METHODS AND DEVICES USING INDIVIDUALLY ADDRESSABLE MICROGEL ELECTROPHORESIS LANE SYNTHESIS FOR

BIOASSAYS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of USSN 62/663,164, filed on

April 26, 2018, which is incorporated herein by reference in its entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

[0002] This invention was made with government support under Grant No.

CA225296 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

[0003] In clinical pathology and molecular biology, determination of genomic sequences has shifted the research focus on the interpretation of genomic sequence data in terms of the structure, function, and control of biological pathways. Genome mapping has enabled linking the cellular state to the gene expression at the mRNA level and is used for identifying clusters of genes for specific biological mechanisms (see, e.g., Jiang (2011) Genome Res. 21(9), 1543-1551). However, genome mapping provides information about the expression of the genomic material in protein level (see, e.g, Arzalluz-Luque el a/. (2017)

Int. J. Biochem. Cell Biol., 90, 161-166) Therefore, quantitative analysis of protein expression is established as a complementary method for obtaining more accurate information about biological pathways by directly measuring the number of the proteins and protein isoforms, which are formed by alternative splicing events, post-translational modifications, and co- translational modifications such as phosphorylation, glycosylation, and protein cleavage (see, e.g., Smith el al. (2013) Nature Meth. 10(3): 186). Here, separation of protein isoforms is crucial since expression of certain isoforms determine the drug resistance, and progression of tumors (see, e.g., Sinkala et al. (2017) Nat. Comm. 8: 14622).

[0004] Separation methods combined with affinity interactions are key to measuring the content and amount of proteins. Such methods include zymography (see, e.g, Frederiks & Mook (2004) ./. Histochem. & Cytochem. 52(6): 711-722; Dhawan etal. (2005) J. Clin. Invest. 115(7): 1765-1776), polyacrylamide gel electrophoresis (PAGE) (see, e.g.,

Shevchenko et al. (1996) Proc. Natl. Acad. Sci. USA, 93(25): 14440-14445; Link etal. (1999). Nature Biotech. 17(7): 676), immunoaffmity electrophoresis (see, e.g., Phillips (2004) Electrophoresis , 25(10-11): 1652-1659), mass spectrometry (MS)-based sequence identification of separated protein species, immunocytochemistry (see, e.g, Taniguchi et al. (2010) Science 329: 533-538; Mahmood & Yang (2012) A'. Am. J. Med. Sci. 4: 429-434; Ashton et al. (2012) Nat. Neurosci. 15: 1399-1406), flow/mass cytometry (see, e.g., Bendall et al. (2011) Science, 332: 687-696; Perfetto et al. (2004) Nat. Rev. Immunol. 4: 648-655), CyTOF (see, e.g, Bruggner et al. (2014) Proc. Natl. Acad. Sci. USA, 111 : E2770-E2777), enzyme-linked-immunosorbent-assays (see, e.g,. Bonner et al. (1972) Rev. Sci. Inst., 43 : 404-409), western blotting (see, e.g., Burnette ( 198 1 ) Anal Biochem. 112(2) 195-203), and immuoaffmity electrophoresis that aim for improving the information obtained from biological samples in terms of content, specificity and selectivity.

[0005] Zymographgy has been introduced as a functional approach for the analysis of proteolytic activity in a cell or tissue extracts (see, e.g, Jiang (2011) Genome Res. 21(9), 1543-1551). Zymography is based on separations in sodium dodecyl sulfate (SDS) polyacrylamide gels containing gelatin, casein, or fibrin as substrate that is brought into contact with a tissue section or cell sample. Substrate and the sample are incubated and go under an enzymatic reaction, which results in white spots in a dark background or as black spots in a fluorescent background (see, e.g., Dhawan etal. (2005) J. Clin. Invest. 115(7): 1765- 1776). Polyacrylamide gel electrophoresis involves separation of heat-induced denatured proteins in the presence of a detergent (often SDS) by the application of an electric field. ^[7][8] This method can be applied in uniform gels, or gradient gels to separate a wider range of proteins at a time. Immunoaffmity electrophoresis, isolation of the target proteins is achieved by immunoaffmity capture using a panel of immobilized antibodies. The captured proteins are then labeled and electrophoresed in gel (see, e.g., Phillips (2004) Electrophoresis , 25(10- 11): 1652-1659). Mass spectrometry based separation involves injection of ionized form of the proteins in an electric or magnetic field (see, e.g., Taniguchi et al. (2010) Science 329: 533-538). This process can be performed by electrospray ionization (and matrix-assisted laser desorption/ionization. In immunocytochemistry, cell samples are selectively stained using colored antigens for visualization based on specific binding of antibody-antigen pairs (see, e.g., Ashton et al. (2012) Nat. Neurosci. 15: 1399-1406). Distribution and localization of differentially expressed proteins can be detected in tissue samples. Flow and mass cytometry as well as CyTOF are the techniques where proteins are labelled with fluorophores or metal ion tags, and are sent through often a laser detector (see, e.g. , Bendall et al. (2011) Science, 332: 687-696; Perfetto et al. (2004) Nat. Rev. Immunol. 4: 648-655; Bruggner et al. (2014) Proc. Natl. Acad. Sci. USA, 111 : E2770-E2777). These techniques provide information about physical and chemical characteristics of proteins. Enzyme-linked- immunosorbent-assay is used for determining the presence or concentration of a protein via an antibody-antigen reaction (see, e.g., Bonner el al. (1972 ) Rev. Sci. Inst., 43: 404-409).

The antibody-antigen reaction results in a colorimetric output which is then measured using spectrometer. Western blotting has been the workhorse as a confirmatory test for bioassays in clinics. Western blotting is based on electrophoresing protein species through a gel, transferring the separated proteins to a membrane by electric field application, and probing the transferred proteins with primary and secondary antibodies for detecting target protein species (Burnette (1981) Anal. Biochem. 112(2): 195-203). Because of the reliability and simplicity, western blotting has been used extensively for bioassays.

[0006] While zymography has a limited sensitivity, requires specific inhibitors for detection of the proteins, and has to be combined with immunohistochemistry, PAGE, immunoaffmity assays, enzyme-linked-immunosorbent-assays, and western blotting are sequential, labor intensive, and difficult to automate. One of the most significant flaw of the abovementioned techniques is the bias toward detection of highly abundant proteins, and lower abundance regulatory proteins (e.g, transcription factors, protein kinases) and protein isoforms are rarely detected when total cell lysates are analyzed. Also, sensitivity (protein extraction from ~ 10 ⁶ cells to meet hundreds of nanograms sample requirement), and selectivity limits are the major measurement problems of these techniques (see, e.g,

Frederiks & Mook (2004) J. Histochem. & Cytochem. 52(6): 711-722; Dhawan et al. (2005) J. Clin. Invest. 115(7): 1765-1776;. Shevchenko et al. (1996) Proc. Natl. Acad. Sci. USA, 93(25): 14440- 14445; Link et al. (1999). Nature Biotech. 17(7): 676; Phillips (2004) Electrophoresis, 25(10-11): 1652-1659; Taniguchi et al. (2010) Science 329: 533-538; Mahmood & Yang (2012) A. Am. J. Med. Sci. 4: 429-434; Ashton et al. (2012) Nat. Neurosci. 15: 1399-1406; Bendall et al. (2011 ) Science, 332: 687-696; Perfetto et al. (2004) Nat. Rev. Immunol. 4: 648- 655; Bruggner et al. (2014) Proc. Natl. Acad. Sci. USA, 111 : E2770-E2777; Bonner et al. (1972) Rev. Sci. Inst., 43: 404-409; Burnette (1981 ) Anal Biochem. 112(2): l95-203). ^{[5] [17]} Thus, these techniques are not compatible with the analysis of protein isoforms from single cells and there is a great need for a simple and quantitative technology for proteome analysis.

[0007] Efforts have been made to miniaturizing the large-scale western blotting in order to reduce the measurement costs and sample consumption, as well as increasing the readout throughput by introducing polymeric structures with protein bands from single cells on them. Single-cell analysis is important to unveil the unnoticed characteristics of rare cell populations, e.g. a circulating tumor cells is found in 1 mL of blood, especially for the proteomic analysis of protein isoforms that lead to different outcomes in a cell’s fate.

SUMMARY

[0008] Direct measurement of proteins and other analytes from single cells has been realized at the microscale level using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing.

[0009] As described herein we provide a novel a hybrid microfluidic format that toggles between a planar microgel array and a suspension of microparticles. In certain embodiments the planar array comprises an array of microgel electrophoresis structures stippled in a thin sheet of polymeric (e.g, polyacrylamide) gel. The planar array of microgel electrophoresis structures, provides for, inter alia, efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separated microgel electrophoresis structures (i.e., separation- encoded microparticles). The suspended microparticles provide for significantly more efficient analyte detection (e.g, more efficient immunoprobing) due to enhanced mass transfer. In certain embodiments, dehydrating the microparticles offers improved analytical sensitivity owing to in-gel concentration of the fluorescence signal for, e.g, high-throughput single-cell targeted proteomics.

[0010] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:

[0011] Embodiment 1 : A substantially planar array of microgel electrophoresis structures, said array comprising:

[0012] a plurality of microgel electrophoresis structures (pGel(s)) wherein each microgel electrophoresis structure comprises:

[0013] a free-standing polymeric gel that forms said microgel electrophoresis structure where said polymeric gel is a gel compatible with electrophoretic separation or isolation of one or more analytes and configured to separate a sample along a directional axis; and

[0014] one or more sample receiving wells disposed in said microgel electrophoresis structure; and

[0015] a separation zone delineating each microgel electrophoresis structure perimeter where said separation zone is structured to facilitate separation of the microgel electrophoresis structures from each other.

[0016] Embodiment 2: The array of microgel electrophoresis structures of embodiment 1, wherein said separation zone is structured to facilitate recovery of at least 50%, or at least 60%, or at least 70% , or at least 80% , or at least 90% , or at least 95% of the pGels comprising the array.

[0017] Embodiment 3: The array of microgel electrophoresis structures according to any one of embodiments 1-2, wherein said separation zone is structured to facilitate separation of the microgel electrophoresis structures from each other while retaining sufficient physical integrity to permit interrogation of separated analyte(s) in the released microgel electrophoresis structures.

[0018] Embodiment 4: The array of microgel electrophoresis structures according to any one of embodiments 1-3, wherein said separation zone comprises troughs around each microgel electrophoresis structure.

[0019] Embodiment 5: The array of microgel electrophoresis structures of embodiment 4, wherein said troughs comprise a trough floor formed from the same polymeric gel material that forms the microgel electrophoresis structures.

[0020] Embodiment 6: The array of microgel electrophoresis structures of embodiment 4, wherein said troughs comprise a trough floor formed from a different polymeric material than the material that forms the microgel electrophoresis structures.

[0021] Embodiment 7: The array of microgel electrophoresis structures according to any one of embodiments 4-6, wherein said troughs comprise a trough floor that is sufficiently thin to facilitate mechanical separation of the microgel electrophoresis from each other by mechanical pulling or shearing.

[0022] Embodiment 8: The array of microgel electrophoresis structures according to any one of embodiments 1-7, wherein said separation zone comprises a plurality of perforations that facilitate separation of the microgel electrophoresis structures from each other.

[0023] Embodiment 9: The array of microgel electrophoresis structures according to any one of embodiments 1-8, wherein said separation zone comprises one or more scoring lines that facilitate separation of the microgel electrophoresis structures from each other. [0024] Embodiment 10: The array of microgel electrophoresis structures according to any one of embodiments 1-9, wherein said separation zone comprises a material comprising a photocleavable linker that facilitates degradation of the trough when exposed to a wavelength of light that cleaves said photocleavable linker.

[0025] Embodiment 11 : The array of microgel electrophoresis structures according to any one of embodiments 1-9, wherein said separation zone comprises a material that is hydrolyzed by a reagent that does not hydrolyze the microgel electrophoresis structures.

[0026] Embodiment 12: The array of microgel electrophoresis structures according to any one of embodiments 1-9, wherein said separation zone comprises a material that preferentially absorbs laser radiation as compared to the microgel electrophoresis structures and degrades when exposed to said laser radiation.

[0027] Embodiment 13 : The array of microgel electrophoresis structures according to any one of embodiments 1-12, wherein said array comprise at least 500, or at least 1,000, or at least 1,500, or at least 2,000, or at least 2,500, or at least 3,000, or at least 3,500, or at least 4,000, or at least 5,000 microgel electrophoresis structures.

[0028] Embodiment 14: The array of microgel electrophoresis structures according to any one of embodiments 1-13, wherein said microgel electrophoresis structures are disposed within a surface area of 20 cm ² or less, or 15 cm ² or less or 10 cm ² or less, or 5 cm ² or less, or 3 cm ² or less or 2 cm ² or less or 1 cm ² or less.

[0029] Embodiment 15: The array of microgel electrophoresis structures according to any one of embodiments 1-14, wherein each of said microgel electrophoresis structures range in volume from about 10 pL, or from about 50 pL, or from about 100 pL, or from about 250 pL, or from about 500 pL, or from about 1000 pL up to 10,000 pL, or up to about 5,000 pL, or up to about 4,000 pL, or up to about 3,000 pL, or up to about 2000 pL.

[0030] Embodiment 16: The array of microgel electrophoresis structures according to any one of embodiments 1-15, wherein each of said microgel electrophoresis structures has an area ranging from about from about 1,000 pm ² , or from about 5,000 pm ² , or from about 10,000 pm ² , or from about 20,000 pm ² , or from about 30,000 pm ² , or from about 40,000 pm ² up to about 500,000 pm ² , or up to about 250,000 pm ² , or up to about 100,000 pm ² , or up to about 80,000 pm ² , or up to about 60,000 pm ² . [0031] Embodiment 17: The array of microgel electrophoresis structures according to any one of embodiments 1-16, wherein said microgel each microgel electrophoresis structure is substantially rectangular.

[0032] Embodiment 18: The array of microgel electrophoresis structures of embodiment 17, wherein:

[0033] said microgel electrophoresis structures each have a length ranging from about 500 pm, or from about 600 pm, or from about 700 pm, or from about 800 pm, or from about 900 pm up to about 3,000 pm, or up to about 2,500 pm, or up to about 2,000 pm, or up to about 1,500 pm, or up to about 1,000 pm; and/or

[0034] said microgel electrophoresis structures each have a width ranging from about 5 pm, or from about 10 pm, or from about 15 pm, or from about 20 pm, or from about 30 pm, or from about 40 pm up to about 500 pm, or up to about 250 pm, or up to about 200 pm, or up to about 150 pm, or up to about 100 pm, or up to about 80 pm, or up to about 60 pm.

[0035] Embodiment 19: The array of microgel electrophoresis structures of embodiment 18, wherein said microgel electrophoresis structures each have a length of about 950 pm.

[0036] Embodiment 20: The array of microgel electrophoresis structures according to any one of embodiments 18-19, wherein said microgel electrophoresis structures each have a width of about 50 pm.

[0037] Embodiment 21 : The array of microgel electrophoresis structures according to any one of embodiments 1-20, wherein said microgel electrophoresis structures each have a thickness (depth) ranging from about from about 15 pm, or from about 20 pm, or from about 30 pm, or from about 40 pm up to about 200 pm, or up to about 150 pm, or up to about 100 pm, or up to about 80 pm, or up to about 60 pm, or up to about 50 pm.

[0038] Embodiment 22: The array of microgel electrophoresis structures of embodiment 21, wherein said microgel electrophoresis structures each have a thickness of about 40 pm.

[0039] Embodiment 23: The array of microgel electrophoresis structures according to any one of embodiments 1-22, wherein said sample receiving wells range in diameter from about 10 pm, or from about 20 pm, or from about 30 pm up to about 100 pm, or up to about up to about 80 pm, or up to about 70 pm, or up to about 60 pm, or up to about 50 pm. [0040] Embodiment 24: The array of microgel electrophoresis structures of embodiment 23, wherein said sample receiving wells have a diameter of about 30 pm.

[0041] Embodiment 25: The array of microgel electrophoresis structures according to any one of embodiments 1-24, wherein said sample receiving wells are sized to each contain a single cell.

[0042] Embodiment 26: The array of microgel electrophoresis structures

embodiment 25, wherein said sample wells are sized to contain a single vertebrate cell.

[0043] Embodiment 27: The array of microgel electrophoresis structures according to any one of embodiments 1-26, wherein said sample receiving wells each have a depth ranging from about from about 15 pm, or from about 20 pm, or from about 30 pm, or from about 40 pm up to about 200 pm, or up to about 150 pm, or up to about 100 pm, or up to about 80 pm, or up to about 60 pm, or up to about 50 pm.

[0044] Embodiment 28: The array of microgel electrophoresis structures of embodiment 27, wherein said sample wells each have a depth of about 40 pm.

[0045] Embodiment 29: The array of microgel electrophoresis structures according to any one of embodiments 1-28, wherein said polymeric gel comprises a hydrogel.

[0046] Embodiment 30: The array of microgel electrophoresis structures of embodiment 29, wherein said polymeric gel comprises a material selected from the group consisting of acrylamide, Ethyl Acrylate, Ethyl Methacrylate, Ethyleneglycol Dimethacrylate, Hydroxy ethyl Acrylate, Isobutyl Methacrylate, Lauryl Methacrylate, Methacrylic Acid, Methyl Acrylate, Methyl Methacrylate, N,N-methylene-bisacryl-amide, n-Butyl

Methacrylate, pentaerythritol tetraacrylate, pentaerythritol triacrylate, Pentaerythritol

Triacrylate, Polyethylene Glycol (200) Diacrylate, Phenyl acrylate, poly(ethylene glycol diacrylate), polypropylene diacrylate), l,3-Butanediol Dimethacrylate, l,4-Butanediol Dimethacrylate, l,6-Hexanediol Dimethacrylate, 2,2,3,3,4,4,5,5-Octafluoropenty Acrylate, 2.2.2-Trifluoroethyl 2-methylacrylate, 2-Ethyl Hexyl Acrylate, 2-Hydroxy ethyl Methacrylate, 2-Hydroxypropyl Acrylate, 4-Hydroxybutyl Acrylate, agarose, Allyl Methacrylate, Benzyl Methylacrylate, Butyl Acrylate, collagen type I, collagen type IV., Diethyleneglycol

Diacrylate, Diethyleneglycol Dimethacrylate, dimethacrylate, Divinyl benzene, divinyl benzene (DVB), Monoethylene Glycol, poly(ethylene glycol), polypropylene glycol), Polyethylene Glycol (200) Dimethacrylate, Polyethylene Glycol (400) Diacrylate,

Polyethylene Glycol (400) Dimethacrylate, Polyethylene Glycol (600) Diacrylate,

Polyethylene Glycol (600) Dimethacrylate, Stearyl Methacrylate, Triethylene Glycol, Triethylene Glycol Dimethacrylate, Trimethylolpropane Triacrylate, gelatin, and the like, and mixtures or copolymers thereof.

[0047] Embodiment 31 : The array of microgel electrophoresis structures of embodiment 30, wherein said polymeric gel comprises acrylamide.

[0048] Embodiment 32: The array of microgel electrophoresis structures of embodiment 31, wherein said polymeric gel comprises 7% up to 14% acrylamide.

[0049] Embodiment 33: A method of separating and detecting one or more analytes in a sample, said method comprising:

[0050] providing an array of microgel electrophoresis structures according to any one of embodiments 1-32 with said sample disposed in one or more of said sample receiving wells;

[0051] placing said array in an electric field where said electric field is sufficient to cause migration of said one or more analytes from the sample receiving wells into and at least partially through the polymeric gel that forms said microgel electrophoresis structure(s);

[0052] contacting the microgel electrophoresis structures with a reagent comprising a detectable label where the reagent associates with one or more of said analytes; and

[0053] detecting and/or localizing said detectable label in said microgel electrophoresis structures.

[0054] Embodiment 34: The method of embodiment 33, wherein said sample comprises a sample selected from the group consisting of a cell, a tissue sample, a biological fluid, and an environmental sample.

[0055] Embodiment 35: The method of embodiment 34, wherein said sample comprises a cell.

