A method for separating analytes in a separation matrix

Abstract

Described herein are methods for separating analytes by applying an electric field to one or more samples in a liquid or semi-liquid separation matrix shared by the one or more samples.

Description

[0001] A METHOD FOR SEPARATING ANALYTES IN A SEPARATION MATRIX

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 722,356 filed on November 19, 2024, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Methods and devices for detecting analytes are important tools for characterizing analytes in biological and industrial applications. In many applications, there is a need to detect the presence of one or more analytes in a sample. For example, rapid detection of an analyte, such as a particular protein in a mixture of proteins, is particularly useful in molecular biology protocols, drug development and disease diagnosis.

[0006] Current methods of detecting proteins are inconvenient, expensive, and possess other deficiencies. For example, Western blotting, has been in widespread use for detecting proteins, for more than four decades. In this technique, a separating gel is polymerized between two plates and usually is mounted vertically with the upper edge of the gel accessible to the sample to be assayed. The ends of each gel are placed in running buffer with an anode and cathode electrode placed at opposite ends of the gel. The sample is denatured and applied to wells created along the upper edge of the gel. An electrophoretic potential is applied between the upper and lower edges of the solid of gel. The electrophoretic potential is applied by a DC power supply and may be in the range of 50 to more than 600 volts. The electrophoretic potential is applied for a period of time that allows the proteins in the sample to distribute themselves (i.e. , separate) vertically through the gel, typically for 20-60 minutes, but in some cases considerably longer. The gel is then removed from between its two glass retaining plates and is then placed on a sheet of blotting material such as porous nitrocellulose of length and width dimensions approximately matching those of the sheet of gel, the blotting material having already been soaked in a buffer to hydrate it. Care must be taken at this step to avoid the presence of air bubbles between the gel and the blotting material, which may impede the direct transfer of the distributed proteins from the gel to the blotting material. The gel and membrane stack are then submerged in transfer buffer and placed between two electrodes. The electrode plates should preferably apply a uniform electrophoretic field across the thicknesses of the sheets of gel and blotting material. This electrophoretic field, typically 10-100 volts, transfers the proteins from the gel to the blotting material in the same distribution in which they were captured in the gel matrix. This transfer process takes approximately 5 minutes to 2 hours but can take as much as overnight depending on the voltage used. After the protein adheres to the blotting material, the blotting material is removed from the sandwich and is washed in a buffer containing one or more blocking agents such as skim milk, bovine serum albumin, or casein for 10-60 minutes and then is immersed in a solution of protein-specific primary antibodies. During the immersion the blotting paper is typically agitated by a rocking or circular motion in the plane of the blotting paper. The immersion step typically takes 1-4 hours but can take overnight or longer. The membrane is then washed to remove unbound primary antibody. The membrane is then immersed in a reporter secondary antibody for typically 1 hour. The membrane is once again washed to remove unbound secondary antibody. Detection can be done with a variety of markers such as chemiluminescent substrates, fluorescent dyes, chromogenic substrates depending upon the secondary antibody conjugate and analytical method used. These methods were described by Towbin et al., Proc. Nat. Acad. Sci. USA 76: 4350-4354 (1979), Burnette, Anal. Biochem. 112: 195-203 (1981), and Rybicki & von Wechmarin, J. Viral. Methods, 5: 267-278 (1982).

[0007] Traditional immunoblotting methods, while widely used, have several drawbacks and deficiencies. As the above description makes clear, the processing is very complex and includes multiple distinct steps that can considerably increase the amount of time to obtain a single blot. There is also extensive handling of fragile components during the process that can introduce error and variability. The variability in the method and the handling of several different components ranging from liquids, gels, membranes that have to be assembled and dissembled and configured in specific way can require the process to be repeated several times before obtaining acceptable results. This complexity also impedes the ability to automate the process.

[0008] Eliminating the need for handling gels and membranes, is key to automating western blotting. As such, methods have been developed for automating western blots, that utilize chemically modified capillaries to separate proteins in liquid matrices and then photo-capture separated proteins to the walls of the glass capillaries. Approaches requiring capillaries have some limitations in that each sample and immunodetection needs to be loaded and run in its own individual capillary. Therefore, many capillaries are required to process many samples, which increases the manufacturing and handling complexity of consumables and potentially produce variability due to loading and set up errors.

[0009] What is needed are methods for detecting and characterizing target proteins and multiple samples simultaneously on a simple planar surface. SUMMARY

[0010] Described herein are methods for separating one or more analytes in one or more samples, the method comprising: loading one or more samples comprising one or more analytes into a solid matrix comprising one or more sample wells; applying an electric field to the solid matrix and a liquid separation matrix shared by the one or more samples; and generating a separation pattern of the one or more analytes in the liquid separation matrix. In some embodiments, wherein the analytes are proteins, nucleic acids, or a combination thereof. In some embodiments, two or more samples comprising one or more analytes are loaded into at least two sample wells. In some embodiments, the electric field is uniform across the separation matrix. In some embodiments, the separation matrix comprises a linear polyacrylamide, a dimethylpolyacrylamide, a dextran, a pullulan, an agarose, polyvinyl alcohol, a polyethylene oxide, a polysaccharide, hydroxyethyl cellulose, hydroxylpropyl cellulose, methylcellulose, or a combination thereof. In some embodiments, the method further comprises removing the liquid separation matrix in a direction perpendicular to the electric field. In some embodiments, the solid matrix comprises agarose or acrylamide. In some embodiments, the solid matrix comprises one or more layers comprising a running buffer layer, a stacking buffer layer, or a combination thereof. In some embodiment, the running buffer layer comprises 0.5-1.5% agarose 100 mM Tris base, 100 mM Tricine, 0.1% SDS pH 8.3. In some embodiments, the stacking buffer layer comprises 0.5-1 .5% agarose, 125 mM Tris, pH 8.0. In some embodiments, the solid matrix further comprises a matrix buffer to maintain pH, conduct electricity, stabilizing ionic strength, and prevent heat build-up. In some embodiments, the matrix buffer comprises buffers, detergents, or salts, at a pH of 4-10 for electrophoresis. In some embodiments, the matrix buffer comprises tris, tris-glycine, bis-tris, Tricine, acetate, SDS, or combinations thereof. In some embodiments, one or more analytes in step (b) are separated by size and charge. In some embodiments, the size is molecular weight. In some embodiments, the charge is isoelectric point. In some embodiments, the method further comprises a run buffer. In some embodiments, the solid matrix comprises 12- 48 sample wells. In some embodiments, the method further comprises detecting the separation pattern of the one or more analytes with at least one detecting reagent. In some embodiments, the one or more samples comprise 1-4 different analytes. In some embodiments, the 1-4 different analytes are detected individually or simultaneously. DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows an exemplary apparatus containing a first solid matrix with wells for loading at least one sample, a liquid sample matrix shared by each sample, and a second solid matrix opposite of the first solid matrix.

