Nanoscale reaction chambers and methods of use thereof

Abstract

Nanoscale reaction chambers and methods of using the nanoscale reaction chambers in biological assays are provided.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 174,847, filed April 14, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Current standard assay formats interrogate cell populations and collect data. During the interrogation and data collection process, cell-specific information can be lost due to cell pooling between steps, for example, between assays and / or instruments. Therefore, there is a need for an assay platform that can interrogate and collect data on cell populations and retain cell-specific information that can be associated with the collected data. Summary of the Invention

[0003] There exists an unmet need for assays that can combine functional single-cell-based assay information with genomic, transcriptomic, and / or proteomic single-cell-based assay information. The present disclosure fulfills this unmet need by providing nanoscale reaction chambers (e.g., nanovials) that perform cell-specific assays while maintaining cell-specific information throughout the course of the assay. The nanovial reaction chambers of the present disclosure are unique in that results from assays (e.g., functional assays) performed in the nanovials can be correlated with genomic, transcriptomic, and / or proteomic analyses of the single-cell components of the assay after data analysis is complete, thereby providing highly targeted multidimensional analysis.

[0004] In one aspect, a method is provided for identifying an antibody or fragment thereof that binds to an antigen of interest, the method comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading the cavity of the nanovial with antibody-producing cells; (c) incubating the nanovial such that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent; (d) adding a detection agent to the cavity of the nanovial such that the detection agent binds to the one or more antibodies or fragments thereof; and (e) (f) detecting one or more signals related to the binding of a detection agent to one or more antibodies or fragments thereof; (f) performing a sequencing assay on nucleic acids derived from the antibody-producing cells, thereby generating sequences of the one or more antibodies or fragments thereof; (g) correlating the sequences of the one or more antibodies or fragments thereof with one or more signals related to the binding of a detection agent to the one or more antibodies or fragments thereof; and (h) identifying one or more antibodies or fragments thereof that bind to the antigen of interest based on the correlating step of (g) (e.g., performing one or more of these steps). In some cases, the method further includes, prior to (f), sorting the nanovial based on one or more signals related to the binding of the detection agent to the antibody. In some cases, the cavity of the nanovial further includes one or more cell capture agents immobilized thereon. In some cases, the one or more cell capture agents include an antibody or fragment thereof that binds to a protein expressed on the surface of the antibody-producing cells. In some cases, the one or more cell capture agents bind to a label present on the surface of the antibody-producing cells. In some cases, the label is streptavidin or biotin. In some cases, the one or more cell capture agents are antigens to which antibody-producing cells can bind. In some cases, the affinity agent is an antibody capture moiety. In some cases, the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, Protein A, and Protein G. In some cases, the affinity agent is an antigen of interest.In some cases, the affinity agent is an antigen-presenting cell expressing the antigen of interest on its surface. In some cases, the detection agent is the antigen of interest. In some cases, the detection agent is an antibody or a fragment thereof. In some cases, the detection agent is directly or indirectly labeled with a detectable label. In some cases, the detectable label is selected from the group consisting of a fluorescent label, a protein affinity tag, an oligonucleotide tag, and a magnetic particle. In some cases, the detectable label is a fluorescent label. In some cases, the detecting step (e) includes determining the amount of detection agent bound to the one or more antibodies or fragments thereof. In some cases, the amount of detection agent bound to the one or more antibodies or fragments thereof corresponds to the level of fluorescence. In some cases, the method further includes, prior to (f), sorting the nanovials based on the level of fluorescence above a background level. In some cases, the sorting step includes performing a flow-based sorting method. In some cases, the flow-based sorting method is fluorescence-activated cell sorting (FACS). In some cases, the detectable label is an oligonucleotide tag. In some cases, the method further includes sequencing the oligonucleotide tag. In some cases, the detecting step (e) comprises counting the number of oligonucleotide tags bound to one or more antibodies or fragments thereof. Optionally, the method further comprises lysing the antibody-producing cells prior to (f). Optionally, the method further comprises reverse transcribing mRNA released from the antibody-producing cells after lysis to generate cDNA. Optionally, the method further comprises sequencing the cDNA. Optionally, the sequencing assay comprises single-cell RNA sequencing. Optionally, the method further comprises encapsulating the nanovial within the droplet prior to (c). Optionally, the cavity of the nanovial comprises an opening to the surface of the nanovial. Optionally, the method further comprises adding one or more blocking particles to block the opening or reduce the size of the opening prior to (c). Optionally, the method further comprises washing the nanovial prior to (d).In some cases, the method involves determining a desired dissociation constant (K) for the antigen of interest. D ) for the antigen of interest. In some cases, the one or more antibodies or fragments thereof have a desired K of less than about 1 μM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. D In some cases, the method further comprises adding a detection agent to the plurality of nanovials at one or more different concentrations. In some cases, the one or more different concentrations have a desired K D , within an order of magnitude. In some cases, the method is configured to identify one or more antibodies or fragments thereof having a desired specificity for the antigen of interest. In some cases, the method further includes adding a plurality of different detection agents to the plurality of nanovials, at least one of the different detection agents having a detectable label. In some cases, the method further includes identifying one or more antibodies or fragments thereof that specifically bind to one of the different detection agents based on measuring the detectable label. In some cases, the detection agent is the antigen of interest.

[0005] In another aspect, a method is provided for identifying one or more antibodies or fragments thereof that modulate a signal transduction pathway of interest, the method comprising (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein; (b) loading antibody-producing cells and antigen-producing cells within the cavity of the nanovial; (c) incubating the nanovial such that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and modulate the signal transduction pathway of interest in the antigen-producing cells; (d) detecting one or more signals related to modulation of the signal transduction pathway of interest; (e) performing a sequencing assay on nucleic acid derived from the antibody-producing cells, thereby generating sequences of the one or more antibodies or fragments thereof; (f) associating the sequences of the one or more antibodies or fragments thereof with one or more signals related to modulation of the signal transduction pathway of interest; and (g) identifying one or more antibodies or fragments thereof that modulate the signal transduction pathway of interest based on the associating step of (f) (e.g., performing one or more of these). In some cases, the method further comprises, prior to (e), sorting the nanovial based on one or more signals related to modulation of a signaling pathway of interest. In some cases, modulation of a signaling pathway of interest comprises activation of the signaling pathway of interest. In some cases, modulation of a signaling pathway of interest comprises inhibition of the signaling pathway of interest. In some cases, the one or more antibodies or fragments thereof modulate the signaling pathway of interest by binding to an antigen on the surface of the antigen-producing cells. In some cases, the one or more antibodies or fragments thereof modulate the signaling pathway of interest by interfering with binding of a ligand to the antigen on the surface of the antigen-producing cells. In some cases, the cavity of the nanovial further comprises one or more cell capture agents immobilized thereon. In some cases, the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of the antibody-producing cells, the antigen-producing cells, or both.In some cases, the one or more cell capture agents bind to a label present on the surface of the antibody-producing cells, the antigen-producing cells, or both. In some cases, the one or more cell capture agents are antigens to which the antibody-producing cells, the antigen-producing cells, or both can bind. In some cases, the antigen-producing cells are engineered to express one or more detectable labels upon modulation of the signal transduction pathway of interest. In some cases, the one or more detectable labels include a fluorescent reporter protein. In some cases, the detecting step (d) includes detecting a level of fluorescence in the nanovial, wherein the level of fluorescence corresponds to modulation of the signal transduction pathway of interest. In some cases, the method further includes, prior to (e), sorting the nanovial based on a level of fluorescence above a background level. In some cases, the sorting step includes performing a flow-based sorting method. In some cases, the flow-based sorting method is fluorescence-activated cell sorting (FACS). In some cases, the detecting step (d) includes detecting a change in one or more mRNA levels in the antigen-producing cells. In some cases, the detecting step includes performing single-cell RNA sequencing on nucleic acids derived from the antigen-producing cells. In some cases, the associating step of (f) includes linking the mRNA levels of the antigen-producing cells with nucleic acid sequence information from the antibody-producing cells based on the shared oligonucleotide barcode. In some cases, the shared oligonucleotide barcode includes one or more unique oligonucleotide tags associated with the nanovial. In some cases, the method further includes lysing the antibody-producing cells and the antigen-producing cells prior to (e). In some cases, the method further includes reverse transcribing the mRNA released from the antibody-producing cells after lysis to generate cDNA. In some cases, the method further includes sequencing the cDNA. In some cases, the sequencing assay includes single-cell RNA sequencing. In some cases, the method further includes encapsulating the nanovial within a droplet prior to (c). In some cases, the cavity of the nanovial includes an opening to the surface of the nanovial.In some cases, the method further comprises, prior to (c), adding one or more blocking particles to block the opening or reduce the size of the opening. In some cases, the method further comprises, prior to (d), washing the nanovial. In some cases, the antibody-producing cells are selected from the group consisting of B cells, plasmablasts, plasma cells, hybridomas, and engineered producer cells. In some cases, the nanovial contains a single antibody-producing cell. In some cases, the method further comprises performing the method on a plurality of nanovials, each of the nanovials containing an individual antibody-producing cell within the cavity of the nanovial. In some cases, the plurality of nanovials comprises at least 20,000 nanovials, at least 100,000 nanovials, or at least 500,000 nanovials. In some cases, each individual antibody-producing cell produces a different antibody. In some cases, the nanovial contains a crosslinked hydrogel. In some cases, the crosslinked hydrogel is a PEG-based crosslinked hydrogel. In some embodiments, the cavity of the nanovial includes a liquid disposed therein. In some embodiments, the nanovial is suspended in an oil phase. In some embodiments, the cavity of the nanovial has a volume of about 100 fL to about 10 nL. In some embodiments, the cavity of the nanovial has a linear dimension of about 5 μm to about 250 μm.

[0006] In yet another embodiment, a method for linking functional single-cell information with genomic, transcriptomic, and / or proteomic information is provided, the method comprising: (a) performing a functional assay on a single cell located in a cavity of a nanovial, wherein the functional assay is associated with a first barcode or a first label; (b) performing a genomic, transcriptomic, and / or proteomic assay on the single cell in the nanovial, wherein the genomic, transcriptomic, and / or proteomic assay is associated with a second barcode or a second label; and (c) linking the functional single-cell information with the genomic, transcriptomic, and / or proteomic information by associating the first barcode or first label with the second barcode or second label. Optionally, the method further comprises, prior to (b), classifying the nanovial based on a signal generated by the functional assay. In some cases, the functional assay is an antibody secretion screening assay. In some cases, the functional assay is an antibody affinity assay. In some cases, the functional assay is an antibody specificity assay. In some cases, the genomic assay is a single-cell RNA sequencing assay. In some cases, the nanovial is associated with a unique oligonucleotide tag. In some cases, the unique oligonucleotide tag includes a poly-dT capture region. In some cases, the first barcode or first label includes an oligonucleotide having a polyA region.

[0007] In yet another aspect, a method for preventing diffusion of a biological agent from a cavity of a nanovial is provided, the method comprising: (a) providing or obtaining a nanovial, the nanovial including a cavity formed therein and including an opening to a surface of the nanovial, and one or more biological agents disposed within the cavity; and (b) adding blocking particles such that the blocking particles interact with the opening of the cavity and substantially block or reduce the size of the opening, thereby inhibiting diffusion of the biological agent from the cavity when the nanovial is placed in a fluid. Optionally, the maximum diameter of the blocking particles is greater than the diameter of the opening of the cavity. Optionally, the blocking particles are in contact with the opening of the cavity. Optionally, the blocking particles surround the opening of the cavity. Optionally, the blocking particles are sized to maintain more than 50% of the volume of the cavity after (b). Optionally, the blocking particles have an average diameter of about 20 micrometers to about 50 micrometers. In some cases, the blocking particles are spherical or substantially spherical in shape. In some cases, the blocking particles include a polymer hydrogel. In some cases, the polymer hydrogel is polyethylene glycol (PEG). In some cases, the blocking particles are coated with one or more affinity agents. In some cases, the one or more affinity agents include an antibody capture moiety. In some cases, the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, protein A, and protein G. In some cases, the one or more affinity agents include an antigen. In some cases, the one or more affinity agents include a nucleic acid. In some cases, the nucleic acid is an oligonucleotide tag or an oligonucleotide barcode. In some cases, the nanovial and / or blocking particles include a mesh-like structure. In some cases, the mesh-like structure includes an average pore size. In some cases, the average pore size is sized such that biological agents having a desired size are retained within the cavities and biological agents having a size smaller than the desired size can diffuse through the mesh-like structure.In some cases, the average pore size is sized to allow transport of fluids and reagents into the cavity of the nanovial. In some cases, the fluids and reagents are selected from the group consisting of stains, surfactants, detergents, lysis buffers, drugs, cytokines, chemokines, culture media, conditioned media, and buffers. In some cases, (b) includes adding a plurality of blocking particles such that the plurality of blocking particles interact with the opening of the cavity and substantially block the opening of the cavity or reduce the size of the opening. In some cases, each of the plurality of blocking particles has an average diameter of about 2 micrometers to about 20 micrometers. In some cases, each of the plurality of blocking particles has a density greater than that of saline at room temperature. In some cases, the number of blocking particles to nanovials is greater than 100:1. In some cases, the plurality of blocking particles is comprised of a polymer selected from the group consisting of PEG, polystyrene, polymethylmethacrylate, glass, and metal.

[0008] In another aspect, a method is provided for concentrating nanovials containing one or more cells, one or more beads, or both, comprising: (a) providing or obtaining a plurality of nanovials, each containing at least one cell, at least one bead, or both disposed within a cavity of the nanovial, and a mixture containing (i) a plurality of suspended cells, suspended beads, or both, (ii) a plurality of empty nanovials, or (iii) both (i) and (ii); (b) suspending the mixture in a solution; (c) adjusting the density of the solution such that the plurality of nanovials containing at least one cell, at least one bead, or both are disposed within a cavity of a nanovial separate from the plurality of suspended cells, suspended beads, or both of (i), the plurality of empty nanovials of (ii), or both of (i) and (ii); and (d) collecting the plurality of nanovials containing at least one cell, at least one bead, or both disposed within the cavity of the nanovial. In some cases, (c) includes adjusting the density of the solution such that the plurality of nanovials containing at least one cell, at least one bead, or both disposed within the cavity of the nanovial have a density greater than the density of the solution. In some cases, (c) includes adjusting the density of the solution such that the plurality of nanovials containing at least one cell, at least one bead, or both disposed within the cavity of the nanovial have a density less than the density of the solution. In some cases, the method further includes performing (a)-(d) one or more times to further concentrate the plurality of nanovials containing at least one cell, at least one bead, or both disposed within the cavity of the nanovial. In some cases, the density of the solution is different in each round of (a)-(d). In some cases, the method further includes, prior to (a), removing the plurality of floating cells, floating beads, or both of (i) or the plurality of empty nanovials of (ii).

[0009] In yet another aspect, a method is provided for identifying a drug or compound that modulates a signal transduction pathway in a cell of interest, the method comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein, the cavity comprising (i) a drug or compound, and (ii) a barcode or label associated with the drug or compound; (b) loading a cell of interest within the cavity of the nanovial; and (c) releasing the drug or compound into a liquid volume within the cavity of the nanovial, such that the drug or compound interacts with the cell of interest. (d) exposing the nanovial to a stimulus to release a signal transduction pathway of interest in the cell of interest; (d) incubating the nanovial so that the drug or compound modulates the signal transduction pathway of interest in the cell of interest; (e) detecting one or more signals related to modulation of the signal transduction pathway in the cell of interest; (f) associating a barcode or label with the one or more signals related to modulation of the signal transduction pathway of interest; and (g) identifying the drug or compound that modulates the signal transduction pathway of interest based on the associating step of (f) (e.g., performing one or more of these). In some cases, the method further includes, prior to (f), classifying the nanovial based on the one or more signals related to modulation of the signal transduction pathway of interest. In some cases, modulation of the signal transduction pathway of interest includes activation of the signal transduction pathway of interest. In some cases, modulation of the signal transduction pathway of interest includes inhibition of the signal transduction pathway of interest. In some cases, the cavity of the nanovial further includes one or more cell capture agents immobilized thereon. In some cases, the one or more cell capture agents include an antibody or fragment thereof that binds to a protein expressed on the surface of the cell of interest. In some cases, one or more cell capture agents are bound to the label present on the surface of the cell of interest.In some cases, the cell of interest is engineered to express one or more detectable labels when the signal transduction pathway of interest is regulated.In some cases, one or more detectable labels comprise fluorescent reporter protein.In some cases, the detecting step (e) includes detecting a level of fluorescence in the nanovial, wherein the level of fluorescence corresponds to modulation of the signaling pathway of interest. In some cases, the method further includes, prior to (f), sorting the nanovial based on a level of fluorescence above a background level. In some cases, the sorting step includes performing a flow-based sorting method. In some cases, the flow-based sorting method is fluorescence-activated cell sorting (FACS). In some cases, the detecting step (e) includes detecting a change in one or more mRNA levels in the cell of interest. In some cases, the detecting includes performing single-cell RNA sequencing on nucleic acids derived from the cell of interest. In some cases, the associating step (f) includes linking the mRNA levels of the cell of interest to a drug or compound based on a shared oligonucleotide barcode. In some cases, the shared oligonucleotide barcode includes one or more unique oligonucleotide sequences associated with a barcode associated with the drug or compound. In some cases, the method further includes lysing the cell of interest prior to (e). In some cases, the method further comprises reverse transcribing the mRNA released from the cells of interest after lysis to generate cDNA. In some cases, the method further comprises sequencing the cDNA. In some cases, the sequencing assay comprises single-cell RNA sequencing. In some cases, the method further comprises, prior to (c), encapsulating the nanovial within the droplet.

[0010] In yet another aspect, a method is provided for identifying a functional chimeric antigen receptor (CAR) or T cell receptor (TCR) for an antigen of interest, the method comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading a CAR-expressing cell or a TCR-expressing cell within the cavity of the nanovial; (c) incubating the nanovial such that one or more cytokines are secreted from the CAR-expressing cell or the TCR-expressing cell and bind to the affinity agent; and (d) incubating the nanovial such that a detection agent binds to the one or more cytokines. (e) adding a detection agent to the cavity of the nanovial; (e) detecting one or more signals related to binding of the detection agent to one or more cytokines; (f) performing a sequencing assay on nucleic acid derived from the CAR-expressing or TCR-expressing cells, thereby generating a CAR or TCR sequence; (g) correlating the CAR or TCR sequence with one or more signals related to binding of the detection agent to one or more cytokines; and (h) identifying a CAR or TCR that functionally interacts with the antigen of interest based on the correlating step of (g) (e.g., performing one or more of these). In some cases, the method further includes sorting the nanovial based on one or more signals related to binding of the detection agent to one or more cytokines prior to (f). In some cases, the cavity of the nanovial further comprises one or more cell capture agents immobilized thereon. In some cases, the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of the CAR-expressing or TCR-expressing cell. In some cases, one or more cell capture agents bind to a label present on the surface of CAR-expressing cells or TCR-expressing cells.In some cases, the label is streptavidin or biotin.In some cases, one or more cell capture agents are antigens or MHC-presented antigens that CAR-expressing cells or TCR-expressing cells can bind to.In some cases, one or more cell capture agents are antigen-presenting cells that express the antigen of interest on their surface or present the antigen in an MHC complex.In some cases, the affinity agent is an antibody capture moiety. In some cases, the antibody capture moiety is one or more selected from the group consisting of anti-IL2, anti-TNF-α, and anti-IFN-γ. In some cases, the detection agent is a secondary antibody or antibody fragment specific to one or more cytokines. In some cases, the detection agent is directly or indirectly labeled with a detectable label. In some cases, the detectable label is selected from the group consisting of a fluorescent label, an oligonucleotide tag, and a magnetic particle. In some cases, the detectable label is a fluorescent label. In some cases, the detecting step (e) includes determining the amount of detection agent bound to the one or more cytokines. In some cases, the amount of detection agent bound to the one or more antibodies corresponds to the level of fluorescence. In some cases, the method further includes, prior to (f), sorting the nanovials based on the level of fluorescence above background levels. In some cases, the sorting step includes performing a flow-based sorting method. In some cases, the flow-based sorting method is fluorescence-activated cell sorting (FACS). In some cases, the detectable label is an oligonucleotide tag. In some cases, the method further includes sequencing the oligonucleotide tag. In some cases, the detecting step in (e) comprises counting the number of oligonucleotide tags bound to one or more cytokines. In some cases, the method further comprises lysing the CAR-expressing or TCR-expressing cells prior to (f). In some cases, the method further comprises reverse transcribing the mRNA released from the CAR-expressing or TCR-expressing cells after lysis to generate cDNA. In some cases, the method further comprises sequencing the cDNA. In some cases, the sequencing assay comprises single-cell RNA sequencing. In some cases, the method further comprises encapsulating the nanovial within the droplet prior to (c). In some cases, the cavity of the nanovial comprises an opening to the surface of the nanovial. In some cases, the method further comprises adding one or more blocking particles to block the opening or reduce the size of the opening prior to (c). In some cases, the method further comprises washing the nanovial prior to (d).

[0011] In another embodiment, nanovials are provided having a size, shape, surface chemistry, density, or any combination thereof that is adapted to fit into and be used with a plurality of different types of instruments designed to analyze particles and / or cells. In some cases, the plurality of different types of instruments designed to analyze particles and / or cells are selected from the group consisting of flow cytometers, fluorescence-activated cell sorters, imaging flow cytometers, image-activated cell sorters, Coulter counters, particle counters, microfluidic droplet generators, microwell arrays, microfluidic chips with microvalves, microfluidic SlipChips, optofluidic microdevices, microfluidic droplet generators, and microfluidic channels. In some cases, the nanovials are less than about 100 μm in diameter and include capped reactive functional groups. In some cases, the nanovials are less than about 60 μm in diameter. In some cases, the nanovials are configured to be flowed through glass capillaries, cuvettes, and / or microfluidic channels. In some cases, the nanovials are configured to be analyzed by optical, electrical, and / or magnetic excitation. In some cases, the size, surface chemistry, and / or buoyancy of the nanovial are such that the nanovial can flow rapidly through the instrument without clogging or substantial clogging. In some cases, the nanovial can flow through the instrument at greater than 10 cm / sec or greater than 1 m / sec without clogging or substantial clogging. In some cases, the nanovial is configured to flow through a channel having a diameter greater than 90 μm. In some cases, the nanovial is configured to be used with a first instrument designed to analyze particles and / or cells and a second instrument designed to subsequently analyze particles and / or cells. In some cases, the nanovial is configured to be sorted by a fluorescence-activated cell sorter, an image-activated cell sorter, or an optofluidic microdevice and then introduced into a microfluidic droplet generator.

