure is defined by the words of the claims set forth at the end of this patent. The detailed description is to be construed as exemplary only and does not describe every possible embodiment, as describing every possible embodiment would be impractical, if not impossible. One could implement numerous alternate embodiments, using either current technology or technology developed after the filing date of this patent, which would still fall within the scope of the claims. It should also be understood that, unless a term is expressly defined in this patent using the sentence “As used herein, the term ‘ ’ is hereby defined to mean . . . ” or a similar sentence, there is no intent to limit the meaning of that term, either expressly or by implication, beyond its plain or ordinary meaning, and such term should not be interpreted to be limited in scope based on any statement made in any section of this patent (other than the language of the claims). To the extent that any term recited in the claims at the end of this patent is referred to in this patent in a manner consistent with a single meaning, that is done for sake of clarity only so as to not confuse the reader, and it is not intended that such claim term be limited, by implication or otherwise, to that single meaning. Finally, unless a claim element is defined by reciting the word “means” and a function without the recital of any structure, it is not intended that the scope of any claim element be interpreted based on the application of 35 U.S.C. § 112, sixth paragraph. | 90,757 |
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