[0012] FIG. 2 shows an exemplary workflow for the electrophoretic separation of a sample with a liquid matrix.

[0013] FIG. 3 shows an exemplary workflow for immunodetection of analytes in a sample using electrophoretic separation of a sample with a liquid matrix.

[0014] FIG. 4A-C show exemplary gel images obtained after electrophoretic separation in a liquid separation matrix. FIG. 4A shows a separation pattern after electrophoretic separation in a liquid separation matrix containing 5% PDMA. FIG. 4B shows a separation pattern after electrophoretic separation in a liquid separation matrix containing 3% solids LPA and 0.08% PDMA. FIG. 4C shows a separation pattern after electrophoretic separation in a liquid separation matrix containing 15% pullulan.

[0015] DETAILED DESCRIPTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0017] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0018] As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0019] As used herein, the term “or” can be conjunctive or disjunctive.

[0020] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0021] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or 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, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0022] As used herein, “or” may be used to describe the conjunction and disjunctive.

[0023] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

[0024] As described herein, “antibody” has its standard meaning and is intended to refer to full- length as well antibody fragments, as are known in the art, including Fab, Fab2, single chain antibodies (Fv for example), monoclonal, polyclonal, chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized using recombinant DNA technologies.

[0025] As described herein, “detect” and “detection” have their standard meaning and are intended to encompass detection including the presence or absence, measurement, and / or characterization of an analyte.

[0026] As described herein, “label” as used herein refers to a detectable moiety. As will be appreciated by those in the art, suitable labels encompass a wide variety of possible moieties. In general, labels include, but are not limited to, (a) isotopic labels, which may be radio-active or heavy isotopes; (b) immune labels, which may be antibodies or antigens; (c) optical dyes, including colored or fluorescent dyes; (d) enzymes such as alkaline phosphatase and horseradish peroxidase, (e) particles such as colloids, magnetic particles, etc., or combinations thereof, such as fluorescent labeled antibodies or chemiluminescent labeled antibodies.

[0027] Described herein are methods for detecting one or more analytes in one or more samples. The methods may include loading one or more samples comprising one or more analytes into a solid matrix comprising one or more sample wells. The method may further comprise applying an electric field to the solid matrix and a liquid separation matrix shared by the one or more sample to generate a separation pattern the one or more analytes in the liquid separation matrix.

[0028] The sample used in the methods described herein may be homogenous or heterogeneous. The sample may contain naturally occurring biological analytes (e.g., endogenous analytes) or man-made analytes (e.g., non-endogenous analytes). Exemplary biological sample include, but are not limited to, a single cell, a plurality of cells, a blood sample, a tissue sample, a skin sample, a urine sample, a water sample, or a soil sample. Exemplary cells may be from a living organism including, but not limited to, a eukaryote, a prokaryote, a mammal, a human, a yeast, or a bacterium. In some embodiments, the single cell or pluralities of cells may include a virus. In some embodiments, the sample may comprise at least one analyte from a single cell and at least one analyte from a plurality of cells.

[0029] The analyte may be any analyte selected by the user. Exemplary analytes include, but are not limited to, proteins, antibodies, antibody fragments, oligopeptides and peptides, derivatives, or analogs, including proteins containing non-naturally occurring amino acids or amino acid analogs. Other exemplary analytes include, but are not limited to, carbohydrates, polysaccharides, glycoproteins, viruses, metabolites, cofactors, nucleotides, polynucleotides, transition state analogs, inhibitors, drugs, nutrients, electrolytes, hormones, growth factors and other biomolecules as well as non-biomolecules, as well as fragments and combinations thereof. In some embodiments, the sample may contain one or more analytes. In some embodiments, the analytes may include one or more distinct proteins.

[0030] As will be appreciated by those skilled in the art, virtually any processing may be used to prepare a sample comprising one or more analytes prior to separating the sample. Exemplary means of processing a sample prior to separation, include but are not limited to, lysing, denaturing, heating, purifying, precipitating, immunoprecipitating, column chromatography, or centrifugation. Processing the one or more samples prior to separation may be performed by any means known to a person of ordinary skill in the art. In some embodiments, the sample containing analytes may undergo denaturation to expose an internal hydrophobic group prior to separation. The methods described herein may comprise resolving or separating one or more analytes in a separation matrix. As described herein, “separation matrix” refers to the medium in which the separation of analytes occurs. In some embodiments, the separation matrix may be liquid or semi-liquid. As described herein, “liquid” refers to a substance in which solutes are completely dissolved in a solution and is characterized by the substance lacking shape or form beyond the shape or form of its container at room temperature. A “liquid,” as used herein, may also be used to describe a material possessing the ability to free-flow. As described herein, “semi-liquid” or “gel” refers to a substance in which solutes are partially dissolved in a solution and is characterized by the substance possessing a degree of shape or form in the absence of a container at room temperature. A “semi-liquid” or “gel” may possess an ability to flow; however, the ability to flow will be less than a liquid. In preferred embodiments, the separation matrix may be homogenous, uniform, or a combination thereof.