[0012] In yet another aspect, a method is provided for detecting cell-killing function of chimeric antigen receptor (CAR)-T cells or T cell receptor (TCR)-expressing T cells in response to binding of an antigen of interest, the method comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading a CAR-expressing T cell or a TCR-expressing T cell within the cavity of the nanovial; (c) contacting the CAR-expressing T cell or the TCR-expressing T cell with a target cell that produces the antigen of interest; (d) capturing on the nanovial one or more cell-killing markers released from the target cell upon lysis or permeabilization of the target cell; and (e) detecting the one or more cell-killing markers directly or with a detection agent to obtain a cell-killing signal. In some cases, the method further includes (f) performing a sequencing assay on nucleic acid derived from the CAR-expressing or TCR-expressing cell, thereby generating a CAR or TCR sequence; (g) linking the CAR or TCR sequence to one or more cell-killing signals; and (h) identifying a CAR or TCR that functionally interacts with the antigen of interest based on the linking step of (g). In some cases, the method further includes, after (e), sorting the nanovial based on the one or more cell-killing signals. In some cases, the cavity of the nanovial further includes one or more cell capture agents immobilized thereon. In some cases, the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of the CAR-expressing or TCR-expressing cell. In some cases, the one or more cell capture agents bind to a label present on the surface of the CAR-expressing or TCR-expressing cell. In some cases, the label is streptavidin or biotin. In some cases, the one or more cell capture agents are an antigen or MHC-presented antigen to which the CAR-expressing or TCR-expressing cell can bind. In some cases, one or more of the cell capture agents are antigen-presenting cells that express the antigen of interest on their surface or present the antigen in an MHC complex. In some cases, the affinity agent is an antibody capture moiety.In some cases, the antibody capture moiety has affinity for a fluorescent protein. In some cases, the detection agent is an antibody or oligonucleotide having affinity for an intracellular biomolecule from the target cell. In some cases, the detection agent is directly or indirectly labeled with a detectable label. In some cases, the detectable label is selected from the group consisting of a fluorescent label, an oligonucleotide tag, and a magnetic particle. In some cases, the detectable label is a fluorescent label. In some cases, the detecting step (e) includes determining the amount of detection agent bound to the one or more cell kill markers. In some cases, the amount of detection agent bound to the one or more cell kill markers corresponds to the level of fluorescence. In some cases, the method further includes, following (e), sorting the nanovials based on the level of fluorescence above background levels. In some cases, the sorting step includes performing a flow-based sorting method. In some cases, the flow-based sorting method is fluorescence-activated cell sorting (FACS). In some cases, the detectable label is an oligonucleotide tag. In some cases, the method further includes sequencing the oligonucleotide tag. In some cases, the detecting step (e) includes counting the number of oligonucleotide tags bound to the one or more cell kill markers. In some cases, the method further comprises lysing the CAR-expressing or TCR-expressing cells following (e). In some cases, the method further comprises reverse transcribing the mRNA released from the CAR-expressing or TCR-expressing cells after lysis to generate cDNA. In some cases, the method further comprises sequencing the cDNA. In some cases, the sequencing assay comprises single-cell RNA sequencing. In some cases, the method further comprises encapsulating the nanovial within the droplet prior to (d). In some cases, the cavity of the nanovial comprises an opening to the surface of the nanovial. In some cases, the method further comprises adding one or more blocking particles to block the opening or reduce the size of the opening prior to (d). In some cases, the method further comprises washing the nanovial prior to (e). [Brief explanation of the drawings]

[0013] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0014] [Figure 1A] 1 depicts experimental data demonstrating the fabrication and characterization of 40 micron nanovials using various UV power / intensity settings, according to various embodiments of the present disclosure. [Figure 1B] 1 depicts experimental data demonstrating the fabrication and characterization of 40 micron nanovials using various UV power / intensity settings, according to various embodiments of the present disclosure. [Figure 1C] 1 depicts experimental data demonstrating the fabrication and characterization of 40 micron nanovials using various UV power / intensity settings, according to various embodiments of the present disclosure. [Figure 2A] 1 depicts experimental data demonstrating the fabrication and characterization of 60 micron nanovials using various UV power / intensity settings, according to various embodiments of the present disclosure. [Figure 2B] 1 depicts experimental data demonstrating the fabrication and characterization of 60 micron nanovials using various UV power / intensity settings, according to various embodiments of the present disclosure. [Figure 2C] 1 depicts experimental data demonstrating the fabrication and characterization of 60 micron nanovials using various UV power / intensity settings, according to various embodiments of the present disclosure. [Figure 3A] 1 depicts a biotinylated nanovial labeled with fluorescent streptavidin, according to various embodiments of the present disclosure. [Figure 3B] 1 depicts a biotinylated nanovial labeled with fluorescent streptavidin, according to various embodiments of the present disclosure. [Figure 4A] 1 depicts scattering-based removal of nanovials of polystyrene beads according to various embodiments of the present disclosure. [Figure 4B]1 depicts scattering-based removal of nanovials of polystyrene beads according to various embodiments of the present disclosure. [Figure 4C] 1 depicts scattering-based removal of nanovials of polystyrene beads according to various embodiments of the present disclosure. [Figure 5] FIG. 1 depicts a schematic diagram of buoyancy enrichment of nanovials with cells or empty nanovials of free cells, according to various embodiments of the present disclosure. [Figure 6A] 1 depicts a schematic representation of a binding assay using the nanovial platform compared to a pooled assay. [Figure 6B] 1 depicts a schematic representation of a binding assay using the nanovial platform compared to a pooled assay. [Figure 7A] Depicts 40 micron nanovials carried in droplets for single-cell sequencing using the 10X Genomics Chromium Next GEM chip G. [Figure 7B] Depicts 40 micron nanovials carried in droplets for single-cell sequencing using the 10X Genomics Chromium Next GEM chip G. [Figure 7C] Depicts 40 micron nanovials carried in droplets for single-cell sequencing using the 10X Genomics Chromium Next GEM chip G. [Figure 7D] Depicts 40 micron nanovials carried in droplets for single-cell sequencing using the 10X Genomics Chromium Next GEM chip G. [Figure 8] 1 depicts a non-limiting example of a workflow for a single-cell antibody affinity assay using a fluorescent readout, according to various aspects of the present disclosure. [Figure 9] 1 depicts a non-limiting example of a workflow for a single-cell antibody affinity assay using barcoded antibodies and single-cell sequencing, according to various aspects of the present disclosure. [Figure 10] 1 depicts a non-limiting example of a workflow for a single-cell antibody specificity assay using FACS and / or sequencing techniques according to various embodiments of the present disclosure. [Figure 11] 1 depicts a non-limiting example of a workflow for an antigen-presenting cell assay according to various aspects of the present disclosure. [Figure 12A] 1 depicts a non-limiting example of an assay workflow for screening biologics that modulate signaling pathways, according to various embodiments of the present disclosure. [Figure 12B] 1 depicts non-limiting examples of oligonucleotide barcodes that can be used in assays for screening biologics that modulate signaling pathways, according to various aspects of the present disclosure. [Figure 13A] 1 depicts loading of multiple objects into a nanovial, according to various aspects of the present disclosure. [Figure 13B] 1 depicts loading of multiple objects into a nanovial, according to various aspects of the present disclosure. [Figure 14A] 1 depicts steady-state diffusion simulations of molecules released from cells on particles of different shapes, according to various aspects of the present disclosure. [Figure 14B] 1 depicts steady-state diffusion simulations of molecules released from cells on particles of different shapes, according to various aspects of the present disclosure. [Figure 14C] 1 depicts steady-state diffusion simulations of molecules released from cells on particles of different shapes, according to various aspects of the present disclosure. [Figure 14D] 1 depicts steady-state diffusion simulations of molecules released from cells on particles of different shapes, according to various aspects of the present disclosure. [Figure 14E] 1 depicts steady-state diffusion simulations of molecules released from cells on particles of different shapes, according to various aspects of the present disclosure. [Figure 15A] 10 depicts steady-state diffusion simulations of molecules released from cells onto nanovials with beads or blocking particles positioned at different distances from the cavity opening, according to various aspects of the present disclosure. [Figure 15B] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with beads or blocking particles positioned at different distances from the cavity opening, according to various aspects of the present disclosure. [Figure 15C] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with beads or blocking particles positioned at different distances from the cavity opening, according to various aspects of the present disclosure. [Figure 15D] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with beads or blocking particles positioned at different distances from the cavity opening, according to various aspects of the present disclosure. [Figure 15E] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with beads or blocking particles positioned at different distances from the cavity opening, according to various aspects of the present disclosure. [Figure 15F] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with beads or blocking particles positioned at different distances from the cavity opening, according to various aspects of the present disclosure. [Figure 16A] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with various cavity morphologies, according to various aspects of the present disclosure. [Figure 16B] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with various cavity morphologies, according to various aspects of the present disclosure. [Figure 16C] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with various cavity morphologies, according to various aspects of the present disclosure. [Figure 16D] 10 depicts steady-state diffusion simulations of molecules released from cells in nanovials with various cavity morphologies, according to various aspects of the present disclosure. [Figure 16E] 1 depicts steady-state diffusion simulations of molecules released from cells onto nanovials with various cavity morphologies, according to various aspects of the present disclosure. [Figure 16F] 1 depicts steady-state diffusion simulations of molecules released from cells onto nanovials with various cavity morphologies, according to various aspects of the present disclosure. [Figure 17] 1 depicts the use of beads or other blocking particles to reduce leakage or transport of molecules from nanovials, according to various embodiments of the present disclosure. [Figure 18] 1 depicts a microscope image of a smaller nanovial blocking the cavity of a larger nanovial, according to various embodiments of the present disclosure. [Figure 19A] 1 depicts a non-limiting example of a workflow for a screening assay using a DNA-encoded library according to various embodiments of the present disclosure. [Figure 19B] 1 depicts a non-limiting example of a workflow for a screening assay using a DNA-encoded library according to various embodiments of the present disclosure. [Figure 20A] 1 depicts a non-limiting example of analysis of nanovials using imaging flow cytometry, such as an ImageStreamX® MKII Imaging Flow Cytometer, according to various embodiments of the present disclosure. [Figure 20B] 1 depicts a non-limiting example of analysis of nanovials using imaging flow cytometry, such as an ImageStreamX® MKII Imaging Flow Cytometer, according to various embodiments of the present disclosure. [Figure 20C] 1 depicts a non-limiting example of analysis of nanovials using imaging flow cytometry, such as an ImageStreamX® MKII Imaging Flow Cytometer, according to various embodiments of the present disclosure. [Figure 20D] 1 depicts a non-limiting example of analysis of nanovials using imaging flow cytometry, such as an ImageStreamX® MKII Imaging Flow Cytometer, according to various embodiments of the present disclosure. [Figure 21] 1 depicts a non-limiting example of a fluorescence microscope image of nanovials carried in a microwell array, according to various aspects of the present disclosure. [Figure 22] 1 depicts non-limiting examples of microscopic images of free cells, empty nanovials, and cell-loaded nanovials being separated by adjusting solution density and centrifugation, according to various aspects of the present disclosure. [Figure 23] 1 depicts non-limiting examples of experimental data for various conditions for loading cells into nanovials, including the effect of mixing and the ratio between the number of cells and the number of nanovials, according to various aspects of the present disclosure. [Figure 24A] 1 depicts non-limiting examples of brightfield and fluorescence microscopy images of antigen-specific signals from IgG-secreting hybridomas loaded in nanovials and incubated at various temperatures to accumulate secretions in both emulsified and non-emulsified states, according to various embodiments of the present disclosure. [Figure 24B] 1 depicts non-limiting examples of brightfield and fluorescence microscopy images of antigen-specific signals from IgG-secreting hybridomas loaded in nanovials and incubated at various temperatures to accumulate secretions in both emulsified and non-emulsified states, according to various embodiments of the present disclosure. [Figure 25] 1 depicts non-limiting examples of gating strategies for identifying empty nanovials, free cells, and cell-laden nanovials using scatter signals, according to various aspects of the present disclosure. [Figure 26A] 1 depicts non-limiting examples of IgG signals from cell-loaded nanovials, free cells, and empty nanovials compared to blank nanovials after performing a single-cell secretion assay, according to various embodiments of the present disclosure. [Figure 26B] 1 depicts non-limiting examples of IgG signals from cell-loaded nanovials, free cells, and empty nanovials compared to blank nanovials after performing a single-cell secretion assay, according to various embodiments of the present disclosure. [Figure 27] 1 depicts a non-limiting example of 35 micron nanovials loaded into droplets with GEM beads and then labeled with oligo-barcoded PE streptavidin using a 10X chromium instrument according to various embodiments of the present disclosure. [Figure 28A] 1 depicts non-limiting examples of time-lapse images showing the encapsulation of both 35 micron and 60 micron nanovials with spherical gel beads using 10X Chomium chip G, according to various embodiments of the present disclosure. [Figure 28B] 1 depicts non-limiting examples of time-lapse images showing the encapsulation of both 35 micron and 60 micron nanovials with spherical gel beads using 10X Chomium chip G, according to various embodiments of the present disclosure. [Figure 29A]

[0023] Figures 1A-1C depict non-limiting examples of experimental data demonstrating the recovery of human and mouse transcripts from nanovial-loaded cells, comparison of recovered transcripts and genes from nanovial-loaded or suspension-loaded cells, and the recovery and linkage of nanovial-attached feature barcodes to single-cell transcripts using both streptavidin- and antibody-labeled 10X Chromium single-cell sequencing platforms, according to various embodiments of the present disclosure. [Figure 29B]

[0023] Figures 1A-1C depict non-limiting examples of experimental data demonstrating the recovery of human and mouse transcripts from nanovial-loaded cells, comparison of recovered transcripts and genes from nanovial-loaded or suspension-loaded cells, and the recovery and linkage of nanovial-attached feature barcodes to single-cell transcripts using both streptavidin- and antibody-labeled 10X Chromium single-cell sequencing platforms, according to various embodiments of the present disclosure. [Figure 30] FIG. 1 depicts a non-limiting example of experimental data demonstrating the loading of two different cell types, one with a fluorescent tag and one without a fluorescent tag, into the same nanovial, according to various embodiments of the present disclosure. [Figure 31] 1 depicts non-limiting examples of bright field and fluorescent images of nanovials with captured IgG selectively enriched by magnetic separation, according to various embodiments of the present disclosure. [Figure 32]1 depicts non-limiting examples of experimental results of nanovials enriched by labeling IgG-specific nanovials with IgG-specific magnetic particles under various mixing techniques and separation using magnetic forces, according to various embodiments of the present disclosure. [Figure 33] 1 depicts a non-limiting example of a cell killing assay workflow that can be used for screening functional CAR and TCR constructs according to various embodiments of the present disclosure. [Figure 34] FIG. 1 depicts non-limiting examples of experimental results of a cell killing assay showing the capture of red fluorescent protein (RFP) onto nanovials from lysed cells and the observation of captured RFP fluorescence on the nanovials by flow cytometry analysis, according to various embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0015] Provided herein are methods and systems for linking functional single-cell characteristics (e.g., secretions) with genomic, proteomic, and / or transcriptomic characteristics of cells using nanovials as information carriers. Further provided herein are methods and systems for improving the specificity and / or sensitivity of assays performed in nanovials. Additionally provided herein are methods and systems for concentrating, packaging, and storing nanovials. Additionally provided herein are integrated systems and software for nanovial analysis and classification.

[0016] method The disclosure herein provides methods for performing assays (e.g., single-cell-based assays) in nanovials (e.g., within the cavity of a nanovial). In some embodiments, the disclosure provides methods for linking information, such as linking functional information with transcriptome, genome, and / or proteome single-cell information. FIGS. 6A and 6B demonstrate the difference between methods for performing pooled assays (FIG. 6A) and nanovial-linked assays (FIG. 6B). During pooled assays (FIG. 6A), cell-specific information can be lost due to pooling of cells between assays and / or when cell populations are transferred between instruments. Advantageously, using nanovial systems (e.g., as provided herein) allows information to be stored directly on the nanovial associated with the cell being investigated, and that information can be retained across multiple processes (e.g., linking functional (e.g., secretion) and sequence (e.g., transcriptome) information), enabling direct linkages between the information. As depicted in Figure 6B, information obtained from a first assay (Assay 1) may be combined with information obtained from a second assay (Assay 2), which may then be combined with information obtained from a third assay (Assay 3). In a non-limiting example, the combined nanovial assays may include, for example, a screening assay that investigates the affinity and / or specificity of a secreted antibody produced from a single cell of interest located in or on the nanovial (e.g., within the cavity of the nanovial). The assay information obtained from the single cell of interest (e.g., affinity, specificity, or any combination thereof) may be combined with genomic, transcriptomic, and / or proteomic information of the single cell of interest contained in the nanovial (e.g., by analyzing the mRNA of the single cell of interest to determine the unique sequence of the secreted and highly functional antibody).

[0017] In various aspects, the method includes performing a first assay on (e.g., single or individual) cells located in or on the nanovial (e.g., within a cavity of the nanovial) to generate first assay information. In some cases, the first assay is a functional cell-based assay described herein (e.g., an antibody secretion assay). The functional cell-based assay can include any functional cell-based assay, including any functional cell-based assay described herein. In some embodiments, the functional cell-based assay is an antibody secretion screening assay. In some embodiments, the functional cell-based assay is an antibody affinity assay. In some embodiments, the functional cell-based assay is an antibody specificity assay. In some embodiments, the functional cell-based assay is a (e.g., DNA) encoded library screening assay. In some embodiments, the functional cell-based assay is a T-cell secretion phenotype assay. In some embodiments, the functional cell-based assay is associated with a first barcode or a first label. In various embodiments, the method further includes performing a second assay on the (e.g., single or individual) cells in or on the nanovial (e.g., within the cavity of the nanovial) to generate second assay information. In some cases, the second assay can be a cell surface marker staining, a viability assay, an intracellular staining assay, and / or a growth assay. The second assay can be a genomic assay, a transcriptomic assay, and / or a proteomic assay (e.g., single-cell sequencing). In some cases, the genomic assay can include a single-cell RNA sequencing assay. The second assay can be associated with a second barcode or a second label. In various embodiments, the method further includes linking the functional single-cell information with the genomic single-cell, transcriptomic single-cell, and / or proteomic single-cell information by associating the first barcode or first label with the second barcode or second label.

[0018] The nanovial can contain various types of barcodes or labels that independently address and link information between different analytical formats. Usually, the first barcode or first label (associated with the first assay, for example, a functional cell-based assay) and the second barcode or second label (associated with the second assay, for example, a genome assay, a transcriptome assay, and / or a proteome assay) are distinguishable so that the first barcode or label can be distinguished from the second barcode or label. Examples of different types of barcodes or labels that can be used for the first barcode or label and the second barcode or label are provided throughout. In one embodiment, the first barcode or first label comprises an oligonucleotide having a polyA region.

[0019] Furthermore, each nanovial may further include a unique nanovial-associated barcode or label so that each individual nanovial can be distinguished from other nanovials. Non-limiting examples of suitable nanovial barcodes or labels include unique nucleic acid or oligonucleotide barcodes, unique peptide barcodes, optical barcodes (e.g., dyes, fluorophores), unique scattering signatures observable by, for example, forward or side scatter flow cytometry, isotopes, or mass barcodes (e.g., readable by CyTOF or mass cytometry). In non-limiting embodiments, the nanovial is associated with a unique oligonucleotide barcode, label, or tag. In some cases, the unique oligonucleotide barcode, label, or tag includes a poly-dT capture region (e.g., for capturing polyA regions, e.g., on the first barcode or first label and / or from mRNA released from cells).

[0020] Additionally, or alternatively, nanovials may have unique shapes and / or sizes that are recognizable by image cytometry or image-activated cell sorting techniques. By using an optical source to bleach or activate fluorophores embedded within the nanovials, nanovials can be further tagged or barcoded after imaging. In particular, specific barcodes or labels of one type can be linked to specific barcodes or labels of another type to transfer and link information between two or more analytical modes. For example, a nanovial having an oligonucleotide barcode containing a first nucleotide sequence (e.g., GACTTCC) may additionally contain a specific level of fluorophore intensity (e.g., AlexaFluor 488 at an intensity greater than 1000 times background intensity). In the same example, another nanovial having an oligonucleotide barcode containing a second distinguishable nucleotide sequence (GCTAACC) may additionally contain a different level of fluorophore intensity (e.g., AlexaFluor 488 at an intensity 100 times background intensity). Any combination of barcode or label types (e.g., as provided herein) can be used (e.g., to associate information) in the methods provided herein. In some embodiments, two or more barcodes or labels can be associated (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more barcodes or labels).

[0021] In another embodiment, first assay information (e.g., functional single-cell-based assay information) can be combined with second assay information (e.g., genomic, transcriptomic, and / or proteomic single-cell-based assay information) through the use of index sorting using fluorescence-activated cell sorting (FACS) or other single-cell analysis and preparation techniques (e.g., using commercial instruments such as Namocell, Nodexus, Nanocellect, etc.). In this embodiment, nanovials containing cells can be analyzed and sorted based on nanovials associated with fluorescent signals on the cells and / or secretions or releases from the cells. Sorting can be performed based on sort gates for various parameters (e.g., fluorescence intensity peak height, fluorescence intensity width, fluorescence intensity area, fluorescence color, scattering intensity peak height, scattering intensity width, scattering intensity area, etc.). In some cases, single nanovials within the sort gate can be sorted into separate wells of a plate (e.g., a 96-well or 384-well plate) using a flow cytometer (e.g., BD FACS Aria II, III, Sony SH800, or other FACS systems compatible with index sorting). Sorting-based information (e.g., fluorescence intensity associated with the nanovial) can be linked to the specific well in the plate into which it was sorted and can then be linked to downstream information obtained from subsequent genomic, transcriptomic, and / or proteomic assays.

[0022] In a non-limiting example, the second assay (e.g., a genomic assay, a transcriptome assay, and / or a proteome assay) comprises sequencing (e.g., nucleic acids present in the cells of interest). When an oligonucleotide barcode or label is used, the method further comprises sequencing the oligonucleotide barcode or label. Any known sequencing technology may be suitable for use in the methods provided herein. For example, sequencing may include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc.

[0023] In some embodiments, the method further comprises sorting the nanovials based on the signal generated by the first assay (e.g., a functional assay) prior to the second assay (e.g., a genomic assay, a transcriptomic assay, and / or a proteomic assay). In non-limiting embodiments, the nanovials are sorted using a FACS-based sorting technique or any other particle, cell, or droplet sorting technique.

