Single cell proteomics using degradable hydrogels

Abstract

The present disclosure is directed to methods for measuring proteomes of single cells. In one aspect, methods comprise disposing cells on a surface, synthesizing hydrogel chambers around each of the cells, lysing the cells to release intracellular proteins for adsorption onto the surface bounded by the hydrogel chambers, depolymerizing the hydrogel chambers, and quantifying the adsorbed proteins with detection antibodies to determine the single cell proteomes.

Description

SINGLE CELL PROTEOMICS USING DEGRADABLE HYDROGELSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 624,611 filed January 24, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] A hallmark of biology is degeneracy, Edelman et al, Proc. Natl. Acad. Sci., 98(24): 13763-13768 (2001). Degeneracy is the ability of elements, such as cells, that are structurally different to perform the same function or yield the same output, and to perform a different function or yield a different output depending on the context in which it is expressed. Examples of degenerate systems are widespread in biology, and include development, immune responses, cancer and functional neuro-anatomy. To understand the operation of such degenerate systems it is necessary to be able to characterize and measure properties of individual cells, such as, their transcriptomes, proteomes, metabolomes, and the like. Techniques that provide only cellular averages of such properties are unable to reveal the cellular heterogeneity necessary for a full understanding of such biological processes, e.g. Dittrich et al, Anal. Bioanal. Chem., 406: 6957- 6961 (2014); Lindstrom et al, editors, Single-Cell Analysis: Methods and Protocols (Humana Press, 2012). Over the last decade, striking progress has been made in transcriptome analysis of single cells and tissues, e.g. Saliba et al, Nucleic Acids Research, 42(14): 8845-8860 (2014); Wang et al, Molecular Cell, 58: 598-609 (2015); Lee et al, Experimental & Molecular Medicine, 52: 1428-1442 (2020); and the like. However, comparable progress has not occurred in proteome analysis, Vistain et al, Trends Biochemical Sciences, 46(8): 661-672 (2021).

[0003] In view of the above, the availability of new methods and apparatus for efficient and convenient analysis of single cell proteomes would advance our understanding of complex degenerate systems in biology.SUMMARY

[0004] A method for determining a location and an identity of a target molecule includes exposing the target molecules, adsorbed onto a spatially barcoded surface, to a detection antibody, under binding conditions for the detection antibody, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, the antibody barcode having a free 5’ end. The antibody barcode corresponds to an epitope of the target molecules for which the detectionantibody is specific. The detection antibody is configured to bind to the epitope. The spatial barcodes of the spatially barcoded surface comprise free 3’ ends. The spatially barcoded surface under hybridization conditions is exposed to a linker oligonucleotide. A free 5’ end region of the linker oligonucleotide hybridizes to the free 5’ end region of the antibody barcode. A free 3’ end region of the linker oligonucleotide hybridizes to a free 3’ end region of a spatial barcode on the spatially barcoded surface. The 3’ end of the spatial barcode can be joined to the 5’ end of the antibody barcode.

[0005] In one embodiment, the present disclosure provides a method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a second handle; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, wherein the antibody barcode comprises a first handle; joining the first handle of the spatial barcode and the second handle of the antibody barcode with a splint, wherein the splint includes (i) a first handle complement configured to hybridize to the first handle and (ii) a second handle complement configured to hybridize to the second handle; and ligating the first handle to the second handle.

[0006] In one aspect, the capture antibody is coupled to the spatially barcoded surface. In another aspect, the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors. In a further aspect, the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells. In an additional aspect, the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously. In a certain aspect, the one or more polymer precursors and the one or more cells are introduced into the flow cell separately. In a particular aspect, the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

[0007] In some aspects, the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface. In one such aspect, the capture antibody is coupled to the top surface. In a specific aspect, the method further includes cleaving the cleavable group to release the antibody barcode before the joining the first handle of the spatial barcode and the second handle of the antibody barcode with the splint.

[0008] In particular aspects, the flow cell further includes a bottom surface, the bottom surface having an opposing orientation with respect to the spatially barcoded surface. In some such aspects, the capture antibody is coupled to the bottom surface. In certain aspects, the method further comprises cleaving the cleavable group to release the antibody barcode before the joining the first handle of the spatial barcode and the second handle of the antibody barcode with the splint. In select aspects, the antibody barcode is coupled to the detection antibody by a scissile bond or by hybridization to an oligonucleotide coupled to the detection antibody.

[0009] Additional embodiments of the present disclosure provide a method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising a handle complement, wherein the handle complement is hybridized with a blocker oligonucleotide that is configured to prevent the handle complement from hybridizing with the handle of the spatial barcode; removing the blocker oligonucleotide from the handle complement; cleaving the cleavable group, thereby allowing the handle to hybridize with the handle complement; and extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

[0010] In some aspects, the capture antibody is coupled to the spatially barcoded surface. In further aspects, the capture antibody is coupled to beads, and wherein the beads are introducedinto the flow cell along with the one or more polymer precursors. In additional aspects, the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells. In certain aspects, the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

[0011] In one aspect, the one or more polymer precursors and the one or more cells are introduced into the flow cell separately. In another aspect, the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

[0012] In particular aspects, the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface. In one such aspect, the capture antibody is coupled to the top surface. In select aspects, the method further comprises cleaving the cleavable group to release the antibody barcode before the allowing the handle to hybridize with the handle complement.

[0013] An additional embodiment of the present disclosure provides a method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the one or more cells in the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising the handle complement, wherein the detection antibody is configured to couples to the secreted biomolecule; removing the blocker oligonucleotide from the handle of the spatial barcode; cleaving the cleavable group to release the antibody barcode, thereby allowing the handle to hybridize with the handle complement; and extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

[0014] In one aspect, the capture antibody is coupled to the spatially barcoded surface. In another aspect, the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors. In an additional aspect, the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells. In a further aspect, the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously. In an additional aspect, the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

[0015] In certain aspects, the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel. In additional aspects, the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface. In particular aspects, the capture antibody is coupled to the top surface. In select aspects, the method further comprises cleaving the cleavable group to release the antibody barcode before the allowing the handle to hybridize with the handle complement.

[0016] In certain aspects, the one or more secreted biomolecules is one of a protein, a cytokine, and a monoclonal antibody. In further aspects, the capture antibody includes a cleavable group so that the capture antibody is configured to separate from a surface.

[0017] Additional embodiments of the present disclosure provide a method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more of polymer precursors into a flow cell, the flow cell comprising a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes comprising a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a third handle complement; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, wherein the detection antibody is configured to couple to the secreted biomolecules; inputting a label into the flow cell such that the label permeates into the one ormore gel chambers, wherein a label barcode of the label hybridizes to the antibody barcode of the detection antibody, and detecting the label. In some aspects, the detecting comprises measuring an optical signal from the label.

[0018] In a further embodiment, the present disclosure provides a method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes comprising a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the oen or more cells in the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and also couples with the secreted biomolecule; wherein the detection antibody is coupled to a label: i) through a scissile or chemically cleavable bond, or ii) by hybridization to a barcode coupled to the detection antibody; detecting a signal associated with the label from the one or more gel chambers, releasing the label from the detection antibody, and detecting a decrease in the signal from the one or more gel chambers.

[0019] In certain aspects, the one or more gel chambers are hydrogel chambers. In further aspects, the one or more gel chambers are formed by selective polymerization of the one or more polymer precursors. In additional aspects, the selective polymerization comprises selectively applying light to the flow cell. In further aspects, the polymer precursors comprise (i) one or more cleavable crosslinkers, and (ii) a photo-initiator.

[0020] In regard to any of the methods, the joining the 3’ end of the spatial barcode to the 5’ end of the antibody barcode can include extending the 3’ end of the spatial barcode to the 5’ end of the antibody barcode to form an extended spatial barcode; and ligating a 3’ end of the extended spatial barcode to the 5’ end of the antibody barcode.

[0021] In regard to any of the methods, before exposing the target molecules, adsorbed onto the spatially barcoded surface, a mixture of polymer precursors and cells can be flowed into a channel of a flow cell. One or more gel chambers can be synthesized enclosing each of the one or more cells disposed in the channel. The cells can be lysed so that the target molecules of eachcell are released into the chamber and adsorbed onto the spatially barcoded surface. The detection antibodies can be bound to the adsorbed target molecules.

[0022] In regard to any of the methods, they can further include collecting optical signals from the one or more cells disposed in the channel; and determining, prior to the synthesizing, the position of each of the one or more cells based on the optical signals.

[0023] In regard to any of the methods, they can further include depolymerizing said one or more gel chambers prior to the binding.

[0024] In regard to any of the methods, they can further include de-adsorbing the adsorbed target molecules prior to the depolymerizing.

[0025] In regard to any of the methods, the spatially barcoded surface can include particles of a plurality of types disposed thereon, wherein each type of particle comprises a surface with different protein adsorption characteristics, and wherein particles of each type have a size and a quantity such that each of the hydrogel chambers encloses particles of every type.

[0026] In regard to any of the methods, the location of the target molecule is determined from a nucleotide sequence of the spatial barcode, and wherein the identity of the target molecule is determined from a nucleotide sequence of the antibody barcode.

[0027] In regard to any of the methods, the spatially barcoded surface can include a plurality of spatial barcodes.

[0028] In regard to any of the methods, the spatially barcoded surface can include a plurality of spatial barcodes, wherein each spatial barcode includes a first primer.

[0029] In regard to any of the methods, the spatial barcode of the spatial barcodes includes a nucleotide sequence deposed on a surface of a channel corresponding to a predetermined location of the spatially barcoded surface.

[0030] In regard to any of the methods, the free 5’ end of the antibody barcode is in a pendant format and not directly attached to the detection antibody before the exposing the spatially barcoded surface under hybridization conditions to the linker oligonucleotide,

[0031] In regard to any of the methods, the antibody barcode corresponds to the target molecule and the target molecule is a predetermined target molecule.

[0032] In regard to any of the methods, the antibody barcode may include a second primer.

[0033] In regard to any of the methods, the target molecule is a protein.

[0034] In regard to any of the methods, the cleavable group can include a disulfide or a diol.

[0035] Another method for determining a location and an identity of a target molecule includes exposing the target molecule, under binding conditions, to one or more detection antibodies, wherein a detection antibody of the one or more detection antibodies is coupled to an antibodybarcode that corresponds to an epitope of the target molecule for which the detection antibody is specific, the antibody barcode having a free 5’ end. A spatially barcoded surface can be exposed, under hybridization conditions, to a linker oligonucleotide. A free 5’ end region of the linker oligonucleotide hybridizes to the free 5’ end region of the antibody barcode. A free 3’ end region of the linker oligonucleotide hybridizes to a free 3’ end region of a spatial barcode on the spatially barcoded surface. The spatially barcoded surface is part of a channel and the spatially barcoded surface comprises (i) one or more spatial barcodes that include free 3’ ends, and (ii) the target molecule that is adsorbed onto the spatially barcoded surface. The 3’ end of the spatial barcode can be joined to the 5’ end of the antibody barcode.

[0036] In regard to any of the methods, the joining the 3’ end of the spatial barcode to the 5’ end of the antibody barcode can include extending the 3’ end of the spatial barcode to the 5’ end of the antibody barcode to form an extended spatial barcode; and ligating a 3’ end of the extended spatial barcode to the 5’ end of the antibody barcode.

[0037] Another method of measuring a secreted biomolecule from a cell includes flowing a mixture of polymer precursors and cells into a channel of a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a second handle. One or more gel chambers can be synthesized enclosing each of the one or more cells disposed in the channel. The cells enclosed in the one or more gel chambers can be incubated so that the cells output the secreted biomolecules. The secreted biomolecules can be captured in the one or more gel chambers, wherein the secreted biomolecule binds with a capture antibody. A detection antibody can be inputted into the channel of the flow cell where the detection antibody permeates into the one or more gel chambers and also binds with the secreted biomolecule. The detection antibody is coupled to an antibody barcode via a cleavable group. The antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific. The antibody barcode includes a first handle. The detection antibody is configured to bind to the secreted biomolecules. The first handle of the spatial barcode and the second handle of the antibody barcode can be joined with a splint. The splint includes a first handle complement configured to hybridize to the first handle and a second handle complement configured to hybridize to the second handle. The first handle can be ligated to the second handle.

[0038] In regard to any of the methods, the capture antibodies can be coupled to the spatially barcoded surface.

[0039] In regard to any of the methods, the capture antibodies can be coupled to beads and the beads are included in the mixture so that the beads are flowed into the channel along with the polymer precursors and the cells.

[0040] In regard to any of the methods, the flow cell can further includes a top surface having an opposing orientation with respect to the spatially barcoded surface and the capture antibodies are coupled to the top surface. Alternatively, the spatially barcoded surface can be a top surface of the flow cell with an opposing orientation to a bottom flow cell surface where the bottom flow cell surface contains capture antibodies or another material or species configured to bind to cellular proteins. As an additional possible configuration, the capture antibodies or other material or species configured to bind to cellular proteins can be disposed on the spatially barcoded surface. While in many embodiments the spatially barcoded surface that optionally contains the capture antibodies or other material or species configured to bind to cellular proteins is a top or bottom surface of the flow cell, the spatially barcoded surface can also be a sidewall, platform, or other surface of the flow cell. The method further includes cleaving the cleavable group to release the antibody barcode before the joining the first handle end of the spatial barcode and the second handle end of the antibody barcode with the splint.

[0041] Another method of measuring a secreted biomolecule from a cell includes flowing a mixture of polymer precursors and cells into a channel of a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a third handle. One or more gel chambers can be synthesized enclosing each of the one or more cells disposed in the channel. The cells enclosed in the one or more gel chambers are incubated so that the cells output the secreted biomolecules. The secreted biomolecules are captured in the one or more gel chambers, wherein the secreted biomolecule binds with a capture antibody. A detection antibody can be inputted into the channel of the flow cell where the detection antibody permeates into the one or more gel chambers and also binds with the secreted biomolecule. The detection antibody can be coupled to an antibody barcode via a cleavable group. The antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific. The antibody barcode can include a third handle complement, wherein the third handle complement is hybridized with a blocker oligonucleotide preventing the third handle complement from hybridizing with the third handle of the spatial barcode. The detection antibody is configured to bind to the secreted biomolecules. The blocker oligonucleotide is removed from the third handle complement. The cleavable group can be cleaved and then allowing the third handle to hybridize with the thirdhandle complement. The spatial barcode can be extended to an end of the antibody barcode to form an extended spatial barcode.