[0031] The separation matrix used in the methods described herein may not contain sample wells or capillaries. Separation of analytes in a fluid path of a capillary have been described. See e.g., U.S. Patent Nos. 7,935,489; 7,935,308; 7,846,676; 9,304,133; 7,935,479; 7,935,479; and 9,400,277. There are several key distinctions between capillary microfluidic technology and methods described herein. With capillary electrophoretic technology, one individual capillary is used to separate analytes in a single sample. In other words, the separation of analytes cannot be performed on more than one sample with a single capillary. To analyze multiple samples with capillary electrophoretic technology, multiple capillaries can be run simultaneously; however, the capillaries exist separately. Therefore, a capillary itself serves as a physical barrier between each sample in a capillary. Since there is a physical barrier formed by the capillaries themselves, the capillaries and the samples therein do not share a separation matrix. In contrast to physical barrier imposed by the capillaries, the one or more samples of the methods described herein may be separated in a shared, uniform liquid matrix. In some embodiments, the separation matrix may be shared by one or more samples. In some embodiments, the separation matrix may be shared by two or more samples.

[0032] The separation matrix used in the methods described herein may comprise a polymer. In some embodiments, the separation matrix may comprise a linear polyacrylamide, a dimethylpolyacrylamide, a dextran, a pullulan, polyvinyl alcohol, a polyethylene oxide, a polysaccharide, hydroxyethyl cellulose, hydroxylpropyl cellulose, methylcellulose, or a combination thereof. In some embodiments, the separation matrix may comprise polydimethyl acrylamide (PDMA), dimethylacetamide (DMA), Tris base, tricine, and water. In preferred embodiments, the separation matrix may include linear polyacrylamide (LPA), Tris base, tricine, and water. In preferred embodiments, the separation matrix may comprise pullulan, Tris base, tricine, and water. In further preferred embodiments, the separation matrix may comprise linear polyacrylamide (LPA), polydimethyl acrylamide (PDMA), tricine, and water.

[0033] The separation matrix may be removed perpendicular to the electric field. In some embodiments, the removal of the separation matrix in the perpendicular direction may reduce disruption to the separation pattern of the separated analytes. In some embodiments, the reduction of disruption of the separation pattern may enhance the signal produced by the one or more analytes.

[0034] In addition to the separation matrix, a solid matrix may be used in the methods described herein. As described herein a “solid matrix” refers to a material that at room temperature has structure and does not freely flow. In some embodiments, the solid matrix is “semi-liquid” or “gel.” In some embodiments, the solid matrix contains one or more layers comprising a running buffer layer, a stacking buffer layer, or a combination thereof. In some embodiment, the solid matrix comprises agarose or acrylamide. In preferred embodiments, a solid matrix may be used to create sample wells, in which the one or more samples comprising the one or more analytes may be added. In preferred embodiments, the solid matrix allows for current and ions to flow through said matrix. In further preferred embodiments, the solid matrix allows for a homogenous electric field across a separation matrix. In preferred embodiments, the solid matrix creates a barrier to prevent leakage of the separation matrix, the detecting reagents, or a combination thereof.

[0035] The solid matrix may also be used to contain the separation matrix. In some embodiments, the solid matrix may exist on one-, two-, three-, or four sides of the separation matrix. In some embodiments, a first solid matrix may be 180° opposite of a second solid matrix.

[0036] The methods described herein may further comprise a matrix buffer. As described herein “matrix buffer” may be used interchangeably with “loading buffer” and refers to buffers, detergents, or salts, at a pH appropriate for electrophoresis. In preferred embodiments, the matrix buffer comprises tris(hydroxymethyl)aminomethane (Tris), Tris-glycine, bis-Tris, tricine, acetate, sodium dodecyl sulfate (SDS), or a combination thereof.

[0037] Matrix buffers and running buffers used in the methods described herein may share similar components at different concentrations. In some embodiments, the matrix buffer and running buffer may include similar component at different pH values. In some embodiments, the run buffers and matrix buffer may create a gradient. In some embodiments, the use of a matrix and run buffer may create a stable long lasting electric field when a current is applied. In some embodiments, the matrix buffer maintains pH, conducts electricity, stabilizes ionic strength, and prevents heat build-up. The matrix buffer may provide support electrophoresis. As described herein, “electrophoresis” refers to the movement of suspended or dissolved molecules through a fluid or gel under the action of an electromotive force applied to electrodes in contact with a fluid. In various embodiments, resolving one or more analytes may comprise isoelectric focusing (IEF) a sample. Without being bound by theory, an analyte will migrate towards the pole (cathode or anode) that carries a charge opposite to the net charge carried by the analyte. The net charge may depend in part on the pH of the medium in which the molecule is migrating. One exemplary, non-limiting, electrophoretic procedure is to establish solutions having different pH values at each end of an electric field, with a gradient range of pH in between. At a certain pH, the isoelectric point of a molecule is obtained, and the molecule carries no net charge. As the molecule crosses the pH gradient, it reaches a spot where its net charge is zero (for example, isoelectric point) and it is thereafter immobile in the electric field. In some embodiments, the electrophoresis procedure may separate molecules according to their different isoelectric points.

[0038] The methods described herein may optionally comprise immobilizing one or more resolved analytes to an immobilization surface. As used described, “immobilizing” refers to substantially reducing or eliminating the movement of analytes after electrophoretic separation. The immobilization may be via covalent bonds or non-covalent means such as by hydrophobic or ionic interaction. A key element of immobilization is that it should not interfere with or compromise the electrophoretic separation of the proteins and only result in immobilization after separation has occurred.