[0024] In the above embodiment, data from a single nanovial / single cell may be linked in a single data structure that contains these linked data sets as columns or entries linked to the same single nanovial or single cell identifier.

[0025] Single-cell secretion assay Provided herein is a single-cell secretion assay that can be used in a nanovial system. The nanovial can serve as a substrate, for example, where release from a cell of interest is associated with the nanovial as part of an assay, in a manner similar to conventional workflows performed in multiwell plates. For example, in a typical workflow, target cells, target molecules, or capture molecules can be immobilized on the surface of the nanovial. The cell of interest can then adhere to the surface of the nanovial (including the immobilized target cell or molecule). The cell of interest can secrete or release a biological product or molecule over an incubation period that is evaluated in the nanovial assay. An assay readout signal (e.g., fluorescence, coded oligonucleotide, magnetic, or any combination thereof) can then be obtained on or within the nanovial or on or within the target cell bound to the nanovial and correlated with the function of the biological product from the cell of interest. Washing steps can be performed between assay steps. Multiple such nanovials containing multiple cells of interest with different biological products can be analyzed and / or classified on the platform based on the functional information of the biological products.

[0026] In some embodiments, provided herein are methods for identifying an antibody or fragment thereof that binds to an antigen of interest. In some cases, the method includes one or more of the following steps: (a) providing or obtaining a nanovial, the nanovial including a cavity formed therein and an affinity agent immobilized therein; (b) loading antibody-producing cells into the cavity of the nanovial; (c) incubating the nanovial so that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent; (d) adding a detection agent to the cavity of the nanovial so that the detection agent binds to the one or more antibodies or fragments thereof; (e) detecting one or more signals relating to binding of the detection agent to the one or more antibodies or fragments thereof; (f) performing a sequencing assay on nucleic acids derived from the antibody-producing cells, thereby generating sequences of the one or more antibodies or fragments thereof; (g) associating the sequences of the one or more antibodies or fragments thereof with one or more signals relating to binding of the detection agent to the one or more antibodies or fragments thereof; and (h) identifying one or more antibodies or fragments thereof that bind to the antigen of interest based on the associating step (g). It should be understood that the steps provided above do not necessarily have to be performed in the exact order in which they appear. The steps provided above may be performed in any order and may be modified to meet the needs of a user. It should also be understood that not all steps provided above are required or need to be performed by a single user. For example, in one embodiment, any of steps (a) through (e) may be performed by a first user, and any one of steps (f) through (h) may be performed by a second, different user. For example, the method may include obtaining nanovials and performing steps (f) through (h) after step (e) has been performed (e.g., by a different user).

[0027] In some embodiments, a method of providing or obtaining a nanovial is provided, wherein the nanovial comprises a cavity formed therein. The nanovial can be any nanovial described herein. In some cases, the nanovial is empty (e.g., does not contain antibody-producing cells). In other cases, the nanovial contains antibody-producing cells (e.g., single cells or individual cells). In some cases, the nanovial can contain more than one antibody-producing cell (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells).

[0028] In some cases, the cavity of the nanovial is coated with or has an affinity agent immobilized thereon. The affinity agent may be immobilized on the surface of the cavity of the nanovial and / or may be within the porous matrix of the nanovial. In some cases, the resulting or provided nanovial is not coated with an affinity agent, and the method includes coating the nanovial with an affinity agent.

[0029] In some embodiments, the affinity agent is an antibody capture moiety (e.g., for binding to and capturing antibodies secreted by antibody-producing cells). Optionally, the antibody capture moiety is selected from the group consisting of an anti-Fc antibody or fragment thereof, Protein A, Protein G, and any combination thereof. Optionally, the antibody capture moiety is not specific to a particular type of antibody, such that all or substantially all antibodies secreted by antibody-producing cells can bind to the antibody capture moiety. In other embodiments, the affinity agent is an antigen of interest. In such cases, antibodies secreted from antibody-producing cells that are capable of binding to the antigen of interest are captured on the nanovial. In another embodiment, the affinity agent is an antigen-presenting cell, and the antigen of interest is expressed or presented on the surface of the antigen-presenting cell. In such cases, antibodies secreted from antibody-producing cells that are capable of binding to the antigen of interest are captured on the antigen-presenting cell (e.g., contained within the cavity of the nanovial).

[0030] In some embodiments, the method further includes loading antibody-producing cells into the cavity of the nanovial. In other embodiments, the method includes obtaining or providing a nanovial with antibody-producing cells already loaded into the cavity. The antibody-producing cells can be any cell that produces an antibody. In some cases, the antibody-producing cells can be B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof. In some cases, the cavity of the nanovial includes one or more cell capture agents immobilized thereto. The one or more cell capture agents can be immobilized on the surface of the cavity of the nanovial and / or can be within the porous matrix of the nanovial. In some cases, the obtained or provided nanovial is not coated with a cell capture agent, and the method includes coating the nanovial with a cell capture agent.

[0031] In some embodiments, the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of the antibody-producing cells. In some embodiments, the one or more cell capture agents bind to a label present on the surface of the antibody-producing cells. For example, the cell capture agent may be biotin, and the antibody-producing cells may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the antibody-producing cells may be coated or labeled with biotin. In some embodiments, the one or more cell capture agents are antigens to which the antibody-producing cells can bind (e.g., via the B cell receptor). In some embodiments, the one or more cell capture agents comprise single-stranded DNA (ssDNA), and the antibody-producing cells may be coated or labeled with complementary ssDNA that hybridizes with the cell capture agent.

[0032] In various embodiments, the method further includes incubating the nanovial so that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent. Any suitable conditions for incubating the nanovial can be used, including any temperature, time, buffer, or medium conditions. Typically, the incubating step involves incubating under conditions such that the antibody-producing cells remain viable for a sufficient period of time to produce and secrete antibodies into the cavity of the nanovial. In some cases, prior to incubation, the nanovial can be encapsulated (e.g., in a droplet or emulsion) so that the contents of the nanovial cannot diffuse or substantially cannot diffuse out of the cavity of the nanovial. In other cases, one or more blocking particles can be added to block or reduce the size of the opening of the cavity of the nanovial (e.g., as described herein).

[0033] In various aspects, the method further includes adding a detection agent to the cavity of the nanovial such that the detection agent binds to one or more antibodies or fragments thereof. In some embodiments, the detection agent is an antigen of interest. In some embodiments, the detection agent is an antibody or fragment thereof. In some embodiments, the detection agent is directly or indirectly labeled with a detectable label (e.g., using a secondary detection agent comprising a detectable label that binds to the primary detection agent). The detectable label may be selected from the group consisting of a fluorescent label, a protein affinity tag, an oligonucleotide tag, a magnetic particle, and any combination thereof. In certain aspects, the detectable label is a fluorescent label (e.g., a fluorescent dye, a fluorophore, etc.).

[0034] In various embodiments, the method further comprises detecting one or more signals relating to the binding of the detection agent to the one or more antibodies or fragments thereof. In some cases, the detecting step comprises determining the amount of detection agent bound to the one or more antibodies or fragments thereof. In one embodiment, the detection agent comprises a fluorescent label, and the method comprises detecting the level of fluorescence, wherein the amount of detection agent bound to the one or more antibodies or fragments thereof correlates with the level of fluorescence. In another embodiment, the detection agent comprises an oligonucleotide label. In this example, the method further comprises sequencing the oligonucleotide label. In some cases, the detecting step comprises counting the number of oligonucleotide tags bound to the one or more antibodies or fragments thereof.

[0035] In some embodiments, the method further comprises sorting the nanovials based on one or more signals related to the binding of the detection agent to the antibody. In some cases, the method comprises sorting the nanovials based on the level of fluorescence (e.g., above background level). The sorting step can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescent signals. In a non-limiting example, the sorting step is a flow-based sorting method (e.g., FACS). In some cases, when the detection agent includes a magnetic particle label, the sample is sorted using magnetic force or magnetically activated cell sorting (e.g., MACS).

[0036] In various embodiments, the method further comprises performing a sequencing assay on nucleic acid derived from antibody-producing cells, thereby generating the sequence of one or more antibodies or fragments thereof. In some cases, the method comprises obtaining or obtaining a sorted nanovial or a plurality of sorted nanovials (e.g., after any of the above methods have been performed), and performing a sequencing assay (e.g., as described herein) on nucleic acid derived from antibody-producing cells. The method further comprises obtaining or obtaining data or data output (e.g., secretion assay data) related to any of the above methods, so that the data can be associated with downstream sequencing data. Any known sequencing technology can be suitable for use in the methods provided herein. For example, sequencing can include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc.

[0037] In one embodiment, the method includes lysing the antibody-producing cells prior to sequencing. In another embodiment, the method includes obtaining or obtaining a lysate of the antibody-producing cells (e.g., after a secretion assay is performed and the nanovials are sorted based on one or more signals associated with the secretion assay) prior to sequencing. In some examples, the method further includes reverse transcribing mRNA released from the antibody-producing cells to generate cDNA, and then sequencing the cDNA.

[0038] In various aspects, the method may further comprise washing the nanovial. Any suitable washing method may be used, including any suitable washing solution or buffer.

[0039] In some embodiments, the method includes performing any of the above methods on a plurality of nanovials, each nanovial containing antibody-producing cells. In some examples, each nanovial contains antibody-producing cells that produce different antibodies (e.g., a single antibody-producing cell). In such methods, the plurality of nanovials can be screened for antibodies that target an antigen of interest. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0040] In various embodiments, the method further includes associating sequences of the one or more antibodies or fragments thereof with one or more signals related to the binding of a detection agent to the one or more antibodies or fragments thereof. The associating step may include associating one or more barcodes or labels associated with a first assay (e.g., a secretion assay) with one or more barcodes or labels associated with a second assay (e.g., a sequencing assay). The method may further include identifying one or more antibodies or fragments thereof that bind to the antigen of interest based on the associating step.

[0041] Antibody affinity assay In some embodiments, the methods provided herein may include analyzing the binding affinity of antibodies secreted from antibody-secreting cells (e.g., B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof) to characterize single-cell nucleotide sequence information associated with this binding affinity, e.g., as depicted in Figures 8 and 9. In some aspects, the methods may include providing or obtaining (or obtaining) a nanovial, where the nanovial includes a cavity formed therein. In some examples, the nanovial may include an opening to the surface of the nanovial. The nanovial may be any nanovial described herein. In some cases, the nanovial is empty (e.g., does not contain antibody-producing cells). In other cases, the nanovial includes antibody-producing cells (e.g., single cells or individual cells). In some cases, the nanovial may include more than one antibody-producing cell (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells).

[0042] In some cases, the cavity of the nanovial is coated with or has an affinity agent immobilized thereon. The affinity agent may be immobilized on the surface of the cavity of the nanovial and / or may be within the porous matrix of the nanovial. In some cases, the resulting or provided nanovial is not coated with an affinity agent, and the method includes coating the nanovial with an affinity agent.

[0043] In some embodiments, the affinity agent is an antibody capture moiety (e.g., for binding to and capturing antibodies secreted by antibody-producing cells) (18, 44). In some cases, the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, protein A, protein G, and any combination thereof. In some cases, the antibody capture moiety is not specific to a particular type of antibody, such that all or substantially all antibodies secreted by the antibody-producing cells can bind to the antibody capture moiety. In other embodiments, the affinity agent is an antigen of interest (20, 46). In such cases, antibodies secreted from the antibody-producing cells that are capable of binding to the antigen of interest are captured on the nanovial.

[0044] In some embodiments, the method further includes loading antibody-producing cells into the cavity of the nanovial. In other embodiments, the method includes obtaining or providing a nanovial with antibody-producing cells already loaded into the cavity. The antibody-producing cells can be any cell that produces an antibody. In some cases, the antibody-producing cells can be B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof. In some cases, the cavity of the nanovial includes one or more cell capture agents immobilized thereto. The one or more cell capture agents can be immobilized on the surface of the cavity of the nanovial and / or can be within the porous matrix of the nanovial. In some cases, the obtained or provided nanovial is not coated with a cell capture agent, and the method includes coating the nanovial with a cell capture agent.

[0045] In some embodiments, one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of antibody-producing cells. In some embodiments, one or more cell capture agents bind to a label present on the surface of antibody-producing cells. For example, the cell capture agent may be biotin, and the antibody-producing cells may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the antibody-producing cells may be coated or labeled with biotin. In some embodiments, one or more cell capture agents is an antigen to which the antibody-producing cells can bind. In some embodiments, one or more cell capture agents comprise ssDNA, and the antibody-producing cells may be coated or labeled with complementary ssDNA that hybridizes with the cell capture agent.

[0046] Optionally, the method further includes encapsulating the nanovial or blocking the opening of the cavity of the nanovial. In some embodiments, the method may include adding one or more blocking particles to block the opening of the cavity of the nanovial or reduce the size of the opening (e.g., Figure 17 (120, 122)). In some examples, the nanovial used in the affinity assay may be emulsified in oil (22, 30, 50, 52). In some cases, the nanovial may be encapsulated within a droplet. In some cases, the nanovial may include a cavity with a small opening (118). Alternatively, the nanovial may not be encapsulated (23, 51).

[0047] In various embodiments, the method further comprises incubating the nanovial so that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent (26, 28, 32, 54, 56, 58). Any suitable conditions for incubating the nanovial can be used, including any temperature, time, medium buffer conditions, etc. Typically, the incubating step involves incubating under conditions such that the antibody-producing cells remain viable for a period of time sufficient to produce and secrete antibodies into the cavity of the nanovial. In some cases, the incubating step can include incubating the antibody-producing cells for a period of time. In some examples, the period can include at least 30 minutes (e.g., at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, etc.). In some cases, the period can include up to 24 hours.

[0048] In various aspects, the method may further include transferring the emulsified nanovial to an aqueous phase, thereby removing the emulsified oil film. Optionally, the aqueous phase nanovial may then be washed before adding the detection agent to the cavity of the nanovial. In other embodiments where blocking particles are used, the method may further include removing excess blocking particles by washing, straining, physical disruption, chemical disruption, buoyancy, magnetic force, or any combination thereof.

[0049] In various embodiments, the method further includes adding a detection agent to the cavity of the nanovial such that the detection agent binds to one or more antibodies or fragments thereof. In some cases, the detection agent can include an antibody or fragment thereof (34, 62, 60, 68, 42). In some examples, the detection agent can include an antigen of interest (40, 38, 64, 66). In some cases, the detection agent can be directly or indirectly labeled with a detectable label. The detectable label can include a fluorescent label (34, 36, 38, 42), a protein affinity tag, an oligonucleotide tag (60, 62, 64, 68), a magnetic particle, or any combination thereof.

[0050] In various embodiments, the method can include adding a detection agent to the plurality of nanovials at one or more different concentrations, the one or more different concentrations being consistent with a desired dissociation constant (K) for the antigen of interest. D ) can be within one order of magnitude of the desired K D can be less than about 1 μM, less than about 100 nM, less than about 10 nM, or less than about 1 nM.

[0051] In various embodiments, the method further comprises detecting one or more signals relating to binding of a detection agent to the one or more antibodies or fragments thereof. In some cases, the detecting step comprises determining the amount of detection agent bound to the one or more antibodies or fragments thereof. In one embodiment, the detection agent comprises a fluorescent label, and the method comprises detecting the level of fluorescence (41), wherein the amount of detection agent bound to the one or more antibodies or fragments thereof correlates with the level of fluorescence. In another embodiment, the detection agent comprises an oligonucleotide label. In this example, the method further comprises sequencing the oligonucleotide label. In some cases, the detecting step comprises counting the number of oligonucleotide tags bound to the one or more antibodies or fragments thereof (70).

[0052] In some embodiments, the method further includes sorting the nanovials based on one or more signals related to binding of the detection agent to the antibody (41). In some cases, the method includes sorting the nanovials based on the level of fluorescence (e.g., above background level) or within a gate of fluorescence intensity (41). The sorting can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescent signals. In a non-limiting example, the sorting is a flow-based sorting method (e.g., FACS). In some cases, when the detection agent includes a magnetic particle label, the sample is sorted using magnetic force or magnetically activated cell sorting (e.g., MACS).

[0053] In various embodiments, the method further comprises performing a sequencing assay on nucleic acids derived from the antibody-producing cells, thereby generating sequences for one or more antibodies or fragments thereof (70). In some cases, the method comprises obtaining or obtaining a sorted nanovial or a plurality of sorted nanovials (e.g., after any of the above methods have been performed) and performing a sequencing assay (e.g., as described herein) on nucleic acids derived from the antibody-producing cells. The method further comprises obtaining or obtaining data or data output (e.g., secretion assay data) related to any of the above methods so that the data can be associated with downstream sequencing data. Any known sequencing technology may be suitable for use in the methods provided herein. For example, sequencing may include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc.

[0054] In one embodiment, the method includes lysing the antibody-producing cells prior to sequencing. In other embodiments, the method includes obtaining or obtaining a lysate of the antibody-producing cells (e.g., after a secretion assay is performed and the nanovials are sorted based on one or more signals associated with the secretion assay) prior to sequencing. In some examples, the method further includes reverse transcribing mRNA released from the antibody-producing cells to generate cDNA, and then sequencing the cDNA.

[0055] In various aspects, the method can further include washing the nanovial. Any suitable washing method can be used, including any suitable washing solution or buffer.

[0056] In some embodiments, the method includes performing any of the above methods on a plurality of nanovials, each nanovial containing antibody-producing cells. In some examples, each nanovial contains antibody-producing cells that produce different antibodies (e.g., a single antibody-producing cell). In such methods, the plurality of nanovials can be screened for antibodies that target an antigen of interest. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0057] In various embodiments, the method further includes associating sequences of the one or more antibodies or fragments thereof with one or more signals related to the binding of a detection agent to the one or more antibodies or fragments thereof. The associating step may include associating one or more barcodes or labels associated with a first assay (e.g., a secretion assay) with one or more barcodes or labels associated with a second assay (e.g., a sequencing assay). The method may further include identifying one or more antibodies or fragments thereof that bind to the antigen of interest based on the associating step.

[0058] In various embodiments, the methods provided herein are suitable for screening and identifying antibodies with a desired affinity for an antigen of interest. The methods provided herein can be used to screen for and identify antibodies with a K for the antigen of interest of less than about 1 μM, less than about 100 nM, less than about 10 nM, or less than about 1 nM. D The antibody can be used to screen for and identify antibodies having

[0059] As provided herein, Figure 8 depicts a non-limiting example of an antibody affinity assay workflow. In this example, nanovials are loaded with cells and coated with an affinity agent, such as an antibody against IgG (18) or an antigen of interest (20). The nanovials are optionally emulsified (22, 30) to seal them and reduce crosstalk. The nanovials are then incubated, and secreted antibodies are captured on the nanovial surface (26, 28, 32). The emulsified nanovials are then returned to the aqueous phase, and background is washed. The sample is then incubated with the antigen of interest (40) to identify high-affinity versus low-affinity antibodies, and then labeled with a fluorescently tagged antibody specific for the antigen (42). The sample is then analyzed and / or sorted (41) using flow cytometry, FACS, or other single-cell fluorescent readout instrumentation. In another embodiment, the nanovials with captured, secreted antibodies are incubated with a fluorescently tagged antigen (38). In both approaches, fluorescently tagged antibodies against IgG may be used to normalize the amount of secreted antibody (36). In another embodiment, nanovials are directly labeled with the antigen of interest (20). Secreted antibodies are labeled based on their relative affinity for binding to the antigen and captured with fluorescently tagged antibodies, such as those against IgG (34). Samples are analyzed and / or sorted using flow cytometry, FACS, or other single-cell fluorescence analysis and sorting instruments (41).

[0060] As provided herein, Figure 9 depicts another non-limiting example of an antibody affinity assay workflow. In this example, nanovials are loaded with cells and coated with an affinity agent, such as an antibody against IgG (44) or an antigen of interest (46). The nanovials are optionally emulsified (50, 52) to seal them and reduce crosstalk. The nanovials are then incubated, and secreted antibodies are captured on the nanovial surface by binding to an affinity agent, such as an antibody against IgG (54, 56) or the antigen of interest (58). The emulsified nanovials are returned to the aqueous phase, and background is washed. The sample is then incubated with the antigen of interest (66) to identify high-affinity versus low-affinity antibodies, and then labeled with an oligonucleotide-tagged antibody (68). The sample is then sequenced (70), for example, using a single-cell sequencing platform, to measure the relative affinities of antibodies produced by different cells, as well as mRNA expression profiles and antibody sequence information. In another embodiment, nanovials capturing secreted antibodies are incubated with oligonucleotide-tagged antigens (64). The samples can then be sequenced to identify antibody-secreting cell mRNA expression profiles containing antibody sequence and affinity information based on counting the oligonucleotide sequences of the tagged antigens along with the associated information provided by the single-cell sequencing barcodes (70). In both approaches, oligonucleotide-tagged antibodies against IgG may be used to normalize the amount of secreted antibody (62). In another embodiment, nanovials are directly labeled with the antigen of interest (46). Secreted antibodies are captured based on relative affinity and labeled with oligonucleotide-tagged antibodies against the secreted antibody (60). Single-cell sequencing using this workflow (70) may use oligonucleotide barcodes or nanovial labels (NLs) associated with the nanovials. The NLs may be located in the nanovials or on separate beads in fluid communication with the nanovials and the cells therein.The separate beads can be oligonucleotide-conjugated beads (e.g., GEM beads or Rhapsody beads) used as part of a single-cell RNA sequencing system. The NL-containing oligonucleotide barcode can further include an oligo-dT sequence (dT) for capturing mRNA from antibody-producing cells via a polyA region, and an antigen or antibody oligonucleotide tag. Reverse transcription is performed to convert the mRNA from the antibody-producing cells and the antigen and antibody oligonucleotide tags into cDNA flanked by NL and, optionally, other oligonucleotide sequences containing a universal sequence and a unique molecular index sequence. The cDNA is amplified and sequenced, for example, using next-generation sequencing. In this way, the same NL code present in the amplified cDNA is used to link antibody sequences from antibody-producing cells to secretion and affinity signals from the oligonucleotide-tagged antigen and antibody. In this workflow, antibody binding and affinity can be assayed and further linked to the antibody sequences (e.g., heavy and light chains) of antibody-secreting cells by searching for the number of sequencing reads of oligonucleotide tag barcodes tethered to the same NL barcode. In some embodiments, selecting antibody sequences with affinity above a threshold comprises identifying NL-barcode-containing reads that contain oligonucleotide-tagged antigen barcode sequences above a threshold level or number of reads, or a threshold fraction of oligonucleotide-tagged antibody barcode sequence reads, and then identifying antibody sequences (e.g., VH and VL) associated with the same NL-barcode reads.