[0056] Embodiment 36: The method of embodiment 35, wherein said sample comprises a mammalian cell.

[0057] Embodiment 37: The method according to any one of embodiments 34-36, wherein said sample comprises a single cell disposed in each loaded sample receiving well.

[0058] Embodiment 38: The method of embodiment 34, wherein said sample comprises a biological fluid, a cellular extract or digest, or a tissue extract or digest disposed in each loaded sample receiving well. [0059] Embodiment 39: The method according to any one of embodiments 33-38, wherein said placing said array in an electric field comprises placing said array in an electrophoresis apparatus.

[0060] Embodiment 40: The method according to any one of embodiments 33-39, wherein said electric field is produced by a voltage difference ranging from about 10 V cm ¹ , or from about 20 V cm ¹ , or from about 30 V cm ¹ , or from about 40 V cm ¹ up to about 100 V cm ¹ , or up to about 90 V cm l, or up to about 80 V cm ¹ , or up to about 70 V cm ¹ , or up to about 60 V cm ¹ .

[0061] Embodiment 41 : The method of embodiment 40, wherein said electric filed is produced by a voltage difference of about 40 V cm ¹ .

[0062] Embodiment 42: The method according to any one of embodiments 33-41, wherein said electric field is substantially uniform across said array.

[0063] Embodiment 43: The method according to any one of embodiments 33-42, wherein said detecting comprises using a device selected from the group consisting of a photomultiplier tube, a charge-coupled device, an intensified charge coupled device, a complementary metal-oxide-semiconductor sensor, visual colorimetric readout, a photodiode, and an imaging microscope system.

[0064] Embodiment 44: The method according to any one of embodiments 33-43, wherein said detectable label is selected from the group consisting of a fluorescent label, a colorimetric label, a chemiluminescent label, an enzymatic label comprising an enzyme substrate or an enzyme, a radiolabel, silver particles, gold particles, and a magnetic label.

[0065] Embodiment 45: The method according to any one of embodiments 33-44, wherein said method comprises dehydrating the microgel electrophoresis structures to concentrate the signal produced by the detectable label.

[0066] Embodiment 46: The method according to any one of embodiments 33-45, wherein detecting comprises detecting the analyte in a substantially intact planar array.

[0067] Embodiment 47: The method of embodiment 46, wherein said detecting comprise visualizing the array with an image analysis system.

[0068] Embodiment 48: The method according to any one of embodiments 33-45, wherein detecting comprises detecting the analyte(s) after the microgel electrophoresis structures (pGel(s)) are separated from each other. [0069] Embodiment 49: The method of embodiment 48, wherein said detecting comprises using flow cytometry to detect the detectable labels associated with separated microgel electrophoresis structures.

[0070] Embodiment 50: The method according to any one of embodiments 33-49, wherein said one or more analytes comprise an analyte selected from the group consisting of a peptide, a nucleic acid, a virus or virus fragment, a bacterium or bacterial fragment, a cytokine, a toxin, an antibody, a peptide nucleic acid, an aptamer, a lectin, an antigen, an enzyme, a small organic molecule, and a cellular receptor binding protein.

[0071] Embodiment 51 : The method of embodiment 50, wherein said analyte comprises a protein.

[0072] Embodiment 52: The method according to any one of embodiments 33-51, wherein said method comprises immobilizing the analyte(s) in the microgel electrophoresis structures after the analytes have migrated into the polymeric gel.

[0073] Embodiment 53: The method of embodiment 52, wherein said immobilizing is by application of UV light and/or by chemical cross-linking.

[0074] Embodiment 54: The method according to any one of embodiments 33-53, wherein said contacting and detecting comprises contacting said sample with a primary antibody that binds to an analyte that is to be detected where the primary antibody is attached to said detectable label.

[0075] Embodiment 55: The method according to any one of embodiments 33-53, wherein said contacting and detecting comprises contacting said sample with a primary antibody that binds to an analyte that is to be detecting and contacting said primary antibody with a secondary antibody that binds to said primary antibody where said secondary antibody is attached to said detectable label.

[0076] Embodiment 56: The method according to any one of embodiments 33-55, wherein said method comprises a western blot.

[0077] Embodiment 57: The method according to any one of embodiments 33-55, wherein said method comprises a differential detergent fractionation.

[0078] Embodiment 58: The method according to any one of embodiments 33-50, and 54-55, wherein said analyte comprises a nucleic acid. [0079] Embodiment 59: The wherein said detecting comprises detecting contacting said nucleic acid with an oligonucleotide probe that hybridizes to said nucleic acid where said oligonucleotide probe is attached to said detectable label.

[0080] Embodiment 60: A method of making a substantially planar array of microgel electrophoresis structures, said method comprising:

[0081] providing a mold surface patterned with features that define the shapes and size a pGel array;

[0082] loading the mold surface with precursors of a polymeric gel;

[0083] covering the loaded gel with a substantially planar surface;

[0084] polymerizing the precursors to form a polymerized polymer gel; and

[0085] releasing the polymerized polymer gel from the mold surface to provide a pGel array disposed on said substantially planar surface.

[0086] Embodiment 61 : The method of embodiment 60, wherein said mold surface is configured to pattern the features of a pGel array according to any one of embodiments 1-29.

[0087] Embodiment 62: The method according to any one of embodiments 60-61, wherein said mold surface comprises an SET-8 surface.

[0088] Embodiment 63 : The method according to any one of embodiments 60-62, wherein said substantially planar surface comprises a glass surface.

[0089] Embodiment 64: The method of embodiment 63, wherein said substantially planar surface comprises a glass slide.

[0090] Embodiment 65: The method according to any one of embodiments 63-64, wherein said substantially planar surface is silanized.

[0091] Embodiment 66: The method according to any one of embodiments 60-65, wherein said polymerization comprises photopolymerization by exposing said precursors to a light source capable of inducing polymerization.

[0092] Embodiment 67: The method of embodiment 66, wherein light source comprises a ETV light source.

[0093] Embodiment 68: The method according to any one of embodiments 60-65, wherein said polymerization comprises chemical photopolymerization.

[0094] Embodiment 69: The method according to any one of embodiments 60-68, wherein said precursors comprises a material selected from the group consisting of acrylamide, Ethyl Acrylate, Ethyl Methacrylate, Ethyleneglycol Dimethacrylate,

Hydroxy ethyl Acrylate, Isobutyl Methacrylate, Lauryl Methacrylate, Methacrylic Acid, Methyl Acrylate, Methyl Methacrylate, N,N-methylene-bisacryl-amide, n-Butyl

Methacrylate, pentaerythritol tetraacrylate, pentaerythritol triacrylate, Pentaerythritol

Triacrylate, Polyethylene Glycol (200) Diacrylate, Phenyl acrylate, poly(ethylene glycol diacrylate), polypropylene diacrylate), l,3-Butanediol Dimethacrylate, l,4-Butanediol Dimethacrylate, l,6-Hexanediol Dimethacrylate, 2,2,3,3,4,4,5,5-Octafluoropenty Acrylate, 2.2.2-Trifluoroethyl 2-methylacrylate, 2-Ethyl Hexyl Acrylate, 2-Hydroxy ethyl Methacrylate, 2-Hydroxypropyl Acrylate, 4-Hydroxybutyl Acrylate, agarose, Allyl Methacrylate, Benzyl Methylacrylate, Butyl Acrylate, collagen type I, collagen type IV., Diethyleneglycol

Diacrylate, Diethyleneglycol Dimethacrylate, dimethacrylate, Divinyl benzene, divinyl benzene (DVB), Monoethylene Glycol, poly(ethylene glycol), polypropylene glycol), Polyethylene Glycol (200) Dimethacrylate, Polyethylene Glycol (400) Diacrylate,

Polyethylene Glycol (400) Dimethacrylate, Polyethylene Glycol (600) Diacrylate,

Polyethylene Glycol (600) Dimethacrylate, Stearyl Methacrylate, Triethylene Glycol, Triethylene Glycol Dimethacrylate, Trimethylolpropane Triacrylate, gelatin, and the like, and mixtures or copolymers thereof.

[0095] Embodiment 70: The method of embodiments 69, wherein said precursors comprise precursors for an acrylamide gel.

[0096] Embodiment 71 : The method of embodiment 70, wherein said precursors comprise N,N,N',N'-tetramethylethylenediamine (TEMED) as a catalyst.

[0097] Embodiment 72: The method according to any one of embodiments 70-71, wherein said precursors comprise ammonium persulfate (APS) as an initiator.

[0098] Embodiment 73 : The method according to any one of embodiments 60-72, wherein the microgel array is incubated in distilled or deionized water prior to releasing the planar substrate from the mold.

[0099] In various embodiments the arrays of polymeric microgel electrophoresis structures pGel array) expressly exclude collections of microcapillary tubes and the like.

Definitions.

[0100] The terms "microgel electrophoresis structures", "pGel", "individually addressable microgel electrophoresis lane" and "separations-encoded microparticle" are used interchangeably. As described herein the microgel electrophoresis structures can exist in two forms: 1) As a substantially planar array of microgel electrophoresis structures; and 2) When separated from each other, as microparticles. Particularly where one or more analytes have been separated in the microgel electrophoresis structures, the presence of the analyte(s) act as a bar code effectively encoding the microparticles (microgels) when separated from each other and may be regarded as "separations-encoded" microparticles. Additionally as described herein when the microgel electrophoresis structures are present as an array ( e.g ., a substantially planar array) it is possible to direct samples and/or regents into any particular microgel electrophoresis structure or into an particular group of electrophoresis structures. Thus the microgel electrophoresis structures may be viewed as "individually addressable" microgel electrophoresis structures (lanes).

[0101] The term "sample" refers to any substance that is to be assayed, e.g., using the methods described herein.

[0102] The term "biological sample" refers to sample that is a sample of biological tissue, cells, or fluid. Such samples include, but are not limited to, cultured cells, acute cell preparations, sputum, amniotic fluid, blood, blood cells (e.g, white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom.

Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. In certain embodiments the biological sample is from a mammal, e.g, a human. However, samples can be from any mammal such as dogs, cats, sheep, cattle, and pigs, etc. and need not be from any mammals. Accordingly biological samples can be from vertebrates, invertebrates, bacteria, protozoans, fungi, and the like. The sample may be pretreated as necessary by dilution in an appropriate buffer solution or concentrated, if desired.

[0103] The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g, proteins, nucleic acids, etc.). In certain embodiments preferred small organic molecules range in size up to about 5000 Da, or up to about 4000 kDa, or up to about 3,000 kDa, or up to about 2000 Da, or up to about 1000 Da.

[0104] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a

corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In certain embodiments the term "peptide" refers to a polymer of amino acid residues typically ranging in length from 2 to about 50 or about 60 residues. In certain embodiments the peptide ranges in length from about 4, 5, 6, 7, 8, 9, 10, or 11 residues to about 60, 50, 45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments the peptide ranges in length from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25 residues. In certain embodiments the amino acid residues comprising the peptide are "L-form" amino acid residues, however, it is recognized that in various embodiments, "D" amino acids can be incorporated into the peptide or the peptide can be all "D" amino acids. Peptides also include amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, "modified linkages" ( e.g ., where the peptide bond is replaced by an a-ester, a b- ester, a thioamide, phosphonamide, carbomate, hydroxylate, and the like (see, e.g., Spatola (1983) Chem. Biochem. Amino Acids and Proteins 7: 267-357), where the amide is replaced with a saturated amine (see, e.g, Skiles et /., U.S. Pat. No. 4,496,542, which is incorporated herein by reference, and Kaltenbronn et al, (1990) Pp. 969-970 in Proc. 1 lth American Peptide Symposium, ESCOM Science Publishers, The Netherlands, and the like)).

[0105] The term“nucleic acid” refers to a nucleotide polymer, and unless otherwise limited, includes known analogs of natural nucleotides that can function in a similar manner (e.g, hybridize) to naturally occurring nucleotides. The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; and RNA. The term nucleic acid encompasses double- or triple-stranded nucleic acid, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). The term nucleic acid also encompasses any chemical modification thereof, such as by methylation and/or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and

functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as T -position sugar modifications, 5- position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitutions of 5-bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like. More particularly, in certain embodiments, nucleic acids, can include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing nonnucleotidic backbones, for example, polyamide ( e.g ., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oregon, as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses locked nucleic acids (LNAs), which are described in U.S. Patent Nos. 6,794,499, 6,670,461, 6,262,490, and 6,770,748.

[0106] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of

immunoglobulin genes or derived therefrom that is capable of binding (e.g., specifically binding) to a target (e.g, to a target polypeptide). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0107] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to these light and heavy chains respectively.

[0108] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' ₂ a dimer of Fab which itself is a light chain joined to V _H -C _H l by a disulfide bond. The F(ab)' ₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab') ₂ dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Certain preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (sFv or scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. The single chain Fv antibody is a covalently linked VH-V _L heterodimer which may be expressed from a nucleic acid including V _H - and VL- encoding sequences either joined directly or joined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. While the V _H and V _L are connected to each as a single

polypeptide chain, the V _H and V _L domains associate non-covalently. The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv), however, alternative expression strategies have also been successful. For example Fab molecules can be displayed on a phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the important point is that the two antibody chains in each Fab molecule assemble post- translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p (see, e.g, U.S. Patent No: 5733743). The scFv antibodies and a number of other structures converting the naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three- dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g, U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778). In certain embodiments antibodies should include all that have been displayed on phage (e.g, scFv, Fv, Fab and disulfide linked Fv (see, e.g, Reiter et al. (1995) Protein Eng.

8: 1323-1331) as well as affibodies, unibodies, and the like.

[0109] The term "free standing" refers to a three-dimensional structure that is able to maintain its three dimensional integrity without external supporting structures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 schematically illustrates an embodiment of the array of microgel electrophoresis structures (pGel array) 100 described herein. In the illustrated embodiment the array comprises a plurality of microgel electrophoresis structures (pGel(s)) 102 disposed on a substrate 104. Each microgel comprises one or more sample receiving wells 106. The array also comprises a separation zone (separation zones) 108 delineating each microgel electrophoresis structure perimeter where the separation zone is structured to facilitate separation of the microgel electrophoresis structures from each other. In certain

embodiments the separation zone comprises perforations 110 that facilitate separation of the microgel electrophoresis structures from each other. One of the pGels is illustrated containing separated analytes 112.

[0111] Figure 2, panel A, illustrates a planar array of polymeric microgel

electrophoresis structures consisting of approximately 1500 units (microgel electrophoresis structures (pGel(s)) fabricated on a half glass slide. Fig. 2, panel B, illustrates the array fabricated on a glass slide using chemical- or photo- polymerization. As illustrated, each pGEL contains a 30 pm diameter (well sample receiving well) where single cells are settled in. Fig. 2, panel C, illustrates single-cell resolution western blotting assay that was run on the planar array format of hydrogel pGELs, which were subsequently released and collected for image analysis. Multiple analytes ( e.g ., protein markers) can be probed in a single pGEL. Proteins with a minimum of 20% mass difference can be resolved using this system. Fig. 2, panel D, shows a schematic view of fabrication workflow for chemical- and photo- polymerization. Fig. 2, panel E, shows a schematic view of single-cell resolution western blotting workflow in free-floating hydrogel pGELs. After performing the cell settling, electrophoresis, protein photocapture to the gel, and immunoprobing, individual hydrogel pGELs are released from the microscope glass slide by the help of a blade. Bands of separated proteins in each pGEL are visualized using a fluorescence scanner for further analysis.

[0112] Figure 3, panel A, shows microscopy images of planar array of the hydrogel pGELs, after fabrication and after probing for two housekeeping protein markers (PTubulin- red and GAPDH-green), respectively. Multiplexed protein detection is shown for hydrated and dried hydrogel pGELs. PTubulin (50kDa) and GAPDH (35kDa) proteins from U251 cells were separated in single pGELs. Fig. 3, panel B, shows free floating hydrogel pGELs released as individual units, after fabrication and after probing for two housekeeping protein markers (PTubulin and GAPDH), respectively. Fig. 3, panel C, illustrates characterization of swelling properties of hydrogel pGELs and efficiency of mask transfer. Epifluorescence images of fluorescently labelled hydrogel pGELs in dry and hydrated states. Fig. 3, panel D, illustrates characterization of surface area changes in dry, hydrated versus attached, released conditions. In the box plot, surface area normalized to attached hydrated condition is shown, n = 2483. Fig. 3, panel E, shows a comparison of the edge effect in fluorescently labelled hydrated/dried, non-labelled hydrated, and fluorescently labelled plain gel with no hydrogel pGELs are shown in the line graph. Inset represents the non-labelled hydrated pGELs. Fig. 3, panel F, shows an epifluorescence image of damaged and non-damaged hydrogel pGELs, and characterization of the effect of the releasing step on hydrogel pGEL damage, n = 2483.

[0113] Figure 4, panel A, shows the resolution between the proteins was calculated as shown, n = 121. Fig. 4, panel B, illustrates a polymeric pGEL with separated PTubulin and GAPDH proteins showing the region of interest for quantification of the results including resolution, area under curve, and peak width. Fig. 4, panel C, shows the area under curve quantified for dried, hydrated, attached, released hydrogel pGEL combinations for two housekeeping proteins (PTubulin and GAPDH) were characterized. Red lines in box plot shows the median RFEi values for each type of gel, n = 121. Fig. 4, panel D, shows the fluorescence intensity quantified for dried, hydrated, attached, released hydrogel pGEL combinations for two housekeeping proteins (PTubulin and GAPDH) were characterized.

Red lines in box plot shows the median RFEI values for each type of gel, n = 121. Fig. 4, panel E, shows the peak width quantified for dried, hydrated, attached, released hydrogel pGEL combinations for two housekeeping proteins (PTubulin and GAPDH) were

characterized. Red lines in box plot shows the median RFU values for each type of gel, n = 121

[0114] Figure 5, panel A, provides confocal microscopy images showing the chemically-polymerized polymeric microgel electrophoresis structure (pGEL) with dimensions and uniformity appropriate for performing electrokinetic protein separations, according to embodiments of the present disclosure. Fig. 5, panel B, provides confocal microscopy images showing the photo-polymerized polymeric pGEL with dimensions and uniformity appropriate for performing electrokinetic protein separations, according to embodiments of the present disclosure. The height of the pGEL and polymerized fluid layer is 60 pm and 30 pm, respectively.

[0115] Figure 6, illustrates the fabrication of polymeric pGELs using a mask based photolithography process, according to certain embodiments of the present disclosure. The desired micropattem was defined by a mylar photo-mask, which determined the portions of the reservoir that were polymerized by a UV light source. Fabrication was completed in 10 min and included three steps: 1) Polymer precursor solution was sandwiched between two solid supports, one with posts one it; 2) The solution was exposed to UV light through a photo-mask; and 3) Excess precursor solution was washed away.

[0116] Figure 7 panel A illustrates characterization of cell settling in the hydrogel microgel electrophoresis structure (pGel) array. Bar graph of cell settling efficiency in the planar array of hydrogel pGELs. Fig. 7, panel B, shows bright field images of wells in the hydrogel pGELs and separation of PTubulin and GAPDH proteins based on cell settling condition.

[0117] Figure 8, panels A-F, illustrates the design and operation of the separations- encoded microparticles (pGELs) for high specificity protein isoform analysis. Panel A) Separations-encoded microparticle array consisting of approximately 3500 releasable units fabricated on a half glass slide. Panel B) Schematic view of single-cell resolution western blotting workflow in microparticles. After performing the single-cell settling,

electrophoresis, protein photocapture to the gel (immobilization), and immunoprobing, microparticles are released from the microscope glass slide by the help of a blade. Bands of separated proteins in each microparticle are visualized using a fluorescence scanner for quantification. Panel C) The array is comprised of a thin layer of polyacrylamide gel. In the illustrated embodiment, each microparticle (pGEL) contains a 30 pm diameter well where single cells are housed. Panel E) Single-cell resolution western blotting assay is ran on microparticles, which can be released (Panel D) and collected for downstream analysis. Multiple protein markers can be probed in individual microparticles, the image shows false- colored micrographs of microparticles. Panel F) False-colored microscopy images of microparticle array attached on and released from a glass slide was probed and imaged in both hydrated and dehydrated states. Microparticles were probed for two housekeeping protein markers b-Tubulin (50 kDa, magenta) and GAPDH (35 kDa, blue) from single U251 cells. Dehydrating microparticles boosts analytical sensitivity thanks to the geometry- enhanced concentration of fluorescence signal.