[0039] The immobilization surface may comprise any material that allows the immobilization and subsequent detection of the analyte. The immobilization surface may include but is not limited to glass, plastic, silicon, fused silica, gel, or the like. The immobilization surface may vary as to dimensions, width, depth, and cross-section. In some embodiments, the immobilization surface comprises one or more reactive moieties. A reactive moiety may be used to covalently immobilize the resolved analyte or analytes. The reactive moiety may comprise any reactive group that can form a covalent linkage with a corresponding reactive group of individual molecules of the sample. Thus, the reactive moiety can comprise any reactive group known in the art, so long as it is compatible with the methods described herein. In preferred embodiments, the reactive moiety on the immobilization surface forms a covalent linkage only with an analyte in the sample.

[0040] In some embodiments, the reactive moiety may be attached directly to the immobilization surface. The reactive moiety can line the immobilization surface or, in another embodiment, may be present in a linear or cross-linked pattern. A wide variety of reactive moieties suitable for covalently linking two molecules together are well-known and may be used in the methods described herein. Exemplary reactive moieties include, but are not limited to, photoreactive groups, chemical reactive groups, and thermoreactive groups.

[0041] The one or more analytes may be immobilized to the immobilization surface by photoimmobilization. Photo-immobilization can be accomplished by the activation of one or more photoreactive groups. A photoreactive group may comprise one or more latent photoreac-tive groups that upon activation by an external energy source, forms a covalent bond with other molecules. Without being bound by theory, photoreactive groups may generate active species - such as free radicals and particularly nitrenes, carbenes, and excited states of ketones - upon absorption of electromagnetic energy. Photoreactive groups may be chosen due to their responsiveness to various portions of the electromagnetic spectrum, such as ultraviolet, infrared, and visible portions of the spectrum. In some embodiments, upon exposure to a light source, a photoreactive group may be activated to form a covalent bond with an adjacent molecule. Nonlimiting photoreactive groups include, but are not limited to, aryl ketones, azides, diazos, diazirines, or quinones.

[0042] In some embodiments, the photoreactive group may comprise aryl ketones including, but not limited to, benzophenone, acetophenone, anthraquinone, diphenylketene, anthrone, or anthrone-like heterocycles or their substituted derivatives. In a preferred embodiment the photoreactive group may be a benzophenone. In some embodiments, the photoreactive group comprises azides including, but not limited to, arylazides including, but not limited to, phenyl azide, 4-fluoro-3-nitrophenyl azide, acyl azides including, but not limited to, benzoyl azide and p- methylbenzoyl azide, sulfonyl azides including, but not limited to, benzenesulfonyl azide, and phosphoryl azides including, but not limited to, diphenyl phosphoryl azide and diethyl phosphoryl azide. In some embodiments, the photoreactive group may comprise diazo compounds and include diazoalkanes including, but not limited to, diazomethane and diphenyldiazomethane, diazoketones including, but not limited to, diazoacetophenone and 1-trifluoromethy 1-1-diazo-2- pentanone, diazoacetates including, but not limited to, f- butyl diazoacetate and phenyl diazoacetate, and [3-keto-a-diazoacetates including, but not limited to, f-butyl-a- diazoacetoacetate. In some embodiments, the photoreactive group may comprise diazirines including, but not limited to, 3-trifluoromethyl-3-phenyldiazirine. In some embodiments, the photoreactive group comprises a A / -((2-pyridyldithio)ethyl)-4-azidosalicylamide, 4-azido-2, 3,5,6- tetrafluorobenzoic acid, 4-azido-2,3,5,6-tetrafluorobenzyl amine, benzophenone-4-maleimide, benzophenone-4-isothiocyanate, or 4-benzoylbenzoic acid.

[0043] In addition to the use of photoactivatable chemistries described above, additional chemical or thermal activation may also be employed. The methods described herein may optionally further comprise contacting one or more analytes with one or more detection reagents. Detection reagents may be capable of binding to or interacting with the analyte to be detected. The process by which one or more analytes of interest are contacted or treated with a detection reagent may be by any method known in the art, so long as it is compatible with the methods described herein. Non-limiting examples for conveying detection reagents through the separation matrix include, but are not limited to, hydrodynamic flow with positive or negative pressure or combinations of the two.

[0044] Detection reagents used in the methods described herein may comprise any organic or inorganic molecule capable of binding to interact with the analyte to be detected. Exemplary detection reagents include, but are not limited to, proteins, peptides, antibodies, enzyme substrates, transition state analogs, cofactors, nucleotides, polynucleotides, aptamers, lectins, small molecules, ligands, inhibitors, drugs, and other biomolecules as well as non-biomolecules capable of binding the analyte to be detected. In various embodiments, two or more different detection agents, which bind to or interact with different analytes, can be detected simultaneously. In some embodiments, the detection reagents may be one or more immunoassay detection reagents. In preferred embodiments, the detection reagents may be antibodies.

[0045] Detection reagents may comprise one or more labeled moieties. In embodiments employing two or more label moieties, each label moiety can be the same, or some, or all, of the label moieties may differ. In some embodiments, the label moiety may comprise a chemiluminescent label. The chemiluminescent label may comprise any entity that provides a light signal and that can be used in accordance with the methods described herein. A wide variety of such chemiluminescent labels are known in the art. See e.g., U.S. Pat. Nos. 6,689,576; 6,395,503; 6,087,188; 6,287,767; 6,165,800; and 6,126,870, which are incorporated by reference herein for such teachings. Without being bound by theory, the use of chemiluminescent labels may require a protein, such as an enzyme capable of reacting with a chemiluminescent substrate in such a way that photon emission by chemiluminescence is induced. Exemplary enzymes include, but are not limited to, peroxidase, [3-galactosidase, phosphatase, or others for which a chemiluminescent substrate is available. In some embodiments, the chemiluminescent label may be selected from any of a variety of classes including, but not limited, a luminol label or an isoluminol label. In preferred embodiments, the detection agents comprise chemiluminescent labeled antibodies.