[0042] In regard to any of the methods, the capture antibodies are coupled to the spatially barcoded surface.

[0043] In regard to any of the methods, the capture antibodies are coupled to beads and the beads are included in the mixture so that the beads are flowed into the channel along with the polymer precursors and the cells.

[0044] In regard to any of the methods, the flow cell further includes a top surface having an opposing orientation with respect to the spatially barcoded surface, wherein the capture antibodies are coupled to the top surface. Alternatively, the spatially barcoded surface can be a top surface of the flow cell with an opposing orientation to a bottom flow cell surface that contains capture antibodies or another material or species configured to bind to cellular proteins. The method further includes cleaving the cleavable group to release the antibody barcode before the allowing the third handle to hybridize with the third handle complement.

[0045] Another method of measuring a secreted biomolecule from a cell includes flowing a mixture of polymer precursors and cells into a channel of a flow cell. The flow cell includes a spatially barcoded surface, wherein the spatially barcoded surface includes a plurality of spatial barcodes, each of the spatial barcodes including a third handle hybridized with a blocker oligonucleotide preventing the third handle from hybridizing with a third handle complement. One or more gel chambers can be synthesized to enclose each of the one or more cells disposed in the channel. The cells enclosed in the one or more gel chambers are incubated so that the cells output the secreted biomolecules. The secreted biomolecules are captured in the one or more gel chambers, wherein the secreted biomolecule binds with a capture antibody. A detection antibody can be inputted into the channel of the flow cell where the detection antibody permeates into the one or more gel chambers and also binds with the secreted biomolecule. The detection antibody is coupled to an antibody barcode via a cleavable group. The antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific. The antibody barcode includes a third handle complement. The detection antibody is configured to bind to the secreted biomolecules. The blocker oligonucleotide can be removed from the third handle of the spatial barcode. The cleavable group can be cleaved to release the antibody barcode and then allowing the third handle to hybridize with the third handle complement. The spatial barcode can be extended to an end of the antibody barcode to form an extended spatial barcode.

[0046] In regard to any of the methods, the capture antibodies are coupled to the spatially barcoded surface.

[0047] In regard to any of the methods, the capture antibodies are coupled to beads and the beads are included in the mixture so that the beads are flowed into the channel along with the polymer precursors and the cells.

[0048] In regard to any of the methods, the flow cell further includes a top surface having an opposing orientation with respect to the spatially barcoded surface, wherein the capture antibodies are coupled to the top surface, the method further comprising cleaving the cleavable group to release the antibody barcode before the allowing the third handle to hybridize with the third handle complement.

[0049] In regard to any of the methods, the secreted biomolecule is one of a protein, a cytokine, and a monoclonal antibody.

[0050] In regard to any of the methods, the capture antibody includes a cleavable group so that the capture antibody is configured to separate from a surface.BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the systems and methods described herein are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0052] FIGs. 1A-1C illustrate embodiments for analyzing the proteomes of single cells.

[0053] FIGs. 2A-2C illustrate an embodiment for linking an antibody barcode of a bound antibody with an adjacent spatial barcode.

[0054] FIG. 2D illustrates an embodiment in which antibody barcodes of two bound antibodies are separately linked to adjacent spatial barcodes, wherein a readout of the barcodes provides a proximity assay of an adsorbed protein.

[0055] FIG. 2E illustrates the embodiment of Figs. 2A-2C wherein released proteins are captured by antibodies attached to a surface.

[0056] FIGS. 3A-3B illustrate an embodiment wherein proteins are adsorbed onto bead surfaces having different physical and / or chemical characters.

[0057] FIGS. 4A-4B illustrate in greater detail instruments for detecting cells and synthesizing hydrogel chambers.

[0058] FIGS. 5A-5B illustrate a flow cell with multiple channels for use with some embodiments described herein.

[0059] FIGs. 6A-6C illustrate another embodiment for linking an antibody barcode of a bound antibody with an adjacent spatial barcode. The antibody barcode has a free 5’ phosphate end and the spatial barcode having a free 3’ end.

[0060] FIG. 6D illustrates another embodiment in which antibody barcodes of two bound antibodies are separately linked to adjacent spatial barcodes, wherein a readout of the barcodes provides a proximity assay of an adsorbed protein. Each of the antibody barcodes has a free 5’ phosphate end and each of the spatial barcode has a free 3’ end.

[0061] FIG. 6E illustrates the embodiment of FIGs. 6A-6C wherein released proteins are captured by antibodies attached to a surface.

[0062] FIG. 7A illustrates an embodiment of a spatial barcode bound to a surface where the spatial barcode has a second handle at a free 3’ end region.

[0063] FIG. 7B illustrates an embodiment of a labeled detection antibody including a detection antibody and an antibody barcode.

[0064] FIG. 7C illustrates an embodiment of a splint configured to couple an end of the spatial barcode and an end of the antibody barcode. The antibody barcode has a free 5’ end.

[0065] FIG. 7D illustrates the splint coupling of the end of the spatial barcode and the end of the antibody barcode.

[0066] FIGs. 8A-8B is a schematic illustrating a secretomic measurement where cells, polymer precursors, and beads coated with capture antibodies were inputted into a flow cell having a bottom surface coated with spatial barcodes. A splint is used for coupling an antibody barcode and a spatial barcode.

[0067] FIGs. 9A-9B is a schematic illustrating a secretomic measurement where cells and polymer precursors were inputted into a flow cell having a bottom surface coated with spatial barcodes and capture antibodies. A splint is used for coupling an antibody barcode and a spatial barcode.

[0068] FIGs. 10A-10B is a schematic illustrating a secretomic measurement where cells and polymer precursors were inputted into a flow cell having a bottom surface coated with spatial barcodes and a top surface coated with capture antibodies. A splint is used for coupling an antibody barcode and a spatial barcode.

[0069] FIG. 11A illustrates another embodiment of a spatial barcode bound to a surface where the spatial barcode has a third handle at a free 3’ end region.

[0070] FIG. 11B illustrates another embodiment of a labeled detection antibody including a detection antibody, an antibody barcode, and a blocking group.

[0071] FIG. 11C illustrates the another embodiment of the labeled detection antibody of FIG. 11B without the blocking group. The antibody barcode has a free ’ end.

[0072]

[0073] FIG. 11D illustrates a third handle complement (Z3’) of the labeled detection antibody of FIG. 11C hybridized with the third handle (Z3) of the spatial barcode of FIG. 11 A.

[0074] FIGs. 12A-12B is a schematic illustrating a secretomic measurement where cells, polymer precursors, and beads coated with capture antibodies were inputted into a flow cell having a bottom surface coated with spatial barcodes. A blocking group on the antibody barcode is used to reduce antibody barcode binding to a spatial barcode in the absence of the antigen.

[0075] FIGs. 13A-13B is a schematic illustrating a secretomic measurement where cells and polymer precursors were inputted into a flow cell having a bottom surface coated with spatial barcodes and capture antibodies. A blocking group on the antibody barcode is used to reduce antibody barcode binding to a spatial barcode in the absence of the antigen.

[0076] FIGs. 14A-14B is a schematic illustrating a secretomic measurement where cells and polymer precursors were inputted into a flow cell having a bottom surface coated with spatial barcodes and a top surface coated with capture antibodies. A blocking group on the antibody barcode is used to reduce antibody barcode binding to a spatial barcode in the absence of the antigen.

[0077] FIG. 15 illustrates the spatial barcode of FIG. 11 A bound to a surface where a third handle (Z3) is hybridized and blocked with a third handle complement (Z3’).

[0078] FIGs. 16A-16B is a schematic illustrating a secretomic measurement where cells, polymer precursors, and beads coated with capture antibodies were inputted into a flow cell having a bottom surface coated with spatial barcodes. A blocking group on the spatial barcode is used to reduce antibody barcode binding to the spatial barcode in the absence of the antigen.

[0079] FIGs. 17A-17B is a schematic illustrating a secretomic measurement where cells and polymer precursors were inputted into a flow cell having a bottom surface coated with spatial barcodes and capture antibodies. A blocking group on the spatial barcode is used to reduce antibody barcode binding to the spatial barcode in the absence of the antigen.

[0080] FIGs. 18A-18B is a schematic illustrating a secretomic measurement where cells and polymer precursors were inputted into a flow cell having a bottom surface coated with spatial barcodes and a top surface coated with capture antibodies. A blocking group on the spatialbarcode is used to reduce antibody barcode binding to the spatial barcode in the absence of the antigen.

[0081] FIG. 19 is a schematic illustrating a secretomic measurement in which cellular proteins are captured, the cellular proteins are coupled to barcoded antibodies, and the barcoded antibodies are coupled to detectable labels.DETAILED DESCRIPTION

[0082] The practice of the present disclosure may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such conventional techniques include, but are not limited to, preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, and the like. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Renault and Duchateau, Editors, Site- directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bomscheuer, Editors, Protein Engineering Handbook (Wiley-VCH, 2009); and the like. Guidance for selecting materials and components to carry out particular functions may be found in available treatises and references on scientific instrumentation including, but not limited to, Moore et al, Building Scientific Apparatus, Third Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013); and like references.

[0083] The present disclosure is directed to methods for measuring the proteomes of single cells. As used herein, “proteome” generally means a complete set of proteins expressed by a single cell. In some embodiments, the term “proteome” encompasses a complete set of proteins expressed by a single cell including their identities and their quantities or relative quantities. In some embodiments, the term “proteome” means a defined subset of proteins expressed by a single cell. In some embodiments, such a subset may be defined by a set of antibodies used to capture and / or detect proteins of the subset. The size of such a defined subset may vary widely. In some embodiments, such a subset may comprise from 2 to 100 different proteins, or from 10 to 50 different proteins, or from 10 to 20 different proteins. The term “antibody” is intended toencompass any specific binding compound; that is, a molecule (usually a macromolecule), that specifically binds to a given protein. In particular, the term “antibody” is intended to encompass antibody fragments, aptamers, and like compounds. By “specific binding” in reference to a specific binding compound (such as an antibody) means that the specific binding compound binds (under physiological conditions) solely to its intended target protein with little or no crossbinding to (i.e. cross-reaction with) other proteins of a designated subset.

[0084] In one aspect, methods described herein comprise (a) synthesizing a gel enclosure, or chamber, around each of a plurality of cells disposed on a surface of a channel, (b) lysing the enclosed cells to release proteins which are adsorbed onto the portion of the channel surface enclosed by the gel chamber, (c) depolymerizing the gel chambers, and (d) detecting with detection antibodies the adsorbed proteins at the locations of the depolymerized gel chambers. In some embodiments, methods described herein further comprise collecting optical signals from cells disposed on the surface, including the position of each cell on the surface, prior to synthesizing the gel enclosures. The term “gel enclosure” is used interchangeably with term “gel chamber.” The composition of such gel enclosures or gel chambers may vary widely, as discussed further below. In some embodiments, such gel enclosures or gel chambers comprise hydrogels.

[0085] In some embodiments, the present disclosure comprises fixing and permeabilizing cells. In some embodiments, cells are fixed and permeablilized after synthesizing chambers and before application of binding compounds specific for such intracellular targets. In some embodiments, fixing and permeabilizing is performed in place of lysing. Fixing and permeablization of cells may be carried out by conventional protocols, such as used in flow cytometry. Typically such protocols include a steps of treating cells with a fixing agent followed by a step of treating cells with a permeabilizing agent. A fixing step typically immobilizes intracellular cellular targets, while retaining cellular and subcellular architecture and permitting unhindered access of antibodies and / or hybridization probes to all cells and subcellular compartments. Wide ranges of fixatives are commercially available, and the correct choice of method will depend on the nature of the targets being examined and on the properties of the antibody and / or hybridization probes used. Fixation methods fall generally into two classes: organic solvents and cross-linking reagents. Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture. Cross-linking reagents (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Cross-linkers preserve cell structure better than organic solvents, but may reduce the antigenicity of some cell components, and require the addition of apermeabilization step, to allow access of the antibodies and / or hybridization probes to the intracellular targets. Fixing and permeabilizing steps include, but are not limited to, methanolacetone fixation (fix in cooled methanol, 10 minutes at -20°C; permeabilize with cooled acetone for 1 min at -20°C); paraformaldehyde-triton fixation (fix in 3-4% paraformaldehyde for 10-20 min; rinse with phosphate buffered saline (PBS); permeabilize with 0.5% Triton X-100 for 2-10 min); paraformaldehy de-methanol fixation (fix in 3-4% paraformaldehyde for 10-20 min; rinse with PBS; permeabilize with cooled methanol for 5-10 min at -20°C). Permeabilizing agents include, but are not limited to, detergents saponin, Triton X-100, Tween-20, NP40.Permeabilizing agents may also include proteinases, such as proteinase K, streptolysin O, and the like.

[0086] In some embodiments, methods described herein comprise synthesizing one or more gel chambers enclosing each of the one or more cells disposed on a surface of a channel; fixing and permeabilizing the cells so that intracellular proteins of each cell are accessible to detection antibodies; and detecting the intracellular proteins with the detection antibodies. In some embodiments, such one or more cells may be nonadherent cells. In some embodiments such methods further comprise collecting optical signals from the one or more cells disposed on the surface; and determining, prior to synthesizing, the position of each of the one or more cells from the optical signals.