[0061] Antibody specificity assays In some embodiments, the methods provided herein may include identifying one or more antibodies or fragments thereof secreted from antibody-producing cells (e.g., B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof) that have a desired specificity to an antigen of interest, as found in FIG. 10, and characterizing the sequence information of the antibodies associated with the desired specificity.

[0062] In some embodiments, the method may include providing or obtaining (or obtaining) a nanovial, where the nanovial includes a cavity formed therein. In some examples, the nanovial may include an opening to the surface of the nanovial. The nanovial may be any nanovial described herein. In some cases, the nanovial is empty (e.g., does not contain antibody-producing cells). In other cases, the nanovial contains antibody-producing cells (e.g., single cells or individual cells). In some cases, the nanovial may contain more than one antibody-producing cell (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells).

[0063] In some cases, the cavity of the nanovial is coated with or has an affinity agent immobilized thereon. The affinity agent may be immobilized on the surface of the cavity of the nanovial and / or may be within the porous matrix of the nanovial. In some cases, the resulting or provided nanovial is not coated with an affinity agent, and the method includes coating the nanovial with an affinity agent.

[0064] In some embodiments, the affinity agent is an antibody capture moiety (e.g., for binding to and capturing antibodies secreted by antibody-producing cells) (72). In some cases, the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, protein A, protein G, and any combination thereof. In some cases, the antibody capture moiety is not specific to a particular type of antibody, such that all or substantially all antibodies secreted by antibody-producing cells can bind to the antibody capture moiety.

[0065] In some embodiments, the method further includes loading antibody-producing cells into the cavity of the nanovial. In other embodiments, the method includes obtaining or providing a nanovial with antibody-producing cells already loaded into the cavity. The antibody-producing cells can be any cell that produces an antibody. In some cases, the antibody-producing cells can be B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof. In some cases, the cavity of the nanovial includes one or more cell capture agents immobilized thereto. The one or more cell capture agents can be immobilized on the surface of the cavity of the nanovial and / or can be within the porous matrix of the nanovial. In some cases, the obtained or provided nanovial is not coated with a cell capture agent, and the method includes coating the nanovial with a cell capture agent.

[0066] In some embodiments, one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of antibody-producing cells. In some embodiments, one or more cell capture agents bind to a label present on the surface of antibody-producing cells. For example, the cell capture agent may be biotin, and the antibody-producing cells may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the antibody-producing cells may be coated or labeled with biotin. In some embodiments, one or more cell capture agents is an antigen to which the antibody-producing cells can bind. In some embodiments, one or more cell capture agents comprise ssDNA, and the antibody-producing cells may be coated or labeled with complementary ssDNA that hybridizes with the cell capture agent.

[0067] Optionally, the method further includes the step of encapsulating the nanovial or blocking the opening of the cavity of the nanovial. In some embodiments, the method may include adding one or more blocking particles to block the opening of the cavity of the nanovial or reduce the size of the opening (e.g., Figure 17 (120, 122)). In some examples, the nanovial used in the assay may be emulsified in oil (74). In some cases, the nanovial may be encapsulated in a droplet. In some cases, the nanovial may include a cavity with a small opening (118). Alternatively, the nanovial may not be encapsulated.

[0068] In various embodiments, the method further includes incubating the nanovial (76) so that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent. Any suitable conditions for incubating the nanovial can be used, including any temperature, time, medium, buffer conditions, etc. Typically, the incubating step involves incubating under conditions such that the antibody-producing cells remain viable for a period of time sufficient to produce and secrete antibodies into the cavity of the nanovial. In some cases, the incubating step can include incubating the antibody-producing cells for a period of time. In some examples, the period can include at least 30 minutes (e.g., at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, etc.). In some cases, the period can include up to 24 hours.

[0069] In various aspects, the method may further include transferring the emulsified nanovial to an aqueous phase, thereby removing the emulsified oil film. Optionally, the aqueous phase nanovial may then be washed before adding the detection agent to the cavity of the nanovial. In other embodiments where blocking particles are used, the method may further include removing excess blocking particles by washing, straining, physical disruption, chemical disruption, buoyancy, magnetic force, or any combination of the foregoing.

[0070] In various aspects, the method further includes adding a detection agent to the cavity of the nanovial such that the detection agent binds to one or more antibodies or fragments thereof. In some cases, the method includes adding multiple different detection agents to the nanovial (78, 80). In some cases, the method includes identifying one or more antibodies or fragments thereof that specifically bind to one of the different detection agents. In some cases, the detection agent may include multiple labeled antigens (78), where a subset of the multiple antigens can bind with high specificity to an epitope of an antibody secreted from the antibody-producing cell. In some cases, the detection agent may be directly or indirectly labeled with a detectable label. The detectable label may include a fluorescent label (78), a protein affinity tag, an oligonucleotide tag (80), a magnetic particle, or any combination thereof. In some cases, a normalized antibody may be used to account for potential differences in the secreted or bound antibodies in the nanovial. In some embodiments, the normalized antibody may include a fluorescent label (84). Alternatively, the normalized antibody may include an oligonucleotide barcode label (82).

[0071] In various embodiments, the method further includes detecting one or more signals related to binding of the detection agent to the one or more antibodies or fragments thereof. In some cases, the detecting step includes determining the amount of detection agent bound to the one or more antibodies or fragments thereof. In one embodiment, the detection agent includes a fluorescent label, and the method includes detecting the level of fluorescence (86), where the amount of detection agent bound to the one or more antibodies or fragments thereof correlates with the level of fluorescence. In another embodiment, the detection agent includes an oligonucleotide label. In this example, the method further includes sequencing the oligonucleotide label (88). In some cases, the detecting step includes counting the number of oligonucleotide tags bound to the one or more antibodies or fragments thereof.

[0072] In some embodiments, the method further includes sorting the nanovials based on one or more signals related to binding of the plurality of detection agents to the antibodies (86). In some cases, the method includes sorting the nanovials based on the level of fluorescence (e.g., above background levels) in one or more fluorescent channels (86). The sorting can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescent signals. In a non-limiting example, the sorting is a flow-based sorting method (e.g., FACS). In some cases, if the detection agents include magnetic particle labels, the sample is sorted using magnetic force or magnetically activated cell sorting (e.g., MACS).

[0073] In various embodiments, the method further comprises performing a sequencing assay on nucleic acid derived from the antibody-producing cells, thereby generating sequences for one or more antibodies or fragments thereof (88). In some cases, the method comprises obtaining or obtaining a sorted nanovial or a plurality of sorted nanovials (e.g., after any of the above methods have been performed) and performing a sequencing assay (e.g., as described herein) on nucleic acid derived from the antibody-producing cells. The method further comprises obtaining or obtaining data or data output (e.g., secretion assay data) related to any of the above methods, such that the data is associated with downstream sequencing data. Any known sequencing technology may be suitable for use in the methods provided herein. For example, sequencing may include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc.

[0074] In one embodiment, the method includes lysing the antibody-producing cells prior to sequencing. In other embodiments, the method includes obtaining or obtaining a lysate of the antibody-producing cells (e.g., after a secretion assay is performed and the nanovials are sorted based on one or more signals associated with the secretion assay) prior to sequencing. In some examples, the method further includes reverse transcribing mRNA released from the antibody-producing cells to generate cDNA, and then sequencing the cDNA.

[0075] In various aspects, the method can further include washing the nanovial. Any suitable washing method can be used, including any suitable washing solution or buffer.

[0076] In some embodiments, the method includes performing any of the above methods on a plurality of nanovials, each nanovial containing antibody-producing cells. In some examples, each nanovial contains antibody-producing cells that produce different antibodies (e.g., a single antibody-producing cell). In such methods, the plurality of nanovials can be screened for antibodies that target an antigen of interest. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0077] In various embodiments, the method further includes associating sequences of the one or more antibodies or fragments thereof with one or more signals related to the binding of a detection agent to the one or more antibodies or fragments thereof. The associating step may include associating one or more barcodes or labels associated with a first assay (e.g., a secretion assay) with one or more barcodes or labels associated with a second assay (e.g., a sequencing assay). The method may further include identifying one or more antibodies or fragments thereof that bind to one or more antigens of interest based on the associating step.

[0078] As provided herein, Figure 10 depicts a non-limiting example of the workflow of an antibody affinity assay. In this example, nanovials carrying cells are coated with an affinity agent, such as an antibody against IgG (72). The nanovials are optionally emulsified to reduce crosstalk and increase signal (74). The nanovials with cells therein are then incubated, and secreted antibodies are captured on the surface of the nanovials (76). If the nanovials were optionally emulsified, they are de-emulsified. The nanovials are then washed. In one embodiment, the nanovials, associated cells, and captured antibodies are incubated with fluorescently coded multiple antigens (78). Fluorescent antibodies, such as those against IgG, are optionally used to normalize the measured signal to the total amount of captured secreted material (84). The samples are then read and / or sorted using flow cytometry or FACS. FACS is used to isolate antibody-secreting cells that secrete antibodies that bind to one or more fluorescently coded antigens based on gating in one or more fluorescent channels (86). In another embodiment, the nanovials are instead incubated with multiple antigens coded with unique oligonucleotide barcodes (80). Optionally, distinct oligonucleotide-tagged antibodies, such as those against IgG, are used for normalization (82). The samples are then analyzed using various single-cell sequencing techniques to link the secreted antibody sequences with the antigen-binding counts of the unique oligonucleotide barcodes (88). This workflow utilizes oligonucleotide barcodes or nanovial labels (NLs) associated with the nanovials. The NLs may be located on separate beads in fluid communication with the nanovials or the cells therein. The separate beads may be oligonucleotide-conjugated beads (e.g., GEM beads or Rhapsody beads) used as part of a single-cell RNA sequencing system.The NL-containing oligonucleotide barcode may further contain an oligo-dT sequence (dT) for capturing mRNA from antibody-producing cells via a polyA region, an oligonucleotide tag for the antigen, and optionally, an antibody. Reverse transcription is performed to convert the mRNA from the antibody-producing cells, the oligonucleotide tag for the antigen, and optionally the antibody into cDNA flanked by the NL and other oligonucleotide sequences, optionally including a universal sequence and a unique molecular index sequence. The cDNA is amplified and sequenced, for example, using next-generation sequencing. In this way, the same NL code present in the amplified cDNA is used to link the antibody sequence from the antibody-producing cells to the specificity and secretion signals from the oligonucleotide-tagged antigen and antibody. In this workflow, antibody specificity can be assayed and further linked to the antibody sequences (e.g., heavy and light chains) of antibody-secreting cells by examining the number of sequencing reads of oligonucleotide barcodes associated with different antigens tethered to the same NL barcode. In some embodiments, selecting antibody sequences with specificity above a threshold involves identifying NL barcode-containing reads that contain oligonucleotide-tagged antigen barcode sequences of one type above a threshold level or number of reads, while another type is below a threshold number of reads, or reads normalized to antibody barcode sequence reads that exceed a threshold number of reads. Then, identifying antibody sequences (e.g., VH and VL) associated with the same NL barcode reads. In some embodiments, selecting antibody sequences with specificity above a threshold involves identifying NL barcode-containing reads that contain oligonucleotide-tagged antigen barcode sequences of one type above a threshold level or number of reads, while another type also exceeds a threshold number of reads. Or, reads normalized to antibody barcode sequence reads that exceed a threshold fraction of reads for one type, but also exceed a threshold fraction of reads for another type, and then identifying antibody sequences (e.g., VH and VL) associated with the same NL barcode reads.

[0079] Antigen-producing cell assay In some embodiments, as depicted in FIG. 11, the methods provided herein may include, for example, identifying one or more antibodies or fragments thereof secreted from antibody-producing cells (e.g., B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof) that have a desired affinity for an antigen expressed or displayed on the surface of an antigen-presenting or antigen-producing cell.

[0080] In some embodiments, the method may include providing or obtaining (or obtaining) a nanovial, where the nanovial includes a cavity formed therein. In some examples, the nanovial may include an opening to the surface of the nanovial. The nanovial may be any nanovial described herein. In some cases, the nanovial is empty (e.g., does not contain antibody-producing cells). In other cases, the nanovial contains antibody-producing cells (e.g., single cells or individual cells). In some cases, the nanovial may contain one or more antibody-producing cells (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells).

[0081] In some cases, the cavity of the nanovial has antigen-presenting cells immobilized therein (90). The antigen-presenting cells can be immobilized on the surface of the cavity of the nanovial. In some cases, the resulting or provided nanovial does not have antigen-presenting cells immobilized therein, and the method includes attaching or immobilizing antigen-presenting cells to the surface of the cavity of the nanovial. In some embodiments, the antigen-presenting cells can be immobilized in the cavity of the nanovial prior to loading with the antibody-producing cells. In other embodiments, the antibody-producing cells can be loaded in the cavity of the nanovial prior to immobilizing the antigen-presenting cells in the cavity of the nanovial. The antigen-presenting cells can be loaded in the cavity of the nanovial at a concentration such that, on average, at least one antigen-presenting cell is loaded in the cavity of the nanovial (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5 or more).

[0082] The surface of the nanovial can be coated with a cell-capturing moiety that captures and fixes antigen-presenting cells to the surface of the nanovial. For example, the surface of the nanovial can be coated with an antibody or fragment thereof that interacts with a protein expressed on the surface of the antigen-presenting cell, or the nanovial can be coated with a cell adhesion peptide (e.g., RGD), or the nanovial can be coated with an extracellular matrix protein.

[0083] Antigen-presenting cells are usually selected so that they produce, present, or express an antigen of interest on their surface. In some cases, antigen-presenting cells are living cells. In other cases, antigen-presenting cells are dead cells (e.g., fixed cells). Antigen-presenting cells can be prokaryotic or eukaryotic cells. Antigen-presenting cells can be bacterial cells, yeast cells, fungal cells, insect cells, or mammalian cells. Antigen-presenting cells can be engineered (e.g., recombinantly) to express an antigen of interest on their surface at a higher level than normal to maximize signaling.

[0084] In some embodiments, the method further comprises loading antibody-producing cells into the cavity of the nanovial. In other embodiments, the method comprises obtaining or providing a nanovial with antibody-producing cells already loaded into the cavity. In some embodiments, the method comprises obtaining or providing a nanovial with antigen-presenting cells already loaded into the cavity prior to loading the antibody-producing cells. In other embodiments, the method comprises obtaining or providing an empty nanovial, and the antigen-presenting cells and antibody-producing cells are loaded into the cavity of the nanovial in any order. The antibody-producing cells can be any cell that produces antibodies. In some cases, the antibody-producing cells can be B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof. In some cases, the cavity of the nanovial comprises one or more cell capture agents immobilized thereon. The one or more cell capture agents can be immobilized on the surface of the cavity of the nanovial and / or within the porous matrix of the nanovial. In some cases, the obtained or provided nanovial is not coated with a cell capture agent, and the method includes coating the nanovial with a cell capture agent.

[0085] In some embodiments, one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of antibody-producing cells. In some embodiments, one or more cell capture agents bind to a label present on the surface of antibody-producing cells. For example, the cell capture agent may be biotin, and the antibody-producing cells may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the antibody-producing cells may be coated or labeled with biotin. In some embodiments, one or more cell capture agents is an antigen to which the antibody-producing cells can bind. In some embodiments, one or more cell capture agents comprise ssDNA, and the antibody-producing cells may be coated or labeled with complementary ssDNA that hybridizes with the cell capture agent.

[0086] Optionally, the method further includes encapsulating the nanovial or blocking the opening of the cavity of the nanovial. In some embodiments, the method may include adding one or more blocking particles to block the opening of the cavity of the nanovial or reduce the size of the opening (e.g., FIG. 17 (120, 122)). In some examples, the nanovial may be emulsified in oil (92). In some cases, the nanovial may be encapsulated within a droplet (92). In some cases, the nanovial may include a cavity with a small opening (118). Alternatively, the nanovial may not be encapsulated.

[0087] In various embodiments, the method may further include incubating the nanovial so that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the antigen of interest (e.g., expressed on the surface of the antigen-presenting cells) (94). Any suitable conditions for incubating the nanovial may be used, including any temperature, time, medium, or buffer conditions. Typically, the incubating step requires incubation under conditions such that the antibody-producing cells remain viable for a period of time sufficient to produce and secrete antibodies into the cavity of the nanovial. In some cases, the incubating step may include incubating the antibody-producing cells for a period of time. In some instances, the period may include at least 30 minutes (e.g., at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, at least 3 hours, etc.). In some cases, the period may include more than 3 hours (e.g., more than 3 hours, more than 5 hours, more than 10 hours, more than 15 hours, or more than 20 hours or more). In some cases, the period may be up to 24 hours.

[0088] In various aspects, the method may further include transferring the emulsified nanovial to an aqueous phase, thereby removing the emulsified oil film. Optionally, the aqueous phase nanovial may then be washed before adding the detection agent to the cavity of the nanovial. In other embodiments where blocking particles are used, the method may further include removing excess blocking particles by washing, straining, physical disruption, chemical disruption, buoyancy, magnetic force, or any combination thereof.

[0089] In various embodiments, the method further includes adding a detection agent to the cavity of the nanovial such that the detection agent binds to one or more antibodies or fragments thereof (e.g., bound to the antigen of interest, e.g., the surface of an antigen-presenting cell). In some cases, the detection agent may include an antibody or fragment thereof (96, 98). For example, the detection agent may be a secondary antibody specific for the secreted antibody (e.g., anti-mouse H&L or anti-mouse Fc for mouse B cells or other antibody-secreting cells). In some cases, the detection agent may be directly or indirectly labeled with a detectable label. The detectable label may include a fluorescent label (98), a protein affinity tag, an oligonucleotide tag (96), a magnetic particle, or any combination thereof.

[0090] In various embodiments, the method further includes detecting one or more signals related to binding of the detection agent to the one or more antibodies or fragments thereof. In some cases, the detecting step includes determining the amount of detection agent bound to the one or more antibodies or fragments thereof. In one embodiment, the detection agent comprises a fluorescent label (98), and the method includes detecting the level of fluorescence (102), where the amount of detection agent bound to the one or more antibodies or fragments thereof correlates with the level of fluorescence. In another embodiment, the detection agent comprises an oligonucleotide label (96). In this example, the method further includes sequencing the oligonucleotide label (102). In some cases, the detecting step includes counting the number of oligonucleotide tags bound to the one or more antibodies or fragments thereof.

[0091] In some embodiments, the method further includes sorting the nanovials based on one or more signals related to binding of a detection agent to the antibody or fragment thereof (102). In some cases, the method includes sorting the nanovials within a gate based on the level of fluorescence (e.g., above background levels) or based on fluorescence intensity (102). The sorting can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescence signals. In a non-limiting example, the sorting is a flow-based sorting method (e.g., FACS). For example, secreted antibodies bound to antibody-secreting cells and antigen-presenting cells of interest can be sorted using FACS or MACS prior to downstream sequencing (e.g., of B cell receptors (paired VH and VL sequences)). A threshold for the fluorescence intensity of the secondary antibody fluorophore (98) can be used to sort the nanovials (and associated antibody-secreting cells of interest) (102) to select for antibody-secreting cells that specifically produce and secrete antibodies specific to the antigen of the antigen-producing cells.

[0092] In various embodiments, the method further comprises performing a sequencing assay on nucleic acids derived from the antibody-producing cells, thereby generating sequences of one or more antibodies or fragments thereof (102). In some cases, the method comprises obtaining or obtaining a sorted nanovial or a plurality of sorted nanovials (e.g., after any of the above methods have been performed) and performing a sequencing assay (e.g., as described herein) on nucleic acids derived from the antibody-producing cells. The method further comprises obtaining or obtaining data or data output (e.g., secretion assay data) related to any of the above methods, such that the data is associated with downstream sequencing data. Any known sequencing technology may be suitable for use in the methods provided herein. For example, sequencing may include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc.

[0093] In one embodiment, the method includes lysing the antibody-producing cells prior to sequencing. In other embodiments, the method includes obtaining or obtaining a lysate of the antibody-producing cells (e.g., after a secretion assay is performed and the nanovials are sorted based on one or more signals associated with the secretion assay) prior to sequencing. In some examples, the method further includes reverse transcribing mRNA released from the antibody-producing cells to generate cDNA, and then sequencing the cDNA.

[0094] In various aspects, the method can further include washing the nanovial. Any suitable washing method can be used, including any suitable washing solution or buffer.

[0095] In various embodiments, the method further includes associating sequences of one or more antibodies or fragments thereof with one or more signals related to the binding of a detection agent to the one or more antibodies or fragments thereof. The associating step may include associating one or more barcodes or labels associated with a first assay (e.g., a secretion assay) with one or more barcodes or labels associated with a second assay (e.g., a sequencing assay). The method may further include identifying one or more antibodies or fragments thereof that bind to the antigen of interest based on the associating step. For example, when labeled with barcoded oligonucleotides (96), the nanovials (and associated cells) can be introduced into a single-cell RNA sequencing workflow (e.g., 10X Genomics Chromium). This workflow utilizes oligonucleotide barcodes or nanovial labels (NLs) associated with the nanovials. For example, NLs can be associated with biotin-functionalized nanovials through the use of oligonucleotide barcodes conjugated to streptavidin (e.g., TotalSeq™-C0971, product number 405271). The NL may be located in a nanovial or in separate beads that are in fluid communication with the nanovial and the cells therein. The separate beads may be oligonucleotide-conjugated beads (e.g., dissolvable GEM beads or Rhapsody beads) used as part of a single-cell RNA sequencing system. The oligonucleotide barcode containing the NL may further include an oligo-dT sequence (dT) for capturing mRNA from antibody-producing cells via a polyA region, an oligonucleotide tag for antigen-presenting cells or antigen-producing cells, and an antibody. Reverse transcription is performed to convert the mRNA from antibody-producing cells, the oligonucleotide tag for antigen-presenting cells, and the antibody into cDNA linked adjacent to the NL, and optionally other oligonucleotide sequences including a universal sequence and a unique molecular index sequence. The cDNA is amplified and sequenced, for example, using next-generation sequencing.In this way, the same NL code present in the amplified cDNA is used to link antibody sequence cDNA from antibody-producing cells, antigen expression levels in antigen-presenting cells, and secretion and affinity signals from oligonucleotide-tagged antibodies. In this workflow, antibody binding can be assayed and further linked to antibody sequences (e.g., heavy and light chains) in antibody-secreting cells by examining the number of sequencing reads of oligonucleotide barcodes tethered to the same NL barcode. In some embodiments, selecting antibody sequences with affinity above a threshold involves identifying NL barcode-containing reads containing oligonucleotide-tagged antibody barcode sequences above a threshold level or number of reads, or above a threshold fraction of reads normalized to the antigen cDNA reads, and then identifying antibody sequences (e.g., VH and VL) associated with the same NL barcode reads.