[0046] In some embodiments, the label moiety may comprise a bioluminescent compound. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent compound is determined by detecting the presence of luminescence. Exemplary bioluminescent compounds include, but are not limited to luciferin, luciferase, and aequorin.

[0047] In some embodiments, the label moiety may comprise a fluorescent dye. The fluorescent dye may comprise any entity that provides a fluorescent signal and that can be used in accordance with the methods described herein. In various embodiments, the fluorescent dye may comprise a resonance-delocalized system or aromatic ring system that absorbs light at a first wavelength and emits fluorescent light at a second wave-length in response to the absorption event. A wide variety of such fluorescent dye molecules are known in the art and may be used with the methods described herein. Exemplary fluorescent dyes can be selected from any of a variety of classes of fluorescent compounds, non-limiting examples include xanthenes, rhodamines, fluoresceins, cyanines, phthalocyanines, squaraines, bodipy dyes, coumarins, oxazines, and carbopyronines. In some embodiments, for example, where detection agents contain fluorophores, such as fluorescent dyes, fluorescence detection may occur by exciting the fluorophore with an appropriate light source and monitoring the resulting fluorescence with a detector sensitive to the characteristic fluorescence emission wavelength.

[0048] Analyte detection may be performed by any method known in the art, so long as it is compatible with the methods described herein. Exemplary instruments that may be used to detect an analyte or a label on an analyte include, but are not limited to, a photodetector, an array of photodetectors, or a charged coupled device (CCD) array. A signal may be a continuously monitored, in real time, to allow the user to rapidly determine whether an analyte is present in the sample, and optionally, the amount or activity of the analyte. In some embodiments, the signal may be measured from at least two different time points. In some embodiments, the signal may be monitored continuously or at several selected time points. Alternatively, the signal may be measured in an end-point embodiment in which a signal is measured after a certain amount of time, and the signal is compared against a control signal (sample without analyte), threshold signal, or standard curve.

[0049] The separation obtained by the methods described herein may produce enhanced separation as compared to methods that use capillaries. The use of capillaries involves loading sample directly onto the capillary through capillary action. The dimensions of a typical capillary are generally small in diameter and may cause proteins to separate and appear as “roundish’ areas that are distinctly different than a typical western blot. This output from capillaries reduces resolution. The use of individual capillaries introduces errors and variation in separation since certain capillaries may run at a different rate than others. Specifically, bubbles introduced during the loading process may result in a lane failure. In addition, the individual nature created by the use of capillaries means that detection reagents must be prepared and loaded separately through each capillary, resulting in potential detection variability due to slightly different concentrations or volumes of antibodies and detection reagents moving through each individual capillary.

[0050] Contrastingly, the methods described herein involve loading one or more samples into larger rectangular sample wells. An electric field is applied to the one or more samples and the one or more samples travel through the sample well and stacking gel (e.g. solid matrix) and into the liquid separation matrix. The use of solid gel wells and a stacking gel (e.g., solid matrix) combined interfaced with a liquid separation matrix produces a more band like appearance, increasing resolution and providing an appearance more consistent with a traditional Western blot. Additionally, the use of a single area for the liquid separation followed by subsequent immune detection may allow the immobilized samples to be exposed to the same pool and concentrations of detection reagents. This may minimize the impact of turbulent flow when subsequent reagents and washes are performed. Without being bound by theory, this may improve the resolution achieved as compared to alternative methods.

[0051] The methods described herein may comprise loading the one or more samples comprising one or more analytes into one or more samples wells. In some embodiments, the method may further comprise applying an electric field to the one or more samples, generating separated analytes in a separation matrix; wherein the separation matrix is a liquid matrix shared by the one or more samples. In some embodiments, the method may further comprise immobilizing separated analytes on an immobilization surface. In some embodiments, the method may further comprise removing the separation matrix. In some embodiments, the method may further comprise introducing one or more wash reagents, blocking reagents, and detecting reagents onto the immobilization surface, wherein the detecting reagents are capable of binding to one or more analytes and generating a detectable signal. In some embodiments, the method may further comprise detecting one or more analytes by measuring at least one signal produced from the detecting reagents.

[0052] In some embodiments, the analytes detected by the method may be proteins, nucleic acids, or a combination thereof. In some embodiments, two or more samples comprising one or more analytes may be loaded into two sample wells during the method. In some embodiments, the separation matrix used in the method does not comprise separation wells. In some embodiments, the electric field applied during the method may be uniform across the separation matrix. In some embodiments, the separation matrix used in the method may comprise a linear polyacrylamide, a dimethylpolyacrylamide, a dextran, a pullulan, an agarose, polyvinyl alcohol, a polyethylene oxide, a polysaccharide, hydroxyethyl cellulose, hydroxyl propyl cellulose, methylcellulose, or a combination thereof.

[0053] In some embodiments, the method may include removing the separation matrix in a direction perpendicular to the electric field. In some embodiments, the method may further comprise a solid matrix. In some embodiments, the solid matrix used in the method may allow current and ion flow to generate a uniform electric field. In some embodiments, the solid matrix used in the method may create a barrier to prevent leakage of the separation matrix, the detecting reagents, or a combination thereof. In some embodiments, the solid matrix used in the method may comprise agarose or acrylamide. In some embodiments, the solid matrix used in the method may comprise a matrix buffer to support electrophoresis. In some embodiments, the matrix buffer used in the method may comprise buffers, detergents, or salts, at a pH appropriate for electrophoresis. In some embodiments, the matrix buffer used in the method may comprise tris, tris-glycine, bis-tris, Tricine, acetate, SDS, or combinations thereof. In some embodiments, the matrix buffer used in the method may comprise one or more layers comprising a running buffer layer, a stacking buffer layer, or a combination thereof. In some embodiments, the running buffer layer used in the method may comprise 0.5-1.5% agarose 100 mM Tris base, 100 mM Tricine, 0.1 % SDS pH 8.3. In some embodiments, the stacking buffer layer used in the method may comprise 0.5-1.5% agarose, 125 mM Tris, pH 8.0.

