Single cell proteomics using degradable hydrogels

Abstract

The present disclosure relates to methods for measuring proteomes of single cells. In one aspect, a method includes disposing cells on a surface, synthesizing a hydrogel chamber around each cell, lysing the cells to release intracellular proteins for adsorption onto the surface defined by the hydrogel chamber, depolymerizing the hydrogel chamber, and quantifying the adsorbed proteins with a detection antibody to determine a single cell proteome.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 390,512, filed on July 19, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by reference

[0004] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. Background Art

[0005] One characteristic of biology is degeneracy, Edelman et al., Proc. Natl. Acad. Sci., 98 (24): 13763-13768 (2001). Degeneracy is the ability of structurally different elements (such as cells) to perform the same function or produce the same output and to perform different functions or produce different outputs according to the environment in which they are expressed. Examples of degenerate systems are very extensive in biology and include development, immune response, cancer, and functional neuroanatomy. In order to understand the operation of this degenerate system, it is necessary to be able to characterize and measure the properties of individual cells (such as its transcriptome, proteome, metabolome, etc.). Technology that only provides cell averages of such properties cannot reveal the cell heterogeneity necessary to fully understand such biological processes, such as Dittrich et al., Anal. Bioanal. Chem., 406: 6957-6961 (2014); Lindstrom et al., Single-Cell Analysis: Methods and Protocols (Humana Press, 2012). In the past decade, significant progress has been made in the analysis of transcriptomes of single cells and tissues, for example, 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); etc. However, there has been no comparable progress in proteome analysis, Vistain et al., Trends Biochemical Sciences, 46(8):661-672 (2021).

[0006] In view of the above, new methods and equipment that can be used to efficiently and conveniently analyze the proteome of single cells will advance our understanding of complex degenerate systems in biology. Summary of the invention

[0007] The present disclosure relates to a method for determining the proteome of a single cell, comprising: (a) synthesizing one or more gel chambers that encapsulate each of one or more cells arranged on a channel surface; (b) lysing the cells so that the cellular proteins of each cell are released into its hydrogel chamber and at least a portion is adsorbed onto the surface encapsulated by the gel chamber; and (c) detecting the adsorbed proteins with a detection antibody. In some embodiments, the method further comprises collecting optical signals from one or more cells arranged on the surface; and determining the position of each of the one or more cells from the optical signals before synthesis. In some embodiments, the method further comprises depolymerizing the hydrogel chamber before detection. In some embodiments, the method further comprises desorbing the adsorbed proteins before depolymerization.

[0008] In some embodiments, a method for determining the proteome of a single cell includes (a) providing a fluidic device comprising: (i) a channel comprising a surface, (ii) a spatial energy modulation element optically connected to the surface, and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element, the detector collecting one or more optical signals for identifying cells and determining their positions on the surface; (b) loading the channel with cells and one or more polymer precursors so that the cells are arranged on or near the surface; (c) synthesizing one or more hydrogel chambers encapsulating each of one or more cells by projecting light into the channel using the spatial energy modulation element so that the projected light causes cross-linking of one or more polymer precursors to form a hydrogel chamber, wherein the position of the hydrogel chamber in the channel is determined by the position of the cell encapsulated thereby as identified by the detector; (d) lysing the cells so that [at least a portion of] the cellular proteins of each cell are released into its hydrogel chamber and adsorbed onto the surface [encapsulated by the hydrogel chamber]; (e) depolymerizing the hydrogel chambers; and (f) loading detection antibodies for identifying the adsorbed proteins.

[0009] Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages described herein will be obtained by referring to the following detailed description setting forth illustrative embodiments (in which the principles of the systems and methods described herein are utilized) and the accompanying drawings (also referred to herein as "Figures"), in which:

[0011] Figure 1A-Figure 1C An embodiment for analyzing the proteome of a single cell is illustrated.

[0012] Figure 2A-2C An embodiment of an antibody barcode and an adjacent spatial barcode for linking bound antibodies is illustrated.

[0013] Figure 2D An embodiment is shown in which the antibody barcodes of two bound antibodies are separately linked to adjacent spatial barcodes, where readout of the barcodes provides a proximity determination of the adsorbed protein.

[0014] Figure 2E Pictured Figure 2A-2C An embodiment wherein the released protein is captured by antibodies attached to the surface.

[0015] Figure 3A-3B Embodiments in which proteins are adsorbed onto bead surfaces having different physical and / or chemical properties are illustrated.

[0016] Figure 4A-4B The apparatus used to detect cells and synthesize the hydrogel chamber is shown in more detail.

[0017] Figure 5A-Figure 5B A flow cell having multiple channels for use with some embodiments described herein is illustrated. DETAILED DESCRIPTION

[0018] Unless otherwise indicated, the practice of the present disclosure may employ conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, all of which are within the art. Such conventional techniques include, but are not limited to, the preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. A specific description of suitable techniques can be obtained by reference to the examples below. However, other equivalent routine procedures may also be used, of course. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Volumes I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Renault and Duchateau, ed., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, ed., Protein Engineering Handbook (Wiley-VCH, 2009); etc. Guidance for selecting materials and components to perform particular functions can be found in available treatises and references on scientific instrumentation, including but not limited to Moore et al., Building Scientific Apparatus, 3rd ed. (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd ed. (Academic Press, 2013); and other references.

[0019] The present disclosure relates to methods for measuring the proteome of a single cell. 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 its identity and its quantity or relative quantity. In some embodiments, the term "proteome" means a defined subset of proteins expressed by a single cell. In some embodiments, this subset can be defined by a set of antibodies used to capture and / or detect the proteins of the subset. The size of this defined subset can vary greatly. In some embodiments, this subset can include 2 to 100 different proteins, or 10 to 50 different proteins, or 10 to 20 different proteins. The term "antibody" is intended to encompass 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 compounds such as antibody fragments, aptamers, etc. "Specific binding" with respect to a specific binding compound (such as an antibody) means that the specific binding compound uniquely binds (under physiological conditions) its intended target protein and hardly or not cross-binds (i.e., cross-reacts with) other proteins in a specified subset.