[0118] Figure 9, panels A-D, illustrates characterization of ERa isoform expression in microparticle assay. Panel A) Measured ERa expression from the same array after a multi step probing (including one stripping round), and after probing of designated microparticles (p>0.05, n = 40 microparticles). Panel B) Fluorescence micrographs of microparticles for Actinin, b-Tubulin, GAPDH and ERa isoforms in MCF 7 cells. RFU, relative fluorescence units. The off-target peak (via ERa antibody) does not coincide with the ERa isoform bands. Panel C) Log-linear plot of species molecular weight against migration distance in 8%T PAG the fluorescently labeled species in panel A. (x-axis error bars within point size (± S.D., n = 3 separations); GAPDH, 35 kDa; ERa46, 46 kDa; b- Tubulin, 50 kDa; ERa66, 66 kDa; Actinin, 100 kDa). Panel D) Box plot demonstrating the separation resolution between ERa isoforms (n = 34 cells).

[0119] Figure 10, panels A-C illustrates characterization of microparticles with different morphologies. Panel A) Separation performance, as measured by peak width, does not significantly change with dehydrated and hydrated states of microparticles. Mann- Whitney EG-test p value was found to be lower than 0.05 (n = 121). Panel B) Fluorescence intensity comparison of b-tubulin and GAPDH proteins suggests that no statistically significant difference exists between the combinations of dehydrated and hydrated states of microparticles. Mann-Whitney U-test p value was found to be lower than 0.05 (n = 121). Panel C) Quantified b-Tubulin and GAPDH expression in microparticles from EG251 glioblastoma cells shows that released and attached microparticles in hydrated and dehydrated states do not demonstrate statistically significant differences. For all

combinations, Mann-Whitney U-test p value was found to be lower than 0.05 (n = 121).

[0120] Figure 11, panels A-C, shows that expression of ERa isoforms change over confluency of cell culture. MCF 7 cells are shown as the model ERa positive organisms, whereas MDA MB 231 and HEK 293 serve as the ERa negative control cell lines. Panel A) Color-coded beeswarm graphs show single-cell protein measurements in subsequent cell culturing days for MCF 7, MDA MB 231, and HEK 293 cell lines. The black bars present the median protein expression level for each group. Panel B) Signal-to-noise ratio (SNR) for ERa isoforms. Red dashed line presents SNR = 3, above which protein quantification was employed for all measurements, n _to tai = 447. Panel C) The grouped box plots show fluctuations in ERa isoform expression over 14 days in MCF 7 cells (n = 478 cells).

Microparticles reported an gradual increase in the number of cells expressing ERa46 (green), while the expression of ERa66 (blue) dropped gradually over 14 days.

[0121] Figure 12, panels A-B, shows a conceptual description of the workflow of separations-encoded microparticle fabrication. Panel A) A microscope slide is silanized to attach microparticle array on the glass. Panel B) The silanization process is followed by polyacrylamide gel synthesis based on chemical polymerization. Hydrogel precursor is sandwiched between the silanized microscope slide and a silicon wafer with SU-8 posts. After releasing the microscope slide with microparticle patterns from the silicon wafer, single-cell analysis is employed. Individual microparticles can be released from the microscope slide using a razor blade.

[0122] Figure 13, panels A-B, illustrates damage analysis in microparticles. Panel A)

False-color fluorescence image of damaged and undamaged microparticles. Panel B) Bar graph shows the percentage of damaged microparticles at hydrated and dehydrated states after and before releasing, p<0.000l (two sample t-test), n = 2483 microparticles.

[0123] Figure 14, panels A-B, illustrates characterization of cell settling in the microparticle array. Panel A) Bar graph summarizes the distribution of settled cells in microwells (n = 3 devices with 3500 units each). Panel B) Bright field images (top) and fluorescence intensity graphs (bottom) of microparticles. Separation of b-Tubulin and GAPDH proteins are shown and the mean antibody fluorescence intensity from two-cell occupancy microparticles was ~5.5x higher than that of single-cell occupancy microparticles (CV = 0.28, n = 10 wells). The intensity was not doubled (not linear) in two-cell occupancy microparticles due to the cell-size related bias.

[0124] Figure 15, panels A-B, illustrates mobility measurements for b-Tubulin and

GAPDH proteins are performed in 8%T 2.6%C polyacrylamide gel microparticles fabricated on partially and fully silanized glass slides. Panel A) Montage of fluorescence micrographs of b-Tubulin at different electrophoresis times on partially and fully silanized glass slides. Panel B) Changes in b-Tubulin (50 kDa) and GAPDH (35 kDa) mobilities in microparticles attached on partially and fully silanized glass slides, n _to tai ⁼ 40 microparticles.

[0125] Figure 16, panels A-C, illustrates a comparison of fluorescence intensities in microparticles. Panel A) Background intensity reduction in released microparticles suggests the geometry-enhanced mass transport during antibody probing and washout steps. Panel B) Characterization of degree of microwell circularity changes in combinations of hydrated, dehydrated, attached, and released states. Surface area normalized to attached hydrated condition is shown in the box plot, p < 0.00001 between all groups, n _totai = 5090. Panel C) Fluorescence intensity graph shows signal intensity changes in hydrated/probed, hydrated/not probed, dehydrated/probed microparticles The inset shows a close-up intensity profile of hydrated/not probed microparticles.

[0126] Figure 17, panels A-C shows cell confluency level changes in three cell lines and change of ERa isoform expression over confluency of MCF 7 cells in slab gel westerns. Panel A) The bar graph shows the confluency levels of estrogen sensitive (MCF 7) and resistant (MDA MB 231) breast cancer cell lines as well as HEK 293 cells over 7 and 14 days. MDA MB 231 and HEK 293 cells reached > 90% confluency after 7 days of cell culture. Slab gel western blots (15 mg protein) were performed to confirm microparticle assay results. The scatter graph of (panel B) ERa46 and (panel C) ERa66 to GAPDH expression ratio shows measured increase and decrease in protein expression levels over 14 days (n = 2 slab gel western runs).

[0127] Figure 18, panels A-B, shows a comparison of cell confluency and ERa expression levels. Panel (A) Bright-field images of MCF 7, MDA MB 231, and HEK 293 cells in corresponding culture days and counted cells per mL that are used to calculate confluency levels. Panel B) Identification of ERa isoforms in MCF 7, MDA MB 231, and HEK 293 cells by conventional slab gel Western blotting. Western blotting images were used in quantification of ERa isoforms in MCF 7 cells. No detectable signal was observed in MDA MB 231, and HEK 293 cells, as expected.

[0128] Figure 19, panels A and B, illustrates finite element simulation of electric field distribution. Electric currents module of COMSOL Multiphysics software was operated by applying steady-state constant electric field through the matrix, where hydrogel and solution conductivities were set to 4304.0 pS m ¹ estimated experimentally. Panel A) A section of microparticle array geometry and the cross section along which the electric field was simulated. Panel B) Electric field distribution in the section at dashed black line in panel A shows that microparticles do not disturb electric field distribution.

DETAILED DESCRIPTION

[0129] In various embodiments an electrophoretic substrate and/or device is provided that provides a novel a hybrid microfluidic format capable of toggling from a planar microgel analyte separation array and a suspension of microparticles. In certain embodiments the electrophoretic substrate comprises a substantially planar array of microgel electrophoresis structures (pGel(s)) stippled in a thin sheet of polymeric gel (e.g, polyacrylamide). By way of example, for single-cell electrophoresis, a thin-layer hydrogel disposed on a substrate (e.g, on a glass slide) is stippled with microwells for receiving cells (or other samples). Each microwell abuts a region for polyacrylamide gel electrophoresis (PAGE) and immobilization (e.g, via photocapture) of separated analytes (e.g, proteins) to the gel matrix.

[0130] In certain embodiments the array elements (microgel electrophoresis structures

(pGels)) are defined by perforations around each rectangular single-cell PAGE region which, upon mechanical release, toggles into a separations-encoded microparticle (pGel). In an embodiment illustrated in Example 1, the microparticles measure estrogen receptor and 5 protein targets in hundreds of single breast cancer cells and kidney cells. Placing the released separations-encoded microparticles (pGels)into suspension for e.g, immunoassays, enhances diffusion into the microgel(s) making the assays significantly more efficient. For example, the efficiency of single-cell immunoblotting steps (e.g, immunoprobe introduction and wash out) is improved by 4x. Additionally, dehydrating the microparticles (pGels) from suspension results in isotropic shrinkage of each particle (10% in length), yielding a l.6x increase in immunoblot fluorescence signal with no penalty on separation resolution.

Designed for optimal mass transport and scaling at each assay stage, separations-encoded microparticles (pGels) provide an adaptable new form factor for precision single-cell analysis.

[0131] One embodiment of the substantially planar array of microgel electrophoresis is schematically illustrated Figure 1. In the illustrated embodiment the array comprises a plurality of microgel electrophoresis structures (pGel(s)) 102 disposed on a substrate 104. Each microgel comprises one or more sample receiving wells 106. The array also comprises a separation zone (separation zones) 108 delineating each microgel electrophoresis structure perimeter where the separation zone is structured to facilitate separation of the microgel electrophoresis structures from each other. In certain embodiments the separation zone comprises perforations 110 (or other features) that facilitate separation of the microgel electrophoresis structures from each other.

[0132] Figure 2, panels A-C, shows photographs of one illustrative array of polymeric microgel electrophoresis structures (pGels). While illustrated pGels are rectangular, the pmicrogels need not be limited to this shape. Essentially any shape can be attached and include regular as well as irregular shapes. In certain embodiments the pGel shape is selected to facilitate the particular assay at issue.

[0133] The separation zone(s) are structured to facilitate separation of the microgel electrophoresis structures (pGels) from each other. In certain embodiments the separation zones are structured to facilitate recovery of a significant number of separated microgel electrophoresis structures (pGels). In certain embodiments the separation zones are structured to facilitate recovery of at least 50%, or at least 60%, or at least 70% , or at least 80% , or at least 90% , or at least 95% of the pGels comprising the array. In certain embodiments the separation zone(s) are structured to facilitate separation of the microgel electrophoresis structures from each other while as retaining sufficient physical integrity of the released separations-encoded microparticles (pGels) to permit interrogation of the microparticles (pGels) to detect the separated analyte(s).

[0134] In certain embodiments the separation zone(s) are structured to facilitate separation of the microgel electrophoresis structures (pGels) from each other while retaining sufficient physical integrity of the released microgels to permit interrogation of the separations-encoded microparticle(s) (pGel(s)) to detect the separated analyte(s). In certain embodiments the separation zone(s) are structured to facilitate separation of the microgel electrophoresis structures (pGels) from each other forming microparticles that substantially retain the physical integrity of each of the separated microgel electrophoresis structures.

[0135] The separation zones can be structured with any of a number features to facilitate separation. In one illustrative approach, the separation zones comprise a trough between adjacent microgel electrophoresis structures. In certain embodiments the thickness of material in the trough is less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 15%, or less than 10%, or less than 5%, or less than 3% the thickness of the polymeric gel forming the microgel electrophoresis structures. In certain embodiments the trough extends all the way to the surface of a substrate on which the microgel electrophoresis structures are deposited. In certain embodiments the troughs comprise a trough floor formed from the same polymeric gel material that forms the microgel electrophoresis structures. In certain embodiments the troughs comprise a trough floor formed from a different polymeric material than the material that forms the microgel electrophoresis structures.

[0136] In certain embodiments the troughs comprise a trough floor that is sufficiently thin to facilitate mechanical separation of the microgel electrophoresis structures (pGels) from each other by mechanical pulling or shearing. In certain embodiments the separation zone (comprising troughs or not) comprises a plurality of perforations that facilitate separation of the microgel electrophoresis structures from each other. In certain

embodiments the separation zone (comprising troughs or not) comprises one or more scoring lines that facilitate separation of the microgel electrophoresis structures from each other.

[0137] In certain embodiments the separation zone (comprising troughs or not) comprises a material comprising a photocleavable linker that facilitates degradation of the trough when exposed to a wavelength of light that cleaves said photocleavable linker. Thus, for example, enzymatic and light degradable polymeric hybrid nanogels have been prepared by free radical inverse miniemulsion copolymerization of acrylamide (AAm) with a functional dextran crosslinker containing acrylate moieties attached to the backbone via a photolabile linker, that is, dextranDphotolabile linker□ acrylate (DexDPLD A) (see, e.g. , Klinger & Landfester (2011) J Polymer Sci., 50(6): 1062-1075).

[0138] In certain embodiments the separation zone comprises a material that is hydrolyzed by a reagent that does not hydrolyze the microgel electrophoresis structures. This can be facilitated for example, by the use of a chemically or enzymatically cleavable linkers, or incorporation of a gel forming material that is readily degraded (e.g., a starch). Cleavable linkers are well known to those of skill in the art (see, e.g., Leriche el al. (2012) Bioorg. & Med. Chem. 20(2): 571-582).

[0139] In certain embodiments the separation zone comprises a material that preferentially absorbs laser radiation as compared to the microgel electrophoresis structures and degrades when exposed to said laser radiation. This can readily be accomplished by incorporation of a dye that absorbs at the laser wavelength.

[0140] The arrays of microgel electrophoresis structures described herein are well suited to high throughput analysis (screening). Accordingly, in certain embodiments, the arrays contain a large number of microgel electrophoresis structures (pGels). In certain embodiments the array comprises at least 500, or at least 1,000, or at least 1,500, or at least 2,000, or at least 2,500, or at least 3,000, or at least 3,500, or at least 4,000, or at least 5,000 microgel electrophoresis structures. In certain embodiments the microgel electrophoresis structures are disposed within a surface area of 20 cm ² or less, or 15 cm ² or less or 10 cm ² or less, or 5 cm ² or less, or 3 cm ² or less or 2 cm ² or less or 1 cm ² or less. In certain embodiments each of the microgel electrophoresis structures range in volume from about 10 pL, or from about 50 pL, or from about 100 pL, or from about 250 pL, or from about 500 pL, or from about 1000 pL up to 10,000 pL, or up to about 5,000 pL, or up to about 4,000 pL, or up to about 3,000 pL, or up to about 2000 pL. In certain embodiments illustrative, but non limiting embodiments, the volume of each polymeric pGel (microparticle (when separated from the array) ranges between 10 and 10,000 picoliters. In certain embodiments each of the microgel electrophoresis structures has an area ranging from about from about 1,000 pm ² , or from about 5,000 pm ² , or from about 10,000 pm ² , or from about 20,000 pm ² , or from about 30,000 pm ² , or from about 40,000 pm ² up to about 500,000 pm ² , or up to about 250,000 pm ² , or up to about 100,000 pm ² , or up to about 80,000 pm ² , or up to about 60,000 pm ² . In certain embodiments illustrative, but non-limiting embodiments, the size of each polymeric pGEL ranges between 1000 and 500,000 pm ² . [0141] In certain embodiments each microgel electrophoresis structure is substantially rectangular. In certain embodiments the microgel electrophoresis structures each have a length ranging from about 500 pm, or from about 600 pm, or from about 700 pm, or from about 800 pm, or from about 900 pm up to about 3,000 pm, or up to about 2,500 pm, or up to about 2,000 pm, or up to about 1,500 pm, or up to about 1,000 pm; and/or the microgel electrophoresis structures each have a width ranging from about 5 pm, or from about 10 pm, or from about 15 pm, or from about 20 pm, or from about 30 pm, or from about 40 pm up to about 500 pm, or up to about 250 pm, or up to about 200 pm, or up to about 150 pm, or up to about 100 pm, or up to about 80 pm, or up to about 60 pm. In certain embodiments each microgel electrophoresis structure ranges in length from about 100 pm up to about 1000 pm. In certain embodiments the microgel electrophoresis structures each have a length of about 950 pm. In certain embodiments the microgel electrophoresis structures each have a width of about 50 pm. In certain embodiments the microgel electrophoresis structures each have a thickness (depth) ranging from about from about 15 pm, or from about 20 pm, or from about 30 pm, or from about 40 pm up to about 200 pm, or up to about 150 pm, or up to about 100 pm, or up to about 80 pm, or up to about 60 pm, or up to about 50 pm. In certain

embodiments the microgel electrophoresis structures each have a thickness ranging from about 15 pm up to about 100 pm. In certain embodiments the microgel electrophoresis structures each have a thickness of about 40 pm.

[0142] In certain embodiments the microgel electrophoresis structures (pGels) comprising an array microgel electrophoresis structures (pGel array) all have about the same dimensions (i.e., approximately the same length, width, and thickness). In certain

embodiments different microgel electrophoresis structures (pGels) comprising an array microgel electrophoresis structures (pGel array) differ in one or more dimensions (e.g, they have different lengths, and/or different widths, and/or different thicknesses).

[0143] In certain embodiments the microgel electrophoresis structures (pGels) comprising an array microgel electrophoresis structures (pGel array) all have sample receiving wells with approximately the same dimensions (i.e., approximately the same length, diameter and depth). In certain embodiments different microgel electrophoresis structures (pGels) comprising an array microgel electrophoresis structures (pGel array) have sample wells that differ in one or more dimensions (e.g, they have different diameters, and/or different depths). [0144] In certain embodiments, each of the individually addressable microgel electrophoresis structure(s) (lane(s)) includes a single or plurality of micron-sized well(s) (sample receiving wells) surrounded by the hydrogel frame (body). In certain embodiments each micron-sized well (sample receiving well) can include an open end in fluid

communication with the environment above the pGel array, and an opposing closed end within the polymeric microgel electrophoresis structure (pGel). In various embodiments the micron-sized (sample receiving) well serves as a container for receiving a sample that is to be analyzed ( e.g ., decomposable biological entities for single-cell bioassays and other applications). In certain embodiments the sample receiving wells range in diameter from about 10 pm, or from about 20 pm, or from about 30 pm up to about 100 pm, or up to about up to about 80 pm, or up to about 70 pm, or up to about 60 pm, or up to about 50 pm. In certain embodiments the sample receiving wells have a diameter of about 30 pm. In certain embodiments the sample receiving wells are sized to each contain a single cell (e.g., a vertebrate cell, an invertebrate cell) or a single microorganism (e.g, bacterium or a protozoan). In certain embodiments the sample wells are sized to contain a single vertebrate cell (e.g, a mammalian cell). In certain embodiments the sample receiving wells each have a depth ranging from about from about 15 pm, or from about 20 pm, or from about 30 pm, or from about 40 pm up to about 200 pm, or up to about 150 pm, or up to about 100 pm, or up to about 80 pm, or up to about 60 pm, or up to about 50 pm. In certain embodiments the sample wells each have a depth of about 40 pm.