[0096] In some embodiments, the method includes performing any of the above methods on a plurality of nanovials, each nanovial containing antibody-producing cells and antigen-presenting cells. In some examples, each nanovial contains antibody-producing cells that produce different antibodies (e.g., a single antibody-producing cell). In such methods, the plurality of nanovials can be screened for antibodies that target an antigen of interest. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0097] As described herein, Figure 11 depicts a non-limiting example of the workflow of an antibody affinity assay. In this example, antibody-producing or antibody-secreting cells and antigen-presenting cells are both carried within nanovials (90). The nanovials are optionally emulsified (92) to reduce crosstalk between samples and enhance signal. The sample in the nanovial is incubated (94) to allow the producing cells to secrete the antibodies. Antibodies specific to the antigen of interest bind to the antigen-presenting cells (94). The nanovials are then returned to the aqueous phase and washed. The antibodies bound to the antigen-presenting cells are then labeled with a secondary antibody (e.g., fluorescently labeled (98) and / or containing an oligonucleotide barcode (96)) and then analyzed and / or classified using downstream fluorescent and / or sequencing readout (102). In another embodiment, antibodies can induce the production of fluorescent proteins (e.g., green fluorescent protein (GFP)) in antigen-presenting cells upon binding to antigen, which can be used to read out the function of the secreted antibody and to sort antibody-secreting cells based on antibody function (100).

[0098] Antibody-regulated signaling pathway assays In some embodiments, the methods provided herein can include identifying one or more antibodies or fragments thereof that modulate a signal pathway of interest, for example, as depicted in FIG. 12A. In some aspects, the methods can include providing or obtaining (or obtaining) a nanovial, where the nanovial includes a cavity formed therein. In some examples, the nanovial can include an opening to the surface of the nanovial. The nanovial can be any nanovial described herein. In some cases, the nanovial is empty (e.g., does not contain antibody-producing cells). In other cases, the nanovial contains antibody-producing cells (e.g., single cells or individual cells). In some cases, the nanovial can contain more than one antibody-producing cell (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells).

[0099] In some cases, the cavity of the nanovial has antigen-presenting or antigen-producing cells immobilized therein (104). Antigen-presenting cells can be immobilized on the surface of the cavity of the nanovial. In some cases, the resulting or provided nanovial does not have antigen-presenting cells immobilized therein, and the method includes attaching or immobilizing antigen-presenting cells to the surface of the cavity of the nanovial. In some embodiments, antigen-presenting cells can be immobilized in the cavity of the nanovial prior to loading with antibody-producing cells. In other embodiments, antibody-producing cells can be loaded in the cavity of the nanovial prior to loading with antigen-presenting cells in the cavity of the nanovial. Antigen-presenting cells can be loaded in the cavity of the nanovial at a concentration such that, on average, at least one antigen-presenting cell is loaded in the cavity of the nanovial (e.g., at least one, at least two, at least three, at least four, or at least five or more).

[0100] The surface of the nanovial can be coated with a cell-capturing moiety that captures and fixes antigen-presenting cells to the surface of the nanovial. For example, the surface of the nanovial can be coated with an antibody or fragment thereof that interacts with a protein expressed on the surface of the antigen-presenting cell, or the nanovial can be coated with a cell adhesion peptide (e.g., RGD), or the nanovial can be coated with an extracellular matrix protein.

[0101] The antigen-presenting cell is usually selected so that it produces, presents, or expresses the antigen of interest on its surface. Typically, in these embodiments, the antigen-presenting cell is a living cell (so that modulation of the signal transduction pathway can be evaluated). The antigen-presenting cell can be a prokaryotic cell or a eukaryotic cell. The antigen-presenting cell can be a bacterial cell, a yeast cell, a fungal cell, an insect cell, or a mammalian cell. The antigen-presenting cell can be engineered (e.g., recombinantly) to express the antigen of interest on its surface at a higher level than normal to maximize signaling.

[0102] In some cases, antigen-producing cells are engineered to express one or more detectable markers upon modulation of the signal transduction pathway of interest. For example, antigen-presenting cells can be engineered to express fluorescent reporter proteins (e.g., GFP, EGFP, RFP, fluorescent reporter proteins sensitive to intracellular calcium concentration (e.g., GCaMP (or GCaMP co-expressed with a fluorescent protein (e.g., mCherry)))) for ratiometric measurement of calcium concentration).

[0103] In some embodiments, the method further comprises loading antibody-producing cells into the cavity of the nanovial. In other embodiments, the method comprises obtaining or providing a nanovial with antibody-producing cells already loaded into the cavity. In some embodiments, the method comprises obtaining or providing a nanovial with antigen-presenting cells already loaded into the cavity prior to loading the antibody-producing cells. In other embodiments, the method comprises obtaining or providing an empty nanovial, and the antigen-presenting cells and antibody-producing cells are loaded into the cavity of the nanovial in any order. The antibody-producing cells can be any cell that produces antibodies. In some cases, the antibody-producing cells can be B cells, plasmablasts, plasma cells, hybridomas, genetically engineered producer cells, or any combination thereof. In some cases, the cavity of the nanovial comprises one or more cell capture agents immobilized thereon. The one or more cell capture agents can be immobilized on the surface of the cavity of the nanovial and / or within the porous matrix of the nanovial. In some cases, the obtained or provided nanovial is not coated with a cell capture agent, and the method includes coating the nanovial with a cell capture agent.

[0104] In some embodiments, one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of antibody-producing cells. In some embodiments, one or more cell capture agents bind to a label present on the surface of antibody-producing cells. For example, the cell capture agent may be biotin, and the antibody-producing cells may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the antibody-producing cells may be coated or labeled with biotin. In some embodiments, one or more cell capture agents is an antigen to which the antibody-producing cells can bind. In some embodiments, one or more cell capture agents comprise ssDNA, and the antibody-producing cells may be coated or labeled with complementary ssDNA that hybridizes with the cell capture agent.

[0105] Optionally, the method further includes encapsulating the nanovial or blocking the opening of the cavity of the nanovial. In some embodiments, the method may include adding one or more blocking particles to block the opening of the cavity of the nanovial or reduce the size of the opening (e.g., FIG. 17 (120, 122)). In some examples, the nanovial may be emulsified in oil (106). In some cases, the nanovial may be encapsulated within a droplet (106). In some cases, the nanovial may include a cavity with a small opening (118). Alternatively, the nanovial may not be encapsulated.

[0106] In various aspects, the method may further include incubating the nanovial so that the one or more antibodies or fragments thereof are secreted from the antibody-producing cells and modulate a signal transduction pathway of interest in the antigen-producing cells (108, 110). In some examples, modulating a signal transduction pathway of interest includes activating the signal transduction pathway of interest. In other examples, modulating a signal transduction pathway of interest includes inhibiting the signal transduction pathway of interest. In some embodiments, the one or more antibodies or fragments thereof modulate the signal transduction pathway of interest by binding to an antigen on the surface of the antigen-producing cells (110). In other embodiments, the one or more antibodies or fragments thereof modulate the signal transduction pathway of interest by interfering with binding of a ligand to the antigen on the surface of the antigen-producing cells (108). The ligand may be, for example, an agonist that is separately introduced into the cavity of the nanovial, and the antibody or fragment thereof may inhibit or block the ligand from modulating the signal transduction pathway of interest (e.g., by directly binding to the ligand or by binding to the antigen on the antigen-producing cells, thereby preventing binding of the ligand to the antigen) (108).

[0107] Any suitable conditions for incubating the nanovial can be used, including any temperature, time, medium, buffer conditions, etc. Incubating can include using a medium or buffer containing a suitable amount of a ligand or agonist that interacts with the antigen of the antigen-producing cells. Typically, the incubating step involves incubating under conditions such that the antibody-producing cells remain viable for a period of time sufficient to produce and secrete antibodies into the cavity of the nanovial. In some cases, the incubating step can include incubating the antibody-producing cells for a period of time. In some instances, the period can include at least 30 minutes (e.g., at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, at least 3 hours, etc.). In some cases, the period can include more than 3 hours (e.g., more than 3 hours, more than 5 hours, more than 10 hours, more than 15 hours, or more than 20 hours or more). In some cases, the period can be up to 24 hours.

[0108] In various aspects, the method may further include transferring the emulsified nanovial to an aqueous phase, thereby removing the emulsified oil film. Optionally, the aqueous phase nanovial may then be washed before adding the detection agent to the cavity of the nanovial. In other embodiments where blocking particles are used, the method may further include removing excess blocking particles by washing, straining, physical disruption, chemical disruption, buoyancy, magnetic force, or any combination thereof.

[0109] In various aspects, the method further comprises detecting one or more signals related to the modulation of a signaling pathway of interest. In some embodiments, the method comprises detecting the level of fluorescence in the nanovial, wherein the level of fluorescence corresponds to the modulation of the signaling pathway of interest. For example, the activity of an antibody or its fragment on a downstream signaling pathway can be assessed by the production of a fluorescent protein (e.g., green fluorescent protein, cyan fluorescent protein, red fluorescent protein, etc.) in antigen-producing cells (also referred to as reporter cells) or the fluorescence level of a calcium concentration-dependent fluorescent reporter protein in the reporter cells. In some embodiments, the method comprises detecting changes in one or more mRNA levels in the antigen-producing cells. In some embodiments, the detecting step comprises performing single-cell RNA sequencing on nucleic acids derived from the antigen-producing cells.

[0110] In some embodiments, the method comprises: The method further includes sorting the nanovials. In some cases, the method includes sorting the nanovials based on the level of fluorescence (e.g., above background level) or within a gate of fluorescence intensity. The sorting step can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescent signals. In a non-limiting example, the sorting step is a flow-based sorting method (e.g., FACS). For example, nanovials containing antigen-producing cells with a specific fluorescence level (e.g., above or below a cutoff) or ratio of fluorescence levels can be sorted using FACS.

[0111] In various embodiments, the method further comprises performing a sequencing assay on nucleic acid derived from the antibody-producing cells, thereby generating sequences for one or more antibodies or fragments thereof (114). In some cases, the method comprises obtaining or obtaining a sorted nanovial or a plurality of sorted nanovials (e.g., after any of the above methods have been performed) and performing a sequencing assay (e.g., as described herein) on nucleic acid derived from the antibody-producing cells. The method further comprises obtaining or obtaining data or data output (e.g., secretion assay data) related to any of the above methods, such that the data is associated with downstream sequencing data. Any known sequencing technology may be suitable for use in the methods provided herein. For example, sequencing may include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc. In a particular embodiment, antibody-secreting cells (e.g., B cells) that have been sorted (e.g., based on one or more signals associated with modulation of a signaling pathway) can be analyzed (e.g., by sequencing) to identify antibody sequences (e.g., paired heavy and light chain sequences) that modulate specific signaling pathways via cell surface binding or interaction with an agonist or ligand as discussed herein.

[0112] In one embodiment, the method includes lysing the antibody-producing cells and / or antigen-producing cells prior to sequencing. In other embodiments, the method includes obtaining or obtaining a lysate of the antibody-producing cells (e.g., after a secretion assay is performed and the nanovials are sorted based on one or more signals associated with the secretion assay) prior to sequencing. In some examples, the method further includes reverse transcribing mRNA released from the antibody-producing cells to generate cDNA, followed by sequencing the cDNA.

[0113] In various aspects, the method can further include washing the nanovial. Any suitable washing method can be used, including any suitable washing solution or buffer.

[0114] In various aspects, the method further includes linking the sequences of one or more antibodies or fragments thereof to one or more signals related to the modulation of a signaling pathway of interest. In some embodiments, the linking step includes linking mRNA levels of the antigen-producing cells with nucleic acid sequence information from the antibody-producing cells based on the shared oligonucleotide barcode. The method may further include identifying one or more antibodies or fragments thereof that modulate the signaling pathway of interest based on the linking step. The shared oligonucleotide barcode may include one or more unique oligonucleotide tags associated with the nanovial (104). In some embodiments, multiple signaling pathways may be examined in a single assay (e.g., by any of the methods provided herein) by using antigen-producing cells genetically modified with multiple distinct fluorescent reporter proteins or multiple different antigen-producing cells each bearing a distinct fluorescent reporter specific to a different signaling pathway. In this embodiment, different transcription factors or promoter regions associated with different signaling pathways may be integrated upstream of the genes encoding each of the fluorescent proteins. Non-limiting examples of signaling pathways that can be examined with these methods include the Akt / PKB signaling pathway, the AMPK signaling pathway, the cAMP-dependent pathway, the Eph / Ephrin signaling pathway, the Hedgehog signaling pathway, the Hippo signaling pathway, the insulin signaling pathway, the JAK-STAT signaling pathway, the MAPK / ERK signaling pathway, the mTOR signaling pathway, the Nodal signaling pathway, the Notch signaling pathway or an engineered synNotch signaling pathway, the PI3K / AKT / mTOR signaling pathway, the TGFβ signaling pathway, the TLR signaling pathway, the VEGF signaling pathway, the Wnt signaling pathway, among others.If multiple signaling pathways are being investigated, the nanovials and associated cells can be sorted using standard multicolor FACS to identify, for example, antibodies that activate one particular signaling pathway but not another, or antibodies that activate one or more signaling pathways but not another one or more (e.g., high fluorescence for fluorescent protein 1, low fluorescence for fluorescent protein 2, etc.). Additionally, antigen-producing cells of interest can be engineered to overexpress the antigen of interest on their cell surface to facilitate this embodiment and focus screening methods on antibodies that interact with specific overexpressed cell surface antigens.

[0115] In some embodiments, the method includes performing any of the above methods on a plurality of nanovials, each nanovial containing antibody-producing cells and antigen-producing cells. In some examples, each nanovial contains antibody-producing cells that produce different antibodies (e.g., a single antibody-producing cell). In such methods, the plurality of nanovials can be screened for antibodies that target an antigen of interest. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0116] As described herein, Figure 12A depicts a non-limiting example workflow for an antibody-regulated signaling pathway assay. Antigen- and antibody-producing cells are seeded into nanovials (104), which may optionally contain oligonucleotide barcode molecules. The nanovials with loaded cells are introduced into culture medium along with an agonist molecule that normally interacts with the antigen on the antigen-producing cells (104). The secreted antibody can bind to the antigen on the surface of the antigen-producing cells (110) or can bind to the agonist that normally binds to the antigen on the antigen-producing cells, preventing the agonist from binding (108). Both processes can modulate signaling pathways within the antigen-producing cells. Single-cell sequencing can be used downstream to measure variations in the expression of genes in the signaling pathways of the antigen-producing cells (114). Information about gene expression changes in this signaling pathway can be linked to antibody sequences (e.g., heavy and light chain sequence information) in antibody-secreting cells using oligonucleotide barcodes containing nanovial labels (NLs) or single-cell labels unique to each nanovial or droplet containing a nanovial (e.g., Figure 12B). The NL labels may be located on separate beads that communicate with the nanovials or nanovials and the cells therein. The separate beads may be oligonucleotide-conjugated beads used as part of a single-cell RNA sequencing system. The NL-containing oligonucleotide barcodes may further contain an oligo-dT sequence (dT) for capturing mRNA from antibody- and antigen-producing cells via polyA tails. Reverse transcription is performed to convert the mRNA from antibody- and antigen-producing cells into cDNAs linked adjacent to the NLs and, optionally, other oligonucleotide sequences containing universal and unique molecular index sequences. The cDNAs are amplified and sequenced, for example, using next-generation sequencing. In this way, the same nanovial labeling code present in the amplified cDNA is used to link antibody sequences from antibody-producing cells to functional changes in gene expression in antigen-producing cells in a single workflow.Downstream analysis of the sequencing files can be used to rank the potency of different antibody sequences for the degree of modulation of signaling pathways in antigen-producing cells (Figure 12B).

[0117] T cell secretory phenotype assay Provided herein are methods for identifying functional chimeric antigen receptors (CARs) or T cell receptors (TCRs) against an antigen of interest.

[0118] In some embodiments, the method may include providing or obtaining (or obtaining) a nanovial, wherein the nanovial includes a cavity formed therein. In some examples, the nanovial may include an opening to the surface of the nanovial. The nanovial may be any nanovial described herein. In some cases, the nanovial is empty (e.g., does not contain CAR- or TCR-expressing cells). In other cases, the nanovial contains CAR- or TCR-expressing cells (e.g., single cells or individual cells). In some cases, the nanovial may contain more than one CAR- or TCR-expressing cell (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cells).

[0119] In some cases, the cavity of the nanovial is coated with or has an affinity agent immobilized thereon. The affinity agent may be immobilized on the surface of the cavity of the nanovial and / or may be within the porous matrix of the nanovial. In some cases, the resulting or provided nanovial is not coated with an affinity agent, and the method includes coating the nanovial with an affinity agent.

[0120] In some embodiments, the affinity agent is a cytokine capture moiety (e.g., for binding to and capturing cytokines secreted by CAR- or TCR-expressing cells). In some cases, the cytokine capture moiety is an anti-cytokine antibody, e.g., an anti-IL2 antibody, an anti-TNF-α antibody, or an anti-IFN-γ antibody. In some cases, the affinity agent is a protein aptamer or a nonspecific capture surface, e.g., polylysine, an extracellular matrix protein, etc.

[0121] In various aspects, the method further comprises loading CAR-expressing cells or TCR-expressing cells into the cavity of the nanovial. In other embodiments, the method comprises obtaining or providing a nanovial with CAR-expressing cells or TCR-expressing cells already loaded into the cavity. In some cases, the CAR-expressing cells or TCR-expressing cells are T cells. In some cases, the cavity of the nanovial comprises one or more cell capture agents immobilized thereon. The one or more cell capture agents may be immobilized on the surface of the cavity of the nanovial and / or may be within the porous matrix of the nanovial. In some cases, the obtained or provided nanovial is not coated with a cell capture agent, and the method comprises coating the nanovial with a cell capture agent.

[0122] In some embodiments, the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of a CAR- or TCR-expressing cell and / or antigen-presenting cell. In some embodiments, the one or more cell capture agents may include an anti-CD45 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD3 / anti-CD28 antibody, or any combination thereof. In some embodiments, the one or more cell capture agents are an antigen or an MHC-presented antigen to which a CAR- or TCR-expressing cell can bind. In some embodiments, the one or more cell capture agents are antigen-presenting cells that express an antigen of interest on their surface or present the antigen in an MHC complex. In some embodiments, the one or more cell capture agents bind to a label present on the surface of a CAR- or TCR-expressing cell. For example, the cell capture agent may be biotin, and the CAR- or TCR-expressing cell may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the CAR- or TCR-expressing cell may be coated or labeled with biotin. In some embodiments, one or more cell capture agents are antibodies or fragments thereof that bind to proteins expressed on the surface of antigen-presenting cells. In some embodiments, one or more cell capture agents bind to a label present on the surface of the antigen-presenting cells. For example, the cell capture agent may be biotin, and the antigen-presenting cells may be coated or labeled with streptavidin. In another non-limiting example, the cell capture agent may be streptavidin, and the antigen-presenting cells may be coated or labeled with biotin. In another non-limiting example, the nanovial may be coated with a cell adhesion peptide (e.g., RGD), or the nanovial may be coated with an extracellular matrix protein to which the antigen-presenting cells can bind. In some embodiments, one or more cell capture agents comprise ssDNA, and the antigen-presenting cells may be coated or labeled with complementary ssDNA that hybridizes with the cell capture agent.

[0123] Optionally, the method further includes the step of enclosing the nanovial or blocking the opening of the cavity of the nanovial. In some embodiments, the method may include adding one or more blocking particles to block the opening of the cavity of the nanovial or reduce the size of the opening (e.g., Figure 17 (120, 122)). In some examples, the nanovial used in the affinity assay may be emulsified in oil. In some cases, the nanovial may be encased in a droplet. In some cases, the nanovial may include a cavity with a small opening (118). Alternatively, the nanovial may not be encased.

[0124] In various embodiments, the method may further include incubating the nanovial so that one or more cytokines are secreted from the CAR- or TCR-expressing cells and bind to the affinity agent. Any suitable conditions for incubating the nanovial may be used, including any temperature, time, medium, buffer conditions, etc. Typically, the incubating step involves incubating under conditions such that the CAR- or TCR-expressing cells remain viable for a period sufficient to produce and secrete cytokines into the cavity of the nanovial. In some cases, the incubating step may involve incubating the CAR- or TCR-expressing cells for a period of time. In some examples, the period may include at least 30 minutes (e.g., at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 4 hours, at least 6 hours, etc.). In some cases, the period may include up to 24 hours.

[0125] In various aspects, the method may further include transferring the emulsified nanovial to an aqueous phase, thereby removing the emulsified oil film. Optionally, the aqueous phase nanovial may then be washed before adding the detection agent to the cavity of the nanovial. In other embodiments using blocking particles, the method may further include removing excess blocking particles by washing, straining, physical disruption, chemical disruption, buoyancy, magnetic force, or any combination thereof.

[0126] In various embodiments, the method further comprises adding a detection agent to the cavity of the nanovial so that the detection agent binds to one or more cytokines. In some cases, the detection agent may comprise an antibody or fragment thereof specific to one or more cytokines. In some cases, the detection agent may be directly or indirectly labeled with a detectable label. The detectable label may comprise a fluorescent label, a protein affinity tag, an oligonucleotide tag, a magnetic particle, or any combination thereof.

[0127] In various embodiments, the method further comprises detecting one or more signals relating to the binding of the detection agent to one or more cytokines. In some cases, the detecting step comprises determining the amount of detection agent bound to one or more cytokines. In one embodiment, the detection agent comprises a fluorescent label, and the method comprises detecting the level of fluorescence, wherein the amount of detection agent bound to one or more cytokines correlates with the level of fluorescence. In another embodiment, the detection agent comprises an oligonucleotide label. In this example, the method further comprises sequencing the oligonucleotide label. In some cases, the detecting step comprises counting the number of oligonucleotide tags bound to one or more cytokines.

[0128] In some embodiments, the method further comprises sorting the nanovials based on one or more signals relating to the binding of the detection agent to one or more cytokines. In some cases, the method comprises sorting the nanovials based on the level of fluorescence (e.g., above background level) or fluorescence intensity within a gate. The sorting step can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescent signals. In a non-limiting example, the sorting step is a flow-based sorting method (e.g., FACS). In some cases, when the detection agent includes a magnetic particle label, the sample is sorted using magnetic force or magnetically activated cell sorting (e.g., MACS).