[0020] In one aspect, the methods described herein include (a) synthesizing a gel shell or chamber around each of a plurality of cells arranged on a channel surface, (b) lysing the encapsulated cells to release a protein adsorbed to a portion of the channel surface encapsulated by the gel chamber, (c) depolymerizing the gel chamber, and (d) detecting the adsorbed protein at the location of the depolymerized gel chamber with a detection antibody. In some embodiments, the methods described herein further include collecting optical signals from cells arranged on the surface (including the location of each cell on the surface) before synthesizing the gel shell. The term "gel shell" is used interchangeably with the term "gel chamber". The composition of such a gel shell or gel chamber can vary greatly, as further discussed below. In some embodiments, such a gel shell or gel chamber comprises a hydrogel.

[0021] In some embodiments, the disclosure includes fixing and permeabilizing cells. In some embodiments, after the synthesis chamber and before the application of binding compounds specific for such intracellular targets, the cells are fixed and permeabilized. In some embodiments, fixation and permeabilization are performed to replace lysis. The fixation and permeabilization of cells can be performed by conventional procedures, such as the procedures used in flow cytometry. Such procedures generally include the steps of treating cells with a fixative and the subsequent steps of treating cells with a permeabilizing agent. The fixing step generally fixes the intracellular cell target, while retaining the cell and subcellular framework and allowing antibodies and / or hybridization probes to approach all cells and subcellular compartments without hindrance. A wide range of fixatives are commercially available, and the correct selection of the method will depend on the nature of the target being tested and on the properties of the antibodies and / or hybridization probes used. Fixation methods are generally divided into two categories: organic solvents and cross-linking agents. Organic solvents such as alcohols and acetone remove lipids and dehydrate cells while precipitating proteins on the cell framework. Cross-linking agents (such as paraformaldehyde) generally form intermolecular bridges through free amino groups, thereby building a network of connected antigens. Cross-linking agents preserve cell structures better than organic solvents, but may reduce the antigenicity of some cell components and require the addition of a permeabilization step to allow antibodies and / or hybridization probes to access intracellular targets. Fixation and permeabilization steps include, but are not limited to, methanol-acetone fixation (fixation in cooled methanol at -20°C for 10 minutes; permeabilization with cooled acetone at -20°C for 1 minute); paraformaldehyde-triton fixation (fixation in 3-4% paraformaldehyde for 10-20 minutes; rinse with phosphate buffered saline (PBS); permeabilization with 0.5% Triton X-100 for 2-10 minutes); paraformaldehyde-methanol fixation (fixation in 3-4% paraformaldehyde for 10-20 minutes; rinse with PBS; permeabilization with cooled methanol at -20°C for 5-10 minutes). Permeabilization agents include, but are not limited to, detergent saponin, Triton X-100, Tween-20, NP40. Permeabilization agents may also include proteases, such as proteinase K, streptolysin O, and the like.

[0022] In some embodiments, the methods described herein include synthesizing one or more gel chambers that encapsulate each of one or more cells disposed on a channel surface; fixing and permeabilizing the cells so that the intracellular proteins of each cell are accessible to a detection antibody; and detecting the intracellular proteins with the detection antibody. In some embodiments, such one or more cells may be non-adherent cells. In some embodiments, such methods further include collecting optical signals from one or more cells disposed on a surface; and determining the position of each of the one or more cells from the optical signals prior to synthesis.

[0023] Figure 1A-Figure 1CAn embodiment of the present disclosure is illustrated. Cells (e.g., 101) are arranged on a surface (102) of a channel (100) formed by a surface (102) and a surface (103), which surfaces can be, for example, surfaces of two parallel glass and / or plastic plates, after which the cell position is determined by a detector (104). A gel housing or chamber is synthesized (108) from a photosynthesizable polymer precursor in the channel (100) using a light source (106). In some embodiments, the detector (104) includes a microscope and an image recognition system operatively associated with the light source (106) to allow the gel chamber to be positioned around the cell (101). The detector (104) can also collect and record optical signals from the cell (101), which indicate its type, health status, function, etc., which can be related to the proteome of the cell. In some embodiments, the cell can be mixed with a polymer precursor (the gel is photosynthesized from the polymer precursor) before being loaded together into the channel (100). As described more fully below, gel chambers (e.g., 125a-d and enlarged view 110) can vary widely in size and shape. For simplicity, gel chambers ( Figure 1A 125a-d, etc.) are illustrated as cylindrical solids, but as illustrated by the enlarged view (110), the gel chamber can include walls and have a non-gel interior, as illustrated by the wall (121) having a thickness (116) enclosing the interior space (111) with the encapsulated interior surface (112). In some embodiments, the wall (121) extends from the surface (102) to the surface (103). After the cells are encapsulated in the gel chamber, as in Figure 1B As shown in the topmost top view, it is lysed (130). Lysis can be accomplished using conventional cell lysis techniques, including but not limited to light-based lysis, chemical lysis, heat-based lysis, etc. In some embodiments, the cells are lysed using a chemical lysis agent that readily passes through the walls of the gel chamber. 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 10°C for 15 min; 2) 200 mM KOH, 50 mM dithiothreitol, heated to 65°C for 10 min; 3) for 4 μL of protease-based lysis buffer: 1 μL of 17 μM SDS combined with 3 μL of 125 μg / mL proteinase K, followed by incubation at 37°C for 60 min, followed by incubation at 95°C for 15 min (to inactivate proteinase K); 4) for 10 μL of detergent-based lysis buffer: 2 μL H2O, 2 μL 10 mM EDTA, 2 μL 250 mM dithiothreitol, 2 μL 0.5% N-lauryl sarcosinate solution; 5) 200 mM Tris pH 7.5, 20 mM EDTA, 2% sarcoyl, 6% Ficoll.

[0024] As the cell wall and cell membrane are broken down by the lysing agent, the protein is released into the interior of the chamber and adsorbed onto the interior surface. In some embodiments, the interior surface of the gel chamber can be selected so that the protein is preferentially adsorbed onto the desired interior surface. For example, in some embodiments, the plate or wall comprising the surface (102) can include a material such as a plasma-treated plastic that preferentially adsorbs proteins, and the plate or wall comprising the surface (103) can include a material such as a surface-passivated glass that resists protein adsorption. Such material selection is 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); etc. Similarly, a gel polymer precursor can be selected to form a gel that minimizes nonspecific adsorption of proteins to the gel surface or structure. A variety of materials can be used for protein adsorption surfaces, including but not limited to non-polar surfaces, hydrophobic surfaces, or hydrophilic surfaces. In some embodiments, the surface is formed on plastic. In some embodiments, oxygen plasma can be used to introduce polar functional groups to the surface and make it hydrophilic. In some embodiments, tetrafluoromethane plasma can be used to introduce non-polar functional groups to the surface and make it hydrophobic. In some embodiments, the surface can be part of a commercially available protein adsorption material, such as Materials (ThermoFisher Scientific).