[0087] Figs. 1A-1C illustrate one embodiment of the present disclosure. Cells (e.g. 101) are disposed on surface (102) of channel (100) formed by surfaces (102) and (103), which, for example, may be surfaces of two parallel plates of glass and / or plastic, after which cell positions are determined by detector (104). Gel enclosures, or chambers, are synthesized (108) from photo-synthesizable polymer precursors in channel (100) using light source (106). In some embodiments, detector (104) comprises a microscope and an image recognition system that is operationally associated with light source (106) to permit the positioning of gel chambers around cells (101). Detector (104) may also collect and record optical signals from cells (101) indicative of their type, state of health, function, or the like, which can be correlated with the proteomes of the cells. In some embodiments, cells may be mixed with polymer precursors (from which gels are photosynthesized) prior to being loaded together into channel (100). As described more fully below, gel chambers (e.g., 125a-d and blow-up 110) may vary widely in size and shape. For simplicity, gel chambers (125a-d and others of Fig. 1A) are illustrated as cylindrical solids, but as illustrated by blow-up (110), gel chambers may comprise walls and have non-gel interiors, as illustrated by wall (121) with thickness (116) enclosing interior space (111) with enclosed interior surface (112). In some embodiments, wall (121) extends from surface (102) to surface(103). After cells are enclosed in gel chambers, as shown in the uppermost top view of Fig. IB, they are lysed (130). Lysing may be accomplished using conventional cell lysing techniques including, but not limited to, photo-based lysing, chemical lysing, heat-based lysing, or the like. In some embodiments, cells are lysed using a chemical lysing agent that readily passes through the walls of the gel chambers. Lysis conditions (and / or reagents) may include, but are not limited to, the following: 1) cells in H2O at 96° C. for 15 min, followed by 15 min at 10° C.; 2) 200 mM KOH, 50 mM dithiotheitol, heat to 65° C. for 10 min; 3) for 4 pL protease-based lysis buffer: 1 pL of 17 pM SDS combined with 3 pL of 125 pg / mL proteinase K, followed by incubation at 37° C. for 60 min, then 95° C. for 15 min (to inactivate the proteinase K); 4) for 10 pL of a detergent-based lysis buffer: 2 pL H2O, 2 pL 10 mM EDTA, 2 pL 250 mM dithiothreitol, 2 pL 0.5% N-laurylsarcosin salt solution; 5) 200mM Tris pH7.5, 20mM EDTA, 2% sarcoyl, 6% Ficoll.

[0088] As cell walls and cell membranes are broken down by a lysing agent, proteins are released into the interior of the chambers and adsorb onto interior surfaces. In some embodiments, interior surfaces of gel chambers may be selected so that protein preferentially adsorb onto a desired interior surface. For example, in some embodiments, the plate or wall comprising surface (102) may comprise a material, such as a plasma- treated plastic, that preferentially adsorbs proteins and the plate or wall comprising surface (103) may comprise a material, such as surface-passivated glass, that resists protein adsorption. Such material selections are well-understood in the field of biosensor technology, as exemplified by the following references: Lichtenberg et al, Sensors, 19: 2488 (2019); Reimhult et al, Sensors, 15: 1635-1675 (2015); Recek et al, Molecules, 18: 12441-12463 (2013); and the like. Likewise, gel polymer precursors may be selected to form gels that minimize non-specific adsorption of proteins to gel surfaces or structures. A wide variety of materials may be used for protein adsorption surfaces including, but not limited to, a non-polar surface, a hydrophobic surface, or a hydrophilic surface. In some embodiments, a surface is formed on a plastic. In some embodiments, an oxygen plasma can be used to introduce polar functional groups to a surface and make it hydrophilic. In some embodiments, a tetrafluoromethane plasma can be used to introduce non-polar functional groups to a surface and make it hydrophobic. In some embodiments, a surface may be part of a commercially available protein adsorbing material, such as MaxiSorp® material (ThermoFisher Scientific).

[0089] In some embodiments, the surface may have attached one or more capture antibodies specific for selected proteins. In some embodiments, such selected proteins may be intracellular proteins. In other embodiments, such selected proteins may be cell membrane proteins, or bothintracellular proteins and cell membrane proteins. As used herein, the term “adsorption” includes the specific binding of proteins to antibodies attached to a surface. A wide variety of methods are available for immobilizing or covalently bonding antibodies to surfaces, e.g. reviewed in Trilling et al, Analyst, 138: 1619-1627 (2013); Gao et al, Analytica Chimica Acta, 1189: 338907 (2022).

[0090] In some embodiments, after released proteins are adsorbed onto the desired surface(s), walls (e.g. 134) of gel chambers are depolymerized to enhance access of detection antibodies to the adsorbed proteins. In other embodiments, after released proteins are adsorbed onto the desired surface(s), walls of gel chambers are left intact and wherein the porosity of the gel walls is selected to permit ready access of detection antibodies. Optionally, as illustrated in Fig. 1C, the distribution of adsorbed proteins may be modified by one or more steps of de-adsorption (136) that allow the de-adsorbed proteins to diffuse throughout the interior of their gel chamber to form a layer of more uniform density (138). After such modification, gel chambers may be depolymerized as described above and the adsorbed proteins exposed to detection antibodies (140). Such de-adsorption steps may be implemented by treating adsorbed proteins with heat and / or de-adsorption agents, such as, a non-ionic detergent, like for example, Brij, Triton, Tween, or the like. In some embodiments, de-adsorption may be implemented by increasing the ionic strength of the reaction mixture.

[0091] In some embodiments, adsorbed proteins may be interrogated by one or more panels of detection antibodies constructed from commercially available panels, for example, available from companies, such as Bio-Techne Corp. (Minneapolis, MN); R&D Systems, Inc. (Minneapolis, MN); and like suppliers.

[0092] In some embodiments, adsorbed proteins may be detected by the embodiment illustrated in Figs. 2A-2C. Protein (200) adsorbed on surface (201) is specifically bound by antibody (203) which is linked to the 5’ end of oligonucleotide (205) by cleavable bond (210). Adjacent to its 5’ end, oligonucleotide (205) comprises in order from the 5’ end first primer binding site P7 (which, for example, may be the same or different than the Illumina, Inc. designated primer binding site) and barcode region (“BC1”) (212). Barcode region BC1 (212) comprises a sequence that uniquely identifies the specific target epitope of antibody (203). Surface (201) further comprises (i) second primer binding site oligonucleotides P5 (206)(vertical gray bars, which may be the same or different than the Illumina, Inc., designated primer), (ii) complements to the first primer binding site oligonucleotides P7 (i.e. P7’)(214)(vertical striped bars, which may be the same or different than the Illumina, Inc. designated oligonucleotide), and (iii) oligonucleotide strands (205) attached to surface (201) by its 3’ end, having (in order) second primer binding sitesequence (206), P5, barcode “BC2” at its 3’ end (216), and a 5’- phosphate. Barcode region “BC2” (216) comprises a sequence that uniquely identifies a particular region of, or spatial position on, surface (201). After antibody (203) binds to protein (200), the resulting complex is exposed (232) to linker oligonucleotide (230) that hybridizes to both the 3’ end of oligonucleotide (202) and the 5’ end of surface bound oligonucleotide strand (205). After such hybridization, the 3’ end of oligonucleotide (202) is extended (234) by an appropriate polymerase in the presence of deoxynucleoside triphosphates and ligated to the 5’ end of oligonucleotide strand (205). After ligation, linker oligonucleotide (230) may be washed away and cleavable bond (210) cleaved to give strand (235) bound to surface (201), which may be amplified by bridge amplification using the P5 second primer binding site (206) and P7’complement of the first primer binding site (P7’, 214) on surface (201), shown through its first few cycles in Fig 2C. The resulting amplicons may be sequenced directly (in situ) or cleaved from surface (201) and sequenced by a separate sequencing instrument.

[0093] In some embodiments, surface (201) may comprise capture antibodies (281) as illustrated in Fig. 2E, either with the P5 second primer binding site and the complement of the first primer binding site P7’ , or without. After capture of protein (200), detection antibody (282) may be added and bound to protein (200) after which it may be used to generate a signal, which may be optical, e.g. fluorescent, or physical, such as, an encoded nucleic acid as described above, or a mixture of the two, e.g. multiplex FISH decoder probes. Multiplex fluorescent in situ hybridization probes (and related decoder probes) are well known to those of skill in the art as evidenced by the following references, which are incorporated herein by reference: Cai et al, U.S. patent publication US2015 / 0267251; Gunderson et al, U.S. patent publication US2003 / 0096239; Liehr et al, Histol.Histopathol., 19: 229-237 (2004); Gunderson et al, Genomics Research, 14: 870- 877 (2004); Kramer, US. Patent 7771949; Bayani et al, Curr. Protocols in Cell Biology, 24:22.5:22.5.1-22.5.25 (2004); Anderson, chapter 6, Methods in Molecular Biology, vol. 659: 83- 96 (2010); and the like.

[0094] In some embodiments of the above, proteins (or other target molecules) may be detected on a spatially barcoded surface by the following steps: (a) adsorbing proteins onto the spatially barcoded surface, wherein spatial barcodes on the surface comprise free 5 ’-phosphates, (b) exposing the spatially barcoded surface under binding conditions to detection antibodies, each detection antibody comprising an antibody barcode that identifies an epitope for which the detection antibody is specific, wherein the antibody barcode comprises a free 3’ end; (c) exposing the spatially barcoded surface under hybridization conditions to a linker oligonucleotide whose 3’ end is configured to hybridize to the free 3’ end of the antibodybarcode and the free 5’ end of a spatial barcode on the surface; (d) extending the 3’ end of the antibody barcode to the 5’ end of the spatial barcode; and (e) ligating the 3’ end of the extended antibody barcode to the 5’ end of the spatial barcode. In some embodiments, the 5’ end of the antibody barcode may be cleaved from the detection antibody. In some embodiments, the location and identity of the protein may be determined from the sequences of the extended 3’ end of the antibody barcode and the spatial barcode. In some embodiments, the extended 3’ end of the antibody barcode and the spatial barcode may be amplified. In some embodiments, the extended 3’ end of the antibody barcode and the spatial barcode may be amplified to form clusters that are sequenced using a sequencing-by-synthesis technique. In some embodiments, the extended 3’ end of the antibody barcode and the spatial barcode may be amplified by bridge amplification.

[0095] More specific and sensitive measurements of selected adsorbed proteins may be made using the embodiment of Fig. 2D, wherein two antibodies are used in accordance with the embodiment of Figs. 2A-2C, but the antibodies are specific for separate epitopes on the protein. Since the antibody -identity barcodes will be linked to the same spatial barcode, the embodiment provides the same information as a proximity assay that directly links two antibodies using oligonucleotide hybridization. Referring to Fig. 2D, protein (250) is adsorbed onto surface (252) which comprises P5 second primer binding site (254), complements of the first primer binding site (256, P7’), and oligonucleotide strands (258 and 259) comprising from its 3’ end: a second primer binding site (P5), spatial barcode, BC2, and a 5 ’-phosphate to enabling ligation. After antibodies (260 and 262) bind to epitope 1 (264) and epitope 2 (266), respectively, linker oligonucleotide (268) is added under conditions where the 3’ end of the linker oligonucleotides form duplexes with the 3’ ends of oligonucleotides (261 and 263) of antibodies (260 and 262, respectively) and with the 5’ ends of surface-bound strands (258 and 259). After extension of the 3’ ends of oligonucleotides (261 and 263), ligation of the extended oligonucleotides to strands (258 and 259) and cleaving the cleavable bond to release the antibodies, the resulting oligonucleotide may be amplified and sequenced as described above to identify the two antibodies and the location of the protein they were bound to.

[0096] An embodiment in which released proteins may be adsorbed onto a plurality of different surfaces is illustrated in Figs. 3A-3B. An instrumental arrangement similar to that of Figs. 1 A- 1B is provided, except that at least one surface of a channel has disposed thereon a plurality of different beads each comprising a different material that has different surface properties, for example, hydrophobic, hydrophilic, negatively charged, positively charged, or the like. The different bead types may be identified by fluorescent markers (e.g. as with beads available fromBioLegends (San Diego)). Surface (350) of channel (351) in the top panel of Fig. 3 A represents a surface covered with a closely packed layer of beads, each with different surface properties. This is further illustrated by blow-up (344) of surface (350) where the different bead types are illustrated with different patterns (striped, spotted, solid dark, solid gray). Second surface (353) may be the same as that for the embodiment of Figs. 1 A-1C; namely, a surface of a glass or plastic plate or wall. The functions and operation of detector (345) and light source (347) are as described above. After synthesis (346) of gel chambers, e.g. 325a-i, cells are lysed (349) to release cellular proteins which are adsorbed onto the various bead types enclosed in the chambers along with each cell. After a predetermined incubation time, gel chambers are depolymerized and detection antibodies are loaded into channel (351) where they specifically bind to their target proteins adsorbed onto the surfaces of the different bead types (which for simplicity are shown as clusters (e.g. 360, 362, 364 and 366) of solid spots (e.g. 357), where the locations of the clusters correspond to the locations of the chambers). Each protein will bind to the different surfaces of the beads with a characteristic pattern; namely (for example), a hydrophobic protein will have more representation on a hydrophobic bead surface than on a hydrophilic bead surface, and likewise for other types of bead surfaces. In some embodiments, for each protein, signals may be integrated over each bead type. The results may be displayed as an intensity profile for each protein on each different surface, as illustrated in Fig. 3B.

[0097] FIGs. 6A-6C illustrate another embodiment for linking an antibody barcode region (BC1 or 212) of a bound antibody (203) with an adjacent spatial barcode (216). The antibody barcode (212) of oligonucleotide (202) having a free 5’ phosphate end and the spatial barcode (216) of oligonucleotide (205) having a free 3’ end. The spatially barcoded surface (201) includes a plurality of spatial barcodes (216), each having a second primer binding site (P5). In addition, the spatially barcoded surface (201) also includes a first primer binding site complement (P7’). A method for determining a location and an identity of a target molecule is described. The target molecule can be a protein, antigen, or antibody. The target molecule (200) can be adsorbed onto a spatially barcoded surface (201) and to a detection antibody (203), under binding conditions for the detection antibody (203), as illustrated in FIG. 6A. The detection antibody (203) is coupled to an antibody barcode (212) via a cleavable group (210). The antibody barcode has a free 5’ end is in a pendant format and the free 5’ end is not directly attached to the detection antibody. It should be noted that P-5’ represents a phosphate on the 5’ end. The antibody barcode (212) corresponds to an epitope of the target molecules for which the detection antibody (203) is specific. The detection antibody (203) is configured to bind to the epitope. Since the epitope for the detection antibody is known beforehand, the antibody barcode (212) represents a code thatcan be measured for determining the presence and / or amount of the known epitope in the sample or flow cell.