[0145] In various embodiments the polymeric gel forming the microgel

electrophoresis structure comprises a hydrogel. In certain embodiments the polymeric gel comprises a material selected from the group consisting of acrylamide, Ethyl Acrylate, Ethyl Methacrylate, Ethyleneglycol Dimethacrylate, Hydroxy ethyl Acrylate, Isobutyl Methacrylate, Lauryl Methacrylate, Methacrylic Acid, Methyl Acrylate, Methyl Methacrylate, N,N- methylene-bisacryl-amide, n-Butyl Methacrylate, pentaerythritol tetraacrylate, pentaerythritol triacrylate, Pentaerythritol Triacrylate, Polyethylene Glycol (200) Diacrylate, Phenyl acrylate, poly(ethylene glycol diacrylate), polypropylene diacrylate), l,3-Butanediol Dimethacrylate, l,4-Butanediol Dimethacrylate, l,6-Hexanediol Dimethacrylate, 2, 2, 3, 3, 4, 4,5,5- Octafluoropenty Acrylate, 2.2.2-Trifluoroethyl 2-methylacrylate, 2-Ethyl Hexyl Acrylate, 2- Hydroxyethyl Methacrylate, 2-Hydroxypropyl Acrylate, 4-Hydroxybutyl Acrylate, agarose, Allyl Methacrylate, Benzyl Methylacrylate, Butyl Acrylate, collagen type I, collagen type IV., Diethyleneglycol Diacrylate, Diethyleneglycol Dimethacrylate, dimethacrylate, Divinyl benzene, divinyl benzene (DVB), Monoethylene Glycol, poly(ethylene glycol), poly(propylene glycol), Polyethylene Glycol (200) Dimethacrylate, Polyethylene Glycol (400) Diacrylate, Polyethylene Glycol (400) Dimethacrylate, Polyethylene Glycol (600) Diacrylate, Polyethylene Glycol (600) Dimethacrylate, Stearyl Methacrylate, Triethylene Glycol, Triethylene Glycol Dimethacrylate, Trimethylolpropane Triacrylate, gelatin, and the like, and mixtures or copolymers thereof. In certain embodiments the polymeric gel comprises acrylamide ( e.g ., 7% up to 14% acrylamide).

[0146] While the array of microgel electrophoresis structures is illustrated on a solid support in Figure 1, in certain embodiments, the intact array is released from the solid support while retaining integrity of the planar array. In certain embodiments the microgel

electrophoresis structures comprising the array are separated from each other to provide a suspension of microparticles (suspended pGels).

[0147] The foregoing description of the microgel arrays is illustrative and non limiting. Using the teaching provided herein numerous variations on the electrophoretic substrate will be available to one of skill in the art.

Fabrication of microgel electrophoresis structure arrays.

[0148] A number approaches have been taken to synthesize gel electrophoresis structures on a microscale. Conventionally, individual polymeric electrophoresis gels have been synthesized using batch processes including, for example, 3D printing (Hahn et al. (2006). Adv. Materials , 18(20): 2679-2684), soft lithography (Di Benedetto et al. 92005) Nanotechnology , 16(5): S165), multiphoton lithography (Kaehr & Shear (2008). Proc. Natl. Acad. Sci. USA, 105(26): 8850-8854), and optical lithography ()Suh et al. (2004)

Biomaterials , 25(3): 557-563. Each of these approaches, however introduced limitations in chemical composition, geometry, and throughput of the microparticles. Microcontact printing (Peng et al. (2013) Langmuir , 29(38): 11809-11814) and micromolding (Eng et al. (2013) Proc. Natl. Acad. Sci. USA, 110(12): 4551-4556) have been utilized for synthesis of microscale polymer microparticle synthesis and provide with inexpensive, convenient, and scalable templates. However, the resulting geometry often hinders high-throughput monitoring.

[0149] In various embodiments methods of fabricating the polymeric microgel arrays described herein with various sizes, shapes, morphologies and chemical compositions of the constituent microgel electrophoresis structures and/or separation zones are provided. In various embodiments the pGel arrays can be fabricated by methods involving photopattening (see, e.g, Figure 2, panel D, and Figure 6) and/or chemical polymerization (see, e.g, Figure 2, panel D, and Figure 12, panel B). This approaches reduce disadvantages of conventional techniques by enabling polymeric microgel lane synthesis based on chemically- induced and photo-induced polymerization in a high-throughput and controlled manner.

[0150] In certain illustrative, but non-limiting embodiments the pGELs can be fabricated on partially or fully silanized glass surfaces. Figure 15, panels A-B, shows the effect of a silanized glass surface on the mobility of two different protein species. Silanized or non silanized surfaces do not affect the migration distance of the proteins from single cells, in hydrogel pGELs.

[0151] Accordingly, in certain embodiments a silanized glass surface is utilized for fabrication of the pGel arrays. Methods of silanizing a glass surface are well known to those of skill and illustrated in Figure 12, panel A. As shown therein, in one illustrative

embodiment, salinization proceeds by first cleaning the glass surface ( e.g , with methanol), incubating the glass surface in a silane solution (e.g., for 30 minutes), rinsing the glass surface to remove unattached silane groups (e.g, rinsing with methanol and DI water), and drying the glass surface.

Photo-polymerization.

[0152] In certain embodiments, the pGel arrays described herein are prepared using photo-polymerization methods. In this approach, a patterned surface is provided that acts as a mold for the pGel array features. This "mold surface" has structural features that are the reverse of the desired features in the final pGel array. Methods of making such patterned surfaces are well known to those of skill in the art, particularly in the semiconductor industry and include, but are not limited to photolithography, dry or wet etching, and the like.

[0153] In certain embodiments the precursors of the polymeric gel are placed between the patterned "mold" surface and a second surface. The trapped precursors are irradiated with light having a wavelength sufficient (e.g, blue light) to initiate polymerization of the precursors so as to produce the desired polymerized polymeric gel. The method may further include removing the patterned mold surface such that the second surface (e.g, the support) carries a polymeric array of free standing pGels as described herein.

[0154] In certain embodiments, the structural features on the patterned surface include a plurality of columns. The columns on the patterned surface may include shapes and sizes that correspond to the desired shapes and sizes of pGels and separation zones comprising the pGel array. In certain embodiments the patterned surface can define troughs between the pGels comprising the array and/or can define perforations or score lines in the separation zone(s) comprising the array of pGels.

[0155] In certain embodiments the photopolymerization methods can utilize a mask

(photomask) to further define features and/or to define pGel array features in the absence of a patterned surface (mold). In certain embodiments the a photomask can be used to define shapes, provide gradient shades, discontinuous lines, and continuous lines between pGEL patterns, and the like and polymerization occurs by illuminating the pGel precursor(s) through the mask. In certain embodiments the polymerization is performed on a partially silanized solid support. Mask patterns or shapes can be defined and include, but are not limited to, triangular, quadrilateral, convex and concave polygonal, regular and irregular polygonal, circular shapes surrounded by transparent, semi-transparent, opaque borders to mask or unmask the underlying monomer layer from ultraviolet light exposure. The patterns may also be fabricated in elliptical shape, bullet shape with or without airfoils or wings to control the subsequent flow control depending on the analysis technique. Monomeric material polymerizes upon exposure of a collimated ultraviolet light, leading to polymeric pGEL synthesis.

[0156] In certain embodiments, photo-induced synthesis of pGELs may limit the chemical composition of the pGels since incorporation of other photo-inducible materials that have the same wavelength absorbance within hydrogel composition will not be possible. Additionally, photopolymerization may limit shape of the resulting structures since photolithography can result in spherical-shaped pGELs as the minimization of surface energy dictates this occurrence. In addition, the processing and analysis ( e.g . single cell integration, reagent introduction, imaging) of spatially-disordered individual pGELs can be a major difficulty. Photopolymerization can also limit throughput (the number of pGELs synthesized at a time), and can be another bottleneck for the aforementioned techniques because the distance from the ultraviolet light source greatly limits the resolution and therefore the number of well-defined pGELs that can be synthesized at a time.

Chemical polymerization.

[0157] In certain embodiments the arrays of microgel electrophoresis structures (pGel arrays) described herein are fabricated using chemical polymerization methods. One illustrative, but non-limiting protocol for fabricating pGel arrays using chemical

polymerization methods is shown in Figure 12, panel B. As illustrated, an SU-8 mold is prepared with patterned features that define the features of the pGel array. Hydrogel precursors are introduced onto the mold and the precursor solution is capped with a glass slide or other planar substrate. The system is incubated for a time sufficient to permit polymerization of the gel precursors ( e.g ., at room temperature for 20 minutes). In certain embodiments the glass slide is silanized (see, e.g., 12, panel A), and the mold is not silanized. The glass slide is then released from the SU-8 mold thereby providing a pGel array disposed on the surface of the slide. Detailed protocols for this method are provided in Example 1.

[0158] The pGEL shape(s) and separation zone features are determined by the features (e.g, posts) on the mold, and pGEL chemistry can be defined by the complex chemicals incorporated in the monomer solution. In some embodiments, pGELs are fabricated in a form of planar array with loose polymeric connections in between, and individual pGELs can be obtained after releasing the pGEL array from both solid supports using various techniques, including but not limited to, ultrasonification (sonication), cutting blade, mechanical stretching, or other techniques. In some embodiments, a non-silanized solid support may contain posts with different heights to create micron-sized wells with full height and connection regions with lower height to be tom apart during the releasing moment, therefore, individual polymeric microgel electrophoresis structures (separations- encoded microparticles) are released from the array.

[0159] This high-throughput technique enables superior control over the shape, geometry, and chemical composition of polymeric microgel electrophoresis structures (pGels). A massively -parallel planar array of polymeric pGELs can be synthesized using this technique, and the planar array can be used for a number of applications as a whole, and individual structures can be released for further analysis steps.

[0160] The innovations described herein enable synthesis (fabrication) of arrays of polymeric microgel electrophoresis structures (pGel arrays) based on both chemically- induced and photo-induced synthesis to mitigate chemical composition, geometry, and throughput problems.

Using the arrays or microgel electrophoresis structures.

[0161] Currently available protein separation techniques enable concurrent separation of tens or hundreds of proteins. However, these techniques require large amounts of precious biological samples and other reagents for quantitative analysis. Following separation of the sample constituents, currently available techniques pose difficulties for subsequent extraction and processing of the sample. Alternatively, microfluidic platforms containing enclosed fluidic microchannels can perform the same analysis with higher efficiency and higher speed by utilizing less biological sample and other reagents. However, in such methods, it is challenging to perform high-throughput detection of separated sample constituents because the number of detectable units is limited by the size of microfluidic platform. Increasing the throughput of detectable units can be achieved using polymeric pGEL arrays as described herein.

[0162] The arrays of microgel electrophoresis structures (pGel arrays) described herein are useful for accurate detection and quantification of a wide variety of analytes including, but not limited to biological molecules in both basic research and clinical settings, where high-throughput screening greatly benefits from high speed, cost-effective, and high- precision techniques. In various embodiments the small volumes of the microgel

electrophoresis structures (pGELs) significantly reduce the amount of reagents and waste, while benefitting the high speed of mass transport reactions on the microscale. Performing assays of biological molecules in picoliter to nanoliter volumes has led to significant improvement in assay sensitivity.

[0163] Moreover, in various embodiments, the microgel electrophoresis structures

(pGels) when separated from each other form, monodisperse populations of microgel electrophoresis structures (pGel particles). In various embodiments the population of microgel electrophoresis structures is viewed as monodisperse if the microgels comprising the population have a size distribution where >90% or greater than 95% have a size within 5% of the median size of the microgels (microparticles) in the population. Such

monodisperse populations exhibit a constant and predictable response to external stimuli including the processing steps required for biological assay and detection.

[0164] In certain embodiments the arrays of microgel electrophoresis structures (pGel arrays) can be used for high-throughput biomolecule analysis. Enabling in-parallel quantification of a plurality of proteins, nucleic acids, cytokines, and the like using a single pGEL has significant implications in maximizing obtainable information within minimal analysis and assay time, sample volume, and cost. Also, it is possible to use this invention to group/organize the analyzed samples and treat different groups in various conditions to eliminate batch-to-batch variability of most methods. This aspect is important for clinicians and researchers. Polymeric pGELs can also be used for discovery and clinical applications, particularly in the rapid quantification of biomolecules ( e.g . proteins, nucleic acids) for cancer diagnostics. [0165] In certain embodiments the methods include determining whether an analyte of interest is present in a sample, e.g ., determining the presence or absence of one or more analytes of interest in a sample. In some instances, the pGel arrays are configured to detect the presence of one or more analytes in a sample. In certain embodiments of the methods, the presence of one or more analytes in the sample may be determined qualitatively or quantitatively. Qualitative determination includes determinations in which a simple yes/no result with respect to the presence of an analyte in the sample is provided to a user.

Quantitative determination includes both semi -quantitative determinations in which a rough scale result, e.g. , low, medium high is provided to a user regarding the amount of analyte in the sample and fine scale results in which an exact measurement of the concentration of the analyte is provided to the user.

[0166] A typical polymeric pGEL structure is a porous medium that allows the target analyte molecules to pass through the structure for size-based, or other separations. To achieve that, the sample (e.g, fluid obtained from decomposable biological entities) is disposed in the micron-sized sample receiving wells which are configured to permit the analyte(s) to enter into the polymeric pGEL for an extended observation duration, wherein the sample (e.g, a fluid) passes through the polymeric medium until the components of the fluid separate from each other based on their size (or other) differences. In certain embodiments the different components of the fluid (analytes) are trapped/captured within the polymeric pGEL upon application of an applied stimulus. In some embodiments of the method, the method includes applying an electric field to the planar array of polymeric pGEL for separation of the components found in the sample (e.g, fluid). In some embodiments, the method further includes contacting the separated analytes with one or more secondary reagents. In some embodiments, the contacting includes one or more of diffusion, electrokinetic transport and hydrodynamic transport. In some embodiments, the one or more secondary reagents are selected from an affinity probe, a dye, an antibody, an enzyme, an enzyme substrate and a nucleic acid. For the readout, proteins and other analytes can be stained using silver stain, Coomassie dye stain, zinc stains, fluorescent dye stains, functional- group-specific stains before imaging. In some embodiments, separated fluids are visualized using a detector which can be a photomultiplier tube, a charge-coupled device, an intensified charge coupled device, a complementary metal-oxide-semiconductor sensor, visual colorimetric readout, a photodiode, and the like.

[0167] Illustrative, but non-limiting methods of using the arrays described herein are shown in Figure 2, panel E and Figure 8, panels A-E. In certain embodiments the methods involve: 1) Providing an array of microgel electrophoresis structures as described herein with a sample disposed in one or more of the sample receiving wells in the array; 2) Placing the array in an electric field where the electric field causes migration of one or more analytes from the sample receiving wells into and at least partially through the polymeric gel that forms the microgel electrophoresis structure(s); 3) contacting the microgel electrophoresis structures with a reagent comprising a detectable label where the reagent associates the analytes; and 4) detecting and/or localizing the detectable label in the microgel

electrophoresis structures.

[0168] In certain embodiments, the electric field may be configured to facilitate the separation of the analytes in a sample based on the physical properties of the analytes. For example, the electric field may be configured to facilitate the separation of the analytes in the sample based on the molecular mass, size, charge ( e.g .. charge to mass ratio), isoelectric point, etc. of the analytes. In certain instances, the electric field is configured to facilitate the separation of the analytes in the sample based on the molecular mass of the analytes. In other embodiments, the electric field is configured to facilitate separation of the analytes in the sample based on the isoelectric point (pi) of the analytes.

[0169] In certain embodiments the electric field is substantially uniform across the array of microgel electrophoresis structures. In certain embodiments placing the array in an electric field comprises placing said array in an electrophoresis apparatus. In certain embodiments the electric field is produced by a potential difference (voltage) ranging from about 10 V cm ¹ , or from about 20 V cm ¹ , or from about 30 V cm ¹ , or from about 40 V cm ¹ up to about 100 V cm ¹ , or up to about 90 V cm l, or up to about 80 V cm ¹ , or up to about 70 V cm ¹ , or up to about 60 V cm ¹ . The method of claim 40, wherein said electric filed is produced by a voltage difference of about 40 V cm ¹ . In certain embodiments the electric field is substantially uniform across the array of pGels (see, e.g., Figure 19).

[0170] Samples that may be assayed utilizing the pGel arrays described herein may vary, and include both simple and complex samples. Simple samples are samples that include the analyte of interest and may or may not include one or more molecular entities that are not of interest, where the number of these non-interest molecular entities may be low, e.g, 10 or less, 5 or less. etc. Simple samples may include initial biological or other samples that have been processed in some manner, e.g, to remove potentially interfering molecular entities from the sample. By“complex sample” is meant a sample that may or may not have the analytes of interest, but also includes many different proteins and other molecules that are not of interest. In some instances the complex sample assayed in the subject methods is one that includes 10 or more, such as 20 or more, including 100 or more, e.g., 103 or more, 104 or more (such as 15,000; 20.000 or 25,000 or more) distinct (i.e., different) molecular entities, that differ from each other in terms of molecular structure or physical properties (e.g, molecular weight size charge, isoelectric point, etc.).

[0171] In certain embodiments the sample comprises a sample selected from the group consisting of a cell, a tissue sample, a biological fluid, and an environmental sample.

In certain embodiments the sample comprises a cell (e.g, a mammalian cell). In certain embodiments the samples of interest are biological samples including, but not limited to, urine, blood, serum, plasma, saliva, semen prostatic fluid, nipple aspirate fluid, lachrymal fluid, perspiration, feces, cheek swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue (e.g, samples obtained from laser capture microdissection (LCM)), and the like. The sample can be a biological sample or can be extracted from a biological sample derived from humans, animals, plants, fungi, yeast, bacteria, tissue cultures, viral cultures, or combinations thereof using conventional methods for the preparation of samples. In certain embodiments the sample is a fluid sample such as a solution of analytes in a fluid. The fluid may be an aqueous fluid, such as, but not limited to water, aa buffer, and the like.

[0172] In certain embodiments the sample comprises a cell (e.g, a mammalian cell).

In certain embodiments the sample comprises a single cell disposed in each loaded sample receiving well. Methods of loading single cells into wells are well known to those of skill in the art and described in Example 1, below. In this regard, it is noted that Figure 7 suggests that settling of multiple cells in a single well may occur (see, Figure 7). However, intensity profiles change accordingly and multiple cell loading is therefore readily detected. Moreover it was observed that loading of multiple cells in wells was infrequent compared to single cell loading.

[0173] As described above, the samples that may be assayed include one or more analytes of interest. Such analytes include, but are not limited to nucleic acids (e.g, double or single- stranded DNA, double or single-stranded RNA, DNA-RNA hybrids, DNA aptamers, RNA aptamers, etc.), proteins and peptides, with or without modifications, e.g.. antibodies, diabodies, Fab fragments, scFv, DNA or RNA binding proteins, phosphorylated proteins (phosphoproteomics), peptide aptamers, epitopes, and the like, small molecules such as inhibitors, activators, ligands, etc., oligo or polysaccharides, mixtures thereof, and the like. [0174] In certain embodiments, after the analyte(s) have moved into the polymeric gel comprising the microgel electrophoresis structure(s) the analyte can be immobilized in the gel for detection. Methods of immobilizing analytes in gels are well known to those of skill in the art. Illustrative methods include, but are not limited to photoimmobilization ( e.g ., using UV light) and chemical cross linking.

[0175] The immobilized analyte (or non-immobilized analyte when no

immobilization is performed) can be detected by contacting the microgel electrophoresis structures with a reagent comprising a detectable label where the reagent associates with one or more of analytes in the pGel.

[0176] In various embodiments the detectable labels used in the methods described herein include, but are not limited to, a fluorescent label, a colorimetric label, a

chemiluminescent label, an enzymatic label comprising an enzyme substrate or an enzyme, a radiolabel, silver particles, gold particles, or a magnetic label.