[0025] In some embodiments, the surface may have attached one or more capture antibodies specific for a selected protein. In some embodiments, such selected proteins may be intracellular proteins. In other embodiments, such selected proteins may be cell membrane proteins, or both intracellular proteins and cell membrane proteins. As used herein, the term "adsorption" includes specific binding of proteins to antibodies attached to the surface. Various methods can be used to immobilize or covalently bond antibodies to a surface, for example, as reviewed in Trilling et al., Analyst, 138: 1619-1627 (2013); Gao et al., Analytica Chimica Acta, 1189: 338907 (2022).

[0026] In some embodiments, after the released protein is adsorbed onto the desired surface, the wall of the gel chamber (e.g., 134) is depolymerized to enhance access of the detection antibody to the adsorbed protein. In other embodiments, after the released protein is adsorbed onto the desired surface, the wall of the gel chamber remains intact, and wherein the porosity of the gel wall is selected to allow easy access by the detection antibody. Optionally, as Figure 1C , the distribution of the adsorbed protein can be modified by one or more steps of desorption (136), which allows the desorbed protein to diffuse throughout its interior of the gel chamber to form a layer of more uniform density (138). Following this modification, the gel chamber can be depolymerized as described above and the adsorbed protein exposed to the detection antibody (140). Such a desorption step can be implemented by treating the adsorbed protein with heat and / or a desorbent (such as a non-ionic detergent, such as, for example, Brij, Triton, Tween, etc.). In some embodiments, desorption can be implemented by increasing the ionic strength of the reaction mixture.

[0027] In some embodiments, the adsorbed proteins can be interrogated by one or more panels of detection antibodies constructed from commercially available panels (e.g., available from suppliers such as Bio-Techne Corp. (Minneapolis, MN); R&D Systems, Inc. (Minneapolis, MN); etc.).

[0028] In some embodiments, the adsorbed protein can be Figure 2A-2CThe embodiment shown in the figure is used for detection. The protein (200) adsorbed on the surface (201) is specifically bound by an antibody (203), which is connected to the 5' end of the oligonucleotide (205) by a cleavable bond (210). The oligonucleotide (205) contains a primer binding site P7 (which may be the same or different from the primer binding site specified by Illumina, Inc.) and a barcode region ("BC1") (212) near its 5' end in sequence from the 5' end. The barcode region BC1 (212) contains a sequence that uniquely identifies the specific target epitope of the antibody (203). The surface (201) further comprises (i) a primer oligonucleotide P5 (206) (vertical grey bars, which may be the same or different from the primer specified by Illumina, Inc.), (ii) a complementary sequence to oligonucleotide P7 (i.e., P7') (214) (vertical striped bars, which may be the same or different from the oligonucleotide specified by Illumina, Inc.), and (iii) an oligonucleotide chain (205) attached to the surface (201) via its 3' end, having (in order) the primer binding site sequence P5 and a barcode "BC2" (216) at its 3' end and a 5' phosphate. The barcode region "BC2" (216) comprises a sequence that uniquely identifies a specific region of the surface (201) or a spatial location on the surface (201). After the antibody (203) is bound to the protein (200), the resulting complex is exposed (232) to an adapter oligonucleotide (230) which hybridizes to both the 3' end of the oligonucleotide (202) and the 5' end of the surface-bound oligonucleotide chain (205). After such hybridization, the 3' end of the oligonucleotide (202) is extended (234) by an appropriate polymerase in the presence of deoxynucleoside triphosphates and ligated to the 5' end of the oligonucleotide chain (205). After ligation, the adapter oligonucleotide (230) can be washed away and the cleavable bond (210) cleaved to yield a chain (235) bound to the surface (201), which can be amplified by bridge amplification using the P5 and P7' primers on the surface (201), by Figure 2C The first few cycles are shown in . The resulting amplicons can be sequenced directly (in situ) or cleaved from the surface (201) and sequenced by a separate sequencing instrument.

[0029] In some embodiments, the surface (201) may include Figure 2EThe capture antibody (281) shown in the figure, the P5 and P7' primers may or may not be present. After capture of the protein (200), the detection antibody (282) can be added and bound to the protein (200), which can then be used to generate a signal, which can be optical (e.g., fluorescent) or physical (such as, as described above, encoded nucleic acid) or a mixture of both (e.g., multiplex FISH decoder probes). Multiplex fluorescent in situ hybridization probes (and related decoder probes) are well known to those skilled in the art, as evidenced by the following references 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, U.S. 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); etc.

[0030] In some of the above embodiments, proteins (or other target molecules) can be detected on a spatially barcoded surface by the following steps: (a) adsorbing proteins onto a spatially barcoded surface, wherein the spatial barcode on the surface comprises a free 5' phosphate, (b) exposing the spatially barcoded surface to detection antibodies under binding conditions, each detection antibody comprising an antibody barcode that identifies an epitope specifically targeted by the detection antibody, wherein the antibody barcode comprises a free 3' end; (c) exposing the spatially barcoded surface to a linker oligonucleotide under hybridization conditions, the 3' end of the linker oligonucleotide being configured to hybridize with the free 3' end of the antibody barcode and the free 5' end of the spatial barcode on the surface; (d) extending the 3' end of the antibody barcode to the 5' end of the spatial barcode; and (e) connecting 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 can be cut from the detection antibody. In some embodiments, the location and identity of the protein can be determined from the sequence of the extended 3' end and the spatial barcode of the antibody barcode. In some embodiments, the extended 3' end and the spatial barcode of the antibody barcode can be amplified. In some embodiments, the extended 3' end and the spatial barcode of the antibody barcode can be amplified to form a cluster sequenced using sequencing by synthesis. In some embodiments, the extended 3' end and the spatial barcode of the antibody barcode can be amplified by bridge amplification.