[0098] The spatially barcoded surface includes a plurality of spatial barcodes (216), where each spatial barcode is bound to the surface at a 5’ end and has a free 3’ ends. The spatially barcoded surface (201) can be exposed under hybridization conditions to a linker oligonucleotide (230) that is illustrated as an outcome of step (232) in FIG. 6A. A free 5’ end region of the linker oligonucleotide (230) hybridizes to the free 5’ end region of the antibody barcode (212) and a free 3 ’ end region of the linker oligonucleotide (230) hybridizes to a free 3 ’ end region of a spatial barcode (216) on the spatially barcoded surface (201). Once the linker oligonucleotide (230) has hybridized to both of the antibody barcode and spatial barcode, the 3’ end of the spatial barcode can be extended to the 5’ end of the antibody barcode to form an extended spatial barcode. After the extension, a 3’ end of the extended spatial barcode can be ligated to the 5’ end of the antibody barcode that is illustrated as an outcome of step (234) in FIG. 6A.

[0099] Subsequent to the extension and ligation of step 234, the detection antibody (203) can be released from the target molecule (200) and then the cleavable bond (210) can be cleaved causing strand (235) to be coupled to the surface though the spatial barcode (216), that is illustrated at the top and middle parts of FIG. 6B. In an alternative method, the detection antibody (203) can remain attached to the target molecule (200) and the cleavable bond (210) is cleaved causing strand (235) to be coupled to the surface though the spatial barcode (216). An upper end of strand (235) includes the first primer binding site (P7), as illustrated in the middle part of FIG. 6B, that can bind to a primer complement (P7’) and then undergo bridge amplification step (235), as shown in bottom portion of FIG. 6B. Through the amplification, numerous strands containing both the spatial barcode (BC2 or 216) and the antibody barcode (BC1 or 212) can be generated as exemplified in the steps of FIG. 6C. Complement strands containing complements of the first and second barcodes (BC1’, BC2’), the first primer binding site (P7), and a complement of the second primer binding site (P5’) can also be generated. The resulting amplicons can be sequenced in situ directly on the surface or cleaved from surface (210) and sequenced by a separate instrument.

[0100] In various embodiments, before exposing the target molecules adsorbed onto the spatially barcoded surface, a mixture of polymer precursors and cells can be disposed into a channel of a flow cell. An image can be taken of the flow cell to identify the location of one or more cells of interest disposed in the channel. Optical signals from the one or more cells disposed in the channel can be collected at a detector, prior to the synthesizing the gel chambers,that allows the position of each of the one or more cells to be determined based on the optical signals.

[0101] In some instances, one or more cells of interest will be proximate to a barcoded region of the spatially barcoded surface. One or more gel chambers enclosing each of the one or more cells disposed in the channel can be synthesized such as by irradiating a patterned ring of light around the one or more cells. The cells can then be lysed so that the target molecules of each cell are released into the chamber and adsorbed onto the spatially barcoded surface. A lysing agent can be flowed into the cell where the lysing agent permeates through a wall of the gel chamber so that the cells lyse and the intracellular proteins can adsorb onto the surface and detection antibodies can bind to the adsorbed target proteins. In various embodiments, the adsorbed target molecules can be de-adsorbed prior to the depolymerizing the gel chambers. The one or more cells of interest can be disposed on the spatially barcoded surface; suspended (e.g., within a fluid within a fluidic device channel) above, below, or adjacent to the spatially barcoded surface; or may be disposed on an additional surface opposing the spatially barcoded surface. For example, the one or more cells may be disposed on a bottom planar surface that opposes a top spatially barcoded surface within a fluidic device.

[0102] In various embodiments, the gel chambers can be depolymerized before binding the detection antibody to the target molecule, antigen, or protein. Gel chambers can be depolymerized under certain instances with a thiol reagent to break disulfide bonds or with sodium periodate to break diol bonds. Gel chambers may also be depolymerized using photocleavage, for example by photolyzing a photoinitiator to perform radical cleavage on a gel chamber.

[0103] In various embodiments, the spatially barcoded surface comprises particles of a plurality of types disposed thereon, wherein each type of particle comprises a surface with different protein adsorption characteristics, and wherein particles of each type have a size and a quantity such that each of the hydrogel chambers encloses particles of every type. In some cases, the particles are functionalized with capture antibodies that are configured to bind to cellular proteins. Each particle may be functionalized with a single type of antibody so that each particle binds to a single molecule (e.g., cellular protein), and more specifically to a single epitope of the single molecule, from a complex mixture such as cell lysate. Alternatively, a particle can be coupled to two or more types of antibodies that bind to different molecules or different epitopes of the same molecule. For example, an assay may utilize (i) multiple beads that each includes a different antibody configured to capture a different cellular protein, and (ii) multiple detection antibodies that are separately targeted to each of the cellular proteins targeted by the beads,wherein each detection antibody is coupled to an antibody barcode that corresponds to an epitope of the target molecule for which the detection antibody is specific.

[0104] In various embodiments, the location of the target molecule is determined from a nucleotide sequence of the spatial barcode (216) and the identity of the target molecule is determined from a nucleotide sequence of the antibody barcode (212). The nucleotide sequence of the spatial barcode (216) can be in a look-up table designed during the set-up for making spatial barcoded surface (201). Thus, once the spatial barcode is sequenced, it can be correlated with the look-up table for determining the location of the target molecule. Various nucleotide sequences of the spatial barcodes are designed and then deposited on predetermined areas of the flow cell. Similarly, the nucleotide sequence of the antibody barcode (212) can be in a look-up table designed during the synthesis of the detection antibody coupled to the antibody barcode (212). A detection antibody with a known specificity for a particular epitope can be coupled with a predetermined antibody barcode and then recorded in the look-up table. Thus, once the antibody barcode is sequenced, it can be correlated with the look-up table for determining the identity of the target molecule in a particular gel chamber.

[0105] FIG. 6D illustrates another embodiment in which antibody barcodes (BCla and BClb) of two bound antibodies (260 and 262) are separately linked to adjacent spatial barcodes (258 and 259). A readout of the barcodes provides a proximity assay of an adsorbed protein. Each of the antibody barcodes has a free 5’ phosphate end and each of the spatial barcodes has a free 3 ’ end. In various embodiments, two or more detection antibodies can be used to identify two or more types of target molecules (e.g., epitope 1 and epitope 2) along with the location of the two target molecules in a flow cell.

[0106] FIG. 6E illustrates the embodiment of FIGs. 6A-6C wherein released proteins are captured by antibodies attached to a surface. The spatially barcoded surface (201) includes a capture antibody (281), spatial barcodes (BC2), a second primer binding site oligonucleotide (P5), and a complement to the first primer oligonucleotide P7 (referred to as P7’). A protein molecule (200) is bound to surface capture antibody (281). A detection antibody coupled to an antibody barcode (BC1) can then be added to the flow cell to bind to protein molecule (200) to form a sandwich format. Next, an oligonucleotide linker can be used in a way similar to FIGs. 6A-6C

[0107] FIG. 7A illustrates an embodiment of a spatial barcode (700) bound to a surface (708) where the spatial barcode has a second handle (Z2 or 702) at a free 3’ end region, a second barcode (BC2 or 704), and a second primer (R2 or 706). The spatial barcode (700) can be boundto the surface (708) via a cleavable bond (728). An example of a cleavable bond can be a uracil where a user enzyme can be used to cleave the spatial barcode (700) from the surface.

[0108] FIG. 7B illustrates an embodiment of a labeled detection antibody (710) including a detection antibody (726) and an antibody barcode (724). The antibody barcode (724) can be bound to the detection antibody (726) via a cleavable bond (730). The antibody barcode (724) can include a first handle (Z1 or 712) at a free 5’ end region, a first barcode (BC1 or 714), and a first primer (R1 or 716). It should be noted that P-5’ represents a phosphate on the 5’ end.

[0109] FIG. 7C illustrates an embodiment of a splint (718) configured to couple an end of the spatial barcode (700) and an end of the antibody barcode (710). The split includes an oligonucleotide sequence that contains a first handle complement (Zl’ or 720) and a second handle complement (Z2’ or 722). The first handle complement (720) is configured to bind to the first handle (712) and the second handle complement (722) is configured to bind to the second handle (702).

[0110] FIG. 7D illustrates the splint (718) coupling the end of the spatial barcode (700) and the end of the antibody barcode (710).

[0111] FIGS. 8-18 depict methods for detecting cellular proteins in fluidic devices with barcoded antibodies, spatially barcoded surfaces, and optional capture agents. While these figures depict the spatially barcoded surfaces as bottom surfaces of fluidic devices, spatial barcodes and capture agents may be disposed on top or bottom surfaces of fluidic devices.

[0112] A method of measuring a secreted biomolecule from a cell can include flowing a mixture of polymer precursors, beads coated with capture antibodies, and cells into a channel of a flow cell (800), as illustrated in FIG. 8A. The flow cell includes a top layer (802) and a bottom layer (804). The bottom layer (804) can include a spatially barcoded surface having a plurality of spatial barcodes (700), each having a second handle (Z2). One or more gel chambers can be synthesized to enclose each of the one or more cells disposed in the channel. The gel chamber can be formed from polymerized polymer precursors to form walls (810). As illustrated in FIG. 8A, the walls (810) of the gel chamber encloses a cell (806) and a bead (808) coated with capture antibodies (809). The cell (806) is enclosed in the gel chamber and outputs a secreted biomolecules that can be a protein such as a cytokine or an antibody. The secreted biomolecules or antigen (814) in the gel chamber can follow a secretion pathway (812) and then bind with a capture antibody (809). A labeled detection antibody (710) is inputted into the channel of the flow cell where the labeled detection antibody permeates into the gel chamber and binds with the secreted biomolecule to form a sandwich format. The detection antibody (726) is coupled to an antibody barcode (724) via a cleavable group (730). The antibody barcode (724)corresponds to an identity of the secreted biomolecules for which the detection antibody (726) is specific. The antibody barcode (724) includes a first handle (Zl) and the detection antibody (726) is configured to bind to the secreted biomolecules. After the labeled detection antibody (710) has bound to the antigen (814), the unbound labeled detection antibody (710) can be washed out of the flow cell. A splint and cleaving agent can be added into the flow cell to cleave off the antibody barcode (724) and to join the second handle (Z2) of the spatial barcode (700) and the first handle (Zl) of the antibody barcode (724) with a splint (718), as illustrated in FIG. 8B. The splint (718) includes a first handle complement (Zl’) to a first handle (Zl) of the antibody barcode and a second handle complement (Z2’) to a second handle (Z2) of the spatial barcode. After splint (718) binds to the first handle (Zl) and to the second handle (Z2), the first handle (Zl) and the second handle (Z2) can be ligated together, and then the splint (718) can be optionally denatured off the ligated moiety. The splint (718) may be extended to an end of the antibody barcode (724) to form an extended splint; and then the first handle (Zl) is ligated to the second handle (Z2).

[0113] In various embodiments, the capture antibodies (809) are coupled to the spatially barcoded surface, as illustrated in FIG. 9A. The antibody barcode (724) does not have to be cleaved from the detection antibody (726) when using capture antibodies (809) coupled to the spatially barcoded surface. The antibody barcode (724) can be joined with the spatial barcode (700) using the splint (718) while the detection antibody (726) remains attached to antigen, as illustrated in FIG. 9B.

[0114] In another embodiment, the flow cell (800) further includes a top surface (802) with an opposing orientation with respect to the bottom surface (804) that is the spatially barcoded surface. The capture antibodies (809) are coupled to the top surface (802) and the spatial barcodes (700) are coupled to the bottom surface, as illustrated in FIG. 10A. The cleavable group (730) is cleaved from the labeled detection antibody (710) before the joining of the second handle of the spatial barcode (700) and the first handle of the antibody barcode (724) with the splint (718) , as illustrated in FIG. 10B.

[0115] FIG. 11A illustrates another embodiment of a spatial barcode (1100) bound to a surface (708) where the spatial barcode (1100) can include a third handle (Z3 or 1102) at a free 3’ end region, a second barcode (BC2 or 704), and a second primer (R2 or 706). The spatial barcode (700) can be bound to the surface (708) via a cleavable bond (728). An example of a cleavable bond can be a uracil where a user enzyme can be used to cleave the spatial barcode (700) from the surface.

[0116] FIG. 11B illustrates an embodiment of a labeled detection antibody with blocking group (1104A). Antibody barcode with blocking group (1108A) includes a third handle complement (Z3’ or 1106) at a 3’ end, a first barcode (BC1 or 714), and a first primer (R1 or 716) at a 5’end. Blocking group (1102) hybridizes to the third handle complement (Z3’). The antibody barcode with blocking group (1108A) can be bound to the detection antibody (726) via a cleavable bond (730).

[0117] FIG. 11C illustrates another embodiment of the labeled detection antibody of FIG. 11B without the blocking group (1104B). The antibody barcode without the blocking group is referred to as 1108B.

[0118] FIG. 11D illustrates a third handle complement (Z3’) of the labeled detection antibody of FIG. 11C hybridized with a third handle (Z3) of the spatial barcode of FIG. 11 A.