[0054] In some embodiments, the one or more analytes separated by the method may be separated by size and charge. In some embodiments, the method may separate the one or more analytes by molecular weight. In some embodiments, the method may separate the one or more analytes by isoelectric point.

[0055] In some embodiments the one or more analytes separated by the method described herein may be immobilized simultaneously to the immobilization surface. In some embodiments, the immobilization surface used in the method may comprises at least one chemical functionalization to interact with the one or more analytes. In some embodiments, the at least one chemical functionalization on the immobilization surface may be a cross-linking molecule selected from the group consisting of aryl azides, benzophenones, anthraquinones, diazo compounds, diazirines, psoralens, derivatives thereof, or combinations thereof. In some embodiments, the immobilization surface used in the method may be transparent, translucent, or opaque. In some embodiments, the one or more analytes separated by the method may be arranged in a separation pattern preserved on the immobilization surface. In some embodiments, the one or more analytes may be immobilized to the immobilization surface with photoactive cross-linking, chemical cross-linking, thermal cross-linking, or a combination thereof. In some embodiments, the immobilization surface may be a glass, a polymer, a copolymer, or a combination thereof. In some embodiments, the copolymer of the immobilization may be a cyclic olefin.

[0056] In some embodiments, one or more washing reagents, blocking reagents, and detecting reagents may be applied sequentially or simultaneously. In some embodiments, detecting reagents used in the method may comprise a fluorophore, chemiluminescent, radioactive, or other detectable reporter. In some embodiments, detecting reagents may comprise at least one primary immunoassay detecting reagent and at least one secondary immunoassay detecting reagent. In some embodiments, a primary immunoassay detecting reagent used in the method may recognize a protein or nucleic acid. In some embodiments, a secondary immunoassay detecting reagent used in the method may comprise a fluorophore, a chemiluminescent reporter, or a radioactive isotope. In some embodiments, detecting reagents used in the method may be immunoassay detecting reagents. In some embodiments, the immunoassay detecting reagents used in the method may be an antibody, antibody fragment, single chain antibody, or derivative thereof.

[0057] In some embodiments, the one or more samples used in the method may comprise 1-4 different analytes. In some embodiments, 1-4 different analytes in the one or more samples used in the method may be analyzed individually of simultaneously. In some embodiments, the method is completed in 3-4 hours.

[0058] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, apparata, assemblies, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions, apparatus, assemblies, and methods provided are exemplary and are not intended to limit the scope of any of the disclosed embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, apparatus, assemblies, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences described herein. The compositions, formulations, apparatus, assemblies, or methods described herein may omit any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0059] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0060] Clause 1 . A method for separating one or more analytes in one or more samples, the method comprising:

[0061] (a) loading one or more samples comprising one or more analytes into a solid matrix comprising one or more sample wells;

[0062] (b) applying an electric field to the solid matrix and a liquid separation matrix shared by the one or more samples; and

[0063] (c) generating a separation pattern of the one or more analytes in the liquid separation matrix.

[0064] Clause 2. The method of clause 1 , wherein the analytes are proteins, nucleic acids, or a combination thereof.

[0065] Clause 3. The method of clause 1 or 2, wherein step (a) two or more samples comprising one or more analytes is loaded into at least two sample wells.

[0066] Clause 4. The method of any one of clauses 1-3, wherein the electric field of step (b) is uniform across the separation matrix.

[0067] Clause 5. The method of any one of clauses 1-4, wherein the separation matrix comprises a linear polyacrylamide, a dimethylpolyacrylamide, a dextran, a pullulan, an agarose, polyvinyl alcohol, a polyethylene oxide, a polysaccharide, hydroxyethyl cellulose, hydroxyl propyl cellulose, methylcellulose, or a combination thereof.

[0068] Clause 6. The method of any one of clauses 1-5, wherein the method further comprises removing the liquid separation matrix in a direction perpendicular to the electric field.

[0069] Clause 7. The method of any one of clauses 1-6, wherein the solid matrix comprises agarose or acrylamide.

[0070] Clause 8. The method of any one of clauses 1-7, wherein the solid matrix comprises one or more layers comprising a running buffer layer, a stacking buffer layer, or a combination thereof.

[0071] Clause 9. The method of clause 8, wherein the running buffer layer comprises 0.5-1.5% agarose 100 mM Tris base, 100 mM Tricine, 0.1% SDS pH 8.3.

[0072] Clause 10. The method of clause 8, wherein the stacking buffer layer comprises 0.5-1.5% agarose, 125 mM Tris, pH 8.0. Clause 11. The method of any one of clauses 1-10, the solid matrix further comprises a matrix buffer to maintain pH, conduct electricity, stabilizing ionic strength, and prevent heat buildup.

[0073] Clause 12. The method of any one of clauses 11 , wherein the matrix buffer comprises buffers, detergents, or salts, at a pH of 4-10 for electrophoresis.

[0074] Clause 13. The method of any one of clauses 11 , wherein the matrix buffer comprises tris, tris-glycine, bis-tris, Tricine, acetate, SDS, or combinations thereof.

[0075] Clause 14. The method of any one of clauses 1-13, wherein the one or more analytes in step (b) are separated by size and charge.

[0076] Clause 15. The method of clause 14, wherein the size is molecular weight.

[0077] Clause 16. The method of clause 14, wherein the charge is isoelectric point.

[0078] Clause 17. The method of any one of clauses 1-16, further comprising a run buffer.

[0079] Clause 18. The method of any one of clauses 1-17, wherein the solid matrix comprises 12-

[0080] 48 sample wells.

[0081] Clause 19. The method of any one of clauses 1-18, further comprising detecting the separation pattern of the one or more analytes with at least one detecting reagent.

[0082] Clause 20. The method of any one of clauses 1-19, wherein the one or more samples comprise 1-4 different analytes.