[0031] Can be used Figure 2D to provide a more specific and sensitive measurement of selected adsorbed proteins, wherein according to Figure 2A-2C The embodiment uses two antibodies, but these antibodies are specific for separate epitopes on the protein. Since the antibody identity barcode will be connected to the same spatial barcode, this embodiment provides the same information as the proximity assay that directly connects the two antibodies using oligonucleotide hybridization. As above, the protein (250) is adsorbed onto a surface (252) including a P5 primer (254), a P7' primer (256) and an oligonucleotide chain (258 and 259), which contains from its 3' end: a P5 segment, a spatial barcode BC2 and a 5' phosphate that enables connection. After antibodies (260 and 262) are respectively bound to epitope 1 (264) and epitope 2 (266), the linker oligonucleotide (268) is added under conditions where the 3' end of the linker oligonucleotide forms a duplex with the 3' end of the respective oligonucleotides (261 and 263) of the antibodies (260 and 262) and with the 5' end of the surface-bound chain (258 and 259). After extending the 3' ends of the oligonucleotides (261 and 263), ligating the extended oligonucleotides to chains (258 and 259), and cleaving the cleavable bonds to release the antibodies, the resulting oligonucleotides can be amplified and sequenced as described above to identify the localization of the two antibodies and the proteins to which they bind.

[0032] In embodiments where the released protein can be adsorbed onto a variety of different surfaces Figure 3A-3B Provides Figure 1A-1B A similar device arrangement, except that at least one surface of the channel is disposed with a plurality of different beads comprising different materials having different surface properties (e.g., hydrophobic, hydrophilic, negatively charged, positively charged, etc.). The different bead types can be identified by fluorescent markers (e.g., as with beads available from BioLegends (San Diego)). Figure 3A The surface (350) of the channel (351) in the top inset represents a surface covered with a tightly packed layer of beads, each type of bead having different surface properties. This is further illustrated by the enlarged view (344) of the surface (350), where different bead types are illustrated with different patterns (stripes, spots, solid black, solid grey). The second surface (353) may be Figure 1A-Figure 1C ; i.e., the surface of a glass or plastic plate or wall. The functions and operations of the detector (345) and the light source (347) are as described above. After the gel chambers (e.g., 325a-i) are synthesized (346), the cells are lysed (349) to release the cellular proteins, which are adsorbed to the various bead types encapsulated in the chambers with the individual cells. After a predetermined incubation time, the gel chambers are disassembled and detection antibodies are loaded into the channel (351), where the detection antibodies specifically bind to the target proteins adsorbed to the surfaces of the different bead types (for simplicity, they are shown as clusters (e.g., 360, 362, 364, and 366) of solid spots (e.g., 357), where the positioning of the clusters corresponds to the positioning of the chambers). Each protein will bind to a different surface of the beads having a characteristic pattern; i.e., (for example), hydrophobic proteins will be more expressed on the hydrophobic bead surface than on the hydrophilic bead surface, and the same is true for other types of bead surfaces. In some embodiments, for each protein, signal integration can be performed for each bead type. The results can be displayed as intensity spectra of each protein on each different surface, e.g. Figure 3B As shown in the picture.

[0033] Systems and Instruments

[0034] exist Figure 4A An example of a system for performing the above methods is illustrated in . In some cases, the flow cell (400) is a component of a fluidic device that provides one or more channels and liquid handling components under programmable control to deliver beads and reagents to the channels. In this illustration, four channels (402, 404, 406, and 408) are shown, and an enlarged view (412) of a segment (410) of channel 2 (404) is shown below. Figure 4AIn the abstract view of the flow cell (400), the inlet, outlet and other features of the channel are not shown. On the first surface (414) of channel 2 (404), a plurality of beads, such as (418), can each be encapsulated by a hydrogel chamber, such as (416). In some embodiments, the porosity of the polymer matrix wall of the hydrogel chamber is selected to be impermeable to the beads, but permeable to the reagents used to form the spatial barcode. Thus, reagents can be introduced into the interior of the hydrogel chamber and removed from the interior of the hydrogel chamber by flowing (420) the reagents through the channel, but the beads remain inside. An example of an optical system (421) for synthesizing hydrogel chamber photosensitivity at the location of the beads in the channel is shown below the enlarged view (412) of the channel segment (410). One of ordinary skill in the art will recognize that a device having the same structure as the hydrogel chamber can be used. Figure 4A and Figure 4B Optical systems of different configurations can perform these functions. In some embodiments, one or more DMD-objective subsystems for synthesizing hydrogel structures can be used to increase the synthesis speed by synthesizing multiple structures simultaneously.

[0035] return Figure 4A , for making the hydrogel chamber light energy synthesis, the light source (422) can generate a beam (423) of light of appropriate wavelength (e.g., UV light), which passes through an appropriate photomask or beam shaping or beam pointing (Galvo) system to shape the beam so as to synthesize the desired one or more structures in the channel. In some embodiments, a digital micromirror device (DMD) (424) is used, and in other embodiments, a physical photomask can be used. The chamber position, shape, and polymer matrix wall thickness can be determined at least in part based on the bead position information determined by the image collected by the detector (432). The reflected light from the DMD (424) can be shaped using conventional optical devices (e.g., collimating optical devices (428)) and can be directed through the objective lens system (434) into the channel 2 section (410). The objective lens (434) and the flow cell (400) can be moved relative to each other in the xy direction (436) to make the chamber light energy synthesis at any position in any channel. In some embodiments, the flow cell (400) moves while the optical system (421) is stationary. In some embodiments, the objective lens (434) can also direct the light beam (427) from the light source (429) to a target (such as a cell) on the first surface (414) and collect an optical signal (such as a fluorescent signal) from an assay performed on the first surface (414). Figure 4B The optical signal is collected by a separate objective lens as shown. Figure 4BInformation collected by the counterparts in the embodiments, particularly the cell positions in their corresponding channels, is used by a computer (438) and / or an auxiliary controller to guide the DMD (424) and a translation device that controls the relative positions of the objective lens (434) and the flow cell (400) to synthesize hydrogel chambers of appropriate shape and size at appropriate locations.

[0036] Figure 4B An alternative optical system is illustrated in which a detection portion (450) of the optical system moves (472) independently of movement (468) of a combining portion (452) of the optical system. The detection portion (450) of the optical system may include a detector (456), an objective lens (458), a light source (460), and interconnected optical elements, such as a dichroic mirror (462). Figure 4A As in the embodiment of the present invention, the detector (456) can be operatively associated with the computer (464) and the synthesis portion (452) of the optical system to provide bead position information to the synthesis portion (452). The computer (464) and the computer (438) can also be operatively associated with a stage and / or motor that controls the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the synthesis portion (452) of the optical system is located on the opposite side of the first surface (464) from the detection portion (450). Figure 4A As with the embodiment of the present invention, the synthesis portion (452) includes conventional components such as an objective lens (474), a reflector (476), a collimating optical device (480), a DMD (482) and a light source (478).