[0129] In various embodiments, the method further comprises performing a sequencing assay on nucleic acid derived from CAR-expressing cells or TCR-expressing cells, thereby generating a sequence of CAR or TCR. In some cases, the method comprises obtaining or obtaining a sorted nanovial or a plurality of sorted nanovials (e.g., after any of the above methods have been performed), and performing a sequencing assay (e.g., as described herein) on nucleic acid derived from CAR-expressing cells or TCR-expressing cells. The method further comprises obtaining or obtaining data or data output related to any of the above methods, so that the data is associated with downstream sequencing data. Any known sequencing technology can be suitable for use in the methods provided herein. For example, sequencing can include Sanger sequencing, next-generation sequencing, third-generation sequencing (e.g., long-read sequencing), single-cell sequencing (e.g., single-cell mRNA sequencing), etc.

[0130] In one embodiment, the method includes lysing the CAR-expressing or TCR-expressing cells prior to sequencing. In another embodiment, the method includes obtaining or obtaining a lysate of the CAR-expressing or TCR-expressing cells (e.g., after the nanovials have been sorted based on one or more signals associated with a cytokine secretion assay) prior to sequencing. In some examples, the method further includes reverse transcribing mRNA released from the CAR-expressing or TCR-expressing cells to generate cDNA, followed by sequencing the cDNA.

[0131] In various aspects, the method can further include washing the nanovial. Any suitable washing method can be used, including any suitable washing solution or buffer.

[0132] In various aspects, the method further includes associating the sequence of the CAR or TCR with one or more signals related to the binding of the detection agent to one or more cytokines. The associating step may include associating one or more barcodes or labels associated with a first assay (e.g., a cytokine secretion assay) with one or more barcodes or labels associated with a second assay (e.g., a sequencing assay). In some embodiments, the associating step includes linking the sequence of the CAR or TCR from the CAR-expressing cell and the TCR-expressing cell to the count of the sequence of one or more oligonucleotide labels associated with the detection agent based on the shared oligonucleotide barcode. The shared oligonucleotide barcode may include one or more unique oligonucleotide tags associated with the nanovial. The method may further include identifying a CAR or TCR that functionally interacts with the antigen of interest based on the associating step.

[0133] In some embodiments, the method comprises carrying out any of the above methods on a plurality of nanovials, each nanovial containing a CAR-expressing cell or a TCR-expressing cell. In some examples, each nanovial contains a CAR-expressing cell or a TCR-expressing cell, each producing a different CAR or TCR. In such a method, the plurality of nanovials can be screened to identify a functional CAR or TCR for an antigen of interest. In other examples, each nanovial contains an antigen-presenting cell or an antigen-producing cell, each expressing a different antigen, and a CAR-expressing cell or a TCR-expressing cell, each producing the same CAR or TCR. In such a method, the plurality of nanovials can be screened to identify the antigen (antigen sequence) functionally targeted by the CAR or TCR. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0134] Drugs or compounds that modulate signaling pathway assays For example, provided herein are methods for identifying drugs or compounds that modulate signaling pathways in cells of interest, as depicted in Figure 19A. In some embodiments, disclosed herein are methods using nanovials to discover drugs that modulate target signaling pathways or target protein expression levels in target cells of interest.

[0135] In some embodiments, the method may include providing or obtaining (or obtaining) a nanovial, wherein the nanovial includes a cavity formed therein. In some examples, the nanovial may include an opening to the surface of the nanovial. The nanovial may be any nanovial described herein. In some examples, the cavity of the nanovial includes (i) a drug or compound and (ii) a barcode or label associated with the drug or compound. The drug or compound may include a small molecule, peptide, protein, aptamer, antibody, guide RNA, short hairpin RNA, siRNA, etc. In a non-limiting example, the barcode or label associated with the drug or compound is a unique oligonucleotide barcode including a drug barcode sequence, and the drug or compound is associated with the unique oligonucleotide barcode. In such cases, determining the barcode or label can identify the drug or compound with which the barcode or label is associated. In some examples, the unique oligonucleotide barcode contains a polyA region (e.g., a 30-mer polyA region) that enables capture by a polyT capture region. In other examples, the unique oligonucleotide barcode contains a poly-T capture region for capturing mRNA of a cell of interest. In some embodiments, a plurality of nanovials are provided, each containing a different drug or compound, a combination of two or more drugs or compounds, or a unique amount of one or more drugs or compounds, and a unique oligonucleotide barcode (e.g., a drug barcode sequence) or label associated with the drug or compound, the combination of two or more drugs or compounds, or a unique amount of the drug or compound. In some cases, the drug or compound is bound to the nanovial (e.g., by using a linker). In some cases, the linker is a photocleavable linker, so that when the nanovial is exposed to light (e.g., UV light), the photocleavable linker is cleaved and the drug / compound is released into the cavity of the nanovial. In other cases, the linker is cleavable by temperature, pH, chemicals, etc.The methods may be used, for example, to screen drug / compound libraries (eg, DNA-encoded libraries).

[0136] In various embodiments, the method further includes loading cells of interest into the cavity of the nanovial (124). The cells of interest can be immobilized on the surface of the cavity of the nanovial. In some cases, the obtained or provided nanovial does not have cells of interest immobilized therein, and the method includes attaching or immobilizing cells of interest to the surface of the cavity of the nanovial. The cells of interest can be loaded into the cavity of the nanovial at a concentration such that, on average, at least one cell of interest is loaded into the cavity of the nanovial (e.g., at least one, at least two, at least three, at least four, or at least five or more).

[0137] The surface of the nanovial can be coated with a cell-capturing moiety that captures and fixes the cells of interest to the surface of the nanovial. For example, the surface of the nanovial can be coated with an antibody or a fragment thereof that interacts with a protein expressed on the surface of the cells of interest, or the nanovial can be coated with a cell adhesion peptide (e.g., RGD), or the nanovial can be coated with an extracellular matrix protein.

[0138] Typically, in these embodiments, the cell of interest is a living cell (such that modulation of a signal transduction pathway can be evaluated). The cell of interest can be a prokaryotic or eukaryotic cell. The cell of interest can be a bacterial cell, a yeast cell, a fungal cell, an insect cell, or a mammalian cell. In some cases, the cell of interest is engineered to express one or more detectable labels upon modulation of the signal transduction pathway of interest. For example, the cell of interest can be engineered to express a fluorescent reporter protein (e.g., GFP, EGFP, RFP, a fluorescent reporter protein that is sensitive to intracellular calcium concentration (e.g., GCaMP (or GCaMP co-expressed with a fluorescent protein (e.g., mCherry)) for ratiometric measurement of calcium concentration)).

[0139] Optionally, the method further includes encapsulating the nanovial or blocking the opening of the cavity of the nanovial. In some embodiments, the method may include adding one or more blocking particles to block the opening of the cavity of the nanovial or reduce the size of the opening (e.g., FIG. 17 (120, 122)). In some examples, the nanovial may be emulsified in oil (126). In some cases, the nanovial may be encapsulated within a droplet (126). In some cases, the nanovial may include a cavity with a small opening (118). Alternatively, the nanovial may not be encapsulated.

[0140] The method may further include exposing the nanovial to a stimulus to release the drug or compound into the liquid volume within the cavity of the nanovial so that the drug or compound interacts with the cells of interest. The stimulus may be, for example, a pH change, a temperature change, the addition of a chemical, exposure to light (e.g., UV light), etc. It should be understood that the stimulus used to release the drug or compound into the cavity of the nanovial is selected based on the type of cleavable linker used to attach the drug or compound to the nanovial.

[0141] In various embodiments, the method may further include incubating the nanovial so that the drug or compound modulates the signal transduction pathway of interest in the cell of interest (132, 130). In some examples, modulating the signal transduction pathway of interest includes activation of the signal transduction pathway of interest. In other examples, modulating the signal transduction pathway of interest includes inhibition of the signal transduction pathway of interest.

[0142] Any suitable conditions for incubating the nanovial can be used, including any temperature, time, medium, buffer conditions, etc. Typically, the incubating step involves incubating under conditions such that the cells of interest remain viable for a period of time sufficient for signal transduction pathway modulation or gene expression changes to occur. In some cases, the incubating step can involve incubating the cells of interest for a period of time. In some instances, the period can include at least 30 minutes (e.g., at least 45 minutes, at least 1 hour, at least 1.5 hours, at least 2 hours, at least 2.5 hours, at least 3 hours, etc.). In some cases, the period can include more than 3 hours (e.g., more than 3 hours, more than 5 hours, more than 10 hours, more than 15 hours, or more than 20 hours). In some cases, the period can be up to 24 hours.

[0143] In various aspects, the method may further include transferring the emulsified nanovial to an aqueous phase, thereby removing the emulsified oil film. Optionally, the aqueous phase nanovial may then be washed before adding the detection agent to the cavity of the nanovial. In other embodiments where blocking particles are used, the method may further include removing excess blocking particles by washing, straining, physical disruption, chemical disruption, buoyancy, magnetic force, or any combination thereof.

[0144] In various aspects, the method further comprises detecting one or more signals related to the modulation of the signaling pathway of interest. In some embodiments, the method comprises detecting the level of fluorescence in the nanovial, wherein the level of fluorescence corresponds to the modulation of the signaling pathway of interest. For example, the activity of a drug or compound on a downstream signaling pathway can be evaluated by the production of a fluorescent protein (e.g., green fluorescent protein, cyan fluorescent protein, red fluorescent protein, etc.) in the cells of interest (also referred to as reporter cells) or the fluorescence level of a calcium concentration-dependent fluorescent reporter protein in the reporter cells. In some embodiments, the method comprises detecting changes in one or more mRNA levels in the cells of interest. In some embodiments, the detecting step comprises performing single-cell RNA sequencing on nucleic acids derived from the cells of interest.

[0145] In some embodiments, the method further comprises sorting the nanovials based on one or more signals related to the modulation of the signal transduction pathway of interest. In some cases, the method comprises sorting the nanovials based on the level of fluorescence (e.g., above background level) or the fluorescence intensity of a gate. The sorting step can be any sorting method typically used to sort particles, droplets, and / or cells based on fluorescent signals. In a non-limiting example, the sorting step is a flow-based sorting method (e.g., FACS). For example, nanovials containing cells of interest with a specific fluorescence level (e.g., above or below a cutoff) or a ratio of fluorescence levels can be sorted using FACS.

[0146] In various aspects, the method further comprises associating the barcode or label with one or more signals related to modulation of the signal transduction pathway of interest. In some embodiments, the associating step of (f) comprises linking the mRNA levels of the cells of interest to a drug or compound based on the shared oligonucleotide barcode.

[0147] In some embodiments, the associating step comprises identifying sequence information from a unique oligonucleotide barcode associated with the drug or compound and sequence information for the mRNA expressed by the cell of interest, and linking this sequence information. In other embodiments, the associating step comprises identifying sequence information from a unique oligonucleotide barcode associated with the drug or compound and a fluorescent reporter signal expressed by the cell of interest, and linking this information.

[0148] In various aspects, the method may further include identifying a drug or compound that modulates the signal transduction pathway of interest based on the correlating step. In one embodiment, the method includes identifying a drug or compound that has a significant effect on mRNA expressed by the cell of interest compared to control cells or compared to the cell of interest without exposure to the drug or compound.

[0149] In various embodiments, the method further comprises carrying out sequencing assay on the nucleic acid derived from the cell of interest.The method can further comprise sequencing the oligonucleotide barcode or label associated with drug or compound.Any known sequencing technology can be suitable for use in the method provided herein.For example, sequencing can include Sanger sequencing, next-generation sequencing, third-generation sequencing (for example, long-read sequencing), single-cell sequencing (for example, single-cell mRNA sequencing), etc.

[0150] In one embodiment, the method comprises lysing the cells of interest before sequencing.In another embodiment, the method comprises obtaining or obtaining the lysate of the cells of interest (for example, after a drug or compound acts on the cells of interest) before sequencing.In some examples, the method further comprises reverse transcribing the mRNA released from the cells of interest to generate cDNA, and then sequencing the cDNA.

[0151] In various aspects, the method can further include washing the nanovial. Any suitable washing method can be used, including any suitable washing solution or buffer.

[0152] In some embodiments, the method includes performing any of the above methods on a plurality of nanovials, each containing a different drug or compound associated with a barcode or label. In such methods, a plurality of different drugs or compounds can be screened for activity on cells of interest. In some embodiments, each nanovial contains different amounts of the same drug or compound and has a barcode or label associated with the amount. In some embodiments, each nanovial contains a combination of two or more drugs in varying amounts associated with a barcode or label. The plurality of nanovials can include, but is not limited to, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000, or at least 100,000 or more nanovials.

[0153] As described herein, Figure 19A depicts a non-limiting example workflow for a drug- or compound-modulated signaling pathway assay. A nanovial is provided or obtained (124) containing a drug or compound with a unique barcode or label associated therewith. The drug or compound can be attached within the cavity of the nanovial (124), for example, through the use of a cleavable linker (e.g., a photocleavable linker). The nanovial can further include a nanovial-associated barcode or label (e.g., an oligobarcode) (124). Cells of interest can be seeded within the cavity of the nanovial (124). Optionally, the nanovial can be emulsified (126). The nanovial with the loaded cells is exposed to a stimulus (e.g., UV light) to cleave the cleavable linker and release the drug or compound into the cavity of the nanovial (128). The nanovial is then incubated under appropriate conditions (e.g., sufficient temperature, time, pH, etc.) such that the drug or compound acts on the cells of interest (e.g., to modulate a signaling pathway) and the cells of interest express a reporter protein or undergo changes in mRNA expression. The drug or compound may act on the cells of interest such that a signaling pathway is modulated (130). Additionally, or alternatively, the drug or compound may act on the cells of interest such that the cells of interest secrete a biomolecule into the cavity of the nanovial, which can then be captured in the nanovial with an affinity agent (132). In this embodiment, a detection agent may be added (e.g., as described throughout) to detect one or more signals associated with binding of the secreted biomolecule to the affinity agent (134). The nanovial may then be interrogated using any of the methods provided herein, including flow-based sorting or sequencing methods (136). Figure 19B shows a non-limiting example of oligonucleotide barcodes that may be used in these embodiments and the binding of information by reverse transcription into cDNA resulting from captured oligo(dT) sequences attached to nanovials or beads associated with the nanovials, followed by sequencing of the successive cDNAs.The mRNA levels from the cells of interest and the drug or compound may be linked by a unique barcode (drug barcode sequence, DBC) associated with the drug and a nanovial label (NL1), which is part of a continuous sequence in the produced cDNA. Alternatively, the mRNA levels from the cells of interest and the drug or compound may be linked via a nanovial label (NL1) present on beads associated with a nanovial in a compartment, resulting in a continuous cDNA containing NL1 and DBC and NL1 and signaling pathway cRNA from the cells of interest. Secretions from the cells of interest can be linked to the drug or compound by linking a unique oligo (secretory sequence) adjacent to the same DBC and / or NL1 sequence.

[0154] Buoyancy concentration of nanovials Provided herein are methods for concentrating nanovials containing one or more cells, one or more beads, or both, as depicted in Figure 5. Optionally, the method includes one or more of the following steps: (a) providing or obtaining a mixture containing a plurality of nanovials, each containing at least one cell, at least one bead, or both disposed within a cavity of the nanovial, and (i) a plurality of suspended cells, suspended beads, or both, (ii) a plurality of empty nanovials, or (iii) both (i) and (ii); (b) suspending the mixture in a solution; (c) adjusting the density of the solution such that the plurality of nanovials containing at least one cell, at least one bead, or both are disposed within a cavity of a nanovial separate from the plurality of suspended cells, suspended beads, or both of (i), the plurality of empty nanovials of (ii), or both of (i) and (ii); and (d) collecting the plurality of nanovials containing at least one cell, at least one bead, or both disposed within the cavity of the nanovial.

[0155] In some embodiments, the preparing step includes adjusting the density of the solution so that a plurality of nanovials containing at least one cell, at least one bead, or both, disposed within the cavities of the nanovials have an effective density greater than the density of the solution (10, 14). In some instances, the density of the solution can be adjusted so that the solution can comprise a density greater than the density of an empty nanovial (10). Alternatively, the density of the solution can be adjusted so that the density of the solution can be less than the density of an empty nanovial (14). In some instances, the preparing step includes adjusting the density of the solution so that a plurality of nanovials containing at least one cell, at least one bead, or both, disposed within the cavities of the nanovials have an effective density less than the density of the solution (12, 16). In some instances, the density of the solution can be adjusted so that the solution can comprise a density greater than the density of an empty nanovial (16). Alternatively, the density of the solution can be adjusted so that the density of the solution can be less than the density of an empty nanovial (12). In some cases, the method of concentrating nanovials may include performing steps (a)-(d) one or more times to further concentrate a plurality of nanovials containing at least one cell, at least one bead, or both, disposed within the cavity of the nanovial. In some examples, the density of the solution may include a different density in each round of steps (a)-(d). In some examples, the method of concentrating nanovials may further include, prior to step (a), removing (i) the plurality of floating cells, floating beads, or both, or (ii) the plurality of empty nanovials.

[0156] The density of the solution may be adjusted using biocompatible water-soluble additives, such as Ficoll, Percoll, Optiprep, glycerol, PEG, Dextran, and hyaluronic acid. Additionally or alternatively, the density of the cells may be adjusted, for example, by adjusting the osmolarity of the solution. For example, the use of a hypotonic solution (compared to normal physiological phosphate-buffered saline) allows the cells to swell, reducing their density. Additionally or alternatively, the density of the nanovials may be adjusted. For example, the density of the nanovials can be increased by increasing the concentration of PEG used in the precursor solution, increasing crosslink density by using lower molecular weight PEG molecules (e.g., 5 kD 4-arm PEG), or embedding or attaching higher density microparticles or nanoparticles (e.g., SiO2 microparticles / nanoparticles, iron oxide microparticles / nanoparticles, etc.) within or on the nanovial matrix. Similarly, the density of the nanovials can be reduced by, for example, decreasing the concentration of the PEG precursor solution, increasing the molecular weight of the precursor polymer, or embedding or attaching lower-density microparticles / nanoparticles (e.g., glass bubble particles for buoyancy-activated cell sorting). Alternatively, cells attached to the nanovials may be labeled with particles such as glass, polymer, iron oxide, or metal particles (e.g., 100 nm to approximately 3 μm in diameter) containing cell-specific affinity tags to adjust the effective density of cell-containing nanovials compared to cell-free nanovials. The effective density of the nanovials relative to the cells may be adjusted by using higher (or lower) density additives that are impermeable to cell membranes but can diffuse into the nanovial matrix.

[0157] Figure 5 depicts a non-limiting example of buoyancy concentration of nanovials as provided herein. Figure 5 outlines the general principle for the enrichment of various cell and nanovial subpopulations utilizing density matching and buoyancy. In this example, a concentrated solution of defined molecular weight and density characteristics is added to a sample of nanovials, cells, and / or nanovials with bound cells (top). By adjusting the density of the nanovials and concentrated solution relative to the cells, nanovials with cells can be enriched relative to empty nanovials (10, 12). In other embodiments, nanovials with cells are enriched relative to free cells (14, 16). In other embodiments, multiple enrichment steps are performed sequentially to further enrich the subpopulation of interest.

[0158] Methods for enhanced capture of biomolecules in nanovial cavities In some embodiments, the present disclosure provides a method for preventing a biological agent from diffusing from a cavity of a nanovial. In some cases, the method for preventing a biological agent from diffusing from a cavity of a nanovial can include the following steps: (a) providing or obtaining a nanovial, the nanovial including a cavity formed therein and including an opening to the surface of the nanovial, and one or more biological agents disposed within the cavity; and (b) adding blocking particles such that the blocking particles interact with the opening of the cavity and substantially block or reduce the size of the opening of the cavity, thereby preventing the biological agent from diffusing from the cavity when the nanovial is placed in a fluid. In some cases, the method using blocking particles as described herein can be used as an alternative to the emulsion or encapsulation processes described throughout.

[0159] In some embodiments, the maximum diameter of the blocking particle may include a diameter larger than the opening of the cavity of the nanovial. In some examples, the shape of the blocking particle may be spherical or substantially spherical. In some embodiments, the blocking particle may contact the opening of the cavity of the nanovial. In some cases, the blocking particle may surround the opening of the cavity of the nanovial. In some examples, the blocking particle may be sized to maintain more than 50% of the volume of the cavity after step (b).

[0160] The blocking particles can have an average diameter ranging from about 15 to about 100 micrometers, depending on the size of the opening of the nanovial cavity. The blocking particles may have an average diameter of at least about 15 μm, at least about 20 μm, at least about 25 μm, at least about 30 μm, at least about 35 μm, at least about 40 μm, at least about 45 μm, at least about 50 μm, at least about 55 μm, at least about 60 μm, at least about 65 μm, at least about 70 μm, at least about 75 μm, at least about 80 μm, at least about 85 μm, at least about 90 μm, at least about 95 μm, or at least about 100 μm. The blocking particles may have an average diameter of at most about 15 μm, at most about 20 μm, at most about 25 μm, at most about 30 μm, at most about 35 μm, at most about 40 μm, at most about 45 μm, at most about 50 μm, at most about 55 μm, at most about 60 μm, at most about 65 μm, at most about 70 μm, at most about 75 μm, at most about 80 μm, at most about 85 μm, at most about 90 μm, at most about 95 μm, or at most about 100 μm. The blocking particles may have an average diameter of about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or 100 μm. In a preferred embodiment, the blocking particles may comprise an average diameter of about 20 micrometers to about 50 micrometers.

[0161] In some embodiments, the blocking particles may comprise a polymer hydrogel. In some cases, the hydrogel may comprise polyethylene glycol (PEG). In some examples, the plurality of blocking particles may be comprised of a polymer selected from the group consisting of PEG, polystyrene, polymethylmethacrylate, glass, and metal.

[0162] In some embodiments, the blocking particles may have binding moieties for biomolecules released from cells contained within the nanovial, for example, to capture excess molecules and prevent crosstalk to adjacent nanovials. In some instances, the blocking particles may be coated with adhesion molecules that bind to the nanovial. In some instances, the blocking particles may be coated with one or more affinity agents. In some instances, the one or more affinity agents may comprise antibody capture moieties. In some instances, the antibody capture moieties may be selected from the group consisting of anti-IgG antibodies, anti-Fc antibodies or fragments thereof, protein A, and protein G. In some instances, the one or more affinity agents may comprise antigens. In some instances, the one or more affinity agents may comprise nucleic acids. In some instances, the nucleic acids may comprise oligonucleotide tags or oligonucleotide barcodes, as seen in the beads of Figures 12B and 19B.