[0177] Illustrative fluorescent molecules (fluorophores) include, but are not limited to, fluorescein, fluorescein isothiocyanate, succinimidyl esters of carboxyfluorescein, succinimidyl esters of fluorescein, 5-isomer of fluorescein dichlorotriazine, caged

carboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green 514, Lucifer Yellow, acridine Orange, rhodamine, tetramethylrhodamine, Texas Red, propidium iodide, JC-l (5,5',6,6'-tetrachloro-l,r,3,3'-tetraethylbenzimidazoylcarbo cyanine iodide),

tetrabromorhodamine 123, rhodamine 6G, TMRM (tetram ethyl rhodamine methyl ester), TMRE (tetramethyl rhodamine ethyl ester), tetramethylrosamine, rhodamine B and 4- dimethylaminotetramethylrosamine, green fluorescent protein, blue-shifted green fluorescent protein, cyan-shifted green fluorescent protein, red-shifted green fluorescent protein, yellow- shifted green fluorescent protein, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives, such as acridine, acridine isothiocyanate, 5-(2'- aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphth-alimide-3,5 disulfonate, N-(4-anilino-l-naphthyl)maleimide, anthranilamide, 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-c acid BODIPY, cascade blue, Brilliant Yellow, coumarin and derivatives: coumarin, 7-amino-4- methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumarin 151), cyanine dyes, cyanosine, 4',6-diaminidino-2-phenylindole (DAPI), 5', 5"- dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red), 7-diethylamino-3-(4'- isothiocyanatophenyl)-4-methylcoumarin, diethylenetriaamine pentaacetate, 4,4'- diisothiocyanatodihydro-stilbene-2-,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid, 5-(dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansylchloride), 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin and derivatives: eosin, eosin isothiocyanate, erythrosin and derivatives: erythrosin B, erythrosin, isothiocyanate, ethidium, fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)amino-fluorescein (DTAF), 2',7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC), fluorescamine, IR144, IR1446, Malachite Green isothiocyanate, 4-methylumbelli-feroneortho cresolphthalein, nitrotyrosine, pararosaniline, Phenol Red, B-phycoerythrin, o-phthal dialdehyde, pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1 -pyrene, butyrate quantum dots, Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), 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, tetramethyl hodamine isothiocyanate (TRITC), riboflavin, 5-(2'-aminoethyl) aminonaphthalene-l- sulfonic acid (EDANS), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid, CAL Fluor Orange 560, terbium chelate derivatives, Cy 3, Cy 5, Cy 5.5, Cy 7, IRD 700, IRD 800, La Jolla Blue, phthalo cyanine, and naphthalo cyanine, coumarins and related dyes, xanthene dyes such as rhodols, resorufins, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol, and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquinones, fluorescent europium and terbium complexes, combinations thereof, and the like. Suitable fluorescent proteins and chromogenic proteins include, but are not limited to, a green fluorescent protein (GFP), including, but not limited to, a GFP derived from Aequoria victoria or a derivative thereof, e.g ., a "humanized" derivative such as Enhanced GFP, a GFP from another species such as Renilla reniformis, Renilla mulleri , or Ptilosarcus guernyi , "humanized" recombinant GFP (hrGFP), any of a variety of fluorescent and colored proteins from anthozoan species, combinations thereof.

[0178] As described above, in certain embodiments, detecting the analyte(s) of interest can include contacting the analyte(s) of interest with a reagent comprising a detectable label where the reagent associates with one or more of analytes in the pGel. For example, contacting the analyte of interest with an analyte detection reagent may include applying a solution of the analyte detection reagent to the polymeric gel comprising the microgel electrophoresis structures. The analyte detection reagent may be contacted to any surface of the pGel(s), such as the top or one or more sides of the pGels.

[0179] In some cases, the analyte detection reagent may be moved through the uGel(s) such that the analyte detection reagent contacts analytes of interest immobilized within the pGel(s). For instance, the analyte detection reagent may be moved through the polymeric gel comprising the pGel by applying an electric field to the pGel, applying a pressure, applying a centrifugal force, passive diffusion, and the like.

[0180] In certain embodiments, detecting the analyte of interest includes contacting the analyte of interest with a primary label that specifically binds to the analyte of interest. In certain embodiments, the method includes enhancing the detectable signal from the labeled analyte of interest. For instance, enhancing the detectable signal from the labeled analyte of interest may include contacting the primary label with a secondary label configured to specifically bind to the primary label. In certain instances, the primary label is a primary antibody that specifically binds to the analyte of interest, and the secondary label is a secondary antibody that specifically binds to the primary antibody. As such, enhancing the detectable signal from the labeled analyte of interest may include contacting the primary antibody with a secondary antibody configured to specifically bind to the primary antibody. The use of two or more detectable labels as described above may facilitate the detection of the analyte of interest by improving the signal-to-noise ratio.

[0181] In certain embodiments the method comprises contacting said sample with a primary antibody that binds to an analyte that is to be detecting and contacting the primary antibody with a secondary antibody that binds to said primary antibody where said secondary antibody is attached to said detectable label.

[0182] As noted above, in certain embodiments the released free-floating hydrogel pGELs provide for increasing the readout-throughput of the assay while allowing grouping of the units for different processes ( e.g . probing for different antibodies). For this reason, in certain embodiments, releasing the pGELs is contemplated (see, e.g. , Figure 2, panels A-C).

[0183] In certain embodiments a primary label is applied to the intact planar array of pgels and the secondary label is applied to the pGels after they are separated from each other (e.g., in suspension). In certain embodiments both the primary label and the secondary label are applied to the pGels after they are separated from each other (e.g, in suspension). In certain embodiments the primary label and the secondary label are applied to the intact planar array of pGels. [0184] In certain embodiments, the analyte detection reagent may not specifically bind to an analyte of interest. In some cases, the analyte detection reagent may be configured to produce a detectable signal from the analyte of interest without specifically binding to the analyte of interest. For example, the analyte of interest may be an enzyme ( e.g ., a cellular enzyme) and the analyte detection reagent may be a substrate for the enzyme. In some cases, contacting the analyte detection reagent (e.g., enzyme substrate) to the analyte of interest (e.g, enzyme) may produce a detectable signal as the substrate is converted by the enzyme.

[0185] In certain embodiments the signal produced by the detectable label is enhanced/increased by dehydrating the pGel array or the separated pGels. Figure 3, panels A-B, shows the fabricated pGELs in planar array and free-floating forms. As drying might be used for increasing the signal, dried and hydrated versions of the pGELs are also shown in this figure. In hydrogel pGELs, the particles dry uniformly as shown in Figure 3, panels C-E. Figure 3, panel F, shows the effect of releasing process on the intactness of the pGELs. Figure 4, panels A-E, demonstrates the separated proteins from single cells in pGELs, and the quantitative analysis of the separated protein species. In this case, housekeeping proteins, GAPH and PTubulin, are probed in pGELs.

[0186] In certain embodiments the methods described herein comprise a western blot.

In certain embodiments the methods comprise a differential detergent fractionation.

[0187] In certain embodiments the analyte comprises a nucleic acid and the detecting comprises detecting contacting the nucleic acid with an oligonucleotide probe that hybridizes to said nucleic acid where said oligonucleotide probe is attached to the detectable label.

[0188] In some embodiments, the methods include the uniplex analysis of an analyte in a sample. By "uniplex analysis" is meant that a sample is analyzed to detect the presence of one analyte in the sample. For example, a sample may include a mixture of an analyte of interest and other molecular entities that are not of interest. In some cases, the methods include the uniplex analysis of the sample to determine the presence of the analyte of interest in the sample mixture.

[0189] Certain embodiments include the multiplex analysis of two or more analytes in a sample. By "multiplex analysis" is meant that the presence two or more distinct analytes, in which the two or more analytes are different from each other, is determined. For example, analytes may include detectable differences in their molecular weight, size, charge (e.g, mass to charge ratio), isoelectric point, and the like. In some instances, the number of analytes is greater than 2, such as 4 or more, 6 or more, 8 or more, etc., up to 20 or more, e.g, 50 or more, including 100 or more, distinct analytes. In certain embodiments, the methods include the multiplex analysis of 2 to 100 distinct analytes, such as 4 to 50 distinct analytes, including 4 to 20 distinct analytes.

[0190] In certain embodiments, the method is configured to separate and/or detect constituents of interest in a sample, where the sample size is small. For example, the method may be configured to separate and/or detect constituents of interest in a sample, where the sample size is 1 mL or less, such as 750 pL or less, including 500 pL or less, or 250 pL or less, of 100 pL or less, or 75 pL or less, or 50 pL or less, or 40 pL or less, or 30 pL or less, or 20 pL or less, or 10 pL or less, or 5 pL or less, or 1 pL or less. In some instances, the method is configured to separate and/or detect constituents of interest in a sample, where the sample size is 20 pL or less.

[0191] In certain embodiments, the method includes concentrating, diluting, or buffer exchanging the sample prior to introducing the sample into the sample receiving wells.

Concentrating the sample may include contacting the sample with a concentration medium prior to contacting the sample with the separation medium. The concentration medium may include a small pore size polymeric gel, a membrane ( e.g ., a size exclusion membrane), combinations thereof, and the like. Concentrating the sample prior to contacting the sample with the separation medium may facilitate an increase in the resolution between the bands of analytes in the separated sample because each separated band of analyte may disperse less as the sample traverses through the separation medium. Diluting the sample may include contacting the sample with additional buffer prior to contacting the sample with the separation medium. Buffer exchanging the sample may include contacting the sample with a buffer exchange medium prior to contacting the sample with the separation medium. The buffer exchange medium may include a buffer different from the sample buffer. The buffer exchange medium may include, but is not limited to, a molecular sieve, a porous resin, and the like.

[0192] In certain embodiments, the method is an automated method. As such, the method may include a minimum of user interaction with the microfluidic devices and systems after introducing the sample into the microfluidic device. For example, the step of directing the sample through the separation medium to produce a separated sample may be performed by the microfluidic device and system, such that the user need not manually perform these steps. In some cases, the automated method may facilitate a reduction in the total assay time. For example, embodiments of the method, including the separation and detection of analytes in a sample, may be performed in 30 min or less, such as 20 min or less, including 15 min or less, or 10 min or less, or 5 min or less, or 2 min or less, or 1 min or less.

[0193] The foregoing methods are illustrative and non-limiting. Using the teaching provided herein numerous variations of the assays will be available to one of skill in the art.

Systems.

[0194] In certain embodiments systems for detecting analytes are provided that incorporate and/or utilize the arrays of microgel electrophoresis structures (pGel arrays) described herein. In certain embodiments the system may also include a detector. In some cases, the detector is a detector configured to detect a detectable label. As described above, the detectable label may be a fluorescent label. For example, the fluorescent label can be contacted with electromagnetic radiation ( e.g ., visible, UV, x-ray, etc.), which excites the fluorescent label and causes the fluorescent label to emit detectable electromagnetic radiation (e.g., visible light, etc.). The emitted electromagnetic radiation may be detected with an appropriate detector to determine the presence of the analyte in a sample separated by the separation medium.

[0195] In some instances, the detector may be configured to detect emissions from a fluorescent label, as described above. In certain cases, the detector includes a photomultiplier tube (PMT), a charge-coupled device (CCD), an intensified charge-coupled device (ICCD), a complementary metal-oxide-semiconductor (CMOS) sensor, a visual colorimetric readout, a photodiode, and the like.

[0196] In certain embodiments the detector is a detector compatible with

solution/suspension detection of analytes. Such systems include, for example flow cytometry systems (FACs).

[0197] In certain embodiments, the system includes an environmental chamber. The environmental chamber may be configured to contain an array of microgel electrophoresis structures (pGel array) as disclosed herein. For instance, the environmental chamber may be configured to contain the an array of microgel electrophoresis structures (pGel array) disposed on the surface of a support. The an array of microgel electrophoresis structures (pGel array) may be positioned inside the environmental chamber, such that pGel array is surrounded by the environment provided inside the environmental chamber. In some instances, the environmental chamber contains an environment (e.g, an assay environment) that has a higher humidity than ambient conditions. An assay environment with a higher humidity may facilitate a reduction in evaporation of liquids ( e.g ., buffers, etc.) from the an array of microgel electrophoresis structures (pGel array). Embodiments of the environmental chamber may be made of any suitable material that is compatible with the array of microgel electrophoresis structures (pGel array) and compatible with the samples, buffers, reagents, etc. used in the pGel array. In some cases, the environmental chamber is made of a material that is inert (e.g., does not degrade or react) with respect to the samples, buffers, reagents, etc. used in the subject devices and methods. For instance, the environmental chamber may be made of materials, such as, but not limited to, glass, quartz, polymers, elastomers, paper, combinations thereof, and the like. In certain embodiments, the environmental chamber includes one or more portions that are substantially transparent. In some embodiments, an environmental chamber with one or more transparent areas facilitates detection of analytes in the microgel electrophoresis structures (pGels) that include, produce, or are labeled with a detectable label, such as a fluorescent label.

[0198] In various embodiments the systems may include various other components as desired. For example, the systems may include fluid handling components, such as microfluidic fluid handling components. The fluid handling components may be configured to direct one or more fluids to and/or from the pGel array. In some instances, the fluid handling components are configured to direct fluids, such as, but not limited to, sample solutions, buffers (e.g, wash buffers, electrophoresis buffers, etc.), and the like. In certain embodiments, the microfluidic fluid handling components are configured to deliver a fluid to the pGels, e.g, to the sample receiving wells. In certain embodiments, the fluid handling components may include microfluidic pumps. In some cases, the microfluidic pumps are configured for pressure-driven microfluidic handling and routing of fluids to and/or from the pGel arrays. In certain instances, the microfluidic fluid handling components are configured to deliver small volumes of fluid, such as 1 mL or less, such as 500 pL or less, including 100 pL or less, for example 50 pL or less, or 25 pL or less, or 10 pL or less, or 5 pL or less, or 1 pL or less.

[0199] In certain embodiments, the systems include one or more electric field generators. An electric field generator may be configured to apply an electric field to various regions of the pGel array(s). In certain embodiments the system may be configured to apply an electric field such that the sample is electrokinetically transported to or through the pGel arrays. In some cases, the applied electric field may be aligned with the directional axis of the separation flow path of pGel(s). As such, the applied electric field may be configured to electrokinetically transport the analytes and moieties in a sample through the separation medium. In some cases, the applied electric field is configured to electrokinetically transport selected analytes that have been separated by the pGel. Selected analytes that have been separated by the separation medium may be transported to a second medium ( e.g ., a blotting medium) or a collection reservoir for subsequent analysis by applying an appropriate electric field to the separation medium along a desired directional axis. In some cases, the directional axis is orthogonal to the directional axis of the pGel used during separation of the analytes in the sample. In some instances, the electric field generators are configured to apply an electric field with a strength ranging from 10 V/cm to 1000 V/cm, such as from 100 V/cm to 800 V/cm, including from 200 V/cm to 600 V/cm.

[0200] In certain embodiments, the electric field generators include voltage shaping components. In some cases, the voltage shaping components are configured to control the strength of the applied electric field, such that the applied electric field strength is

substantially uniform across the separation medium. The voltage shaping components may facilitate an increase in the resolution of the analytes in the sample. For instance, the voltage shaping components may facilitate a reduction in non-uniform movement of the sample through the pGel(s). In addition, the voltage shaping components may facilitate a

minimization in the dispersion of the bands of analytes as the analytes traverses the pGel(s).

[0201] In certain embodiments, the systems provide microfluidic devices that may be configured to consume a minimum amount of sample while still producing detectable results. For example, the system may be configured to use a sample volume of 100 pL or less, such as 75 pL or less, including 50 pL or less, or 25 pL or less, or 10 pL or less, for example, 5 pL or less, 2 pL or less, or 1 pL or less while still producing detectable results. In certain embodiments, the system is configured to have a detection sensitivity of 1 nM or less, such as 500 pM or less, including 100 pM or less, for instance, 1 pM or less, or 500 fM or less, or 250 fM or less, such as 100 fM or less, including 50 fM or less, or 25 AM or less, or 10 fM or less. In some instances, the system is configured to be able to detect analytes at a concentration of 1 pg/mL or less, such as 500 ng/mL or less, including 100 ng/mL or less, for example, 10 mg/mL or less, or 5 ng/mL or less, such as 1 ng/mL or less, or 0.1 ng/mL or less, or 0.01 ng/mL or less, including 1 pg/mL or less. In certain embodiments, the system has a dynamic range from 10 ¹⁸ M to 10 M, such as from 10 ¹⁵ M to 10 ³ M, including from 10 ¹² M to 10 ⁶ M.

[0202] In certain embodiments, the microfluidic devices are operated at a temperature ranging from l°C to l00°C, such as from 5°C to 75°C, including from l0°C to 50°C, or from 20°C to 40°C. In some instances, the microfluidic devices are operated at a temperature ranging from 35°C to 40°C.

Illustrative applications.

[0203] In certain embodiments, the devices, systems and methods described herein find use in a variety of different applications where one or more analytes are to be detected in a sample. In various embodiments these analytes can comprise nucleic acids, proteins, peptides, or other biomolecules. For example, the polymeric microgel electrophoresis structures (pGels) described herein can be used to detect pathogens, immune reactions or presence of biochemical agents, environmental and water pathogen monitoring and detection, food safety, and bio-agricultural analysis in a sample. In certain embodiments the microgel electrophoresis structures (pGels) described herein are capable of distinguishing distinct analytes in multiplexed assays and experiments that form a basis for a variety of analytical platforms. Biomolecule capturing substances within the structure of polymeric microgel electrophoresis structures (pGels) described herein can facilitate the production of bioanalytical devices for these multiplexed assays and experiments with a high readout throughput.

[0204] In certain embodiments, the devices, systems and methods described herein find use in rapid detection of molecules of interest in applications such as fundamental biology, medical diagnostics, environmental monitoring, biological defense and

pharmaceutical research. Detection and quantitation of analytes or molecules of interest may include, but not limited to, viruses, peptides, polynucleotides, proteins such as toxins, cytokines, and other small molecules, one or more polyclonal or monoclonal antibodies, a Fab, F(ab') ₂ , scFv, or small chain fragment, peptides or a peptide nucleic acids, aptamers, lectin, one or more small ligands, antigens, enzymes, oligonucleotides, deoxyribonucleic acids, ribonucleic acids, biotin, and cellular receptor binding proteins.

[0205] In certain embodiments, the devices, systems and methods described herein find use in analysis of intact cells and secreted proteins by separations. Processing intact cells ensures the preservation of physiological cell response without external perturbances which occur during the processing, transfer, and preparation steps. In addition, imaging of intact cells prior to the analysis (without separation) can be performed for linking the phenotype to the protein analysis. This step can be important for histology of cancer cells.

[0206] In certain embodiments, the devices, systems and methods described herein find use in differential detergent fractionation (DDF) of cells. DDF yields biochemically and electrophoretically distinct fractions comprising, for example, cytosolic proteins and extractable cytoskeletal elements, membrane and organelle proteins, nuclear membrane proteins and extractable nuclear proteins, and detergent-resistant cytoskeletal filaments and nuclear matrix proteins. The obtained fractions can then be separated, enriched, purified further. For example, after DDF treatment a part of cell, e.g. nuclei, can be preserved in the microwell while separations from cytoplasmic proteins can be achieved. This step can be followed by RNA sequencing of the nuclei to obtain complementary genomic and proteomic comparison of the same cell.

[0207] In certain embodiments, the devices, systems and methods described herein find use in nucleic acid separations, where the nucleic acids include, inter alia, DNA, RNA, DNA derivatives, and RNA derivatives. In various embodiments this usage can provide quantitative nucleic acid analysis from complex samples using, e.g. , one or more target- specific probes. End point analysis of multiple nucleic acid targets can be achieved in a single separation platform that minimizes the possible experimental artefacts of a

regular/conventional nucleic acid analysis.

[0208] In certain embodiments, the devices, systems and methods described herein find use in indirectly capturing single-cell or tissue proteins, that are bound to a specific target protein(s) (or other moieties). This approach is called co-immunoprecipitation, and allows for better understanding of protein-protein interactions (or other interactions) in a complex sample. In addition, in certain embodiments, systems and methods described herein find use in chromatin-immunoprecipitation to measure the interactions between proteins and DNA in biological samples and single cells. To achieve that, microbeads coated with a target protein can be used as loading standard to serve as a measurement quality control given the fact that single-cell resolution measurements are prone to run-to-run variability.

[0209] In certain embodiments, the devices, systems and methods described herein find use in the detection of antibodies (e.g, in the domain of proteins). The detection of antibodies in a sample can be an indication of an underlying disease or condition, where a subject produces such antibodies in response to the disease or condition. As such, polymeric microgel electrophoresis structures (pGels) described herein can find use in the detection and diagnosis of a disease condition in a subject (e.g, human or animal) by detecting antibodies associated with the disease condition.

[0210] In certain embodiments, the devices, systems and methods described herein find use in high-throughput electrophoretic protein separations. For example, the subject devices, systems and methods find use in applications where determination of the presence or absence, and/or quantification of one or more analytes ( e.g ., proteins) in a sample is desired. For example, the subject devices, systems and methods find use in the separation and detection of proteins, peptides, nucleic acids, and the like. In some cases, the subject devices, systems and methods find use in the separation and detection of proteins.