[0037] In some embodiments, a system for implementing the methods described herein includes: (i) a channel comprising a surface; (ii) a spatial energy modulation element optically connected to the surface; and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element, the detector identifying cells and determining their location on the surface. It should be understood that the term "detector" as used herein may include, but is not limited to: a microscope element that collects and optionally magnifies an image of a portion of the channel; and an image analysis element that includes software for identifying cells and associated location information. A computer element may use such information generated by the detector, as well as user input, to generate commands for other elements (such as the spatial energy modulation element) to perform a variety of functions, including, but not limited to, synthesis chambers, "on demand" degradation of chambers, selective photodegradation chambers, and the like. Examples of configurations of such embodiments are described above. Figure 4A-4BIn some embodiments, the channel of the fluid device further includes a second surface (as shown in Figure 2), wherein the first surface and the second surface are arranged relative to each other across the channel, and wherein the polymer matrix wall of the chamber extends from the first surface to the second surface to form a chamber each having an interior. In some embodiments, the chambers in the channel each encapsulate a single cell. In some embodiments, the first wall and the second wall are both made of light-transmitting materials (such as glass, plastic, etc.), and are positioned so that the first surface and the second surface are substantially parallel to each other. The vertical distance between the first surface and the second surface can be in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm.

[0038] In some embodiments, multiple channels can be Figure 5A-Figure 5B As shown, the plurality of channels can be arranged together in a flow channel. In some embodiments, the plurality of channels can be in the range of 2 to 12, or 2 to 8, or 2 to 6, or in the range of 2 to 4. An example of a flow cell (500) is shown in a cross-sectional view and a top view. In some cases, the flow cell (500) has: a bottom wall or first wall (506), the bottom wall or first wall having a first surface (505); a top wall or second wall (502), the top wall or second wall having a second surface (501); and a spacer (504) sealingly sandwiched between the bottom wall or first wall and the top wall or second wall, the longitudinal holes of the spacer forming channels 1-channel 6, one of which is represented by (508) in the cross-sectional view and by (512) in the top view. In some embodiments, the thickness of the spacer (504) can be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm, which determines the inner thickness of the channel. The top wall (502) includes an inlet (514) and an outlet (516) for loading and removing reagents and beads from channel 1-channel 6 individually or collectively. In some embodiments, at least one of the wall (502) and the wall (506) is made of a light-transmitting material (such as glass, plastic, etc.). The flow cell (500) can be operationally associated with a fluid device that delivers reagents and beads to any one of channel 1-channel 6 under programmed control. Guidance for specific designs (including fluid handling and valve adjustment of such fluid systems) can be found in U.S. Patents 8921073, 8173080, and 8900828, etc., which are incorporated herein by reference. Figure 5B A channel of a flow cell (500) is illustrated having randomly distributed (not to scale) hydrogel chambers having a quasi-ring-shaped cross-section, such as (520), on a first surface thereof.

[0039] As described above, any of the first surface, second surface, or polymer matrix wall of the chamber may include capture elements and other functional groups for performing various operations including, but not limited to, capturing beads, capturing cells, capturing analytes (such as mRNA, secreted proteins, intracellular proteins, or genomic sequences), capturing components of analytical reagents (such as oligonucleotide tags from antibodies), etc. Derivatized surfaces for such purposes are well known to those skilled in the art, as evidenced by the following references: Integrated DNA Technologies brochure (cited above); Hermanson (cited above); etc.

[0040] As described above, in some embodiments, the fluid device of the method includes a detector or is operationally associated with a detector, which can share the optical path of the spatial energy modulation element, or can be arranged near the second wall or in an embodiment (such as a groove) with only the first wall and the first surface, arranged on the other side of the first wall compared to the spatial energy modulation element. The detector is positioned so that it can detect optical signals from cells in the channel or detect optical signals near cells in the channel, for example, the cells are distributed on the first surface in the chamber. In some embodiments, the first wall and the second wall each contain a light-transmitting material, for example, so that the spatial energy modulation element can project light energy into the interior of the channel, and so that the detector can detect optical signals (such as fluorescence emission or reflected light from biological components). In some embodiments, the projected energy from the spatial energy modulation element is light energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulation element can have a complex cross-section, thereby allowing (in various embodiments) to synthesize multiple chambers simultaneously. Light-transmitting materials include but are not limited to materials such as glass, quartz, and plastic.

[0041] The spatial energy modulation element for achieving polymerization using light energy may include a physical photomask or a virtual photomask, such as a digital micromirror device (DMD). The following references are incorporated herein by reference and provide guidance in selecting and operating a DMD for achieving photopolymerization of 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); etc.

[0042] As used herein, "channel" generally means a container capable of accommodating a fluid (which may be static or flowing) and having at least one surface on which beads and synthesis chambers may be arranged. In some embodiments, a channel may have a first surface and / or a second surface on which a synthesis chamber may be arranged and / or on which beads or particles may be arranged. As used herein, reference to "surface" without reference to "first" or "second" is intended to include a first surface or a second surface (if two surfaces are present in a fluid device (e.g., including a flow cell)). In some embodiments, a channel may limit the flow of a fluid passing therethrough from an inlet to an outlet. In other embodiments, a channel may contain a non-flowable volume of fluid that may be removed, replaced, or added through an opening or an inlet; that is, in some embodiments, a channel may be a groove or a groove-like structure.

[0043] Gel Room

[0044] A variety of photosynthesized gels and degradable gels can be used to implement the methods described herein. The following references incorporated by reference provide guidance for selecting such gels to obtain desired properties (including but not limited to biocompatibility, porosity, gelation rate, degradation rate, etc.): 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., ACS Appl. 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., US Patent Publication US2016 / 0177030; Shih et al., Biomacromolecules, 13 (7): 2003-2012 (2012); etc. In some embodiments, a photoinitiator is used for free radical polymerization to form a photosynthesized gel. In some embodiments, the photoinitiator includes Irgacure 2959, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) or eosin-Y (see, for example, Choi et al., Biotechniques, 66 (1): 40-53 (2019)). In some embodiments, the hydrogel precursor includes hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, poly (ethylene 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, the polymer precursor includes PEG or multi-arm PEG. In some embodiments, the polymer precursor includes an enzymatically degradable cross-linking agent. In some embodiments, such enzymatically degradable cross-linking agents can be degraded by esterases or peptidases. In some embodiments, the polymer precursor includes a photodegradable cross-linking agent. In some embodiments, such a photodegradable cross-linking agent includes nitrobenzyl.