[0163] In some embodiments, the blocking particle and / or nanovial can include a mesh-like structure (122). In some cases, the mesh-like structure can include an average pore size. In some instances, the average pore size can be sized to retain biological agents having a desired size within the cavity and allow biological agents having a size smaller than the desired size to diffuse through the blocking particle and / or nanovial. In some instances, the average pore size can be sized to allow the transport of fluids and reagents into the cavity of the nanovial. In some instances, the fluids and reagents can be selected from the group consisting of stains, surfactants, detergents, lysis buffers, drugs, cytokines, chemokines, primers, polymerases, culture media, conditioned media, and buffers. In some instances, the average pore size can be sized to retain oligonucleotides, mRNA, proteins, or genomic DNA.

[0164] In some embodiments, step (b) can include adding a plurality of blocking particles (124), such that the plurality of blocking particles interact with the opening of the cavity and substantially block or reduce the size of the opening, as seen in FIG. 17. In some instances, each of the plurality of blocking particles can include an average diameter of about 2 micrometers to about 20 micrometers. In some instances, each of the plurality of blocking particles can include a density greater than that of saline at room temperature. In some instances, the number of blocking particles to nanovials can be greater than 100:1.

[0165] Methods of using nanovials with multiple instruments and microfluidic devices In some embodiments, the present disclosure provides nanovials having a size, shape, surface chemistry, and / or density that are compatible with and configured for use in a number of different types of instruments designed to analyze particles and / or cells. In some cases, the number of different types of instruments designed to analyze particles and / or cells may be selected from the group consisting of flow cytometers, fluorescence-activated cell sorters, imaging flow cytometers, image-activated cell sorters, Coulter counters, particle counters, microfluidic droplet generators, microwell arrays, microfluidic chips with microvalves, microfluidic SlipChips, optofluidic microdevices, microfluidic droplet generators, and microfluidic channels. In some examples, the size of the nanovials may be less than about 100 μm in diameter (e.g., less than about 95 μm, less than about 80 μm, less than about 75 μm, less than about 70 μm, less than about 65 μm, or less). In some cases, the nanovials include capped reactive functional groups. In some cases, the size of the nanovial can be less than 60 μm (e.g., less than about 55 μm, less than about 50 μm, less than about 45 μm, less than about 40 μm, or less). In some examples, the nanovial can be configured to flow through a glass capillary, cuvette, and / or microfluidic channel. In some cases, the nanovial can be configured to be analyzed by optical, electrical, and / or magnetic excitation. In some cases, the size, surface chemistry, and / or buoyancy of the nanovial can be such that the nanovial can flow rapidly through an instrument without clogging or substantial clogging. In some examples, the nanovial can flow through an instrument at greater than 10 cm / sec, or greater than 1 m / sec, without clogging or substantial clogging. In some cases, the nanovial can be configured to flow through a channel having a diameter greater than 90 μm. In some embodiments, the nanovial is configured to be used with a first instrument designed to analyze particles and / or cells, and a second instrument designed to subsequently analyze particles and / or cells.In some embodiments, the nanovials are configured to be sorted by a fluorescence-activated cell sorter, an image-activated cell sorter, or an optofluidic microdevice and then introduced into a microfluidic droplet generator.

[0166] Nanovial Provided herein is a nanovial for use in any of the methods and systems described herein. As generally used herein, the term "nanovial" refers to a microscale particle having a cavity or space that can be used to hold cells and molecules. The space or cavity is in contact with, communicates with, or opens onto the outer surface of the nanovial. As described herein, the space or cavity may be formed as a subtracted space or cavity in the shape of a sphere, resulting in a final nanovial with a crescent-shaped cross section, such as that illustrated in any one of Figures 5, 6B, 8, 9, 10, 11, 12A, 12B, 14A, 14B, 15A, 16A, 16B, 16C, 16D, 17, or 19A. The inscribed void or cavity intersects the spherical or ellipsoidal envelope at its surface to create a pathway for fluid filling (and also allow access to cells, beads, and other microscopic objects). In one preferred aspect, the void or cavity intersects the spherical or ellipsoidal envelope at a narrow opening (e.g., a small percentage of the actual surface area of ​​the nanovial's spherical or ellipsoidal envelope). In some embodiments, this subarea defined by the opening is <33%, in other embodiments <10%, and in further embodiments, <5% of the nanovial's overall spherical / ellipsoidal envelope or surface area. In some embodiments, the surface of the nanovial may be decorated with one or more reactive or binding moieties to associate affinity agents, capture agents, nucleic acids, peptides, proteins, fluorophores, labels, barcodes, particles, cells, etc. For example, reactive or binding moieties may be formed on the surface of nanovias within the void or cavity. Binding or reactive moieties may include, by way of example, nucleic acids, peptides, cell adhesion peptides, catalysts, enzymes, antibodies, primers, antigens, aptamers, fluorophores, biotin, or biotin / streptavidin complexes. Orthogonal reactive chemistries known to those skilled in the art may also be used for conjugation of reactive or binding moieties to nanovials.Fabrication of nanovials can follow procedures, for example, using the aforementioned microfluidic device design shown in WO2020037214A1, the disclosure of which is incorporated herein by reference in its entirety. Nanovials may also be referred to as drop carrier particles, as disclosed in WO2020037214A1. To fabricate nanovials, a microfluidic droplet generator device is used to form a monodisperse emulsion in an oil phase, whereby the internal dispersed phase contains an aqueous two-phase system. Part of the aqueous two-phase system is a crosslinkable hydrogel precursor, such as poly(ethylene glycol) (PEG) or its derivatives. The other part of the aqueous two-phase system is a polymer, such as dextran or gelatin. The two phases of the aqueous two-phase system then separate into distinct regions within the formed droplets. One component of the two-phase system (i.e., the crosslinkable component) is then crosslinked to form the nanovial. Figures 1 and 2 show phase-separated droplets before and after crosslinking with UV light at various power / intensity and wash settings for 40- and 60-micrometer diameter nanovials, respectively. In a preferred embodiment, the aqueous two-phase system contains a crosslinkable component: PEG or a PEG derivative (e.g., 10 kDa 4-arm PEG-norbornene) (using a crosslinker) and uncrosslinked dextran (e.g., 40 kDa). A microfluidic droplet generator device is used to generate an emulsion of the aqueous two-phase system within the oil phase. Droplets containing the two aqueous phase components (e.g., PEG and dextran) separate after droplet formation in a phase separation operation. After phase separation, the PEG or PEG derivative component is crosslinked to form a gel. For example, a crosslinker such as dithiothreitol (DTT) in the presence of a photoinitiator (e.g., Irgacure® 2959, LAP, etc.) within the PEG or PEG derivative component is then exposed to light (e.g., UV excitation) to initiate crosslinking. Of course, other cross-linkers can also be used, such as cysteine-containing peptides or other dithiols or multi-arm cross-linkers. In related embodiments, the PEG and / or either the polymer phase can contain a combination of one or both of a photoinitiator and a cross-linker.Nanovials can be modified to contain biotin, carboxylic acids, amines, oligonucleotides, and other functional groups known in the art for covalent bioconjugation or noncovalent association reactions through the addition of, for example, biotin-PEG-thiol, carboxylic acid-PEG-thiol, amine-PEG-thiol, oligonucleotide-thiol, etc., during the manufacturing process. PEG attached to a functional group can be used to increase the accessibility of the functional group on the nanovial surface. The nanovial can then be conjugated with additional chemistry, for example, through the attachment of streptavidin to a biotin-conjugated nanovial. This can be useful for fluorescently visualizing the nanovial (Figure 3) and / or for binding affinity or cell-capture agents as described herein. After crosslinking, the nanovial can be washed to remove the oil and dextran phases. In some embodiments, the nanovial contains a crosslinked hydrogel. The crosslinked hydrogel can be a PEG-based crosslinked hydrogel. In some embodiments, the cavity of the nanovial contains an aqueous fluid disposed therein. In some embodiments, the nanovials are suspended in an oil phase. In some embodiments, the nanovial cavity has a volume of about 100 fL to about 10 nL. In some embodiments, the nanovial cavity has a linear dimension of about 5 μm to about 250 μm. In embodiments using nanovials with an aqueous solution suspended in an oil phase (e.g., emulsified) as described herein, the formation of an emulsion containing the nanovials is achieved by combining a suspension of the nanovials in the aqueous phase with oil (and optional surfactant) and mixing (e.g., by vortexing, pipetting, etc.) as described in WO2020037214A1. The hydrodynamic shear from the stirring and mixing produces an emulsion of decreasing size. After continued stirring, the nanovials contained within the droplets act as size inhibitors, preventing further droplet shrinkage. Both pipetting and vortexing can be used to encapsulate the nanovials in the oil phase. Both pipetting and vortexing can be used to encapsulate the nanovials in the oil phase.In one embodiment of a uniform nanovial emulsion, substantially all of the nanovial-containing droplets (e.g., >95%, >99%, or >99.9%) are each associated with a single nanovial. Due to their unique size range, the nanovials can be easily filtered from surrounding smaller satellite droplets (e.g., background droplets generated during emulsification) using standard filtration techniques.

[0167] system In some embodiments, aspects of the present disclosure provide systems and software for analyzing and classifying nanovials. In some cases, the systems and software may include an integrated instrument (e.g., a flow cytometer, a fluorescence-activated cell sorter, or any combination thereof) capable of analyzing and classifying nanovials. In some cases, the systems may include a scanner or reader for reading and analyzing a barcode or QR code present on the nanovial container or packaging. In some cases, the barcode or QR code may include information about the size, shape, material, density, or any combination thereof, of the nanovial. In some cases, the barcode or QR code may provide settings for the integrated instrument (e.g., a flow cytometer, a fluorescence-activated cell sorter, or any combination thereof) to optimize the analysis of the nanovials. In some embodiments, the scanner or reader capable of reading and analyzing the barcode or QR code may include a smartphone. In some cases, the smartphone may include an app capable of communicating with the integrated instrument via radio frequency, Bluetooth, Wi-Fi, or any combination thereof.

[0168] In some embodiments, the integrated instrument may include machine-readable instructions or software contained on the integrated instrument's physical or cloud-based memory or server. The software may include machine-readable instructions configured to perform analyses specific to the nanovial assay obtained by scanning the barcode or QR code described above. In some cases, the software may provide machine-executable instructions for performing automated gating of nanovial events. The software may include machine-executable instructions for performing automated gating of nanovials containing cells based on scattering and fluorescence intensity data. The software may analyze the collected nanovial data and generate a graphical interpretation thereof in the form of a histogram and / or a dimension-reduced plot (e.g., visualization via t-distributed stochastic neighbor embedding, ViSNE plot, or a combination thereof).

[0169] In some embodiments, the integrated device can be connected to the Internet or a network. In some examples, the software can upload nanovial analysis data to a server in the cloud or retrieve data from a server in the cloud. In some cases, the software can perform offline calculations in the cloud (e.g., cloud computing). In some cases, the integrated device can connect to a server and immediately update device settings. In some cases, one or more users may upload device settings to the cloud, which can be accessed via the Internet by one or more users performing nanovial analysis. In some examples, the network-connected integrated device can report geographic or other location based on network information where a barcode or QR code is scanned and store this information in anonymized form on a local or cloud-based server.

[0170] Unless otherwise defined, all terminology, notations, and other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from that commonly understood in the art.

[0171] Throughout this application, various embodiments may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges, as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6, as well as individual numerical values ​​within the range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the broadness of the range.

[0172] As used in this specification and claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "sample" includes multiple samples, including mixtures thereof.

[0173] "Determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present (e.g., detecting). Such terms can include quantitative, qualitative, or quantitative and qualitative determinations. Evaluating can be relative or absolute. "Detecting the presence of" can include determining the amount of something present, in addition to determining whether something is present or absent, depending on the context.

[0174] The terms "subject," "individual," or "patient" are often used interchangeably herein. A "subject" can be a biological entity containing expressed genetic material. The biological entity can be, for example, a plant, animal, or microorganism, including bacteria, viruses, fungi, and protozoa. A subject can be tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. A mammal can be a human. A subject can be diagnosed or suspected of being at high risk for a disease. In some cases, a subject is not necessarily diagnosed or suspected of being at high risk for a disease.

[0175] The term "antibody," as used herein, describes various naturally occurring immunoglobulins as well as the immunoglobulin class of scrubs, which are engineered immunoglobulins or immunoglobulin fragments such as bispecific antibodies, BiTE antibodies, DARTs, FAb fragments, minibodies, nanobodies, multivalent nanobodies, half-life extended nanobodies, scFvs, heavy chain antibodies, affibodies, etc. The term "antibody" refers to immunoglobulins and engineered immunoglobulins from human, mouse, llama, rabbit, goat, rat, chicken, monkey, donkey, etc., as well as humanized mouse or other species.

[0176] The term "ex vivo" is used to describe events that occur outside of a subject's body. Ex vivo assays are not performed on a subject. Rather, the assay is performed on a sample that is separate from the subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0177] The term "in vitro" is used to describe events that occur within a container for holding a laboratory reagent such that the reagent is separated from the biological source from which the substance is obtained. In vitro assays can include cell-based assays in which live or dead cells are used. In vitro assays can also include cell-free assays in which no intact cells are used.

[0178] As used herein, a number followed by the term "about" refers to a number that is plus or minus 10% of that number. A range followed by the term "about" refers to a range of minus 10% from the lowest value to plus 10% from the highest value.

[0179] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initial," "next," "then," "before," "after," "finally," and "finally," is not intended to limit the scope of the embodiments disclosed herein, but is exemplary.

[0180] Any systems, methods, software, compositions, and platforms described herein are modular and not limited to sequential processes, and thus terms such as "first" and "second" do not necessarily imply a priority, order of importance, or order of operation.

[0181] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0182] Numbered Embodiments The following embodiments detail non-limiting permutations of feature combinations disclosed herein. Other permutations of feature combinations are also contemplated. In particular, each of these numbered embodiments is contemplated as dependent or related to the preceding or following numbered embodiment, regardless of the order in which they are listed.

[0183] Embodiment 1: A method for identifying an antibody or fragment thereof that binds to an antigen of interest, comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading antibody-producing cells within the cavity of the nanovial; (c) incubating the nanovial such that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent; and (d) adding a detection agent to the cavity of the nanovial such that the detection agent binds to the one or more antibodies or fragments thereof. (e) detecting one or more signals relating to binding of a detection agent to the one or more antibodies or fragments thereof; (f) performing a sequencing assay on nucleic acid derived from the antibody-producing cells, thereby generating sequences of the one or more antibodies or fragments thereof; (g) correlating the sequences of the one or more antibodies or fragments thereof with the one or more signals relating to binding of a detection agent to the one or more antibodies or fragments thereof; and (h) identifying the one or more antibodies or fragments thereof that bind to the antigen of interest based on the correlating step of (g).

[0184] Embodiment 2: The method of embodiment 1, further comprising, prior to (f), sorting the nanovials based on one or more signals related to binding of the detection agent to the antibody.

[0185] Embodiment 3: The method of embodiment 1 or 2, wherein the cavity of the nanovial further comprises one or more cell-trapping agents immobilized thereto.

[0186] Embodiment 4: The method of embodiment 3, wherein the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of the antibody-producing cell.

[0187] Embodiment 5: The method of embodiment 3, wherein the one or more cell capture agents bind to a label present on the surface of the antibody-producing cells.

[0188] Embodiment 6: The method of embodiment 5, wherein the label is streptavidin or biotin.

[0189] Example 7: The method of embodiment 3, wherein the one or more cell capture agents is an antigen to which antibody-producing cells can bind.

[0190] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the affinity agent is an antibody capture moiety.

[0191] Embodiment 9: The method of embodiment 8, wherein the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, Protein A, and Protein G.

[0192] Embodiment 10: The method of any one of embodiments 1 to 7, wherein the affinity agent is an antigen of interest.

[0193] Embodiment 11: The method of any one of embodiments 1 to 10, wherein the affinity agent is an antigen-presenting cell that expresses the antigen of interest on its surface.

[0194] Embodiment 12: The method of any one of embodiments 1 to 11, wherein the detection agent is an antigen of interest.

[0195] Embodiment 13: The method of any one of embodiments 1 to 12, wherein the detection agent is an antibody or a fragment thereof.

[0196] Embodiment 14: The method of any one of embodiments 1 to 13, wherein the detection agent is directly or indirectly labeled with a detectable label.

[0197] Embodiment 15: The method of embodiment 14, wherein the detectable label is selected from the group consisting of a fluorescent label, a protein affinity tag, an oligonucleotide tag, and a magnetic particle.

[0198] Embodiment 16: The method of embodiment 15, wherein the detectable label is a fluorescent label.

[0199] Embodiment 17: The method of embodiment 16, wherein the detecting step (e) comprises determining the amount of detection agent bound to the one or more antibodies or fragments thereof.

[0200] Embodiment 18: The method of embodiment 17, wherein the amount of detection agent bound to the one or more antibodies or fragments thereof corresponds to the level of fluorescence.

[0201] Embodiment 19: The method of any one of embodiments 16 to 18, further comprising, prior to (f), sorting the nanovials based on the level of fluorescence above background levels.

[0202] Embodiment 20: The method of embodiment 19, wherein the classifying step comprises implementing a flow-based classification method.

[0203] Embodiment 21: The method of embodiment 20, wherein the flow-based classification method is fluorescence-activated cell sorting (FACS).

[0204] Embodiment 22: The method of embodiment 15, wherein the detectable label is an oligonucleotide tag.

[0205] Embodiment 23: The method of embodiment 22, further comprising sequencing the oligonucleotide tag.

[0206] Embodiment 24: The method of embodiment 23, wherein the detecting step (e) comprises counting the number of oligonucleotide tags bound to the one or more antibodies or fragments thereof.

[0207] Embodiment 25: The method of any one of embodiments 1 to 24, further comprising, prior to (f), a step of lysing antibody-producing cells.

[0208] Embodiment 26: The method of embodiment 25, further comprising reverse transcribing mRNA released from the antibody-producing cells after lysis to produce cDNA.

[0209] Embodiment 27: The method of embodiment 26, further comprising sequencing the cDNA.

[0210] Embodiment 28: The method of any one of embodiments 1 to 27, wherein the sequencing assay comprises single-cell RNA sequencing.

[0211] Embodiment 29: The method of any one of embodiments 1 to 28, further comprising, prior to (c), encapsulating the nanovials within the droplet.

[0212] Embodiment 30: The method of any one of embodiments 1 to 28, wherein the cavity of the nanovial comprises an opening to the surface of the nanovial.

[0213] Embodiment 31: The method of embodiment 30, further comprising, prior to (c), adding one or more blocking particles to block the openings or reduce the size of the openings.

[0214] Embodiment 32: The method of any one of embodiments 1 to 31, further comprising, prior to (d), washing the nanovial.

[0215] Embodiment 33: The method comprises: immunizing an antigen of interest with a desired dissociation constant (K D33. The method of any one of embodiments 1 to 32, wherein the method is configured to identify one or more antibodies or fragments thereof having:

[0216] Embodiment 34: The one or more antibodies or fragments thereof have a desired K of less than about 1 μM, less than about 100 nM, less than about 10 nM, or less than 1 nM for the antigen of interest. D 34. The method of embodiment 33, comprising:

[0217] Embodiment 35: The method of embodiment 33 or 34, further comprising adding a detection agent to the plurality of nanovials at one or more different concentrations.

[0218] Embodiment 36: One or more different concentrations are used to achieve a desired K D 36. The method of embodiment 35, wherein the α-amino acid sequence is within an order of magnitude of

[0219] Embodiment 37: The method of any one of embodiments 1 to 32, wherein the method is configured to identify one or more antibodies or fragments thereof with a desired specificity for an antigen of interest.

[0220] Embodiment 38: The method of embodiment 37, further comprising adding a plurality of different detection agents to the plurality of nanovials, wherein at least one of the different detection agents comprises a detectable label.

[0221] Embodiment 39: The method of embodiment 38, further comprising identifying one or more antibodies or fragments thereof that specifically bind to one of the different detection agents based on measurement of the detectable label.

[0222] Embodiment 40: The method of any one of embodiments 3 to 39, wherein the detection agent is an antigen of interest.

[0223] Embodiment 41: A method for identifying one or more antibodies or fragments thereof that modulate a signal transduction pathway of interest, comprising: (a) providing or obtaining a nanovial, wherein the nanovial comprises a cavity formed therein; (b) loading antibody-producing cells and antigen-producing cells within the cavity of the nanovial; (c) incubating the nanovial such that the one or more antibodies or fragments thereof are secreted from the antibody-producing cells and modulate the signal transduction pathway of interest in the antigen-producing cells; (d) detecting one or more signals related to modulation of the signal transduction pathway of interest; (e) performing a sequencing assay on nucleic acids derived from the antibody-producing cells, thereby generating sequences of the one or more antibodies or fragments thereof; (f) associating the sequences of the one or more antibodies or fragments thereof with one or more signals related to modulation of the signal transduction pathway of interest; and (g) identifying one or more antibodies or fragments thereof that modulate the signal transduction pathway of interest based on the associating step of (f).

[0224] Embodiment 42: The method of embodiment 41, further comprising, prior to (e), sorting the nanovials based on one or more signals related to modulation of a signaling pathway of interest.

[0225] Embodiment 43: The method of embodiment 41 or 42, wherein the modulation of the signaling pathway of interest comprises activation of the signaling pathway of interest.

[0226] Embodiment 44: The method of embodiment 41 or 42, wherein modulating the signaling pathway of interest comprises inhibiting the signaling pathway of interest.

[0227] Embodiment 45: The method of any one of embodiments 41 to 44, wherein the one or more antibodies or fragments thereof modulate a signal transduction pathway of interest by binding to an antigen on the surface of an antigen-producing cell.

[0228] Embodiment 46: The method of any one of embodiments 41 to 44, wherein the one or more antibodies or fragments thereof modulate a signal transduction pathway of interest by interfering with binding of a ligand to an antigen on the surface of an antigen-producing cell.

[0229] Embodiment 47: The method of any one of embodiments 41 to 46, wherein the cavity of the nanovial further comprises one or more cell-trapping agents immobilized thereon.

[0230] Embodiment 48: The method of embodiment 47, wherein the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of antibody-producing cells, antigen-producing cells, or both.

[0231] Embodiment 49: The method of embodiment 47, wherein the one or more cell capture agents bind to a label present on the surface of antibody-producing cells, antigen-producing cells, or both.

[0232] Embodiment 50: The method of embodiment 47, wherein the one or more cell capture agents are antigens that can be bound by antibody-producing cells, antigen-producing cells, or both.