[0211] In certain embodiments, the devices, systems and methods described herein find use in performing Western blots, e.g., on subcellular fractions of a cell. Subcellular fractions include, but are not limited to, DNA, and organelles found in a cell. The

measurement and analysis of DNA, subcellular organelles that are intact, protein localization, protein expression, and the fractionation of different subcellular compartments in a single cell can be achieved using multifunctional buffers and multidirectional electrophoretic

separations. This system combines sample preparation, protein/subcellular organelle separation and/or analysis.

[0212] The subject devices, systems and methods described herein find use in a variety of different applications where determination of the presence or absence, and/or quantification of one or more analytes in a sample is desired. In certain embodiments, the methods are directed to the detection of nucleic acids, proteins, or other biomolecules in a sample. The methods may include, but are not limited to, the detection of a set of

biomarkers, e.g, two or more distinct protein biomarkers, in a sample. For example, the methods may be used in the rapid, clinical detection of two or more disease biomarkers in a biological sample, e.g, as may be employed in the diagnosis of a disease condition in a Subject, in the ongoing management or treatment of a disease condition in a subject, etc. In addition, the devices, systems and methods described herein may find use in protocols for the detection of an analyte in a sample, such as, but not limited to, Western blotting, Southern blotting, Northern blotting, Eastern, Far-Western blotting, Southwestern blotting, and the like.

[0213] In certain embodiments, the devices, systems and methods described herein find use in detecting biomarkers. In some cases, the devices, systems and methods described herein may be used to detect the presence or absence of particular biomarkers, as well as an increase or decrease in the concentration of particular biomarkers in blood, plasma, serum, or other bodily fluids or excretions, such as, but not limited to, urine, blood, serum, plasma, saliva, semen, prostatic fluid, nipple aspirate fluid, lachrymal fluid, perspiration, feces, cheek Swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue ( e.g ., samples obtained from laser capture microdissection (LCM)), and the like. The presence or absence of a biomarker or significant changes in the concentration of a biomarker can be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual. For example, the presence of a particular biomarker or panel of biomarkers may influence the choices of drug treatment or

administration regimes given to an individual. In evaluating potential drug therapies, a biomarker may be used as a surrogate for a natural endpoint Such as Survival or irreversible morbidity. If a treatment alters the biomarker, which has a direct connection to improved health, the biomarker can serve as a surrogate endpoint for evaluating the clinical benefit of a particular treatment or administration regime. Thus, personalized diagnosis and treatment based on the particular biomarkers or panel of biomarkers detected in an individual are facilitated by the devices, systems and methods described herein. Furthermore, the early detection of biomarkers associated with diseases is facilitated by the high sensitivity of the subject devices and systems, as described above. Due to the capability of detecting multiple biomarkers on a single chip, combined with sensitivity, scalability, and ease of use, the presently disclosed microfluidic devices, systems and methods finds use in portable and point-of-care or near-patient molecular diagnostics.

[0214] In certain embodiments, the devices, systems and methods described herein find use in detecting biomarkers for a disease or disease state. In some cases, the disease is a cellular proliferative disease such as, but not limited to, a cancer, a tumor, a papilloma, a sarcoma, or a carcinoma, and the like. In certain instances, the devices, systems and methods described herein find use in detecting biomarkers for the characterization of cell signaling pathways and intracellular communication for drug discovery and vaccine development. For example, the devices, systems and methods described herein find use in detecting the presence of a disease, such as a cellular proliferative disease, such as a cancer, tumor, papilloma, sarcoma, carcinoma, or the like. In certain instances, particular biomarkers of interest for detecting cancer or indicators of a cellular proliferative disease include, but are not limited to the following: prostate specific antigen (PSA), which is a prostate cancer biomarker; C-reactive protein, which is an indicator of inflammation; transcription factors such as p53, which facilitates cell cycle and apoptosis control; polyamine concentration, which is an indicator of actinic keratosis and squamous cell carcinoma; proliferating cell nuclear antigen (PCNA), which is a cell cycle related protein expressed in the nucleus of cells that are in the proliferative growth phase; growth factors, such as IGF-I; growth factor binding proteins, such as IGFBP-3 : micro-RNAs, which are single-stranded RNA molecules of about 21-23 nucleotides in length that regulate gene expression; carbohydrate antigen CA19.9, which is a pancreatic and colon cancer biomarker, cyclin-dependent kinases;

epithelial growth factor (EGF); vascular endothelial growth factor (VEGF); protein tyrosine kinases; over-expression of estrogen receptor (ER) and progesterone receptor (PR); and the like. For example, the devices, systems and methods described herein may be used to detect and/or quantify the amount of endogenous prostate specific antigen (PSA) in diseased, healthy and benign samples.

[0215] In certain embodiments, the devices, systems and methods described herein find use in detecting biomarkers for an infectious disease or disease state. In some cases, the biomarkers can be molecular biomarkers, such as but not limited to proteins, nucleic acids, carbohydrates, small molecules, and the like. For example, in certain embodiments, the devices, systems and methods described herein may be used to monitor HIV viral load and patient CD4 count for HIV/AIDS diagnosis and/or therapy monitoring by functionalizing the sensor surface with antibodies to HIV capsid protein p24, glycoproteins 120 and 41, CD4+ cells, and the like. Particular diseases or disease states that may be detected by the devices, systems and methods described herein include, but are not limited to, bacterial infections, viral infections, increased or decreased gene expression, chromosomal abnormalities ( e.g . deletions or insertions), and the like. For example, in certain embodiments the devices, systems and methods described herein can be used to detect gastrointestinal infections, such as but not limited to, aseptic meningitis, botulism, cholera, E. colt infection, hand-foot-mouth disease, helicobacter infection, hemorrhagic conjunctivitis, herpangina, myocaditis, paratyphoid fever, polio, shigellosis, typhoid fever, vibrio septicemia, viral diarrhea, etc. In addition, the devices, systems and methods described herein can be used to detect respiratory infections, such as but not limited to, adenovirus infection, atypical pneumonia, avian influenza, Swine influenza, bubonic plague, diphtheria, influenza, measles, meningococcal meningitis, mumps, parainfluenza, pertussis (/.e., whooping cough), pneumonia, pneumonic plague, respiratory syncytial virus infection, rubella, scarlet fever, septicemic plague, severe acute respiratory syndrome (SARS), tuberculosis, etc. In addition, the devices, systems and methods described herein can be used to detect neurological diseases, such as but not limited to, Creutzfeldt-Jakob disease, bovine spongiform encephalaopathy ( i.e ., mad cow disease), Parkinson's disease, Alzheimer's disease, rabies, etc. In addition, the devices, systems and methods described herein can be used to detect urogenital diseases, such as but not limited to, AIDS, chancroid, Chlamydia, condyloma accuminata, genital herpes, gonorrhea,

lymphogranuloma Venereum, non-gonococcal urethritis, syphilis, etc. In addition, the devices, systems and methods described herein can be used to detect viral hepatitis diseases, such as but not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D. hepatitis E. etc. In addition, the devices, systems and methods described herein can be used to detect hemorrhagic fever diseases, such as but not limited to, Ebola hemorrhagic fever, hemorrhagic fever with renal syndrome (HFRS), Lassa hemorrhagic fever, Marburg hemorrhagic fever, etc. In addition, the devices, systems and methods described herein can be used to detect Zoonosis diseases, such as but not limited to, anthrax, avian influenza, brucellosis,

Creutzfeldt-Jakob disease, bovine spongiform encephalopathy ( i.e ., mad cow disease), enterovirulent E. colt infection, Japanese encephalitis, leptospirosis, Q fever, rabies, sever acute respiratory syndrome (SARS), etc. In addition, the devices, systems and methods described herein can be used to detect arbovirus infections, such as, but not limited to, Dengue hemorrhagic fever, Japanese encephalitis, tick- borne encephalitis, West Nile fever, Yellow fever, etc. In addition, the devices, systems and methods described herein can be used to detect antibiotics-resistance infections, such as but not limited to, Acinetobacter baumannii, Candida albicans, Enterococci sp., Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus , etc. In addition, the devices, systems and methods described herein can be used to detect vector-borne infections, such as but not limited to, cat Scratch disease, endemic typhus, epidemic typhus, human Ehrlichosis, Japanese spotted fever, louse-borne relapsing fever, Lyme disease, malaria, trench fever, Tsut Sugamushi disease, etc. Similarly, the Subject devices, systems and methods can be used to detect cardiovascular diseases, central nervous diseases, kidney failures, diabetes, autoimmune diseases, and many other diseases.

[0216] In certain embodiments the devices, systems and methods described herein find use in diagnostic assays, such as, but not limited to, the following: detecting and/or quantifying biomarkers, as described above; screening assays, where samples are tested at regular intervals for asymptomatic subjects; prognostic assays, where the presence and or quantity of a biomarker is used to predict a likely disease course; stratification assays, where a subjects response to different drug treatments can be predicted; efficacy assays, where the efficacy of a drug treatment is monitored; and the like.

[0217] The devices, systems and methods described herein also find use in validation assays. For example, validation assays may be used to validate or confirm that a potential disease biomarker is a reliable indicator of the presence or absence of a disease across a variety of individuals. The short assay times for the Subject devices, systems and methods may facilitate an increase in the throughput for screening a plurality of samples in a minimum amount of time.

[0218] In some embodiments, the devices, systems and methods described herein facilitate sample extraction or downstream processing of the separated sample, for example by subsequent immunological blotting, mass spectrometry, flow cytometry, and the like.

[0219] In some instances, the devices, systems and methods described herein can be used without requiring a laboratory setting for implementation. In comparison to the equivalent analytic research laboratory equipment, the devices systems and methods described herein provide comparable analytic sensitivity in a portable, hand-held system. In some cases, the weight and operating cost are less than the typical stationary laboratory equipment. In certain embodiments, the systems and devices described herein may be integrated into a single apparatus, such that all the steps of the assay, including separation, transfer, labeling and detecting of an analyte of interest, may be performed by a single apparatus. For example, in some instances, there are no separate apparatuses for separation, transfer, labeling and detecting of an analyte of interest. In addition, the systems and devices described herein can be utilized in a home setting for over-the-counter home testing by a person without medical training to detect one or more analytes in Samples. In certain embodiments the systems and devices described herein may also be utilized in a clinical setting, e.g ., at the bedside, for rapid diagnosis or in a setting where stationary research laboratory equipment is not provided due to cost or other reasons.

Illustrative Commercial Applications

[0220] The polymeric microgel electrophoresis structures (pGELs) described herein are useful for a wide range of applications, including protein barcode assays, bioassays, particle- based bioassays, single-cell proteomics, proteomics, micro-electromechanical systems, biomaterials, drug delivery, self-assembly, photonic materials, rheological fluids, and other applications. In certain embodiments the pGELs are defined as structures having features in the size range of 200 pm to 1000 pm. By changing the photomask, monomer composition, or UV exposure- time, precise control is exercised over pGEL geometry and chemistry for a range of applications, which is one unique feature of this system. The post geometry is defined as the volume of the isolated hydrogel pGEL. Advantages and disadvantages in comparison to other devices and methods

[0221] In various illustrative, but non-limiting embodiments a technique is provided where a planar array of polymeric microgel electrophoresis structures (pGel array) is synthesized on an open microfluidic platform via microcontact printing (using a silicon wafer with shaped microposts with different shapes for chemical synthesis), and photolithography (using a photomask for different-shaped pGELs) and collimated ultraviolet light source for photo-induced synthesis).

[0222] The polymeric microgel electrophoresis structure arrays (pGel arrays) (and separated separations-encoded microparticles) described herein have apparent and significant advantages over other techniques and devices for carrying out biological assays. As explained herein, the polymeric microgel electrophoresis structures (pGels) can be physically immobilized in a form of a planar array during the biological assay, and they can be released to provide high-throughput screening during monitoring/analysis process. The released polymeric microgel electrophoresis structures (separations-encoded microparticles)have well- defined boundaries determined by the fabrication process. While the planar array of individual microgel electrophoresis structures provides benefits for faster reaction kinetics, and efficient separation and washing steps in a well-ordered way, disassembly of the planar array results in the formation of individual separations-encoded microparticles (pGels) for easy monitoring/scanning in a flow-through device. In addition, the polymeric structures enable settling different cell types on the same array without mixing them, and probing the selected parts of the array for different biomarkers. This is advantageous because of maintaining the same sample preparation and treatment conditions, which eliminates batch- to-batch variability in bioassays.

Kits

[0223] In certain embodiments, aspects of the present disclosure additionally include kits that comprise one or more devices as described in detail herein ( e.g ., planar arrays of microgel electrophoresis structures as described herein). In certain embodiments, the kit may include packaging configured to contain the device. In certain embodiments the packaging may comprise a sealed packaging, such as a sterile sealed packaging. By“sterile ¹ is meant that there are substantially no microbes (such as fungi, bacteria, viruses, spore forms, etc.).

In some instances, the packaging may be configured to be sealed, e.g., a water vapor-resistant packaging, optionally under an air tight and/or vacuum seal. In certain embodiments the kits may further include a buffer. For instance, the kit may include a buffer, such as an electrophoretic buffer, a sample buffer, and the like. In certain cases, the buffer is an electrophoresis buffer, such as, but not limited to, a Tris buffer, a Tris-glycine, and the like.

In some instances, the buffer includes a detergent (Such as sodium dodecyl sulfate, SDS).

[0224] In certain embodiments the kits may further include additional reagents, such as but not limited to, release reagents, denaturing reagents, refolding reagents, detergents, detectable labels ( e.g ., fluorescent labels, colorimetric labels, chemiluminescent labels, multicolor reagents, enzyme-linked reagents, detection reagents (e.g., avidin-Streptavidin associated detection reagents), e.g, in the form of at least one if not more analyte detection reagents (such as first and second analyte detection reagents), calibration standards, radiolabels, gold particles, magnetic labels, etc.), and the like.

[0225] In certain embodiments, the kit may include an analyte detection reagent, such as a detectable label, as described herein. The detectable label may be associated with a member of a specific binding pair. Suitable specific binding pairs include, but are not limited to: a member of a receptor/ligand pair, a ligand-binding portion of a receptor; a member of an antibody/antigen pair, an antigen-binding fragment of an antibody; a hapten, a member of a lectin/carbohydrate pair; a member of an enzyme/substrate pair; biotin/avidin; biotin/streptavidin; digoxin/antidigoxin; a member of a DNA or RNA aptamer binding pair, a member of a peptide aptamer binding pair; and the like. In certain embodiments, the member of the specific binding pair includes an antibody. The antibody may specifically bind to an analyte of interest in the separated sample bound to the separation medium. For example, the detectable label may include a labeled antibody (e.g., a fluorescently labeled antibody) that specifically binds to the analyte of interest.

[0226] In certain embodiments in addition to one or more of the above components, the subject kits may further include instructions for practicing the methods described herein (e.g, protocols for the use of pGel(s)). These instructions may be present in the kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g, a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Another means would be a computer readable medium, e.g, on which the information has been recorded or stored. Yet another means that may be present is a website address which may be used via the Internet to access the information at a removed site. Any convenient means may be present in the kits. EXAMPLES

[0227] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Separation-Encoded Microparticles For Single-Cell Western Blotting

[0228] Widely used in research and clinical medicine, immunoblots ( e.g ., western blots) provide target specificity by combining an electrophoretic separation of target species with subsequent immunoassaying steps. While miniaturization has benefited immunoblots in both separation efficiency and applicability to single cells, the widely used‘microchannef and capillary devices make access to resolved targets cumbersome. To design a single-cell immunoblot with the most favorable mass transport conditions at each assay stage, we introduce a microfluidic format that toggles from a planar array of single-cell electrophoresis separations to a suspension of microparticles, with each rectangular microparticle housing one separation. For single-cell electrophoresis, a thin-layer hydrogel on a glass slide is stippled with microwells for cell isolation, each abutting a region for polyacrylamide gel electrophoresis (PAGE) and photocapture of separated proteins to the gel matrix. Array elements are defined by perforations around each rectangular single-cell PAGE region which, upon mechanical release, toggles into a separations-encoded microparticle. The

microparticles measure estrogen receptor and 5 protein targets in hundreds of single breast cancer cells and kidney cells. For immunoassays, holding separations-encoded

microparticles in suspension enhances diffusion into the gel making single-cell

immunoblotting steps (e.g., immunoprobe introduction and wash out) more efficient by 4x. Dehydrating the microparticles from suspension results in isotropic shrinkage of each separations-encoded particle (10% in length), yielding a l.6x increase in immunoblot fluorescence signal with no penalty on separation resolution. Designed for optimal mass transport and scaling at each assay stage, separations-encoded microparticles provide an adaptable new form factor for precision single-cell analysis.

Introduction to this example.

[0229] The analysis of proteins in single cells plays a central role in identifying the link between cellular heterogeneity and disease states (Schubert (2011) Nature 480: 133; Chattopadhyay et al. (2014) Nat Immunol 15: 128). Like single-cell genomics and transcriptomics, single-cell protein analysis reports information that is concealed in bulk experiments (Smits et al. (2006) Nat. Rev. Microbiol. 4: 259; Dubnau & Losick (2006) Mol. Microbiol. 61 : 564-572). For detection of protein targets known a priori , immunoassays are widely used in both analysis of large samples and to achieve single-cell resolution.

Historically, heterogeneous immunoassays have been performed using a wide range of immobilizing substrates, including paper strips (Luo et al. (2014) Anal. Chem. 86: 12390- 12397), membranes (U.S. Patent No. 7,182,894), and nozzle arrays (Mao et al. (2012) Anal. Chem. 85: 816-819). Conventional immunoassays ( e.g ., ELISAs (Sevecka & MacBeath (2006) Nature Meth. 3: 825-831), immunohistochemistry (Ng et al. (2015) Nature Commun. 6: 7513)) form the basis for the immobilizing substrates to study single-cell behavior, although specific protein types (such as protein isoforms that are crucial for cancer studies) cannot be detected in the absence of high specificity probes (Ward et al. (2013) Oncogene,

32: 2463). Other conventional immunoassays such as flow cytometry (Perfetto et al. (2004) Nat. Rev. Immunol. 4, 648-655; Perfetto et al. (2004) Nat. Rev. Immunol. 4: 648; Lombards Banek et al. (2016) Angew. Chem. Int. Ed. 55: 2454-2458), and mass cytometry/CyTOF (Bendall et al. (2011) Science, 332: 687-696; Yang et al. (2016) Anal. Chem. 88: 6672-6679) can spatially resolve most protein isoforms, but intracellular protein targets are still difficult to measure in multiplexed runs and therefore analytical sensitivity remains insufficient for detection of key signaling proteins (Spitzer & Nolan (2016) Cell , 165: 780-791). Macroscale immunoassays have been downscaled in order to improve sample detection and analysis capabilities by controlling mass and heat transport. At the microscale, heterogeneous immunoassays have used photopolymerized gel constructs (Duncombe & Herr (2013) Lab Chip, 13: 2115-2123), microfluidic capillaries (Chen et a (2016) Nat. Chem. Biol. 12: 76), enclosed microfluidic channels (Dawod et al. (2017) Analyst, 142: 1847-1866; Prakadan et al. (2017) Nature Rev. Genet. 18: 345; Dugan et al. (2017) Anal. Bioanal. Chem. 409: 169- 178), microwell arrays (Kang et al. (2016) Nature Protoc. 11 : 1508; Wood et al. (2010) Proc. Natl. Acad. Sci. USA, 107: 10008-10013), and immuno-barcoding (Fan et al. (2008) Nature Biotechnol. 26: 1373). These approaches greatly simplified the target labeling process, facilitating a stepwise workflow of protein electrophoresis and antibody probing to discern off-target signals. However, a tradeoff still exists between maintaining satisfactory assay sensitivity and multiplexed analysis of > 1000 samples within the same batch that is required for measuring cell-to-cell variability in large populations (Hughes et al. (2014) Nature Met.