[0045] In some embodiments, such photodegradable cross-linking agents include coumarin moieties. In some embodiments, photodegradable hydrogels are used, for example, because the photodegradation of the hydrogel chambers can be selectively and as required, so that the specified hydrogel chambers can be degraded without affecting the unselected hydrogel chambers. In some embodiments, the hydrogel chambers are non-selectively degraded so that all the hydrogel chambers in a given channel (or other vessels) are degraded simultaneously. In some embodiments, such non-selective degradation is carried out with a cutting agent that specifically cuts unstable bonds in the hydrogel. For example, such cutting agents include reducing agents. In some embodiments, such non-specific degradation is carried out with enzymes that cut bonds or chemical elements in the hydrogel. Chemical elements can include, but are not limited to, peptides, polysaccharides, and oligonucleotides.

[0046] In the accompanying drawings, for convenience, the hydrogel chambers are illustrated as being placed in isolation without being connected to adjacent chambers, and are illustrated as having a cylindrical or ring-like shape; however, the spatial energy modulation element can synthesize chambers of different shapes and sizes, as may be useful for a particular application. In some embodiments of the proliferation assay, each of the synthesized hydrogel chambers has the same shape and area, for example, having a diameter selected from 0.001 to 0.01 mm 2 The inner area of ​​the range is annular.

[0047] Function . A variety of photosynthesizable gels can be used in conjunction with the methods described herein. In some embodiments, hydrogels are used, particularly because they are compatible with living cells and are widely used in formulating gels with desired properties (including but not limited to porosity, degradability, mechanical strength, ease and speed of synthesis, etc.). In some embodiments, the gel or hydrogel is both photosynthesizable and photodegradable. In some embodiments, the gel degradation mechanism is compatible with living cells.

[0048] Porosity. In some embodiments, the hydrogel porosity is selected to allow the passage of selected reagents while preventing other reagents or objects (such as cells or proteins of lysed cells) from passing through. In some embodiments, the polymer chains of the cross-linked hydrogel structure form a hydrogel matrix with pores (i.e., a porous hydrogel matrix). In some embodiments, the average diameter of the pores is from about 2nm to about 25nm, or from about 5nm to about 20nm. In some embodiments, the average pore diameter is selected to prevent cell proteins from passing through. In some embodiments, the average pore diameter is selected to prevent cell proteins with a molecular weight of 1 kilodalton or more from passing through. In some embodiments, the average pore diameter is selected to prevent cell proteins with a molecular weight of 5 kilodalton or more from passing through. In some embodiments, the pore size of the hydrogel structure is tuned by changing the ratio of the concentration of the polymer precursor to the concentration of the cross-linking agent, changing pH, salt concentration, temperature, light intensity, etc. Guidance for selecting materials and conditions to control hydrogel porosity can 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); etc.

[0049] Size and shape of hydrogel chambers. In some embodiments, the polymer matrix wall of the chamber inhibits the passage of a predetermined component, such as a mammalian cell, a bacterial cell, or a protein from a lysed cell. In some embodiments, the polymer matrix wall extends from a first surface to a second surface (parallel to the first surface) to form a chamber in a channel. In some embodiments, the chamber has a polymer matrix wall and an interior. In some embodiments, the size of the interior of the chamber is designed to encapsulate cells, such as mammalian cells. For example, such chambers may include a cylindrical shell or a polygonal shell, which includes an interior space or an interior and a polymer matrix wall. In some embodiments, such chambers may have a cross-section of a ring-like shape. As used herein, the term "ring-like cross-section" means a cross-section that is topologically equivalent to a ring. In some embodiments, the interior space or interior of the chamber has an inner diameter of 1 μm to 500 μm and a volume ranging from 1 picoliter to 200 nanoliters or 100 picoliters to 100 nanoliters or 100 picoliters to 10 nanoliters. In some embodiments, the thickness of the polymer matrix wall is at least 1 μm (micrometer). In some embodiments, the value of the height of the chamber with a quasi-annular cross-section ranges from 10 μm to 500 μm or ranges from 50 μm to 250 μm. In some embodiments, the aspect ratio (i.e., height / width) of the polymer matrix wall with a quasi-annular cross-section is 1 or less. In some embodiments, the aspect ratio and polymer matrix wall thickness are selected to maximize chamber stability against forces (such as reagent flow through a channel), washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix is ​​degradable. In some embodiments, the degradation of at least one polymer matrix is ​​"as needed".

[0050] In some embodiments, the chamber in the channel is discontinuous. In some embodiments, the chamber in the channel can be adjacent to an adjacent chamber. In some embodiments, the chamber can share a polymer matrix wall with each other. In some embodiments, the chamber can be synthetic, and its slit or other orifice is large enough to allow some components (such as beads) to pass through, but small enough to prevent other components (such as cells) from passing through.

[0051] Hydrogel compositions. As mentioned above, hydrogel compositions can vary greatly, and hydrogels can be formed by a variety of methods. Biocompatible hydrogel precursors include, but are not limited to, hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, poly (ethylene 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, hydrogels are formed by light-induced free radical crosslinking. In some embodiments, hydrogels are formed by light-induced thiol-ene reactions.