[0119] A method of measuring a secreted biomolecule from a cell can include flowing a mixture of polymer precursors, beads coated with capture antibodies, and cells into a channel of a flow cell (800), as illustrated in FIG. 12A. The flow cell includes a top layer (802) and a bottom layer (804). The bottom layer (804) can include a spatially barcoded surface having a plurality of spatial barcodes (700), each having a third handle (Z3). One or more gel chambers can be synthesized to enclose each of the one or more cells disposed in the channel. The gel chamber can be formed from polymerized polymer precursors to form walls (810). As illustrated in FIG. 12A, the walls (810) of the gel chamber encloses a cell (806) and a bead (808) coated with capture antibodies (809). The cell (806) is enclosed in the gel chamber and outputs a secreted biomolecules that can be a protein such as a cytokine or an antibody. The secreted biomolecules or antigen (814) in the gel chamber can follow a secretion pathway (812) and then bind with a capture antibody (809). A labeled detection antibody with blocking group (1104A) is inputted into the channel of the flow cell where the labeled detection antibody with blocking group (1104A) permeates into the gel chamber and binds with the secreted biomolecule to form a sandwich format. The detection antibody (726) is coupled to an antibody barcode with blocking group (1108A) via a cleavable group (730). The first barcode (714) corresponds to an identity of the secreted biomolecules for which the detection antibody (726) is specific. The antibody barcode without blocking group (1108B) includes a third handle complement (Z3’) and the detection antibody (726) is configured to bind to the secreted biomolecules. After the labeled detection antibody (1104A) has bound to the antigen (814), the unbound labeled detection antibody (1104A) can be washed out of the flow cell. A cleaving agent can be added into the flow cell to cleave off the antibody barcode (724) and the blocking group can be removed to hybridize the third handle (Z3) of the spatial barcode (1100) and the third handle complement(Z3’) of the antibody barcode (1108B), as illustrated in FIG. 12B. The spatial barcode (1100) is extended to an end of the antibody barcode (1108B) to form an extended spatial barcode.

[0120] In various embodiments, the capture antibodies (809) are coupled to the spatially barcoded surface, as illustrated in FIG. 13A. The antibody barcode without blocking group (1108B) does not have to be cleaved from the detection antibody (726) when using capture antibodies (809) coupled to the spatially barcoded surface. The antibody barcode without blocking group (1108B) can be hybridized with the spatial barcode (700) while the detection antibody (726) remains attached to antigen, as illustrated in FIG. 13B.

[0121] In another embodiment, the flow cell (800) further includes a top surface (802) with an opposing orientation with respect to the bottom surface (804) that is the spatially barcoded surface. The capture antibodies (809) are coupled to the top surface (802) and the spatial barcodes (700) are coupled to the bottom surface, as illustrated in FIG. 14A. The blocking group is removed and the cleavable group (730) is cleaved from the labeled detection antibody without blocking group (1104A) before the hybridization of the third handle of the spatial barcode (1100) and the third handle complement of the antibody barcode without the blocking group (1104B), as illustrated in FIG. 14B.

[0122] FIG. 15 illustrates another embodiment of a spatial barcode with a blocking group (1100A) bound to a surface (708). The spatial barcode with a blocking group (1100A) can include a third handle (Z3 or 1102) at a free 3’ end region, a second barcode (BC2 or 704), and a second primer (R2 or 706). The spatial barcode with a blocking group (1100A) can be bound to the surface (708) via a cleavable bond (728). An example of a cleavable bond can be a uracil where a user enzyme can be used to cleave the spatial barcode with blocking group (1100A) from the surface. It should be noted that the spatial barcode without blocking group is referred to as 1100. The blocking group can have the same functionality as the third handle complement (Z3’).

[0123] A method of measuring a secreted biomolecule from a cell can include flowing a mixture of polymer precursors, beads coated with capture antibodies, and cells into a channel of a flow cell (800), as illustrated in FIG. 16A. The flow cell includes a top layer (802) and a bottom layer (804). The bottom layer (804) can include a spatially barcoded surface having a plurality of spatial barcodes with blocking groups (1100A), each having a third handle (Z3) and third handle complement (Z3’) functioning as a blocking group. One or more gel chambers can be synthesized to enclose each of the one or more cells disposed in the channel. The gel chamber can be formed from polymerized polymer precursors to form walls (810). As illustrated in FIG. 16A, the walls (810) of the gel chamber encloses a cell (806) and a bead (808) coatedwith capture antibodies (809). The cell (806) is enclosed in the gel chamber and outputs secreted biomolecules that can be a protein such as a cytokine or an antibody. The secreted biomolecules or antigen (814) in the gel chamber can follow a secretion pathway (812) and then bind with a capture antibody (809). A labeled detection antibody (1104B) is inputted into the channel of the flow cell where the labeled detection antibody (1104B) permeates into the gel chamber and binds with the secreted biomolecule to form a sandwich format. The detection antibody (726) is coupled to an antibody barcode (1108B) via a cleavable group (730). The first barcode (714) corresponds to an identity of the secreted biomolecules for which the detection antibody (726) is specific. The antibody barcode (1108B) includes a third handle complement (Z3’) and the detection antibody (726) is configured to bind to the secreted biomolecules. After the labeled detection antibody (1104B) has bound to the antigen (814), the unbound labeled detection antibody (1104B) can be washed out of the flow cell. Next, the blocking group is removed and a cleaving agent is added into the flow cell to cleave off the antibody barcode (1108B) and the blocking group can be removed to hybridize the third handle (Z3) of the spatial barcode (1100) and the third handle complement (Z3’) of the antibody barcode (1108B), as illustrated in FIG. 16B. The spatial barcode (1100) is extended to an end of the antibody barcode (1108B) to form an extended spatial barcode.

[0124] In various embodiments, the capture antibodies (809) are coupled to the spatially barcoded surface, as illustrated in FIG. 17A. The antibody barcode (1108B) does not have to be cleaved from the detection antibody (726) when using capture antibodies (809) coupled to the spatially barcoded surface. The antibody barcode (1108B) can be hybridized with the spatial barcode without blocking group (1100) while the detection antibody (726) remains attached to antigen, as illustrated in FIG. 17B.

[0125] In another embodiment, the flow cell (800) further includes a top surface (802) with an opposing orientation with respect to the bottom surface (804) that is the spatially barcoded surface. The capture antibodies (809) are coupled to the top surface (802) and the spatial barcodes (1100A) are coupled to the bottom surface, as illustrated in FIG. 18A. The cleavable group (730) is cleaved from the labeled detection antibody (1104B) before the hybridization of the third handle of the spatial barcode (1100) and the third handle complement of the antibody barcode without the blocking group (1104B), as illustrated in FIG. 18B.

[0126] In some embodiments, a label may be conjugated to an antibody by a scissile or chemically cleavable bond or by hybridization to a barcode coupled to the antibody. The label can be conjugated to the antibody prior to antibody loading into a fluidic device. For example, the label may be hybridized to a barcode coupled to the antibody before the antibody is loadedinto the fluidic device. Alternatively, the label can be conjugated to the antibody after the antibody is loaded into a fluidic device and optionally after the antibody is coupled to a target molecule. In such methods, one or more detection antibodies can be incubated in a flow cell, unbound detection antibodies can be removed from the flow cell, and then the labels can be added to the flow cell under conditions that permit conjugation of the labels to the detection antibodies. The labels may then be detected, wherein local label density is proportional to detection antibody density (and thus target molecule density) within the flow cell. Alternatively, a label may be detached from a detection antibody after the detection antibody is coupled to a target molecule. The label may diffuse away from the detection antibody (which may still be affixed to the target molecule) following label detachment, such that label detachment may decrease local signal intensity at sites populated by the detection antibody.

[0127] FIG. 19 depicts a method in which labels are conjugated to a detection antibody following detection antibody binding to its target. In this example, a flow cell (800) includes capture antibodies (809) on a top surface (802) and a bottom surface (804) opposing the top surface (802). A cell (806) is enclosed within a gel chamber within the fluidic device formed from one or more polymer matrix walls (810). One or more antigens (814) secreted by the cell (806) in the fluidic device bind to the antibodies (809) on the top surface (802) of the flow cell (800). The antibodies (809) may bind a single antigen (814) or multiple different antigens (814) secreted (812) by the cell (806). A detection antibody (1104B) may then be loaded into the flow cell (800) to diffuse through the polymer matrix walls (810) to bind to the antigen or antigens (814) secreted (812) by the cell (806). The flow cell (800) may then be loaded with labels (1105) that include a barcode (1105A) that is configured to hybridize to an antibody barcode of the detection antibody (1104B) and a detectable moiety (1105B) such as a fluorophore. Upon loading into the flow cell (800), the labels (1105) diffuse through the polymer matrix walls (810) and hybridize to barcodes on detection antibodies (1104B) that are bound to antigens (814) which are coupled to antibodies (809) on the top surface (802) of the flow cell (800). The labels (1105) may then be detected.Systems and Instrumentation

[0128] An example of a system for carrying out the above method is illustrated in Fig. 4A.In some cases, flow cell (400) is a component of a fluidic device that provides one or more channels and liquid handling components under programmable control for delivering beads and reagents to the channels. In this illustration, four channels (402, 404, 406, and 408) are shown, with blow-up view (412) of segment (410) of channel 2 (404) shown below. In the abstractedview of flow cell (400) of Fig. 4A, inlets, outlets and other features of the channels are not shown. On first surface (414) of channel 2 (404) a plurality of beads, e.g. (418), can each be enclosed by a hydrogel chamber, e.g. (416). In some embodiments, the porosity of polymer matrix walls of the hydrogel chambers is selected to be impermeable to the beads, but permeable to reagents for forming spatial barcodes. Thus, reagents may be introduced to, and removed from, the interiors of the hydrogel chambers by flowing (420) them through the channels, but beads are retained inside. Below blow-up (412) of channel segment (410) is shown an example of an optical system (421) for photosynthesizing hydrogel chambers at the locations of beads in the channels. One of ordinary skill in the art would recognize that optical systems with different configurations than those of Fig. 4A and 4B may be employed for carrying out these functions. In some embodiments, one or more DMD-objective subsystems for synthesizing hydrogel structures may be employed to increase the speed of synthesis by synthesizing multiple structures simultaneously.

[0129] Returning to Fig. 4A, for photosynthesizing the hydrogel chambers, light source (422) can generate a light beam (423) of appropriate wavelength light (e.g. UV light) that passes through an appropriate photo-mask or beam-shaping or beam steering (Galvo) system for shaping a beam to synthesize a desired structure or structures in a channel. In some embodiments, a digital micromirror device (DMD)(424) is employed, in other embodiments, a physical photomask may be employed. Chamber position, shape and polymer matrix wall thickness can be determined at least in part from bead position information determined from images collected by detector (432). Reflected light from DMD (424) can be shaped using conventional optics, e.g. collimating optics (428), and can be directed through objective lens system (434) into channel 2 segment (410). Objective (434) and flow cell (400) may move relative to one another in the xy- directions (436) to photosynthesize chambers at any position in any of the channels. In some embodiments, flow cell (400) moves and optical system (421) is stationary. In some embodiments, objective (434) may also direct light beam (427) from light source (429) to targets, such as cells, on first surface (414) and collect optical signals, such as fluorescent signals, from assays taking place on first surface (414). Alternatively, optical signal collection may be carried out with a separate objective as shown if Fig. 4B. Information collected by detector (432), or its counterpart in the embodiment of Fig. 4B, particularly cellular positions in their respective channels, is employed by computer (438) and / or subsidiary controllers to direct DMD (424) and translation devices controlling the relative positions of objective (434) and flow cell (400) to synthesize hydrogel chambers of the appropriate shape and size at the appropriate locations.

[0130] Fig. 4B illustrates an alternative optical system in which the detection portion (450) of the optical system moves (472) independently from the movement (468) of the synthesis portion (452) of the optical system. Detection portion (450) of the optical system may comprise detector (456), objective (458), light source (460) and interconnecting optical elements, such as dichroic mirror (462). As with the embodiment of Fig. 4A, detector (456) can be operationally associated with computer (464) and the synthesis portion (452) of the optic system to provide synthesis portion (452) with bead position information. Computer (464) and (438) can also be operationally associated with stages and / or motors controlling the relative positions of the objectives of the optical systems and the position of the flow cell. In this embodiment, synthesis portion (452) of the optical system is located on the other side of first surface (464) from detection portion (450). As with the embodiment of Fig. 4A, the synthesis portion (452) comprises the conventional components objective (474), mirror (476), collimating optics (480), DMD (482) and light source (478).

[0131] In some embodiments, systems for implementing the methods described herein comprise (i) a channel comprising a surface, (ii) a spatial energy modulation element in optical communication with the surface, and (iii) a detector in optical communication with the surface and in operable association with the spatial energy modulating element, the detector identifying cells and determining positions thereof on the surface. It is understood that the term “detector” as used herein may include, but not be limited by, a microscope element that collects and optionally magnifies an image of a portion of a channel and an image analysis element that comprises software for identifying cells and associated position information. A computer element can use such information generated by a detector together with user input to generate commands for other elements, such as, the spatial energy modulating element to carry out a variety of functions including, but not limited to, synthesizing chambers, “on-demand” degrading of chambers, selectively photo-degrading chambers, and the like. Examples of configurations of such embodiments are illustrated in Figs. 4A-4B which are described above. In some embodiments, a channel of a fluidic device further comprises a second surface (e.g. illustrated in Fig. 2) wherein said first surface and the second surface are disposed opposite one another across the channel, and wherein the polymer matrix walls of the chambers extend from the first surface to the second surface to form chambers each having an interior. In some embodiments, chambers in a channel each enclose a single cell. In some embodiments, both the first wall and the second wall are made of optically transmissive materials, such as, glass, plastic, or the like, and are positioned so that the first surface and second surface are substantially parallel to one another. Theperpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm.

[0132] In some embodiments, a plurality of channels may be arranged together in a flow channel as illustrated in Figs. 5A-5B. In some embodiments, the plurality of channels may be in the range of from 2 to 12, or from 2 to 8, or from 2 to 6, or in the range of from 2 to 4. An example of a flow cell (500) is shown in a cross-sectional view and a top view. In some cases, flow cell (500) has bottom, or first, wall (506) with first surface (505); top, or second, wall (502) with second surface (501); and sandwiched sealingly therebetween spacer (504) whose longitudinal holes form channels 1-6, one of which is indicated by (508) in the cross-sectional view, and by (512) in the top view. In some embodiments, spacer (504) may have a thickness in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm, which determines the interior height of the channels. Top wall (502) comprises inlets (514) and outlets (516) for either separately or jointly loading and removing reagents and beads from channels 1-6. In some embodiments, at least one of walls (502) and (506) are made of light transmissive materials, such as glass, plastic, or the like. Flow cell (500) may be operationally associated with a fluidic device that delivers reagents and beads to any of channels 1-6 under programmed control. Guidance for particular designs, including fluid handling and valving for such fluidic systems may be found in U.S. patents 8921073; 8173080; 8900828; and the like, which are incorporated herein by reference. Fig. 5B illustrates channels of flow cell (500) with random distributions (not to scale) of hydrogel chambers with annulus-like cross- sections, such as (520), on their first surfaces.