11 : 749). Microparticles including barcoded hydrogel microparticles (Appleyard et al.

(2011) Nature Protoc. 6: 1761), suspensions of particles (Meiring et al. (2004) Chem. Mater. 16: 5574-5580; Lee et al. (2008) Biomed. Microdevices, 10: 813-822; Moorthy et al. (2007) Anal. Chem. 79: 5322-5327), and droplets (Wen et al. (2016) Molecules, 21 : 881) have also found utility in immunoassays; yet, the detection of target proteins directly from single cells could not be achieved in microparticle systems.

[0230] Miniaturization is well suited to electrophoretic separations owing to favorable scaling of physical phenomena including: 1) Efficient dissipation of Joule heating owing to high surface area to volume ratios found in microscale separation channels; and 2) Precision isolation and manipulation of individual cells (diameters ~30 pm) - even among large populations of cells. For rapid electrophoretic analysis of single cell lysate, microchannel junctions and microwells prove useful for seamless handling of 1-5 pL of cell lysate. When an immunoassay is appended to a completed electrophoretic analysis, several additional advantages of miniaturization accrue. First, mass-based separation of proteins prior to an immunoassay separates any off-target, non-specific signal from that of the target (Mahmood & Yang (2012) N. Am. ./. Med. Sci. 4: 429). Second, controlled mass transport at the microscale shortens the probing and washout times of immunoassays compared to surface- based immunoassay ( e.g ., microtiter plates). Suspended surfaces, such as microparticles, offer even more efficient mass transport owing to 3D access of reagents to the surfaces of the particle and, hence, reduced diffusion-length scales to the interior of the particle. Increasing the concentration of an immunoprobe can enhance immunoassay sensitivity via improved partitioning of immunoprobe into the particle.

[0231] As described herein we introduce separations-encoded microparticles for single-cell immunoblotting, a hybrid approach that brings the selectivity of separations tothe efficient compartmentalization of microparticles. The basis of the separation-encoded microparticles is a hydrogel molding and release technique, in which a planar array of microparticles is created with perforations delineating each microparticle perimeter. After use of the planar array for cell isolation and protein electrophoresis, the arrayed

microparticles are mechanically released to create suspensions of microparticles, each encoded with a single-cell protein separation. By design, the hybrid device is designed for optimal performance at each assay stage. First, the planar hydrogel arrays are well-suited to sample preparation (e.g., isolation of a single cell in each microparticle using a microwell feature molded into each microparticle, in-microwell chemical cell lysis) and PAGE of single-cell lysate from the microwell into the abutting hydrogel and finally photoblotting- based protein target immobilization to hydrogel (Fig. 8, panels A and B). Second, the microparticle form factor is well-suited to heterogeneous immunoassays (e.g,

immunoprobing, washing) and further manipulation of hundreds-to-thousands of single-cell immunoblots. Dehydration of the microparticles shrinks the dimensions, yielding geometry- enhanced analytical sensitivity, with PAGE resolving power scale-invariant after blotting (immobilization). Lastly, we assess oncoprotein isoform expression across a range of cancer cell phenotypes. Separations-encoded microparticles bring performance benefits from both microarrays and microparticles to offer new avenues for high-throughput, high-selectivity protein cytometry.

Results and discussion

Releasable separations-encoded microparticles: Single-cell immunoblots

[0232] Immunoblotting integrates PAGE of each single-cell lysate with a subsequent immunoassay to confer selectivity beyond PAGE alone (Hughes et al. (2014) Nature Met. 11 : 749). In conventional protein immunoblotting, protein peaks are transferred from the PAGE hydrogel onto a hydrophobic membrane using electro-transfer or diffusion (Kinoshita et al. (2009) Nature Protoc. 4: 1513). Hydrophobic interactions immobilize the protein separation on the blotting membrane, thus retaining separation information on a material with larger pore size than polyacrylamide molecular sieving gels. The larger pore size of blotting membranes (polyvinylidene difluoride (PVDF), nitrocellulose) reduces thermodynamic partitioning and enhances transport of immunoreagents to the immobilized protein material, thus underpinning efficient immunoprobing during the immunoassay.

[0233] We developed a method to create a planar array of releasable microparticles comprised of a dual-function hydrogel that, when comprising the planar array, acts as a molecular sieving matrix for electrophoresis and, when in a suspension of microparticles, acts as an immobilization scaffold for heterogeneous immunoassays performed on the separated protein targets. On silanized glass microscope slides (Fig. 12, panel A), we chemically polymerized polyacrylamide on an SU-8 mold to create planar microparticles, with perforations defining the perimeter of each microparticle. In selecting the microparticle shape, we sized each microparticle to house a microwell (15 pm radius, 40 pm depth) for mammalian cell isolation with an abutting region for protein PAGE. After single-cell PAGE analysis and photocapture of proteins to the hydrogel, the microparticles are released from the array by mechanical shearing, using a razor blade (Fig. 12, panel B).

[0234] To determine microparticle geometries, we sought to develop single-cell protein PAGE to analyze five ER-associated cancer signaling protein targets, spanning 35 kDa to 100 kDa (see Table 1) with a minimum mass difference between neighboring targets of 8% ( e.g ., b-tubulin and ERa46). The long axis of the rectangular microparticle (Z _sep ) was determined using two separations-driven design criteria: (i) a target separation resolution (SR, defined as SR = DΏ 4s where AL is the separation length, s is the average peak width) of 0.5 for the closest neighbors (AL = AL _mm ) and (ii) the maximum electromigration distance (/. _max ) for the protein target with the fastest electrophoretic mobility ( m , in-gel mobility, defined as m = m ₀ 10 ^~KT , where K is the retardation coefficient of an analyte and Lis the total acrylamide concentration in the gel (Duncombe & Herr (2013 ) Lab Chip, 13: 2115-2123; Butterman et al. (1988) Electrophoresis, 9: 293-298). According to Ferguson analysis results, estimated electrophoretic mobility of proteins at 40 V cm ^-1 for 30 s in 8%T, 2.6%C gel is determined as shown in Table 1, (more details on electric field distribution in the array in supplemental information below)). Using these design rules, we fabricated planar arrays of rectangular microparticles ~950-pm long, 250-pm wide, and 40-pm thick (Fig. 8, panel C). Upon mechanical release, we observed 94% yield of microparticles at hydrated state (n = 3 chips; 3500 microparticles per chip), with 91.4% of the successfully released microparticles exhibiting damage by visual inspection (Fig. 13). Signal acquisition can be performed on the microparticle array or suspended microparticles (Fig. 8, panels D and E), in either the hydrated or dehydrated state (Fig. 8, panel F).

Table 1. Protein targets in separations-encoded microparticles.

[0235] We next validated the microparticle immunoblots through analysis of a range of breast cancer cell morphologies and types, using a panel of well-established breast cancer and kidney cell lines (breast adenocarcinoma, MCF 7; invasive breast adenocarcinoma, MDA MB 291; embryonic kidney cells, HEK 293). Average diameters of MCF 7, MDA MB 291, and HEK 293 cells were measured as 16 pm, 18 pm, and 16 pm, respectively; microwell diameters of 30 pm were used for all experiments. Based on the distribution of cell diameters in each cell population, cell settling resulted in zero, one, or multiple cells in microwells and perforations. In single-cell handling, we observed an average of 95% single-cell microwell occupancy for MCF 7, MDA MB 291 and HEK 293 cells, with 0.2% spurious isolation of cells in the perforations when a cell suspension of ~l0 ⁶ cells mL ¹ in 1 PBS was introduced to the array (n = 4 devices, 3500 microwells per device), using a 10 min settling period (Fig. 14, panel A). A small number of microwells housed more than one cell (0.4%).

[0236] Two housekeeping protein targets, GAPDH and b-tubulin, were probed in the same microparticle assay using lOx diluted AlexaFluor 647 and AlexaFluor 555,

respectively, and the resulting signal intensities were correlated with microwell occupancy (Fig. 14, panel B). Cells settled in the perforations did not have a detectable signal, which we attribute to rapid lysate dilution by convective flow (Fig. 14, panel B). After settling, in microwell chemical lysis (30 s) and single-cell protein PAGE (20 s, E = 40 V/cm) were completed for GAPDH and b-tubulin across all cell types. Microparticle arrays were immunoprobed with a cocktail of GAPDH and b-tubulin (primary probing duration 3h, secondary probing duration lh, washout periods 20 min; more details in Experimental Procedures). Observed electromigration behavior agreed with estimates (electromigration distances varying 0.9% and 1.1% for GAPDH and b-tubulin, respectively, 2 devices, n = 40 microparticles). With microparticles fabricated on silanized glass slides (Fig. 15), electrophoretic mobilities of GAPDH and b-Tubulin were ~9.0 x 10-9 m2 V-l s-l and ~6.0 x 10-9 m2 V-l s-l, respectively (Fig. 14, panel B), corroborated by previous observations (Duncombe & Herr (2013) Lab Chip, 13: 2115-2123). As expected, no protein signal was detected for empty microwells (n = 2 devices, 3500 microwells per device, Fig. 14, panel B).

[0237] In considering diffusive transport during immunoprobing, we hypothesized that immunoprobing of a suspension of microparticles would be more efficient than immunoprobing of the surface-attached planar microparticle array. The probing and washing steps dominate the planar assay duration. As context, conventional single-cell western blotting sees 75% assay duration devoted to probing and 25% to washout steps. Long durations are required owing to the limited mass-transport of antibody probes into the gel. In contrast, when in suspension the surface area of each microparticle is available for diffusion- based antibody probe introduction; whereas, transport into the hydrogel array is inhibited on the surface side that is attached to the glass slide. Then with T _di ^ _usion (where t _di ff _usion is the transport time, x is the gel thickness, and D is the diffusion coefficient for antibody in an 8 %T gel (Tong & Anderson (1996) Biophys. J. 70: 1505)), x is the half-thickness of the microparticle thickness when the microparticle is in suspension and the full-thickness when the microparticle is anchored to a glass slide in the planar array. The geometric argument applies to all stages of immunoprobing and washout, suggesting that the microparticle format could reduce the duration of the ~4 hr immunoprobing-related steps by 25%. [0238] Background signal is also important to detection performance. Background is dictated by the efficacy of the washout process after probing. In comparing the planar array to the suspended microparticles, we observed background signal reduction of l.3x in microparticles, and we indeed observed effective performance with reduced washout times (~5 min vs. ~20 min; n = 1 device, 3500 microparticles, CV = 0.2) in the suspension of microparticles versus the attached array (Fig. 16, panel A).

[0239] Next, in considering the immunoassay which is a reaction between a protein target and immunoprobe, as well as the transport during immunoprobing, we write

R _eaction = V( ^k _on i ^Ab ] _gei + ^k _o ff) and [Ab] _gel = K[Ab\ ₀ , where r is the reaction time, k _on is the reaction coefficient, k _on is forward reaction rate constant, k _ojj i s backward reaction rate constant, [Ab] _gei is antibody concentration in gel, [Ab] ₀ is antibody concentration in solution— including partitioning coefficient for the hydrogel, K = 0.17 for 8%T PAG

(Vlassakis & Herr (2015) Anal. Chem. 87: 11030-11038). We use the Damkohler number (Da, with Da = t _transport A _reaction ) (Probstein (2005) Physicochemical hydrodynamics: an introduction (John Wiley & Sons)) for low-affinity (Ko~10 ⁶ ), medium-affinity (Ko~l0 ⁹ ), and high-affinity (K _D ~l0 ^u ) immunoprobes.

[0240] Given this physical framework, we estimate that immunoprobing with low- affinity antibodies will be reaction-limited (Da~0.7), while immunoprobing with medium- affinity (Da~280) and high-affinity antibodies is mass-transport-limited (Da~475) (Vlassakis & Herr (2015) Anal. Chem. 87: 11030-11038). From this analysis, we conclude that as long as medium- and high-affinity antibodies are used, the expedited diffusion into a 40-pm thick gel benefits the immunoprobing duration 4x faster in microparticle format when compared to the planar array format, according to t _di ff _usion calculations.

[0241] We next sought to use the suspension of separations-encoded microparticles to overcome an important multiplexing limitation inherent to immunoblotting. In

immunoprobing, an antibody pair is typically used to (i) detect the protein target (unlabeled primary antibody probe) and (ii) detect the unlabeled primary antibody (fluorescently labeled secondary antibody probe). The secondary antibody probe needs to be selective for the animal species in which the primary antibody probe was raised. Herein lies the detection challenge: primary antibodies are raised in just a handful of animal species. If multiple primary antibodies of the same species are used for target detection, the secondary antibody probes must be applied to the PAGE separation serially (not as one cocktail). The serial application demands multiple secondary antibody probing rounds and multiple gel stripping rounds, to ensure selective readout (Chattopadhyay et al. (2014) Nat Immunol 15: 128). For example, two rounds of probing and stripping takes +50 hours for slab gel Westerns, and +9 hrs for conventional single-cell western blotting (Hughes et al. (2014) Nature Met. 11 : 749).

[0242] To overcome this target multiplexing limitation, we fractionate the

microparticle suspension into aliquots and apply distinct antibody probe solutions to each ( e.g ., Era, Actinin). As a negative control, we performed two rounds of probing (for each probing round, 3 h primary and 1 h secondary antibody probing steps with 20 min washing time after the probing steps) and stripping (lh) for ERa and Actinin antibodies separately (see Experimental Procedures). Fig. 9, panel A shows ERa expression level decreases in previously Actinin-probed microparticles, compared to microparticles probed for ERa alone. We calculated a 15.8% decrease in average expression quantified from negative control group (p>0.05, n = 40 microparticles). In multiplexed single-cell immunoassays, off-target probe binding is a substantial challenge (e.g., immunocytochemistry, flow cytometry) (see, e.g, Sevecka & MacBeath (2006) Nature Meth. 3 : 825-83 1 ; Ng et al. (2015) Nature

Commun. 6: 7513; Lombard DBanek et al. (2016) Angew. Chem. Int. Ed. 55: 2454-2458; Bendall et al. (2011) Science, 332: 687-696). Performing a separation, followed by immunoprobing helps to overcome this challenge by spatially separating the off-target signal. We investigated the off-target signal for both the ERa isoform and Actinin in separations- encoded microparticles and observed off-target signals for ERa (Fig. 9, panel B).

[0243] PAGE resolves two protein isoforms reactive to one ERa antibody probe: full- length (ERa66) estrogen receptor isoform (66 kDa) and truncated (ERa46) estrogen receptor isoform (46 kDa) (Kimmerling et al. (2016) Nat. Comm. 7: 10220; Beech et al. (2012) Lab Chip, 12: 1048-1051). We validated the separation of ERa46 and ERa66 isoforms using three housekeeping proteins— Actinin (100 kDa), b-tubulin (50 kDa), and GAPDH (35 kDa)— as reference standards in a Ferguson plot analysis. We observed a linear relationship between migration distance and molecular mass for both the planar array and the suspended microparticles (R ² = 0.97, n = 121 microparticles and R ² = 0.95, n = 34 microparticles, respectively; Fig. 9, panel C). Separation resolution between the two ERa isoforms was 1.77 ± 0.33 (Fig. 9, panel D; n = 34 microparticles), which is considered to be bottom line separated and therefore quantitatively measurable.

Dehydrated separations-encoded microparticles: Single-cell proteoform profiling

[0244] We next sought to explore unique performance gains possible in single-cell immunoblotting using the microparticle system. Specifically, we sought to understand analytical performance enhancements conferred by the isotropic shrinking of separation- encoded microparticles through dehydration of the microparticles from suspension. In a hydrogel microparticle that shrinks isotopically, we expect two potentially favorable scaling phenomena. First, we anticipate no sacrifice in separation resolution (SR, SR = ALl 4s), as both AL and s should shrink by equal factors, assuming a detector with sufficient spatial resolution to resolve the final separation. At the hydrated state, the polymer network in separation-encoded microparticles is distributed uniformly. Shrinking develops upon evaporation of the aqueous phase (in this case water), initiating from the surface uniformly and resulting in a higher density polymer film formation at the outer interfaces (Waje et al. (2005) Braz. J. Chem. Eng. 22: 209-216). Film formation may slow down the subsequent shrinking process under constant temperature and humidity levels, because the volume fraction of the polymer network in the film turns out to be much higher than in other portions of the gel (Waje et al. (2005) Braz. J. Chem. Eng. 22: 209-216; Tokita et al. (1999) J. Phys. Soc. Jpn. 68: 330-333; Suzuki et al. (1999) J. Chem. Phys.111 : 360-367). However, the water molecules inside the gel can still evaporate constantly even after the formation of the film layer since the molecules are small enough (A-scale) compared to the pores in an 8%T 2.6%C polyacrylamide gel (nm-scale). After evaporation of all liquid content, the gel reaches a compact, uniform, and dehydrated state that does not impact SR (Tokita et al. (2000) J. Chem. Phys. 113: 1647-1650). Second, we expect improved analytical sensitivity of an immunoblot as the local concentration of fluorophores is inversely correlated with the volume (//') of microparticles (Caster & Kahn (2012) Cell Logistics, 2: 176-188). The average fluorescence si ·gna il i ·ncrease ( _/ StI _\ ) can i be ca ilcu ilated i as fo illows, S cI _T

where c is the intensity of the fluorescence signal, » is the location of the analyte peak, and bg is background (Lin et al. (2013) Adv. Opt. Mater. 1 : 568-572; Nguyen et al. (2010) Lab Chip, 10: 1623-1626; Zhu & Craighead (2012) Annu. Rev. Biophys. 41 : 269-293).

[0245] To understand the mechanism of hydrogel shrinkage, we performed single-cell immunoblots as described, then dehydrated the microparticle suspension by evaporation through heating on a hot plate. We measured a reduction of 83 ± 8 pm in microparticle length (950 pm to 866 pm; n = 250 microparticles) and 31 ± 5 pm in microparticle width (254 pm to 223 pm; n = 250 microparticles) suggesting isotropic shrinkage of each microparticle. The degree of circularity of the microwells was assessed (i.e.,

Degree of circularity = 2 sfnA/C , where A is the particle area including hole, C is the perimeter of the microwell (Carvalho et al. (2013) PloS one 8: e59387). Accordingly, a circular feature has a degree of circularity of 1.0, with non-circular features having values of <1.0. When comparing suspensions of hydrated microparticles to dehydrated microparticles, we observed no significant difference in the degree of circularity for the microwells (p-value <0.00001, n= 2881, 1823, 276, and 110, respectively; Fig. 16, panel B).

[0246] To next assess the impact of dehydration on the PAGE performance of separations-encoded microparticles, we assessed SR using GAPDH and b-tubulin in hydrated and dehydrated microparticles. We first considered the peak width of each target {4U) probed and measured a 10% and 7% reduction in peak width (140 pm to 125 pm for GAPDH, 106 pm to 99 pm for b-tubulin; n = 121) (Fig. 10, panel A), consistent with observed shrinkage of the microparticle extents. We scrutinized any changes to□ L between the two markers stemming from dehydration-induced shrinkage of the separations-encoded microparticles.

[0247] We found a median fluorescence signal intensity increase of l.6x (CV = 1.2, n

= 121) on the dehydrated microparticles relative to the hydrated ones (Fig. 10, panel B). According to the measured reduction in the dimensions of the dehydrated microparticles, fluorescence signal intensity increase (intensity/pm ³ ) is expected to be l.4x. Therefore, the measured increase is found to be in accordance with the calculated increase. Consequently, the measured SR values for the two markers were SR = 0.72 (CV = 22.86, n = 16

microparticles) in hydrated microparticle suspensions and SR = 0.72 (CV = 21.43, n = 62 microparticles) in dehydrated microparticles from suspension, which are not significantly different (two sample t-test p = 0.18), as anticipated from geometric arguments.

[0248] To understand the impact of microparticle shrinkage on analytical sensitivity and overall detection performance, we assessed the target signal (AUC) and the background signal. The impact of increased local concentration of fluorescence signal in dehydrated microparticles was characterized using GAPDH and b-tubulin. We found the median normalized AUC for dehydrated microparticles was ~ 1.3 to l.7x higher than hydrated microparticles (Mann- Whitney U-test p-value < 0.05 for each antibody type used, Fig. 10, panel C) (see supplemental information below).