[0052] Hydrogel degradation. In some embodiments, the hydrogel chamber is usually degradable or depolymerizable in the channel or "as needed". The usually degradable hydrogel chamber is degraded by treating with a degradation agent or (equivalently) a depolymerizing agent that is exposed to all chambers in the channel. The depolymerizing agent may include, but is not limited to, heat, light, and / or chemical depolymerizing agents (sometimes also referred to as cutting agents or degradation agents). In some embodiments, polymer precursors that allow photocrosslinking and photodegradation may be used as needed for degradation, for example, using different wavelengths for crosslinking and degradation. For example, Eosin Y can be used for free radical polymerization at a defined area using a 500nm wavelength, after which 380nm irradiation may be used to cut the crosslinking agent. In other embodiments, a light cage hydrogel cutting agent may be included in the formation of the polymer matrix wall. For example, an acid-labile crosslinking agent (such as an ester, etc.) may be used to produce a hydrogel, and then UV light may be used to generate local acidic conditions, which in turn degrade the hydrogel. In some embodiments, at least one polymer matrix can be degraded by at least one of the following: (i) contacting at least one polymer matrix with a cleavage agent; (ii) heating at least one polymer matrix to at least 90°C; or (iii) exposing at least one polymer matrix to a certain wavelength of light that cleaves a photo-cleavable cross-linking agent, which cross-links the polymers of at least one polymer matrix. In some embodiments, at least one polymer matrix includes a hydrogel. In some embodiments, the cleavage agent degrades the hydrogel. In some embodiments, the cleavage agent includes a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage agent, or a combination thereof. In some embodiments, the cleavage agent includes 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 can be functionalized by coupling functional groups to the polymer matrix or hydrogel. Some non-limiting examples of functional groups can include capture agents (e.g., picolinaldehyde (PCA)), acrylamide, agarose, biotin, streptavidin, strep-tag II, linkers, functional groups containing aldehydes, phosphates, silicates, esters, acids, amides, aldehyde dithiolanes, PEGs, thiols, olefins, alkynes, azides, or combinations thereof. In some cases, the functionalized polymer matrix can be used to capture biomolecules within a polymer matrix compartment formed adjacent to (e.g., around or on) a biological component.

[0053] Biomolecules can be produced by biological components (e.g., secretomes from cells). Functionalized surfaces of polymer matrices inside compartments can be used to capture reagents or molecules from outside the compartments. Functionalized surfaces can increase the surface area covered by reagents, molecular sensors, or any molecules of interest (e.g., antibodies).

[0054] Photosynthesis. In some embodiments, generating a polymer matrix in a channel or groove of a fluid device includes exposing 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 of 350nm to 800nm. In some embodiments, the light generating device generates light of 350nm to 600nm. In some embodiments, the light generating device generates light of 350nm to 450nm. In some embodiments, the light generating device generates UV light. In some embodiments, generating a polymer matrix in the fluid device is performed using a spatial light modulator (SLM) (i.e., a spatial energy modulation element capable of spatially generating a desired light intensity pattern). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam manipulated using a galvanometer. In some embodiments, the SLM is based on liquid crystal.

[0055] Although the present disclosure has been described with reference to several specific 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 also applicable to various sensor embodiments and other subject matter in addition to those discussed above.

[0056] definition

[0057] Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow standard treatises and texts in the field, e.g., Kornberg and Baker, DNA Replication, 2nd edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd edition (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immuology, 6th edition (Saunders, 2007).

[0058] As used herein, "antibody" generally refers to any binding compound that can specifically bind to a given protein, including but not limited to 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, etc.

[0059] As used herein, "detection antibody" generally refers to an antibody conjugated to a detection moiety that allows identification of the protein to which the antibody binds. The detection moiety may include a fluorescent dye, a barcode, an enzyme, etc. In some embodiments, the detection moiety may be covalently attached to the antibody. In some embodiments, the detection moiety may be conjugated to the antibody via a cleavable bond or via hybridization.

[0060] As used herein, "cell" generally refers to a biological cell that can be determined by the methods and systems described herein, but is not limited to vertebrate cells, invertebrate cells, eukaryotic cells, mammalian cells, microbial cells, protozoan cells, prokaryotic cells, bacterial cells, insect cells or fungal cells. In some embodiments, mammalian cells are determined by the methods and systems described herein. In particular, any mammalian cell that can or has been genetically altered to be used in medicine, industry, environment or treatment processes can be analyzed by the methods and systems described herein. In some embodiments, "cell" as used herein includes genetically modified mammalian cells. In some embodiments, "cell" includes stem cells. In some embodiments, "cell" refers to cells modified by CRISPR Cas9 technology. In some embodiments, "cell" refers 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 particular interest are cytotoxic T lymphocytes engineered for therapeutic applications (such as cancer therapy).

[0061] As used herein, "hydrogel" generally means a gel comprising a cross-linked hydrophilic polymer network that is able to absorb and retain large amounts of water (e.g., 60 to 90 percent water, or 70 to 80 percent) without dissolving due to the establishment of physical or chemical bonds (which may be covalent, ionic, or hydrogen bonds) between polymer chains. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications.

[0062] Hydrogels can include natural or synthetic polymers and can be reversible (i.e., degradable or depolymerizable) or irreversible. Synthetic hydrogel polymers can include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and poly(vinyl alcohol). Natural hydrogel polymers include alginate, hyaluronic acid, and collagen. The following references 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).

[0063] As used herein, "as needed" generally means that operations can be directed to individual, discrete, selected locations (e.g., spatial location of polymer precursor solutions; or selected polymer matrix chambers). Such selections can be based on manual observation of optical signals or data collected by the detector, or such selections can be based on computer algorithms that operate on optical signals or data collected by the detector. Manual observation of optical signals or data collected by the detector can include real-time detection or detection of a certain period of time before modulating unit energy to polymerize polymer precursors or degrading chambers. For example, a subset of chambers (all formed with photodegradable polymer matrix walls) can be preselected based on position information and the value of the optical signal from the analytical determination performed in the chamber for release and removal of its contents. The preselected chamber can be photodegraded by selectively projecting a beam of light of appropriate wavelength characteristics (e.g., with a spatial energy modulation element) to degrade the polymer matrix wall of the preselected chamber. In another example, multiple chambers can be observed in real time (e.g., via fluorescence microscopy) to detect an analyte of interest, and one or more chambers in multiple chambers can be selected in real time after detecting the analyte of interest for degradation.

[0064] As used herein, "polymer matrix" generally refers to a phase material (e.g., continuous phase material) comprising at least one polymer. In some embodiments, polymer matrix refers to at least one polymer and interstitial space not occupied by polymer. Polymer matrix can be composed of one or more types of polymers. Polymer matrix can include straight chain, branched and cross-linked polymer units. Polymer matrix can also include non-polymer species inserted into the interstitial space not occupied by polymer chains. The inserted species can be solid, liquid or gas species. For example, the term "polymer matrix" can encompass dry hydrogels, hydrated hydrogels and hydrogels comprising glass fibers. Polymer matrix can include polymer precursors, which generally refer to one or more molecules that can trigger or initiate polymerization when activated. Polymer precursors can be activated by electrochemical energy, photochemical energy, photons, magnetic energy or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which are polymerized to produce polymer matrix) and cross-linked compounds, which can include photoinitiators, other compounds necessary or useful for generating polymer matrix (especially as polymer matrix of hydrogel).