[0133] As noted above, any of first surfaces, second surfaces or polymer matrix wall of chambers may comprise capture elements and other functional groups for carrying out a variety of operations including, but not limited to, capturing beads, capturing cells, capturing analytes (such as, mRNA, secreted proteins, intracellular proteins, or genomic sequences), capturing constituents of analytical reagents (such as, oligonucleotide labels from antibodies), and the like. Derivatizing surfaces for such purposes is well-known to those skilled in the art, as evidenced by the following references: Integrated DNA Technologies brochure (cited above); Hermanson (cited above); and the like.

[0134] As noted above, in some embodiments, a fluidic device of the method comprises or is operationally associated with a detector that either may share an optical path of the spatial energy modulating element or may be disposed adjacent to the second wall or opposite the first wall from the spatial energy modulating element in embodiments, such as wells, that have only a first wall and first surface. The detector is positioned so that it is capable of detecting optical signalsfrom or adjacent to cells in the channel, for example, distributed over the first surface in chambers. In some embodiments, the first and second walls each comprise optically transmissive material, for example, so that a spatial energy modulating element may project light energy to the interior of the channel, and so that a detector may detect optical signals, such as fluorescent emissions or reflected light from biological components. In some embodiments, the projected energy from the spatial energy modulating element is a light energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulating element may have a complex cross-section that permits (in various embodiments) the simultaneous synthesis of a plurality of chambers. Optically transmissive materials include, but are not limited to, glass, quartz, plastic, and like materials.

[0135] Spatial energy modulating elements using light energy for polymerization may comprise physical photomasks or virtual photomask, such as, a digital micromirror device (DMD). The following references, which are hereby incorporated by reference, provide guidance in selecting and operating a DMD for photopolymering gels: Chung et al, U.S. patent 10464307; Hribar et al, U.S. patent 10351819; Das et al, U.S. patent 9561622; Huang et al, Biomicrofluidics, 5: 034109 (2011); and the like.

[0136] As used herein, “channel” generally means a container capable of holding fluid (which may be static or flowing) and having at least one surface on which beads may be disposed and chambers synthesized. In some embodiments, a channel may have a first surface and / or a second surface on which chambers may be synthesized and / or on which beads or particles may be disposed. As used herein, reference to a “surface” without reference to “first” or “second” is intended to comprise a first surface or a second surface (if two are present in a fluidics device, e.g. comprising a flow cell). In some embodiments, a channel may constrain a flow of fluid therethrough from an inlet to an outlet. In other embodiments, a channel may comprise a nonflowing volume of fluid that may be removed, replaced or added to by way of an opening or inlet; that is, in some embodiments, a channel may be a well or a well-like structure.Gel Chambers

[0137] A wide variety of photo-synthesizable gels and degradable gels are available for implementing the methods described herein. Guidance for selecting such gels for desired properties including, but not limited to, biocompatibility, porosity, gelation speed, degradation speed, and like properties, is provided in the following references, which are incorporated by reference: Kharkar et al, Chem. Soc. Rev., 42: 7335-7372 (2013); Kharkar et al Polymer Chem., 6(31): 5565-5574 (2015); Neumann et al, Acta Biomater., 39: 1-11 (2016); DeForest et al, Nature Chemistry, 3(12): 925-931 (2012); Bowman et al, U.S. patent 9631092; LeValley et al, ACSAppl. Bio. Mater., 3(10): 6944-6958 (2020); Kabb et al, ACS Appl. Mater. Interfaces, 10: 16793-16801 (2018); Fairbanks et al, Macromolecules, 44: 2444-2450 (2011); Fairbanks et al, Adv. Mater., 21(48): 5005-5010 (2009); Sugiura et al, U.S. patent publication US2016 / 0177030; Shih et al, Biomacromolecules, 13(7): 2003-2012 (2012); and the like. In some embodiments, photo-synthesized gels are formed using a photo-initiator for radical polymerization. In some embodiments, photo-initiators comprise Irgacure 2959, Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), or Eosin-Y (e.g. see Choi et al, Biotechniques, 66(1): 40-53 (2019)). In some embodiments, hydrogel precursors comprise hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, polyethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic acid-co-glycolic acid)-b- poly(ethylene glycol)-b-poly(lactic acid-co-glycolic acid) (PLGA-PEG-PLGA), and poly(vinyl alcohol). In some embodiments, polymer precursors comprise PEG or multi-arm PEG. In some embodiments, polymer precursors comprise an enzymatically degradable cross-linker. In some embodiments, such enzymatically degradable cross-linker is degradable by an esterase or a peptidase. In some embodiments, polymer precursors comprise a photo-degradable cross-linker. In some embodiments, such photo-degradable cross-linker comprises a nitrobenzyl group.

[0138] In some embodiments, such photo-degradable cross-linker comprises a courmarin moiety. In some embodiments, photo-degradable hydrogels are used, for example, because photo-degradation of hydrogel chambers may be carried out selectively and on-demand, so that specified hydrogel chambers may be degraded without affecting non-selected hydrogel chambers are unaffected. In some embodiments, hydrogel chambers are degraded non-selectively, so that all hydrogel chambers in a given channel (or other vessel) are degraded simultaneously. In some embodiments, such non- selective degradation is carried out with a cleavage reagent that specifically cleaves a labile bond in a hydrogel. For example, such cleavage agent comprises a reducing agent. In some embodiments, such non-specific degradation is carried out with an enzyme that cleaves a bond or chemical element in a hydrogel. Chemical elements may include, but are not limited to, peptides, polysaccharides and oligonucleotides.

[0139] In the figures, for convenience, hydrogel chambers are illustrated as standing in isolation without connection with adjacent chambers and as having a cylindrical or annular-like shapes; however, a spatial energy modulating element may synthesize chambers of different shapes and sizes, as is useful for particular applications. In some embodiments of the proliferation assay, each hydrogel chamber synthesized has the same shape and area, for example, annular-like with an interior area selected from the range of 0.001 to 0.01 mm2.

[0140] Function. A wide variety of photo-synthesizable gels may be used in connection with the methods described herein. In some embodiments, hydrogels are used in particular because of their compatibility with living cells and the versatility of formulating gels with desired properties including, but not limited to, porosity, degradability, mechanical strength, ease and speed of synthesis, and the like. In some embodiments, gels or hydrogels are both photo-synthesizable and photo-degradable. In some embodiments, gel degradation mechanisms are compatible with living cells.

[0141] Porosity. In some embodiments, hydrogel porosity is selected to permit passage of selected reagents while at the same time preventing the passage of other reagents or objects, such as, a cell or proteins of a lysed cell. In some embodiments, crosslinking the polymer chains of the hydrogel structure forms a hydrogel matrix having pores (i.e., a porous hydrogel matrix). In some embodiments, the pores have an average diameter of from about 2 nm to about 25 nm, or from about 5 nm to about 20. In some embodiments, average pore diameters are selected to prevent the passage of cellular proteins. In some embodiments, average pore diameters are selected to prevent the passage of cellular proteins having a molecular weight of 1 kiloDaltons or greater. In some embodiments, average pore diameters are selected to prevent the passage of cellular proteins having a molecular weight of 5 kiloDaltons or greater. In some embodiments, the pore size of the hydrogel structures is tuned by varying the ratio of the concentrations of polymer precursors to the concentration of crosslinkers, varying pH, salt concentrations, temperature, light intensity, and the like. Guidance for selecting materials and conditions to control hydrogel porosity may be found in the following references: Jung et al, Biochem. Eng. J., 135: 123-132 (2018); Winther et al, Biochim. Biophys. Acta, 1840(2): doi: 10.1016 / j.bbagen.3013.03.031 (2014); Annabi et al, Tissue Engineering, part B, 16(4): 371- 383 (2010); and the like.

[0142] Size and Shape of Hydrogel Chambers. In some embodiments, a polymer matrix wall of a chamber inhibits passage of a predetermined component, such as a mammalian cell, a bacterial cell, or proteins from a lysed cell. In some embodiments, a polymer matrix wall extends from the first surface to a second surface (parallel to the first surface) to form a chamber within a channel. In some embodiments, a chamber has polymer matrix walls and an interior. In some embodiments, the interior of a chamber is sized for enclosing a cell, such as a mammalian cell. For example, such chamber may comprise a cylindrical shell or a polygon shell, comprising an inner space, or interior and a polymer matrix wall. In some embodiments, such chambers may have annular-like cross-sections. As used herein, the term "annular-like cross-section" means a cross- section topologically equivalent to an annulus. In some embodiments, the inner space, orinterior, of a chamber has an inner diameter from 1 m to 500 pm and a volume in the range of from 1 pico liter to 200 nano liters, or from 100 pi co liters to 100 nano liters, or from 100 picoliters to 10 nano liters. In some embodiments, the polymer matrix wall has a thickness of at least 1 pm (micrometer). In some embodiments, the height of a chamber with an annular-like cross section have a value in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, a polymer matrix wall having an annular-like cross-section has an aspect ratio (i.e., height / width) of 1 or less. In some embodiments, aspect ratio and polymer matrix wall thickness are selected to maximize chamber stability against forces, such as reagent flow through the channel, washings, and the like. In some embodiments, the at least one polymer matrix wall is a hydrogel wall. In some embodiments, the at least one polymer matrix is degradable. In some embodiments, the degradation of the at least one polymer matrix is "on demand."

[0143] In some embodiments, chambers in a channel are non-contiguous. In some embodiments, chambers in a channel may be contiguous with adjacent chambers. In some embodiments, chambers may share polymer matrix walls with one another. In some embodiments, chambers may be synthesized with slits or other orifaces large enough to permit passage of certain components, e.g. beads, but small enough to prevent passage of other components, e.g. cells.

[0144] Hydrogel Compositions. As mentioned above, hydrogel compositions may vary widely and hydrogels may be formed by a variety of methods. Biocompatible hydrogel precursors comprise, but are not limited to, hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, polyethylene glycol)-b- polypropylene oxide)-b- poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic acid-co- glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic acid-co-glycolic acid) (PLGA-PEG- PLGA), and poly(vinyl alcohol). In some embodiments, hydrogels are formed by photo- initiated free radical crosslinking. In some embodiments, hydrogels are formed by photo- initiated thiol-ene reactions.

[0145] Hydrogel Degradation. In some embodiments, hydrogel chambers are degradable or depolymerizable either generally within a channel or “on demand” within a channel. Hydrogel chambers that are generally degradable are degraded by treatment with a degradation agent, or equivalently, a depolymerization agent that is exposed to all chambers within channel. Depolymerization agents may include, but are not limited to, heat, light, and / or chemical depolymerization reagents (also sometimes referred to a cleaving reagents or degradation reagents). In some embodiments, on demand degradation may be implemented using polymer precursors that permit photo-crosslinking and photo-degradation, for example, using differentwavelengths for crosslinking and for degradation. For example, Eosin Y may be used for radical polymerization at defined regions using 500 nm wavelength, after which illumination at 380 nm can be used to cleave the cross linker. In other embodiments, photo-caged hydrogel cleaving reagents may be included in the formation of polymer matrix walls. For example, acid labile crosslinkers (such as esters, or the like) can be used to create the hydrogel and then UV light can be used to generate local acidic conditions which, in turn, degrades the hydrogel. In some embodiments, the at least one polymer matrix is degradable by at least one of: (i) contacting the at least one polymer matrix with a cleaving reagent; (ii) heating the at least one polymer matrix to at least 90 °C; or (iii) exposing the at least one polymer matrix to a wavelength of light that cleaves a photo-cleavable cross linker that cross links the polymer of the at least one polymer matrix. In some embodiments, the at least one polymer matrix comprises a hydrogel. In some embodiments, the cleaving reagent degrades the hydrogel. In some embodiments, the cleaving reagent comprises a reducing agent, an oxidative agent, an enzyme, a pH based cleaving reagent, or a combination thereof. In some embodiments, the cleaving reagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3- hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments, the surface of the polymer matrix or hydrogel may be functionalized by coupling a functional group to the polymer matrix or hydrogel. Some nonlimiting examples of functional group may include a capture reagent ( e.g., pyridinecarboxaldehyde (PCA)), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an aldehyde dithiolane, PEG, a thiol, an alkene, an alkyne, an azide, or a combination thereof. In some cases, the functionalized polymer matrix may be used to capture biomolecules inside a polymer matrix compartment formed adjacent to (e.g., around or on) the biological component.

[0146] The biomolecule may be produced by the biological component ( e.g., secretome from a cell). The functionalized surface of the polymer matrix inside the compartment may be used to capture reagents or molecules from outside the compartment. The functionalized surface may increase surface area covered by a reagent, a molecular sensor, or any molecule of interest (e.g., an antibody).

[0147] Photosynthesis. In some embodiments, the generation of a polymer matrix within a channel or well of a fluidic device comprises exposing the one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light at 350 nm to 800 nm. In some embodiments, the light generating device generates light at 350 nm to 600 nm. In someembodiments, the light generating device generates light at 350 nm to 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of a polymer matrix within said fluidic device is performed using a spatial light modulator (SLM) (i.e. a spatial energy modulation element that is capable of generating desired light intensity pattern spatially). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid-crystal based.

[0148] While the present disclosure has been described with reference to several particular example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the systems and methods described herein. The present disclosure is applicable to a variety of sensor implementations and other subject matter, in addition to those discussed above.

[0149] The following embodiments recite illustrative, non-limiting permutations and combinations of features disclosed herein. Other permutations and combinations of features are also contemplated.

[0150] Embodiment 1. A method for determining a location and an identity of target molecules, the method comprising: exposing the target molecules, adsorbed onto a spatially barcoded surface, to a detection antibody, under coupling conditions for the detection antibody, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, the antibody barcode having a free 5’ end, wherein the antibody barcode corresponds to an epitope of the target molecules for which the detection antibody is specific, wherein the detection antibody is configured to couple to the epitope, and wherein spatial barcodes of the spatially barcoded surface comprise free 3’ ends; exposing the spatially barcoded surface under hybridization conditions to a linker oligonucleotide, wherein a free 5’ end region of the linker oligonucleotide hybridizes to the free 5’ end region of the antibody barcode, wherein a free 3’ end region of the linker oligonucleotide hybridizes to a free 3’ end region of a spatial barcode on the spatially barcoded surface; joining the 3’ end of the spatial barcode to the 5’ end of the antibody barcode.