[0249] In understanding the effect of dehydration on the background signal, we compared the background fluorescence signal intensities obtained from hydrated/probed, hydrated/not probed, dehydrated/probed microparticles (Fig. 16, panel C). We probed separations-encoded microparticles with a secondary antibody (labeled with AlexaFluor 647) solution for 3 h and washed the excess solution for 1 h in TBST solution before imaging at 635 nm wavelength. Background fluorescence intensity of dehydrated/probed microparticles was ~2x higher than hydrated/probed microparticles at the central regions, while this difference increases to be ~40x relative to hydrated/not probed microparticles. The increase in the fluorescence intensity resulted in an enhanced SNR, defined as the ratio of the average fluorescence signal minus the mean background signal to the standard deviation of the background (Sharma et al. (2014) Langmuir, 30: 10979-10983). The noise on the dehydrated microparticles was noted to decrease by l.5x (median SNR = 9.8, CV = 0.8, n = 392 microparticles) relative to the hydrated microparticles, SNR = 6.4 (CV = 0.4, n = 342 microparticles). The lower noise in dehydrated microparticles yields an improved analytical sensitivity. This result is in accordance with our observations regarding the effect of microparticle shrinkage on the target detection signal. The geometry -induced performance changes made target signal detectable in 17% more dehydrated microparticles versus detectable in hydrated microparticles. Reduced noise in the separations-encoded

microparticles is attributed to the enhanced mass-transport of antibodies during the washout step, although dehydration (shrinkage) process increases the signal intensity in

microparticles.

[0250] Next, we measured ERa protein isoform expression differences using the separations-encoded microparticles, ERa46 (46 kDa) and ERa66 (66 kDa) isoforms, in MCF 7 (estrogen sensitive), MDA MB 231 (estrogen resistant), and HEK 293 (non-expressing) cells at different confluency levels. For the estrogen-sensitive cell line, we expect an inverse correlation between ERa46 expression and ERa66 expression if hormonal resistance increases with ERa46 expression (Kimmerling et al. (2016) Nat. Comm. 7: 10220). We first confirmed that the gradual increase in cell confluency levels agrees with the gradual increase in housekeeping protein expression levels for all cell lines (Fig. 11 panel A and Fig. 17, panel A). On the basis of separations-encoded microparticle assay results, we compared SNR of ERa46 and ERa66 in MCF 7 cells and found that the average of both SNR values was above SNR = 3 threshold. Particularly, the SNR average was 4.79 for ERa46 (n = 385) and 6.41 for ERa66 (n = 93) (Fig. 11 panel B). Importantly, AUC analysis in separations-encoded microparticles revealed a 2.8x increase in truncated isoform (ERa46) and 6.4x decrease in full-length isoform (ERa66) over a l4-day period in estrogen sensitive cells (Fig. 11 panel C, n = 478 cells); therefore, confirmed that higher confluency increases ERa46 expression that suppresses the expression of ERa66 in MCF 7 cells (Kimmerling et al. (2016) Nat. Comm. 7: 10220; Beech et al. (2012) Lab Chip, 12: 1048-1051). Surprisingly, separations-encoded microparticles reported minute levels of ERa46 isoform-expressing cells in the estrogen resistant cell line, while this population remained masked in slab gel westerns (Fig. 18). ERa66 isoform was not detected in estrogen resistant cell line as it is in the class of highly invasive phenotype that reportedly lacks ERa66 isoform (Beech et al. (2012) Lab Chip, 12: 1048-1051). Neither of the isoforms was detected in the non-expressing cell line, which has been used as a negative control in this experiment.

[0251] Quantification of ERa expression changes in MCF 7 cells benefited from the use of releasable separations-encoded microparticles in two key aspects. First, enhanced mass transport in microparticles helped to reduce the total immunoprobing time from ~50 hours to ~l4 hours, even though we employed two probing and stripping rounds for a total of 5 protein species. Second, we achieved to discriminate minor cell populations representing ERa46 expressing MCF 7 cells, ( e.g . 6 cells on day 3, 54 cells on day 5, and 163 cells on day 7 from a large cell population of 478 cells, see supplemental information below).

[0252] In summary, we have designed, developed, and applied separations-encoded microparticles to advance performance and utility of single-cell immunoblotting, which has been limited by microscale mass transport considerations particularly during immunoprobing. Mass transport limitations are a bottleneck of immunoassays, requiring substantial dedication of assay time to the probing and washout steps. Separations-encoded microparticles reduce mass transport restrictions, boost analytical performance, and bring handling flexibility by combining normally disparate microarray and microparticle formats. Furthermore, protein immunoblotting with separations-encoded microparticles confers selectivity suitable for isoform detection while allowing selective probing and multiplexing by isolation individual cell readouts. Merging microparticles with biomolecular separations surmounts measurement challenges where: 1) immunoassays are insufficient owing to limited probe selectivity and 2) Powerful mass spectrometry tools lack analytical sensitivity and throughput. The unique capabilities of the separations-encoded microparticles should provide a tunable, versatile format for cytometry.

Experimental Procedures

Fabrication of SU-8 mold and separations-encoded microparticles.

[0253] The SU-8 mold fabrication was performed by following the manufacturer’s instructions (MicroChem) and previous publications (Hughes & Herr (2012) Proc. Natl.

Acad. Sci. USA, 109: 21450-21455). Approximately 7000 microparticles (250x 1000x40 pm) can be fabricated from a 70x48-mm mini-slab polyacrylamide gel. Each microparticle contained a microwell (30 pm in diameter), a separation lane (950 pm in length) and 50-pm wide perforations defining the microparticles. 8%T, 2.6%C polyacrylamide gel containing 5 mM BPMAC was layered on an acrylate-silanized microscope glass slide by the help of the SU-8 mold. The gel was synthesized by chemical polymerization using 0.08% APS as the initiator and 0.08% TEMED as the catalyst. The gels were incubated in distilled water for 10 min at room temperature prior to releasing the glass slide from the SET-8 mold. After following single-cell western blotting procedure, microparticles were released from the glass slide by shearing using a razor blade when the microparticles are in the hydrated state.

Thanks to the perforations, individual particles can be released easily.

Single-cell western blotting using separations-encoded microparticles

Single-cell resolution western blotting.

[0254] Approximately 1 x 10 ⁶ EG251 -GFP cells were settled by gravity into the wells located on microparticles for 10 min, and the gel was washed three times with 1 mL 1 ^c PBS to remove excess cells off the gel surface. Then, settled cells were lysed by directly pouring the RIPA-like lysis buffer at 55°C over the slide. Cell lysates were electrophoresed at 40 V cm ^-1 for 30 s, and immediately after protein bands were immobilized by ETV activation (Lightningcure LC5, Hamamatsu) of the BPMAC. The microparticle array was incubated in TBST buffer overnight at room temperature.

Immunoprobing and imaging of separation-encoded microparticles after single-cell western blotting.

[0255] Donkey Anti-Goat IgG (H+L) Cross-Adsorbed Cross- Adsorbed Antibody,

Alexa Fluor 555-labeled antibody (cat. no. A21432), Donkey Anti-Rabbit IgG (H+L) Cross- Adsorbed antibody, Alexa Fluor 555-labeled (cat. ho.A21432), and Donkey Anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary antibody, Alexa Fluor 647-labeled (cat. no. A31573) were purchased from Thermo Fisher Scientific. After the electrophoretic separation and UV- induced covalent attachment of proteins to benzophenone in microparticles, the gel was washed for overnight in l x TBST on an orbital shaker. Separations-encoded microparticles were incubated in 30 pL of a 1 : 10 dilution of primary antibodies in 1 x TBST with 2% BSA solution for three hours, and washed three times for 20 min in 1 x TBST. Secondary antibodies were incubated for two hours in a 1 : 10 dilution and gels were washed three times for 20 min in 1 x TBST and dried using nitrogen stream. After the immunoprobing, the hydrated microparticle array was released from the glass slide surface using a razor blade. During this process, microparticles were completely peeled off from the glass surface without any remaining parts attached to the surface. Separations-encoded microparticles can be imaged in the released format or in the array format. For all cases (attached, released, hydrated, and dehydrated states), we imaged the gel particles using a fluorescence microarray scanner (Molecular Devices, Genepix 4300A) with an Alexa-Fluor 555 filter (532 laser excitation, 650 PMT), and an Alexa-Fluor 647 filter (647 laser excitation, 550 PMT).

Data analysis and simulations.

[0256] Protein bands were quantified using in-house developed MATLAB scripts as described in Kang et al. (2016) Nature Protoc. 11 : 1508. Peak widths were characterized by Gaussian curve fitting in MATLAB (R20l7b, Curve Fitting Toolbox). The integrated intensity of a marker was calculated if its R ² value was larger than 0.7 for the given region of interest. For all quantified results, the protein peaks with Gaussian fitting R ² value larger than 0.7 and SNR value larger than 3 were analyzed. Electric field distribution simulations were performed in COMSOL Multiphysics 5.3a (Burlington, MA) to characterize protein mobility during electromigration in separations-encoded microparticles. A 2D asymmetric model was used. The gel width was 250 pm and the length was 1000 pm. The diameter of the microwell was set to 30 pm. The maximum and minimum mesh element sizes were 30 and 0.3 pm, respectively. The model was solved in stationary mode by applying a constant electric field through the matrix, where hydrogel and solution conductivities were set to 4304.0 pS m ¹ estimated experimentally (Yamauchi & Herr (2017) Microsys. Nanoeng. 3: 16079). The goal of the simulations was to estimate the electric field instabilities caused by discontinuous surface area created by arrayed microparticles.

Supplemental Information.

Experimental procedures

Chemicals and reagents.

[0257] Acrylamide/bis-acrylamide, 30% (wt/wt) solution (Sigma-Aldrich, cat. no.

A3699), sodium dodecyl sulfate (SDS) (cat. no. L3771), sodium deoxycholate (NaDOC) (cat. no. D6750), Triton X-100, N,N,N',N'-tetramethylethylenediamine (TEMED) (cat. no.

T9281), ammonium persulfate (APS) (cat. no. A3678), and bovine serum albumin (BSA)

(cat. no. L3771) were purchased from Sigma Aldrich. Goat anti-GAPDH primary antibody (Sigma-Aldrich, cat. no. SAB2500450), mouse anti- Tubulin (Genetex, cat no. GTX11312), rabbit anti-ERa SP1 (Thermo Scientific, cat. no. RM-9101-S0), rabbit anti-actinin (Cell Signaling, cat no. 6487) were purchased from the suppliers. N-[3-[(3-Benzoylphenyl)- formami do] propyl] methacrylamide (BPMAC) was synthesized by PharmAgra Laboratories. Tris-glycine (10 ^c ) EP buffer was procured from Bio-Rad (25 mM Tris, pH 8.3; 192 mM glycine, cat. no. 1610734). Phosphate-buffered saline was acquired from VWR (10* PBS) (cat. no. 45001-130). Petroleum jelly was purchased from Cumberland Swan Petroleum Jelly (cat. no. 18-999-1829). Tris-buffered saline with Tween (TBST) was obtained from Santa Cruz Biotechnology (20x TBST) (cat. no. 281695). Deionized water (18.2 MW) was obtained from an Ultrapure Millipore filtration system.

Cell culture.

[0258] The MCF 7 cells were cultured in RPMI 1640 media (Therm oFisher

Scientific, cat. no. 11875093) supplemented with 10% Charcoal-stripped serum (Sigma- Aldrich, cat. no. F6765), and 1% penicillin/streptomycin (ThermoFisher Scientific, cat. no. 15140122). MDA MB 231 cells were cultured in the same culture media with MCF 7 cells. U251-GFP cells were cultured in high glucose DMEM (Life Technologies, cat. no. 11965) supplemented with 1% penicillin/streptomycin, 1 x MEM nonessential amino acids

(11140050), 1 mM sodium pyruvate (Life Technologies, cat. no. 11360-070), and 10% FBS. HEK 293 cells were cultured in the same culture media with U251-GFP cells, except 10% Charcoal-stripped serum was used instead of 10% FBS. All cells were grown in a humidified atmosphere containing 5% C02 at 37 °C. All cells were cultured in flasks.

Slab gel western blotting

Imaging of living cells.

[0259] The confluency of the cells was determined by imaging the culture dishes in bright field using an Olympus 1X71 inverted fluorescence microscope equipped with an EMCCD camera (iXon3 885, Andor, Belfast, Ireland), motorized stage (Applied Scientific Instrumentation, Eugene, OR), and automated filter cube turret controlled through

MetaMorph software (Molecular Devices, Sunnyvale, CA).

Cell lysate preparation for slab gel western blotting.

[0260] At the desired confluency level, cells were trypsinized using 1 mL of trypsin

EDTA solution for 1-2 min at 37 °C and 4 mL culture medium was added to the 25 cm ² (T- 25) culture flask. After centrifuging the cells at 1000 rpm for 5 min in a 15 mL tube, the culture medium was aspirated. Cells were resuspended in 1 mL PBS and counted using trypan blue stain. Cells were washed two times more with ice-cold PBS to remove all the loosely bound serum proteins in the media before adding lmL of ice-cold protease inhibitor- contained RIPA buffer (0.15 mmol L ¹ NaCl, 5 mmol L ¹ EDTA, 1% Triton-X 100, 10 mmol L ¹ Tris-HCl. Cells were incubated on ice for 15 min while vortexing in every 5 min. The suspension was transferred to an Eppendorf tube and centrifuged at 25,000 x g for 30 min at 4°C. The supernatant containing 30 pg mL ¹ of cellular proteins were used in slab gel western blotting.

Slab gel western blotting.

[0261] 4-12% gradient gel (Novex WedgeWell Tris-Glycine, cat. no. XP04125BOX ) and a Bio-Rad electrophoresis chamber connected to a Bio-Rad high voltage power supply set at 200 V were utilized for protein separation from cell lysates. Each well was loaded with 30 pg protein labeled with a 9: 1 mixture of 4x Lamelli buffer and 10* NuPAGE reducing agent at 1 : 1 ratio. After the electrophoresis, Pierce PowerBlot Rapid Transfer System (ThermoFisher Scientific, cat. no. PB0112) was used to transfer the proteins to a PVDF membrane according to the manufacturer’s instructions. The PVDF membrane was blocked in 5% BSA (w/v) solution for 30 min and was incubated in 10 mL solution with a 1 : 1000 dilution of primary antibodies in 1 x TBST with 5% BSA solution for overnight at 4 °C. The membrane was rinsed three times with TBST for 10 min each round. Secondary antibodies were incubated in a 5% BSA solution with a 1 : 10000 dilution for 1 h and PVDF membrane was rinsed using TBST three times with TBST for 10 min each round. After the rinsing step, Western Lightning (PerkinElmer, cat. no. NEL120E001EA) was utilized as described in manufacturer’s protocol for obtaining chemiluminescence images using a Chemidoc XRS system (Bio-Rad, cat. no. 170-8265).

Quantitative microparticle analysis of ER protein isoforms from single cells

[0262] Estrogen receptor, ER, is the main contributor to the development of hormone resistance against the therapy in circa 70% of breast tumors (Beech et al. (2012) Lab Chip, 12: 1048-1051). Particularly, truncated (ERa46) estrogen receptor isoform is reported to enhance hormone therapy responses by inhibiting the expression of full-length (ERa66) estrogen receptor isoform (Kimmerling et al. (2016) Nat. Comm. 7: 10220). The relation between cell confluency and ERa expression in hormone-sensitive cell lines has remained unclear, although this finding may suggest new strategies in therapies. Elucidating mechanisms of confluency-dependent expression of these isoforms requires quantitative measurement of ERa isoforms. We hypothesized that cell confluency level would impact expression levels of ERa46 protein in estrogen-sensitive cells, while no expression would be detected in estrogen resistant and non-expressing cell lines. We used separations-encoded microparticles to address the confluency-dependence of ERa46 and ERa66 expression levels in estrogen sensitive (MCF 7) over 14 days.

[0263] We performed slab gel Western blotting assay to validate the microparticle assay results (Fig. 16, panels B and C). A similar trend in ERa isoform expression levels was observed in slab gel Western analysis, except that no expression could be detected on day 3 of MCF 7 cells unlike microparticle analysis results. We did not observe any detectable signal from ERa isoforms expressed in MDA MB 231 in slab gel Westerns, although microparticles reported 6, 12, 20 cells expressing ERa46 on days 3, 5, and 7, respectively (Fig. 18) (n _to tai = 38). We did not observe any ERa isoform signal in HEK 293 cells as shown in Fig. 18. Slab gel Western blot analysis confirmed our major finding on ERa expression level changes in MCF 7 cells using microparticle assay, which provided more insight into isoform expression in cell subpopulations.

Characterization of the electric field distribution in microparticle assay

[0264] A uniform electric field distribution is compulsory for maintaining repeatable and comparable protein separation in microparticle arrays. In a typical workflow, we apply an electric field across the microparticle array to draw proteins through the microwell wall into the central region of microparticles. The uniformity of the field can be ensured by immersing the unidirectionally placed microparticles on top of a silanized glass plate in a conductive buffer where two parallel platinum electrodes are placed in to apply the electric field. We characterized the uniformity of electric field using two approaches: (1) we simulated electric field distribution in the assay, (2) we tracked the peak position

displacement of b-tubulin (50kDa) and GAPDH (35kDa) proteins as a function of time in microparticles placed on a glass slide. Firstly, the electric potential in the microparticle assay was found to be uniform and unaltered between the electrodes according to numerical simulations (Fig. 19). This was an expected outcome because polyacrylamide gel must not result in a discontinued conductivity along the separation axis as long as soaked in the buffer solution during electrophoresis (Yamauchi & Herr (2017) Microsys. Nanoeng. 3: 16079). Simulation result was also confirmed by electrophoresis of b-tubulin and GAPDH proteins obtained from U251 cells (Fig. 15). We also tested electrophoretic mobility of two proteins (b-tubulin and GAPDH) on non-silanized glass slides, as it is known that silanization process does not always result in a uniform monolayer of silane groups on the glass slide (Gumuscu et al. (2015) Biomacromolecules, 16: 3802-3810). Comparison of measured mobilities shown in Fig. 15 suggest that silanization state does not have a statistically significant impact on the electrophoretic mobility of the proteins (two sample t-test, p = 0.0001, n _Siianized glass =

20, Hnon-silanized glass ^— 20).

Cell-occupancy dependent fluorescence signal changes in separations-encoded microparticles

[0265] We compared the antibody fluorescence intensities in multiple-cell occupancy microwells and single-cell occupancy microwells at the central region of microparticles. The mean antibody fluorescence intensity from two-cell occupancy microparticles was ~5.5x higher than that of single-cell occupancy microparticles (CV = 0.28, n = 10 microwells). The intensity was not doubled (therefore not linear) in two-cell occupancy microparticles due to the cell-size related bias. This result is in accordance with our previous findings (Kang et al. (2016) Nature Protoc. 11 : 1508).

Characterization of the fluorescent signal intensity distribution

[0266] As we discussed above, shrinking effect led to increase of the fluorescence signal as the equal number of fluorescence molecules were packed in a smaller volume after dehydration. Interestingly, we observed significantly higher signal at the edges of the central region of separations-encoded microparticles. The edge effect was observed 2 35 higher in hydrated/not probed particles, 5.35x higher in dehydrated particles, 8.1 l x higher in hydrated particles (Fig. 16, panel C). We attribute this occurrence to hydrogel pore size formation at the outer boundaries during polymerization. Polymerization reaction rate is slower at the edges because of the higher oxygen concentrations (oxygen diffuses in the particles passing through the edges first) (Gumuscu et al. (2015) Biomacromolecules, 16: 3802-3810).

Reduced reaction rate leads to formation of larger pores, in which fluorescent molecule concentration can be higher (Pregibon et al. (2007) Science, 315: 1393-1396). The edge effect did not interfere with the of protein measurements since the background intensity in the central region of the particles was uniformly distributed.

[0267] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.