Claims

1. A method for determining the proteome of a single cell, the method comprising: include: (a) synthesizing one or more gel chambers that encapsulate each of one or more cells disposed on a channel surface; (b) lysing the cells such that cellular proteins of each cell are released into its gel compartment and at least a portion is adsorbed onto the surface enclosed by the gel compartment; as well as (c) detecting the adsorbed protein using a detection antibody.

2. The method of claim 1, further comprising collecting an optical signal from the one or more cells disposed on the surface; and determining a position of each of the one or more cells from the optical signal prior to the synthesizing.

3. The method of claim 1, further comprising depolymerizing the one or more gel chambers prior to the detecting. The method according to claim 3 , further comprising desorbing the adsorbed protein prior to the depolymerization.

5. The method of claim 3, wherein the surface comprises a plurality of types of particles disposed on the surface, wherein each type of particle comprises a surface having different protein adsorption properties, and wherein the size and number of each type of particle is such that each of the hydrogel chambers encapsulates each type of particle.

6. The method of claim 3, wherein each of the detection antibodies comprises a label capable of generating a signal indicative of the protein to which such detection antibody is specifically directed.

7. The method of claim 6, wherein the label is capable of generating an optical signal indicative of a protein to which the detection antibody is specifically directed.

8. The method of claim 6, wherein the label comprises a barcode indicating a protein to which the detection antibody is specifically directed.

9. A method for determining the proteome of a single cell, the method include: (a) providing a fluidic device comprising: (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 operably associated with the spatial energy modulation element; (b) loading the channel with cells and one or more polymer precursors such that the cells are disposed on or near the surface; (c) collecting, using the detector, one or more optical signals for identifying the cell and the location of the cell on the surface; (d) synthesizing one or more hydrogel chambers encapsulating each of the one or more cells by projecting light into the channel using the spatial energy modulation element such that the projected light causes cross-linking of the one or more polymer precursors to form a hydrogel chamber, wherein the position of the hydrogel chamber in the channel is determined by the position of the cell encapsulated by the hydrogel chamber identified by the detector; (e) lysing the cells such that at least a portion of the cellular proteins of each cell are released into its hydrogel compartment and adsorbed to a portion of the surface enclosed by the hydrogel compartment; (f) depolymerizing the hydrogel chamber; and (g) loading the channel with detection antibodies for identifying the adsorbed protein.

10. The method of claim 9, wherein the surface comprises a plurality of types of particles disposed on the surface, wherein each type of particle comprises a surface having different protein adsorption properties, and wherein the size and number of each type of particle is such that each of the hydrogel chambers encapsulates each type of particle.

11. The method of claim 9, wherein each of the detection antibodies comprises a label capable of generating a signal indicative of the protein to which such detection antibody is specifically directed.

12. The method of claim 11, wherein the label is capable of generating an optical signal indicative of a protein to which the detection antibody is specifically directed.

13. The method of claim 11, wherein the label comprises a barcode indicating a protein to which the detection antibody is specific.

14. The method of claim 9, further comprising desorbing the adsorbed protein on the surface prior to the depolymerization.

15. A method for determining the location and identity of a target molecule on a surface, the method include: (a) exposing one or more target molecules adsorbed to a spatially barcoded surface to detection antibodies under binding conditions, wherein each detection antibody comprises an antibody barcode that identifies an epitope to which the detection antibody is specific, wherein the antibody barcode comprises a free 3' end, and wherein the spatial barcode of the surface comprises a free 5' phosphate; (b) exposing the spatially barcoded surface to an adapter oligonucleotide under hybridization conditions, the 3' end of the adapter oligonucleotide being configured to hybridize to the free 3' end of the antibody barcode and to hybridize to the free 5' end of the spatial barcode on the surface; (c) extending the 3' end of the antibody barcode to the 5' end of the spatial barcode; as well as (d) ligating the 3' end of the extended antibody barcode to the 5' end of the spatial barcode.

16. The method of claim 15, wherein the location and identity of the target molecule is determined from the nucleotide sequence of the extended 3' end of the antibody barcode and the spatial barcode.

17. The method of claim 16, wherein the target molecule is a protein.

18. A method for determining the proteome of a single cell, the method include: (a) synthesizing one or more gel chambers that encapsulate each of one or more cells disposed on a channel surface; (b) fixing and permeabilizing the cells to make the intracellular proteins of each cell accessible to the detection antibody; as well as (c) detecting the intracellular protein using the detection antibody.

19. The method of claim 18, further comprising collecting an optical signal from the one or more cells disposed on the surface; and determining a position of each of the one or more cells from the optical signal prior to the synthesizing.

20. A method for determining the proteome of one or more cells, the method include: (a) providing a channel having a surface, wherein the surface comprises the one or more cells disposed on the surface; (b) synthesizing a gel chamber encapsulating a cell of the one or more cells; (c) lysing the cells so that cellular proteins of the cells are released into the gel chamber, wherein at least a portion of the cellular proteins are adsorbed to a portion of the surface enclosed by the gel chamber; as well as (d) detecting at least a portion of the adsorbed protein using one or more detection antibodies.

21. A method for determining the location and identity of a target molecule, the method include: (a) providing a channel having a spatially barcoded surface, the spatially barcoded surface comprising one or more spatial barcodes comprising a free 5' end, wherein the spatially barcoded surface comprises the target molecule adsorbed to the spatially barcoded surface; (b) exposing the target molecule to one or more detection antibodies under binding conditions, wherein the detection antibody in the one or more detection antibodies comprises (i) an antibody barcode that identifies the epitope to which the detection antibody is specifically directed and (ii) a free 3' end; (c) exposing the spatially barcoded surface to an adapter oligonucleotide under hybridization conditions, the 3' end of the adapter oligonucleotide being configured to hybridize to the free 3' end of the antibody barcode and to hybridize to the free 5' end of the spatial barcode on the surface; (d) extending the 3' end of the antibody barcode to the 5' end of the spatial barcode; as well as (e) ligating the 3' end of the extended antibody barcode to the 5' end of the spatial barcode.

Images