[0151] Embodiment 2. The method of embodiment 1, further comprising: before the exposing the target molecules to the detection antibody, flowing a mixture of polymer precursors and cells into a channel of a flow cell that comprises the spatially barcoded surface; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the channel; lysing the cells so that the target molecules of each cell are released into the chamber and adsorbed ontothe spatially barcoded surface; and coupling the detection antibodies to the adsorbed target molecules.

[0152] Embodiment 3. The method of embodiment 2, further comprising collecting optical signals from the one or more cells disposed in the channel; and determining, prior to the synthesizing, the position of each of the one or more cells based on the optical signals.

[0153] Embodiment 4. The method of embodiment 3, wherein the positions of the gel chambers are determined by the positions of the cells identified by the detector.

[0154] Embodiment 5. The method of embodiment 4, wherein the synthesizing comprises projecting light into the channel with a spatial energy modulating element in operable communication with the detector, such that the projected light causes cross-linking of one or more polymer precursors to form the one or more gel chambers.

[0155] Embodiment 6. The method of embodiment 3, further comprising determining a cell type of the one or more cells based on the optical signals.

[0156] Embodiment 7. The method of embodiment 2, further comprising depolymerizing said one or more gel chambers prior to the coupling.

[0157] Embodiment 8. The method of embodiment 7, further comprising de-adsorbing the adsorbed target molecules prior to the depolymerizing.

[0158] Embodiment 9. The method of embodiment 8, wherein the de-adsorbed cellular proteins diffuse into the interior of their gel chamber and re-adsorb to the surface prior to the depolymerizing.

[0159] Embodiment 10. The method of embodiment 3, wherein the spatially barcoded surface or a surface in fluidic communication with the spatially barcoded surface comprises particles of a plurality of types disposed thereon, wherein each type of particle comprises a surface with different protein adsorption characteristics, and wherein particles of each type have a size and a quantity such that each of the hydrogel chambers encloses particles of every type.

[0160] Embodiment 11. The method of embodiment 2, wherein the one or more gel chambers are permeable to the detection antibodies.

[0161] Embodiment 12. The method of embodiment 11, wherein the method further comprises inputting the detection antibodies into the one or more gel chambers to the cellular proteins adsorbed onto the surface enclosed by the one or more gel chambers.

[0162] Embodiment 13. The method of embodiment 1, wherein the location of the target molecule is determined from a nucleotide sequence of the spatial barcode, and wherein the identity of the target molecule is determined from a nucleotide sequence of the antibody barcode.

[0163] Embodiment 14. The method of embodiment 1, wherein the spatially barcoded surface comprises a plurality of spatial barcodes.

[0164] Embodiment 15. The method of embodiment 1, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, wherein each spatial barcode includes a first primer.

[0165] Embodiment 16. The method of embodiment 1, wherein the spatial barcode of the spatial barcodes comprises a nucleotide sequence deposed on a surface of a channel corresponding to a predetermined location of the spatially barcoded surface.

[0166] Embodiment 17. The method of embodiment 1, wherein the free 5’ end of the antibody barcode is in a pendant format and not directly attached to the detection antibody before the exposing the spatially barcoded surface under hybridization conditions to the linker oligonucleotide.

[0167] Embodiment 18. The method of embodiment 1, wherein the antibody barcode corresponds to the target molecule and the target molecule was a predetermined target molecule.

[0168] Embodiment 19. The method of embodiment 1, wherein the antibody barcode includes a second primer.

[0169] Embodiment 20. The method of embodiment 1, wherein said target molecule is a protein.

[0170] Embodiment 21. The method of embodiment 1, wherein the cleavable group comprises a disulfide or a diol.

[0171] Embodiment 22. The method of embodiment 1, wherein the spatially barcoded surface is a top surface of the flow cell.

[0172] Embodiment 23. The method of embodiment 1, wherein the spatially barcoded surface is a bottom surface of the flow cell.

[0173] Embodiment 24. The method of embodiment 1, wherein the cellular proteins or the portion of the cellular proteins are adsorbed to a material coupled to the surface.

[0174] Embodiment 25. The method of embodiment 1, wherein the cellular proteins or the portion of the cellular proteins are bound to one or more capture antibodies coupled to the surface.

[0175] Embodiment 26. The method of embodiment 1, wherein the surface is a surface of a particle in the channel.

[0176] Embodiment 27. The method of embodiment 26, further comprising: before the exposing the target molecules to the detection antibody, flowing a mixture of polymer precursors and cells into a channel of a flow cell that comprises the spatially barcoded surface; synthesizing one or more gel chambers enclosing each of the one or more cells and the particle disposed in thechannel; lysing the cells so that the target molecules of each cell are released into the chamber and adsorbed onto the spatially barcoded surface; and coupling the detection antibodies to the adsorbed target molecules, wherein the one or more gel chambers are impermeable to the particle.

[0177] Embodiment 28. The method of embodiment 1, wherein the joining comprises ligating the 3’ end of the spatial barcode to the 5’ end of the antibody barcode.

[0178] Embodiment 29. The method of embodiment 1, wherein prior to the joining the method further comprises extending the spatial barcode, the antibody barcode, or the spatial barcode and the antibody barcode over the linker oligonucleotide.

[0179] Embodiment 30. A method for determining a location and an identity of a target molecule, the method comprising: exposing the target molecule, under coupling conditions, to one or more detection antibodies, wherein a detection antibody of the one or more detection antibodies is coupled to an antibody barcode that corresponds to an epitope of the target molecule for which the detection antibody is specific, the antibody barcode having a free 5’ end; exposing a spatially barcoded surface, under hybridization conditions, to a linker oligonucleotide, wherein a free 5’ end region of the linker oligonucleotide hybridizes to the free 5’ end region of the antibody barcode, wherein a free 3’ end region of the linker oligonucleotide hybridizes to a free 3’ end region of a spatial barcode on the spatially barcoded surface, wherein the spatially barcoded surface is part of a channel and the spatially barcoded surface comprises (i) one or more spatial barcodes that include free 3’ ends, and (ii) the target molecule that is adsorbed onto the spatially barcoded surface; and joining the 3’ end of the spatial barcode to the 5’ end of the antibody barcode.

[0180] Embodiment 31. A method of measuring a secreted biomolecule from a cell, the method comprising: flowing a mixture of polymer precursors and cells into a channel of a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a second handle; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the channel; incubating the cells enclosed in the one or more gel chambers so that the cells output the secreted biomolecules, capturing the secreted biomolecules in the one or more gel chambers, wherein the secreted biomolecule couples with a capture antibody; inputting a detection antibody into the channel of the flow cell where the detection antibody permeates into the one or more gel chambers and also couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, theantibody barcode comprising a first handle, wherein the detection antibody is configured to couple to the secreted biomolecules; joining the first handle of the spatial barcode and the second handle of the antibody barcode with a splint, wherein the splint includes a first handle complement configured to hybridize to the first handle and a second handle complement configured to hybridize to the second handle; and ligating the first handle to the second handle

[0181] Embodiment 32. The method of embodiment 31, wherein the capture antibodies are coupled to the spatially barcoded surface.

[0182] Embodiment 33. The method of embodiment 31, wherein the capture antibodies are coupled to beads and the beads are included in the mixture so that the beads are flowed into the channel along with the polymer precursors and the cells.

[0183] Embodiment 34. The method of embodiment 31, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface, wherein the capture antibodies are coupled to the top surface, the method further comprising cleaving the cleavable group to release the antibody barcode before the joining the first handle end of the spatial barcode and the second handle end of the antibody barcode with the splint.

[0184] Embodiment 35. The method of embodiment 31, wherein the flow cell further includes a bottom surface, the bottom surface having an opposing orientation with respect to the spatially barcoded surface, wherein the capture antibodies are coupled to the bottom surface, the method further comprising cleaving the cleavable group to release the antibody barcode before the joining the first handle end of the spatial barcode and the second handle end of the antibody barcode with the splint.

[0185] Embodiment 36. The method of embodiment 31, wherein the antibody barcode is coupled to the antibody by a scissile bond or by hybridization to an oligonucleotide coupled to the antibody.

[0186] Embodiment 37 A method of measuring a secreted biomolecule from a cell, the method comprising: flowing a mixture of polymer precursors and cells into a channel of a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a third handle; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the channel; incubating the cells enclosed in the one or more gel chambers so that the cells output the secreted biomolecules; capturing the secreted biomolecules in the one or more gel chambers, wherein the secreted biomolecule couples with a capture antibody; inputting a detection antibody into the channel of the flow cell where the detection antibody permeates into the one or more gelchambers and also couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising a third handle complement, wherein the third handle complement is hybridized with a blocker oligonucleotide preventing the third handle complement from hybridizing with the third handle of the spatial barcode, wherein the detection antibody is configured to couple to the secreted biomolecules; removing the blocker oligonucleotide from the third handle complement; cleaving the cleavable group and then allowing the third handle to hybridize with the third handle complement; extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

[0187] Embodiment 38. The method of embodiment 37, wherein the capture antibodies are coupled to the spatially barcoded surface.

[0188] Embodiment 39. The method of embodiment 37, wherein the capture antibodies are coupled to beads and the beads are included in the mixture so that the beads are flowed into the channel along with the polymer precursors and the cells.

[0189] Embodiment 40. The method of embodiment 37, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface, wherein the capture antibodies are coupled to the top surface, the method further comprising cleaving the cleavable group to release the antibody barcode before the allowing the third handle to hybridize with the third handle complement.

[0190] Embodiment 41. A method of measuring a secreted biomolecule from a cell, the method comprising: flowing a mixture of polymer precursors and cells into a channel of a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a third handle hybridized with a blocker oligonucleotide preventing the third handle from hybridizing with a third handle complement; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the channel; incubating the cells enclosed in the one or more gel chambers so that the cells output the secreted biomolecules; capturing the secreted biomolecules in the one or more gel chambers, wherein the secreted biomolecule couples with a capture antibody; inputting a detection antibody into the channel of the flow cell where the detection antibody permeates into the one or more gel chambers and also couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising a third handle complement,wherein the detection antibody is configured to couple to the secreted biomolecules; removing the blocker oligonucleotide from the third handle of the spatial barcode; cleaving the cleavable group to release the antibody barcode and then allowing the third handle to hybridize with the third handle complement; extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

[0191] Embodiment 42. The method of embodiment 41, wherein the capture antibodies are coupled to the spatially barcoded surface.

[0192] Embodiment 43. The method of embodiment 41, wherein the capture antibodies are coupled to beads and the beads are included in the mixture so that the beads are flowed into the channel along with the polymer precursors and the cells.

[0193] Embodiment 44. The method of embodiment 41, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface, wherein the capture antibodies are coupled to the top surface, the method further comprising cleaving the cleavable group to release the antibody barcode before the allowing the third handle to hybridize with the third handle complement.

[0194] Embodiment 45. The method of any one of embodiments 33-44, wherein the secreted biomolecule is one of a protein, a cytokine, and a monoclonal antibody.

[0195] Embodiment 46. The method of any one of embodiments 33-44, wherein the capture antibody includes a cleavable group so that the capture antibody is configured to separate from a surface.

[0196] Embodiment 47. The method of any one of embodiments 1-30, wherein the joining the 3’ end of the spatial barcode to the 5’ end of the antibody barcode comprises: extending the 3’ end of the spatial barcode to the 5’ end of the antibody barcode to form an extended spatial barcode; and ligating a 3’ end of the extended spatial barcode to the 5’ end of the antibody barcode.

[0197] Embodiment 48. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more of polymer precursors into a flow cell, the flow cell comprising a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes comprising a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a third handle complement; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secretedbiomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, wherein the detection antibody is configured to couple to the secreted biomolecules; inputting a label into the flow cell such that the label permeates into the one or more gel chambers, wherein a label barcode of the label hybridizes to the antibody barcode of the detection antibody, and detecting the label.

[0198] Embodiment 49. The method of embodiment 48, wherein the detecting comprises measuring an optical signal from the label.

[0199] Embodiment 50. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes comprising a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the oen or more cells in the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and also couples with the secreted biomolecule; wherein the detection antibody is coupled to a label: i) through a scissile or chemically cleavable bond, or ii) by hybridization to a barcode coupled to the detection antibody; detecting a signal associated with the label from the one or more gel chambers, releasing the label from the detection antibody, and detecting a decrease in the signal from the one or more gel chambers.

[0200] Embodiment 51. The method of any one of embodiments 1-50, wherein the one or more gel chambers are hydrogel chambers.

[0201] Embodiment 52. The method of any one of embodiments 1-51, wherein the one or more gel chambers are formed by selective polymerization of the one or more polymer precursors.

[0202] Embodiment 53. The method of Embodiment 52, wherein the selective polymerization comprises selectively applying light to the flow cell.

[0203] Embodiment 54. The method of any one of Embodiments 1-53, wherein the polymer precursors comprise (i) one or more cleavable crosslinkers, and (ii) a photo-initiator.

[0204] Embodiment 55. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a second handle; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, wherein the antibody barcode comprises a first handle; joining the first handle of the spatial barcode and the second handle of the antibody barcode with a splint, wherein the splint includes (i) a first handle complement configured to hybridize to the first handle and (ii) a second handle complement configured to hybridize to the second handle; and ligating the first handle to the second handle.

[0205] Embodiment 56. The method of embodiment 55, wherein the capture antibody is coupled to the spatially barcoded surface.

[0206] Embodiment 57. The method of embodiment 55, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors.

[0207] Embodiment 58. The method of embodiment 55, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells.

[0208] Embodiment 59. The method of embodiment 55, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

[0209] Embodiment 60. The method of embodiment 55, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

[0210] Embodiment 61. The method of embodiment 55, wherein the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

[0211] Embodiment 62. The method of embodiment 55, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface.