[0083] Clause 21. The method of any one of clauses 20, wherein the 1-4 different analytes are detected individually or simultaneously.

[0084] Clause 22. Use of the method of claim 1-21 , to detect a separation pattern of one or more analytes in one or more samples, wherein the separation pattern comprises resolution between each analyte in the one or more sample.

[0085] EXAMPLES

[0086] Example 1

[0087] Exemplary Cleaning Glass Slides for Silane Treatment

[0088] An acid wash or a vacuum plasma gas treatment were used to clean slides of foreign material and generate surface suitable for silane modification. Briefly, slides were submerged in 0.5 M HCI at RT for 30 minutes. The slides were subsequently rinsed 10 times with DI water. The slides were then rinsed in pure ethanol immediately proceed with silane modification. Plasma treatment involved placing a slide in a cleaning chamber of a Diener Electronic PICO. The slides were subjected to vacuum until under 0.3 mBar and maintained under vacuum for 10 minutes. The chamber was flooded with air re-equalized to 0.2-0.3 mBar. Plasma was generated at 50% power for 5 minutes. Gas was turned off and the chamber was ventilated before removing slide for silane modification.

[0089] Exemplary Glass Surface Modification with a Silane

[0090] A vinyl or acrylate function group were added to the preceding glass surface. A variety of silane compounds may be used with a similar procedure. Briefly, a cleaned glass slide was submerged into freshly made 1% 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) solution in 95% ethanol. The materials were incubated for 2 minutes before removing and rinsing the slides twice with 100% ethanol. The slides were placed in an oven set to 120 °C for 15 min. Slides were either immediately used or placed in a sealed chamber under nitrogen.

[0091] Exemplary Glass Surface Modification with Acrylamide and Photoreactive Groups

[0092] The addition of photoreactive groups to the modified glass surface can be done in a wide variety of ways consistent with methods in the literature by either polymerizing molecules onto the silane modified surface or by grafting preformed polymers onto the surface. Briefly, slides were coated in a solution containing 218.8 ml_ dimethylsulfoxide (DMSO), 0.4 mL 10% ammonium persulfate, 0.05 mL tetramethylethylenediamine (TEMED), 1.29 g of 4-benzoylphenyl acrylate, and 29.46 g of sulfo-acrylamide. After an appropriate period of time, slides were removed from the polymerized solution and washed extensively in DMSO followed by water. Slides were then dried under nitrogen. Slides were stored or immediately used in the following steps of electrophoresis and surface capture of samples.

[0093] Exemplary Preparation of Stacking Gel

[0094] A stacking gel for sized-based protein separation by electrophoresis was prepared. Briefly, a stacking gel composed of 125 mM Tris(hydroxymethyl)aminomethane) (Tris base), pH 8, with 1% SeaKem™ Gold Agarose (Lonza) was heated to 90-100 °C to dissolve and melt the agarose. The stacking gel was maintained at a temperature at 90 °C to keep the gel as a liquid prior to creating the stacking gel layer and sample wells. Table 1 and Table 2 provide exemplary methods for preparing the 125 mM Tris-HCI and agarose solution.

[0095] Table 1. 125 mM Tris-HCI, pH 8, Buffer

[0096] Tris Base 15.14 g

[0097] Ultra- Pure water 800 mL pH to 8.0 with 10 A / HCI and bring up to 1000 mL with ultra pure water

[0098] Total volume 1000 mL Table 2. Agarose

[0099] 1% SeaKem™ Gold Agarose 1 g

[0100] 125 mM Tris-HCI pH 8.0 to lOO mL

[0101] Total volume 100 mL

[0102] Exemplary Preparation of Running Buffer

[0103] Running buffer was prepared as a liquid or solidifying gel. For liquid buffer, a solution of 100 mM Tris base, 100 mM A / -[Tris(hydroxymethyl)methyl]glycine (Tricine), 0.1% sodium dodecyl sulfate (SDS) pH 8.3 or similar was appropriate. A solidifying running buffer gel was prepared by adding 0.5% SeaKem™ Gold Agarose to the liquid buffer above and heat to dissolve. The solution temperature was maintained at 90 °C until ready to pour.

[0104] Table 3. Exemplary Running Buffer

[0105] Tris Base 121 g

[0106] Tricine 179 g

[0107] SDS 10 g

[0108] Ultra- Pure water to 1000 mL

[0109] Exemplary Preparation of the Liquid Separation Matrix

[0110] A liquid separation matrix containing polydimethyl acrylamide (PDMA) was prepared by dissolving 5 mL A / ,A / -Dimethylacrylamide into 95 mL of 100 mM Tris base, 100 mM Tricine, pH 8.3. The solution was placed in a scintillation vial or similar container and capped with an air-tight septum cap. The gasses were removed from the vial with a syringe followed by sparging / bubbling nitrogen or argon through the solution for 2-3 hours to remove dissolved oxygen. To the sparged solution, 50 pL of tetramethylethylenediamine (TEMED) was added. Subsequently, 250 pL to freshly prepared 10% ammonium persulfate solution was added. The resulting solution was mixed, and nitrogen or argon gas was blown over the solution for 30 minutes while the polymer formed. The gas was removed, and the solution was left at room temperature for approximately 16 hours. After the incubation, the solution was degassed under vacuum for 15 minutes before use as the liquid separation matrix. A similar process was used to formulate a linear polyacrylamide (LPA) / PDA and pullulan-based separation matrices. Exemplary formulations are shown in Tables 4-5. Table 4. 3% v / v LPA + 0.08% volume / volume PDMA

[0111] LPA (4.56%) 65.8 mL

[0112] PDMA (5%) 1 .6 mL

[0113] 10x Running Buffer 10 mL

[0114] Ultra-Pure water to 100 mL

[0115] Table 5. 15% w / v Pullulan

[0116] Pullulan 7.5 g

[0117] 10x Tricine Native Buffer 5 mL

[0118] Ultra- Pure water to 50 mL

[0119] Exemplary Sample Preparation

[0120] Samples containing 1 pg of HeLa cell lysate were mixed with reducing lithium dodecyl sulfate sample buffer to create a final mixture of Tris base (141 mM), Tris HCI (106 mM), lithium dodecyl sulfate (2%), ethylenediaminetetraacetic acid (EDTA) (0.51 mM), Coomassie Blue G-250 (0.22 mM), phenol red (0.175 mM), dithiothreitol (50 mM), pH 8.5. The samples were heated at 70 °C for 10 minutes. The sample matrix and polydimethyl acrylamide (PDMA) were subsequently added to the sample wells.