[0212] Embodiment 63. The method of embodiment 62, wherein the capture antibody is coupled to the top surface.

[0213] Embodiment 64. The method of embodiment 63, further comprising cleaving the cleavable group to release the antibody barcode before the joining the first handle of the spatial barcode and the second handle of the antibody barcode with the splint.

[0214] Embodiment 65. The method of embodiment 55, wherein the flow cell further includes a bottom surface, the bottom surface having an opposing orientation with respect to the spatially barcoded surface.

[0215] Embodiment 66. The method of embodiment 65, wherein the capture antibody is coupled to the bottom surface.

[0216] Embodiment 67. The method of embodiment 66, further comprising cleaving the cleavable group to release the antibody barcode before the joining the first handle of the spatial barcode and the second handle of the antibody barcode with the splint.

[0217] Embodiment 68. The method of embodiment 55, wherein the antibody barcode is coupled to the detection antibody by a scissile bond or by hybridization to an oligonucleotide coupled to the detection antibody.

[0218] Embodiment 69. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules forwhich the detection antibody is specific, the antibody barcode comprising a handle complement, wherein the handle complement is hybridized with a blocker oligonucleotide that is configured to prevent the handle complement from hybridizing with the handle of the spatial barcode; removing the blocker oligonucleotide from the handle complement; cleaving the cleavable group, thereby allowing the handle to hybridize with the handle complement; and extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

[0219] Embodiment 70. The method of embodiment 69, wherein the capture antibody is coupled to the spatially barcoded surface.

[0220] Embodiment 71. The method of embodiment 69, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors.

[0221] Embodiment 72. The method of embodiment 69, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells.

[0222] Embodiment 73. The method of embodiment 69, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

[0223] Embodiment 74. The method of embodiment 69, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

[0224] Embodiment 75. The method of embodiment 69, wherein the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

[0225] Embodiment 76. The method of embodiment 69, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface.

[0226] Embodiment 77. The method of embodiment 76, wherein the capture antibody is coupled to the top surface.

[0227] Embodiment 78. The method of embodiment 77, further comprising cleaving the cleavable group to release the antibody barcode before the allowing the handle to hybridize with the handle complement.

[0228] Embodiment 79. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the one or morecells in the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising the handle complement, wherein the detection antibody is configured to couples to the secreted biomolecule; removing the blocker oligonucleotide from the handle of the spatial barcode; cleaving the cleavable group to release the antibody barcode, thereby allowing the handle to hybridize with the handle complement; and extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

[0229] Embodiment 80. The method of embodiment 79, wherein the capture antibody is coupled to the spatially barcoded surface.

[0230] Embodiment 81. The method of embodiment 79, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors.

[0231] Embodiment 82. The method of embodiment 79, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells.

[0232] Embodiment 83. The method of embodiment 79, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

[0233] Embodiment 84. The method of embodiment 79, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

[0234] Embodiment 85. The method of embodiment 79, wherein the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

[0235] Embodiment 86. The method of embodiment 79, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface.

[0236] Embodiment 87. The method of embodiment 86, wherein the capture antibody is coupled to the top surface.

[0237] Embodiment 88. The method of embodiment 86, further comprising cleaving the cleavable group to release the antibody barcode before the allowing the handle to hybridize with the handle complement.

[0238] Embodiment 89. The method of any one of embodiments 55-88, wherein the one or more secreted biomolecules is one of a protein, a cytokine, and a monoclonal antibody.

[0239] Embodiment 90. The method of any one of embodiments 55-89, wherein the capture antibody includes a cleavable group so that the capture antibody is configured to separate from a surface.Definitions

[0240] Unless otherwise specifically defined herein, terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow those of standard treatises and texts in the field, e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Abbas et al, Cellular and Molecular Immunology, 6thedition (Saunders, 2007).

[0241] “Antibody” as used herein generally means any binding compound capable of specifically binding to a given protein including, without limitation, immunoglobulin molecules or fragments thereof and aptamers. Fragments of immunoglobulin molecules include, but are not limited to, Fab, Fv and F(ab’)2, Fab’ fragments, and the like.

[0242] “Detection antibody” as used herein generally means an antibody conjugated with a detection moiety that permits the identification of a protein that the antibody is bound to. A detection moiety may comprise a fluorescent dye, a barcode, an enzyme, or the like. In some embodiments, a detection moiety may be covalently linked to an antibody. In some embodiments, a detection moiety may be conjugated to an antibody by a scissile bond or by hybridization.

[0243] Cells” as used herein generally refer to biological cells that may be assayed by methods and systems described herein, but are not limited to, vertebrate, non-vertebrate, eukaryotic, mammalian, microbial, protozoan, prokaryotic, bacterial, insect, or fungal cells. In some embodiments, mammalian cells are assayed by methods and systems described herein. In particular, any mammalian cell which may be, or has been, genetically altered for use in a medical, industrial, environmental, or remedial process, may be analyzed by methods and systems described herein. In some embodiments, “cells” as used herein comprise genetically modified mammalian cells. In some embodiments, “cells” comprise stem cells. In someembodiments, “cells” refer to cells modified by CRISPR Cas9 techniques. In some embodiments, “cells” refer to cells of the immune system including, but not limited to, cytotoxic T lymphocytes, regulatory T cells, CD4+ T cells, CD8+ T cells, natural killer cells, antigen- presenting cells, or dendritic cells. Of special interest are cytotoxic T lymphocytes engineered for therapeutic applications, such as cancer therapy.

[0244] “Hydrogel” as used herein generally means a gel comprising a crosslinked hydrophilic polymer network with the ability to absorb and retain large amounts of water (for example, 60 to 90 percent water, or 70 to 80 percent) without dissolution due to the establishment of physical or chemical bonds between the polymeric chains, which may be covalent, ionic or hydrogen bonds. Hydrogels exhibit high permeability to the oxygen and nutrients, making them attractive materials for cell encapsulation and culturing applications.

[0245] Hydrogels may comprise natural or synthetic polymers and may be reversible (i.e. degradable or depolymerizable) or irreversible. Synthetic hydrogel polymers may include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate) and poly(vinyl alcohol). Natural hydrogel polymers include alginate, hyaluronic acid and collagen. The following reference describe hydrogels and their biomedical uses: Drury et al, Biomaterials, 24: 4337-4351 (2003); Garagorri et al, Acta Biomatter, 4(5): 1139-1147 (2008); Caliari et al, Nature Methods, 13(5): 405-414 (2016); Bowman et al, U.S. patent 9631092; Koh et al, Langmuir, 18(7): 2459-2462 (2002).

[0246] “ On demand" as used herein generally means an operation may be directed to individual, discrete, selected locations ( e.g. a spatial location of polymer precursor solution; or a selected polymer matrix chamber). Such selection may be based on manual observation of optical signals or data collected by a detector, or such selection may be based on a computer algorithm operating on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include either real-time detection or detection at a time period prior to modulating a unit of energy to polymerize polymer precursors or degrading a chamber. For example, a subset of chambers (all formed with photo-degradable polymer matrix walls) may be pre-selected for releasing and removing their contents based on position information and the values of optical signals from an analytical assay carried out in the chambers. The pre-selected chambers may be photo- degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) to degrade the polymer matrix walls of the pre-selected chambers. In another example, a plurality of chambers may be observed in real-time (e.g. via fluorescent microscopy) fordetection of an analyte of interest and one or more chambers of the plurality of chambers is selected, in real-time, upon detection of the analyte of interest, for degradation.

[0247] "Polymer matrix" as used herein generally refers to a phase material (e.g. continuous phase material) that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the interstitial space not occupied by the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also contain non- polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term "polymer matrix" may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. A polymer matrix may comprise a polymer precursor, which generally refers to one or more molecules that upon activation can trigger or initiate a polymeric reaction. A polymer precursor can be activated by electrochemical energy, photochemical energy, a photon, magnetic energy, or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (that are polymerized to produce a polymer matrix) and crosslinking compounds, which may include photo-initiators, other compounds necessary or useful for generating polymer matrices, especially polymer matrices that are hydrogels.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a second handle; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, wherein the antibody barcode comprises a first handle; joining the first handle of the spatial barcode and the second handle of the antibody barcode with a splint, wherein the splint includes (i) a first handle complement configured to hybridize to the first handle and (ii) a second handle complement configured to hybridize to the second handle; and ligating the first handle to the second handle.

2. The method of claim 1, wherein the capture antibody is coupled to the spatially barcoded surface.

3. The method of claim 1, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors.

4. The method of claim 1, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells.

5. The method of claim 1, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

6. The method of claim 1, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

7. The method of claim 1, wherein the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

8. The method of claim 1, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface.

9. The method of claim 8, wherein the capture antibody is coupled to the top surface.

10. The method of claim 9, further comprising cleaving the cleavable group to release the antibody barcode before the joining the first handle of the spatial barcode and the second handle of the antibody barcode with the splint.

11. The method of claim 1, wherein the flow cell further includes a bottom surface, the bottom surface having an opposing orientation with respect to the spatially barcoded surface.

12. The method of claim 11, wherein the capture antibody is coupled to the bottom surface.

13. The method if claim 12, further comprising cleaving the cleavable group to release the antibody barcode before the joining the first handle of the spatial barcode and the second handle of the antibody barcode with the splint.

14. The method of claim 1, wherein the antibody barcode is coupled to the detection antibody by a scissile bond or by hybridization to an oligonucleotide coupled to the detection antibody.

15. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising a handle complement, wherein the handle complement is hybridized with a blocker oligonucleotide that is configured to prevent the handle complement from hybridizing with the handle of the spatial barcode; removing the blocker oligonucleotide from the handle complement; cleaving the cleavable group, thereby allowing the handle to hybridize with the handle complement; and extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

16. The method of claim 15, wherein the capture antibody is coupled to the spatially barcoded surface.

17. The method of claim 15, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors.

18. The method of claim 15, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells.

19. The method of claim 15, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

20. The method of claim 15, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

21. The method of claim 15, wherein the flow cell comprises a channel, and wherein the one or more cells are disposed in the channel.

22. The method of claim 15, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface.

23. The method of claim 22, wherein the capture antibody is coupled to the top surface.

24. The method of claim 23, further comprising cleaving the cleavable group to release the antibody barcode before the allowing the handle to hybridize with the handle complement.

25. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the one or more cells in the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers,wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode via a cleavable group, wherein the antibody barcode corresponds to an identity of the secreted biomolecules for which the detection antibody is specific, the antibody barcode comprising the handle complement, wherein the detection antibody is configured to couples to the secreted biomolecule; removing the blocker oligonucleotide from the handle of the spatial barcode; cleaving the cleavable group to release the antibody barcode, thereby allowing the handle to hybridize with the handle complement; and extending the spatial barcode to an end of the antibody barcode to form an extended spatial barcode.

26. The method of claim 25, wherein the capture antibody is coupled to the spatially barcoded surface.

27. The method of claim 25, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors.

28. The method of claim 25, wherein the capture antibody is coupled to beads, and wherein the beads are introduced into the flow cell along with the one or more polymer precursors and the one or more cells.

29. The method of claim 25, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell simultaneously.

30. The method of claim 25, wherein the one or more polymer precursors and the one or more cells are introduced into the flow cell separately.

31. The method of claim 25, wherein the flow cell comprises a channel, and wherein the oneor more cells are disposed in the channel.

32. The method of claim 25, wherein the flow cell further includes a top surface, the top surface having an opposing orientation with respect to the spatially barcoded surface.

33. The method of claim 32, wherein the capture antibody is coupled to the top surface.

34. The method of claim 32, further comprising cleaving the cleavable group to release the antibody barcode before the allowing the handle to hybridize with the handle complement.

35. The method of any one of claims 1-34, wherein the one or more secreted biomolecules is one of a protein, a cytokine, and a monoclonal antibody.

36. The method of any one of claims 1-35, wherein the capture antibody includes a cleavable group so that the capture antibody is configured to separate from a surface.

37. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell comprising a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes comprising a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the one or more cells into the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to an antibody barcode, wherein the antibody barcode corresponds to an identity of the secretedbiomolecules for which the detection antibody is specific, wherein the detection antibody is configured to couple to the secreted biomolecule; inputting a label into the flow cell such that the label permeates into the one or more gel chambers, wherein a label barcode of the label hybridizes to the antibody barcode of the detection antibody, and detecting the label.

38. The method of claim 37, wherein the detecting comprises measuring an optical signal from the label.

39. A method of measuring one or more secreted biomolecules from one or more cells, the method comprising: introducing one or more polymer precursors into a flow cell, the flow cell including a spatially barcoded surface, wherein the spatially barcoded surface comprises a plurality of spatial barcodes, each of the spatial barcodes including a handle hybridized with a blocker oligonucleotide, wherein the blocker oligonucleotide is configured to prevent the handle from hybridizing with a handle complement; introducing the one or more cells in the flow cell; synthesizing one or more gel chambers enclosing each of the one or more cells disposed in the flow cell; incubating the one or more cells enclosed in the one or more gel chambers so that the one or more cells output the one or more secreted biomolecules; capturing the one or more secreted biomolecules in the one or more gel chambers, wherein a secreted biomolecule of the one or more secreted biomolecules couples with a capture antibody; inputting a detection antibody into the flow cell such that the detection antibody permeates into the one or more gel chambers and couples with the secreted biomolecule, wherein the detection antibody is coupled to a label: i) through a scissile or chemically cleavable bond, or ii) by hybridization to a barcode coupled to the detection antibody; detecting a signal associated with the label from the one or more gel chambers, releasing the label from the detection antibody, anddetecting a decrease in the signal from the one or more gel chambers.

40. The method of any one of claims 1-39, wherein the one or more gel chambers are hydrogel chambers.

41. The method of any one of claims 1-40, wherein the one or more gel chambers are formed by selective polymerization of the one or more polymer precursors.

42. The method of claim 41, wherein the selective polymerization comprises selectively applying light to the flow cell.

43. The method of any one of claims 1-42, wherein the polymer precursors comprise (i) one or more cleavable crosslinkers, and (ii) a photo-initiator.

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