[0121] Exemplary Electrophoresis conditions

[0122] Samples were separated by applying 50-100 V across the gel until the control dye traveled 20-30 mm (approximately 15 minutes). Exemplary Surface Immobilization

[0123] Immediately after electrophoresis, the samples were subjected to UV light (e.g., 302 nm) at a distance of <10 mm for 5 minutes directed at the separation area through the glass surface of the cartridge. This crosslinked the sample proteins to the benzophenone functionalized glass surface.

[0124] Exemplary Removal of Separation Matrix

[0125] After immobilization of the proteins to the functionalized glass surface, the liquid separation matrix was removed by flowing approximately 30 mL of 25 mM Tris, 0.15 M NaCI, 0.05% poly(oxyethylene)-sorbitan-monolaurate (Tween® 20), pH 7.5 (TBST), until the polydimethyl acrylamide (PDMA) matrix was removed. The functionalized glass surface containing the proteins was rinsed with TBS buffer. Exemplary Immunodetection of Analytes

[0126] The functionalized glass surface containing the separated proteins was blocked by flowing Blocker™ Bovine Serum Albumin (BSA) over the samples and incubated for 5-30 minutes. A primary antibody to the target protein diluted 1 :200 (1 mg / mL stock) in Blocker™ BSA was added to the blocked functionalized glass surface containing the separated proteins. The primary antibody was incubated on the protein containing glass for 1 hour with or without pulsing. The protein containing glass was washed with approximately 10-15 volume exchanges over 10 minutes of T ris buffered saline with Tween® 20 (TBST). A corresponding secondary-horseradish peroxidase antibody conjugate was diluted 1 :200 (1 mg / mL stock) in Blocker™ BSA and added to the protein containing glass. The protein containing glass and secondary antibody were incubated for 1 hour and subsequently washed with approximately 10-15 volume exchanges of Tris buffered saline with Tween® 20 (TBST) over 10 minutes. The sample was treated and exposed to SuperSignal™ West Femto Maximum Sensitivity Substrate and imaged with an imaging system compatible with chemiluminescent western blot imaging. An overview of the method is shown in FIG. 3.

[0127] Exemplary gel images obtained after electrophoretic separation in a liquid separation matrix are shown in FIG. 4A-C.

Claims

CLAIMSWhat is claimed:

1. A method for separating one or more analytes in one or more samples, the method comprising:(a) loading one or more samples comprising one or more analytes into a solid matrix comprising one or more sample wells;(b) applying an electric field to the solid matrix and a liquid separation matrix shared by the one or more samples; and(c) generating a separation pattern of the one or more analytes in the liquid separation matrix.

2. The method of claim 1 , wherein the analytes are proteins, nucleic acids, or a combination thereof.

3. The method of claim 1 , wherein step (a) two or more samples comprising one or more analytes are loaded into at least two sample wells.

4. The method of claim 1 , wherein the electric field of step (b) is uniform across the separation matrix.

5. The method of claim 1 , wherein the separation matrix comprises a linear polyacrylamide, a dimethylpolyacrylamide, a dextran, a pullulan, an agarose, polyvinyl alcohol, a polyethylene oxide, a polysaccharide, hydroxyethyl cellulose, hydroxylpropyl cellulose, methylcellulose, or a combination thereof.

6. The method of claim 1 , wherein the method further comprises removing the liquid separation matrix in a direction perpendicular to the electric field.

7. The method of claim 1 , wherein the solid matrix comprises agarose or acrylamide.

8. The method of claim 1 , wherein the solid matrix comprises one or more layers comprising a running buffer layer, a stacking buffer layer, or a combination thereof.

9. The method of claim 8, wherein the running buffer layer comprises 0.5-1.5% agarose 100 mM Tris base, 100 mM Tricine, 0.1 % SDS pH 8.3.

10. The method of claim 8, wherein the stacking buffer layer comprises 0.5-1.5% agarose, 125 mM Tris, pH 8.0.

11. The method of claim 1 , wherein the solid matrix further comprises a matrix buffer to maintain pH, conduct electricity, stabilizing ionic strength, and prevent heat build-up.

12. The method of claim 11 , wherein the matrix buffer comprises buffers, detergents, or salts, at a pH of 4-10 for electrophoresis.

13. The method of claim 11 , wherein the matrix buffer comprises tris, tris-glycine, bis-tris, Tricine, acetate, SDS, or combinations thereof.

14. The method of claim 1 , wherein the one or more analytes in step (b) are separated by size and charge.

15. The method of claim 14, wherein the size is molecular weight.

16. The method of claim 14, wherein the charge is isoelectric point.

17. The method of claim 1 , further comprising a run buffer.

18. The method of claim 1 , wherein the solid matrix comprises 12-48 sample wells.

19. The method of claim 1 , further comprising detecting the separation pattern of the one or more analytes with at least one detecting reagent.

20. The method of claim 1 , wherein the one or more samples comprise 1-4 different analytes.

21. The method of claim 20, wherein the 1-4 different analytes are detected individually or simultaneously.

22. Use of the method of claim 1 , to detect a separation pattern of one or more analytes in one or more samples, wherein the separation pattern comprises resolution between each analyte in the one or more sample.

Images