[0233] Embodiment 51: The method of any one of embodiments 41 to 50, wherein the antigen-producing cells are engineered to express one or more detectable labels upon modulation of a signaling pathway of interest.

[0234] Embodiment 52: The method of embodiment 51, wherein the one or more detectable labels comprise a fluorescent reporter protein.

[0235] Embodiment 53: The method of embodiment 52, wherein the detecting step (d) comprises detecting a level of fluorescence in the nanovial, wherein the level of fluorescence corresponds to modulation of the signal transduction pathway of interest.

[0236] Embodiment 54: The method of embodiment 53, further comprising, prior to (e), sorting the nanovials based on the level of fluorescence above background levels.

[0237] Embodiment 55: The method of embodiment 54, wherein the classifying step comprises implementing a flow-based classification method.

[0238] Embodiment 56: The method of embodiment 55, wherein the flow-based classification method is fluorescence-activated cell sorting (FACS).

[0239] Embodiment 57: The method of any one of embodiments 41 to 56, wherein the detecting step (d) comprises detecting changes in one or more mRNA levels in the antigen-producing cells.

[0240] Embodiment 58: The method of embodiment 57, wherein detecting comprises performing single-cell RNA sequencing on nucleic acids derived from the antigen-producing cells.

[0241] Embodiment 59: The method of embodiment 58, wherein the associating step (f) comprises linking mRNA levels of antigen-producing cells with nucleic acid sequence information from antibody-producing cells based on shared oligonucleotide barcodes.

[0242] Embodiment 60: The method of embodiment 59, wherein the shared oligonucleotide barcode comprises one or more unique oligonucleotide tags associated with the nanovial.

[0243] Embodiment 61: The method of any one of embodiments 41 to 60, further comprising, before (e), a step of lysing antibody-producing cells and antigen-producing cells.

[0244] Embodiment 62: The method of embodiment 61, further comprising reverse transcribing mRNA released from the antibody-producing cells after lysis to produce cDNA.

[0245] Embodiment 63: The method of embodiment 62, further comprising sequencing the cDNA.

[0246] Embodiment 64: The method of any one of embodiments 41 to 63, wherein the sequencing assay comprises single-cell RNA sequencing.

[0247] Embodiment 65: The method of any one of embodiments 41 to 64, further comprising, prior to (c), encapsulating the nanovials within the droplet.

[0248] Embodiment 66: The method of any one of embodiments 41 to 64, wherein the cavity of the nanovial comprises an opening to the surface of the nanovial.

[0249] Embodiment 67: The method of embodiment 66, further comprising, prior to (c), adding one or more blocking particles to block the openings or reduce the size of the openings.

[0250] Embodiment 68: The method of any one of embodiments 41 to 67, further comprising, prior to (d), washing the nanovial.

[0251] Embodiment 69: The method of any one of embodiments 1 to 68, wherein the antibody-producing cells are selected from the group consisting of B cells, plasmablasts, plasma cells, hybridomas, and engineered producer cells.

[0252] Embodiment 70: The method of any one of embodiments 1 to 69, wherein the nanovial contains a single antibody-producing cell.

[0253] Embodiment 71: The method of any one of embodiments 1 to 70, further comprising carrying out the method on a plurality of nanovials, each of the nanovials containing an individual antibody-producing cell within the cavity of the nanovial.

[0254] Embodiment 72: The method of embodiment 71, wherein the plurality of nanovials comprises at least 20,000 nanovials, at least 100,000 nanovials, or at least 500,000 nanovials.

[0255] Embodiment 73: The method of embodiment 71 or 72, wherein each individual antibody-producing cell produces a different antibody.

[0256] Embodiment 74: The method of any one of embodiments 1 to 73, wherein the nanovial comprises a crosslinked hydrogel.

[0257] Embodiment 75: The method of embodiment 74, wherein the crosslinked hydrogel is a PEG-based crosslinked hydrogel.

[0258] Embodiment 76: The method of any one of embodiments 1 to 75, wherein the cavity of the nanovial comprises a liquid disposed therein.

[0259] Embodiment 77: The method of any one of embodiments 1 to 76, wherein the nanovials are suspended in an oil phase.

[0260] Embodiment 78: The method of any one of embodiments 1 to 77, wherein the cavity of the nanovial has a volume of about 100 fL to 10 nL.

[0261] Embodiment 79: The method of any one of embodiments 1 to 78, wherein the cavity of the nanovial has a linear dimension of about 5 μm to 250 μm.

[0262] Embodiment 80: A method for linking functional single-cell information with genomic, transcriptomic, and / or proteomic information, comprising: (a) performing a functional assay on a single cell located in a cavity of a nanovial, wherein the functional assay is associated with a first barcode or a first label; (b) performing a genomic, transcriptomic, and / or proteomic assay on the single cell in the nanovial, wherein the genomic, transcriptomic, and / or proteomic assay is associated with a second barcode or a second label; and (c) linking the functional single-cell information with the genomic, transcriptomic, and / or proteomic information by associating the first barcode or first label with the second barcode or second label.

[0263] Embodiment 81: The method of embodiment 80, further comprising, prior to (b), sorting the nanovials based on a signal generated by the functional assay.

[0264] Embodiment 82: The method of embodiment 80 or 81, wherein the functional assay is an antibody secretion screening assay.

[0265] Embodiment 83: The method of embodiment 80 or 81, wherein the functional assay is an antibody affinity assay.

[0266] Embodiment 84: The method of embodiment 80 or 81, wherein the functional assay is an antibody specificity assay.

[0267] Embodiment 85: The method of embodiment 80 or 81, wherein the genomic assay is a single-cell RNA sequencing assay.

[0268] Embodiment 86: The method of any one of embodiments 80 to 85, wherein the nanovials are associated with unique oligonucleotide tags.

[0269] Embodiment 87: The method of embodiment 86, wherein the unique oligonucleotide tag comprises a poly-dT capture region.

[0270] Embodiment 88: The method of any one of embodiments 80 to 87, wherein the first barcode or first label comprises an oligonucleotide having a polyA tract.

[0271] Embodiment 89: A method of preventing diffusion of a biological agent from a cavity of a nanovial, the method comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and including an opening to a surface of the nanovial, and one or more biological agents disposed within the cavity; and (b) adding blocking particles such that the blocking particles interact with the opening of the cavity and substantially block the opening of the cavity or reduce the size of the opening, thereby inhibiting diffusion of the biological agent from the cavity when the nanovial is placed in a fluid.

[0272] Embodiment 90: The method of embodiment 89, wherein the maximum diameter of the blocking particles is greater than the diameter of the opening of the cavity.

[0273] Embodiment 91: The method of embodiment 89 or 90, wherein the blocking particles are in contact with the opening of the cavity.

[0274] Embodiment 92: The method of any one of embodiments 89 to 91, wherein the blocking particles surround the opening of the cavity.

[0275] Embodiment 93: The method of any one of embodiments 89 to 92, wherein the blocking particles are sized to maintain more than 50% of the volume of the cavity after (b).

[0276] Embodiment 94: The method of any one of embodiments 89 to 93, wherein the blocking particles have an average diameter of about 20 micrometers to 50 micrometers.

[0277] Embodiment 95: The method of any one of embodiments 89 to 94, wherein the blocking particles are spherical or substantially spherical in shape.

[0278] Embodiment 96: The method of any one of embodiments 89 to 95, wherein the blocking particles comprise a polymer hydrogel.

[0279] Embodiment 97: The method of embodiment 96, wherein the polymer hydrogel is polyethylene glycol (PEG).

[0280] Embodiment 98: The method of any one of embodiments 89 to 97, wherein the blocking particles are coated with one or more affinity agents.

[0281] Embodiment 99: The method of embodiment 98, wherein the one or more affinity agents comprise an antibody capture moiety.

[0282] Embodiment 100: The method of embodiment 99, wherein the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, Protein A, and Protein G.

[0283] Embodiment 101: The method of embodiment 99, wherein the one or more affinity agents comprises an antigen.

[0284] Embodiment 102: The method of embodiment 99, wherein one or more affinity agents comprise a nucleic acid.

[0285] Embodiment 103: The method of embodiment 102, wherein the nucleic acid is an oligonucleotide tag or an oligonucleotide barcode.

[0286] Embodiment 104: The method of any one of embodiments 89 to 103, wherein the nanovials and / or blocking particles comprise a mesh-like structure.

[0287] Embodiment 105: The method of embodiment 104, wherein the mesh-like structure comprises an average pore size.

[0288] Embodiment 106: The method described in embodiment 105, wherein the average pore size is sized so that biological agents having a desired size are retained within the cavities and biological agents having a size smaller than the desired size can diffuse through the mesh-like structure.

[0289] Embodiment 107: The method of embodiment 106, wherein the average pore size is sized to allow transport of fluids and reagents into the cavity of the nanovial.

[0290] Embodiment 108: The method of embodiment 107, wherein the fluids and reagents are selected from the group consisting of stains, surfactants, detergents, lysis buffers, drugs, cytokines, chemokines, culture media, conditioned media, and buffers.

[0291] Embodiment 109: (b) The method of any one of embodiments 89 to 108, comprising adding a plurality of blocking particles such that the blocking particles interact with the opening of the cavity and substantially block the opening of the cavity or reduce the size of the opening.

[0292] Embodiment 110: The method of embodiment 109, wherein each of the plurality of blocking particles has an average diameter of about 2 micrometers to about 20 micrometers.

[0293] Embodiment 111: The method described in embodiment 109 or 110, wherein each of the plurality of blocking particles has a density greater than that of saline at room temperature.

[0294] Embodiment 112: The method of any one of embodiments 109 to 111, wherein the number of blocking particles to nanovials is greater than 100:1.

[0295] Embodiment 113: The method of any one of embodiments 109 to 112, wherein the plurality of blocking particles are composed of a polymer selected from the group consisting of PEG, polystyrene, polymethylmethacrylate, glass, and metal.

[0296] Embodiment 114: A method of concentrating nanovials containing one or more cells, one or more beads, or both, comprising: (a) providing or obtaining a plurality of nanovials, each containing at least one cell, at least one bead, or both disposed within a cavity of the nanovial, and a mixture containing (i) a plurality of suspended cells, suspended beads, or both, (ii) a plurality of empty nanovials, or (iii) both (i) and (ii); (b) suspending the mixture in a solution; (c) adjusting the density of the solution such that the plurality of nanovials containing at least one cell, at least one bead, or both are disposed within a cavity of a nanovial separate from the plurality of suspended cells, suspended beads, or both of (i), the plurality of empty nanovials of (ii), or both of (i) and (ii); and (d) harvesting the plurality of nanovials containing at least one cell, at least one bead, or both disposed within the cavity of the nanovial.

[0297] Embodiment 115: The method of embodiment 114, wherein (c) comprises adjusting the density of the solution such that a plurality of nanovials containing at least one cell, at least one bead, or both, disposed within the cavity of the nanovial have a density greater than the density of the solution.

[0298] Embodiment 116: The method of embodiment 114, wherein (c) comprises adjusting the density of the solution such that a plurality of nanovials containing at least one cell, at least one bead, or both, disposed within the cavity of the nanovial have a density that is lower than the density of the solution.

[0299] Embodiment 117: The method of any one of embodiments 114 to 116, further comprising performing (a) to (d) one or more times to further concentrate a plurality of nanovials containing at least one cell, at least one bead, or both, disposed within the cavity of the nanovial.

[0300] Embodiment 118: The method of embodiment 117, wherein the density of the solution is different in each round of (a) to (d).

[0301] Embodiment 119: The method of any one of embodiments 114 to 118, further comprising, prior to (a), removing (i) the plurality of floating cells, floating beads, or both, or (ii) the plurality of empty nanovials.

[0302] Embodiment 120: A method for identifying a drug or compound that modulates a signal transduction pathway in a cell of interest, the method comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein, the cavity comprising (i) a drug or compound, and (ii) a barcode or label associated with the drug or compound; (b) loading a cell of interest within the cavity of the nanovial; (c) exposing the nanovial to a stimulus to release the drug or compound into a liquid volume within the cavity of the nanovial such that the drug or compound interacts with the cell of interest; (d) incubating the nanovial such that the drug or compound modulates a signal transduction pathway of interest in the cell of interest; (e) detecting one or more signals related to modulation of a signal transduction pathway in the cell of interest; (f) associating a barcode or label with one or more signals related to modulation of the signal transduction pathway of interest; and (g) identifying a drug or compound that modulates the signal transduction pathway of interest based on the associating step of (f).

[0303] Embodiment 121: The method of embodiment 120, further comprising, prior to (f), sorting the nanovials based on one or more signals related to modulation of a signal transduction pathway of interest.

[0304] Embodiment 122: The method of embodiment 120 or 121, wherein the modulation of the signaling pathway of interest comprises activation of the signaling pathway of interest.

[0305] Embodiment 123: The method of embodiment 120 or 121, wherein modulating the signaling pathway of interest comprises inhibiting the signaling pathway of interest.

[0306] Embodiment 124: The method of any one of embodiments 120 to 123, wherein the cavity of the nanovial further comprises one or more cell capture agents immobilized thereon.

[0307] Embodiment 125: The method of embodiment 124, wherein the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of the cells of interest.

[0308] Embodiment 126: The method of embodiment 125, wherein the one or more cell capture agents bind to a label present on the surface of the cells of interest.

[0309] Embodiment 127: The method of any one of embodiments 120 to 126, wherein the cells of interest are engineered to express one or more detectable labels upon modulation of the signaling pathway of interest.

[0310] Embodiment 128: The method of embodiment 127, wherein the one or more detectable labels comprise a fluorescent reporter protein.

[0311] Embodiment 129: The method of embodiment 128, wherein the detecting step (e) comprises detecting a level of fluorescence in the nanovial, wherein the level of fluorescence corresponds to modulation of the signal transduction pathway of interest.

[0312] Embodiment 130: The method of embodiment 129, further comprising, prior to (f), sorting the nanovials based on the level of fluorescence above background levels.

[0313] Embodiment 131: The method described in embodiment 130, wherein the classifying step includes implementing a flow-based classification method.

[0314] Embodiment 132: The method described in embodiment 131, wherein the flow-based classification method is fluorescence-activated cell sorting (FACS).

[0315] Embodiment 133: The method of any one of embodiments 41 to 56, wherein the detecting step (d) comprises detecting a change in one or more mRNA levels in the cells of interest.

[0316] Embodiment 134: The method of embodiment 133, wherein detecting comprises performing single-cell RNA sequencing on nucleic acids derived from the cells of interest.

[0317] Embodiment 135: The method of embodiment 134, wherein the associating step (f) comprises linking the mRNA levels of the cells of interest to a drug or compound based on a shared oligonucleotide barcode.

[0318] Embodiment 136: The method of embodiment 135, wherein the shared oligonucleotide barcode comprises one or more unique oligonucleotide sequences associated with a barcode associated with a drug or compound.

[0319] Embodiment 137: The method of any one of embodiments 120 to 136, further comprising, prior to (e), lysing the cells of interest.

[0320] Embodiment 138: The method of embodiment 137, further comprising reverse transcribing the mRNA released from the cells of interest after lysis to produce cDNA.

[0321] Embodiment 139: The method of embodiment 138, further comprising sequencing the cDNA.

[0322] Embodiment 140: The method described in any one of embodiments 120 to 139, wherein the sequencing assay comprises single-cell RNA sequencing.

[0323] Embodiment 141: The method of any one of embodiments 120 to 140, further comprising, prior to (c), encapsulating the nanovials within the droplet.

[0324] Embodiment 142: A method of identifying a functional chimeric antigen receptor (CAR) or T cell receptor (TCR) for an antigen of interest, comprising: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading a CAR-expressing cell or a TCR-expressing cell within the cavity of the nanovial; (c) incubating the nanovial such that one or more cytokines are secreted from the CAR-expressing cell or the TCR-expressing cell and bind to the affinity agent; and (d) a detection agent binds to the one or more cytokines. (e) detecting one or more signals relating to binding of the detection agent to one or more cytokines; (f) performing a sequencing assay on nucleic acid derived from the CAR-expressing cell or TCR-expressing cell, thereby generating a sequence of the CAR or TCR; (g) associating the sequence of the CAR or TCR with the one or more signals relating to binding of the detection agent to one or more cytokines; and (h) identifying a CAR or TCR that functionally interacts with the antigen of interest based on the associating step of (g).

[0325] Embodiment 143: The method of embodiment 142, further comprising, prior to (f), sorting the nanovials based on one or more signals related to binding of the detection agent to one or more cytokines.

[0326] Embodiment 144: The method of embodiment 142 or 143, wherein the cavity of the nanovial further comprises one or more cell capture agents immobilized thereon.

[0327] Embodiment 145: The method of embodiment 144, wherein the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of a CAR-expressing cell or a TCR-expressing cell.

[0328] Embodiment 146: The method of embodiment 144, wherein the one or more cell capture agents bind to a label present on the surface of the CAR-expressing or TCR-expressing cell.

[0329] Embodiment 147: The method of embodiment 146, wherein the label is streptavidin or biotin.

[0330] Embodiment 148: The method of embodiment 144, wherein the one or more cell capture agents are antigens or MHC-presented antigens to which CAR-expressing or TCR-expressing cells can bind.

[0331] Embodiment 149: The method of embodiment 144, wherein one or more cell capture agents are antigen-presenting cells that express the antigen of interest on their surface or present the antigen in an MHC complex.

[0332] Embodiment 150: The method of any one of embodiments 142 to 149, wherein the affinity agent is an antibody capture moiety.

[0333] Embodiment 151: The method of embodiment 150, wherein the antibody capture moiety is one or more selected from the group consisting of anti-IL2, anti-TNF-α, and anti-IFN-γ.

[0334] Embodiment 152: The method of any one of embodiments 142 to 151, wherein the detection agent is a secondary antibody or antibody fragment specific for one or more cytokines.

[0335] Embodiment 153: The method of any one of embodiments 142 to 152, wherein the detection agent is directly or indirectly labeled with a detectable label.

[0336] Embodiment 154: The method of embodiment 153, wherein the detectable label is selected from the group consisting of a fluorescent label, an oligonucleotide tag, and a magnetic particle.

[0337] Embodiment 155: The method of embodiment 154, wherein the detectable label is a fluorescent label.

[0338] Embodiment 156: The method of embodiment 155, wherein the detecting step (e) comprises determining the amount of detection agent bound to the one or more cytokines.

[0339] Embodiment 157: The method of embodiment 156, wherein the amount of detection agent bound to the one or more cytokines corresponds to the level of fluorescence.

[0340] Embodiment 158: The method of any one of embodiments 155 to 157, further comprising, prior to (f), sorting the nanovials based on the level of fluorescence above background levels.

[0341] Embodiment 159: The method described in embodiment 158, wherein the classifying step includes implementing a flow-based classification method.

[0342] Embodiment 160: The method described in embodiment 159, wherein the flow-based classification method is fluorescence-activated cell sorting (FACS).

[0343] Embodiment 161: The method of embodiment 153, wherein the detectable label is an oligonucleotide tag.

[0344] Embodiment 162: The method of embodiment 161, further comprising sequencing the oligonucleotide tag.

[0345] Embodiment 163: The method of embodiment 162, wherein the detecting step (e) comprises counting the number of oligonucleotide tags bound to the one or more cytokines.

[0346] Embodiment 164: The method of any one of embodiments 142 to 163, further comprising, prior to (f), lysing the CAR-expressing cells or TCR-expressing cells.

[0347] Embodiment 165: The method of embodiment 164, further comprising reverse transcribing the mRNA released from the CAR-expressing or TCR-expressing cells after lysis to produce cDNA.

[0348] Embodiment 166: The method of embodiment 165, further comprising sequencing the cDNA.

[0349] Embodiment 167: A method according to any one of embodiments 142 to 166, wherein the sequencing assay comprises single-cell RNA sequencing.

[0350] Embodiment 168: The method of any one of embodiments 142 to 167, further comprising, prior to (c), encapsulating the nanovials within the droplet.

[0351] Embodiment 169: The method of any one of embodiments 142 to 167, wherein the cavity of the nanovial comprises an opening to the surface of the nanovial.

[0352] Embodiment 170: The method of embodiment 169, further comprising, prior to (c), adding one or more blocking particles to block the openings or reduce the size of the openings.

[0353] Embodiment 171: The method of any one of embodiments 142 to 170, further comprising, prior to (d), washing the nanovial.

[0354] Embodiment 172: A nanovial having a size, shape, surface chemistry, density, or any combination thereof, adapted to fit into and be used with a number of different types of instruments designed to analyze particles and / or cells.

[0355] Embodiment 173: The nanovial described in embodiment 172, wherein the plurality of different types of instruments designed to analyze particles and / or cells are selected from the group consisting of flow cytometers, fluorescence-activated cell sorters, imaging flow cytometers, image-activated cell sorters, Coulter counters, particle counters, microfluidic droplet generators, microwell arrays, microfluidic chips with microvalves, microfluidic SlipChips, optofluidic microdevices, microfluidic droplet generators, and microfluidic channels.

[0356] Embodiment 174: The nanovial of embodiment 172 or 173, wherein the size of the nanovial is less than about 100 μm in diameter and comprises a capped reactive functional group.

[0357] Embodiment 175: The nanovial of any one of embodiments 172 to 174, wherein the size of the nanovial is less than about 60 μm in diameter.

[0358] Embodiment 176: A nanovial described in any one of embodiments 172 to 175, wherein the nanovial is configured to be flowed through a glass capillary, cuvette, and / or microfluidic channel.

[0359] Embodiment 177: A nanovial according to any one of embodiments 172 to 176, wherein the nanovial is configured to be analyzed by optical, electrical, and / or magnetic excitation.

[0360] Embodiment 178: A nanovial described in any one of embodiments 172 to 177, wherein the size, surface chemistry, and / or buoyancy of the nanovial are such that the nanovial can flow rapidly through the device without clogging or substantial clogging.

[0361] Embodiment 179: The nanovial of embodiment 178, wherein the nanovial can flow through the device at greater than 10 cm / sec, or greater than 1 m / sec, without clogging or substantially without clogging.

[0362] Embodiment 180: A nanovial according to any one of embodiments 172 to 179, wherein the nanovial is configured to be flowed through a channel having a diameter greater than 90 μm.

[0363] Embodiment 181: A nanovial described in any one of embodiments 172 to 173, wherein the nanovial is configured to be used with a first instrument designed to analyze particles and / or cells and subsequently with a second instrument designed to analyze particles and / or cells.

[0364] Embodiment 182: The nanovial described in embodiment 181, wherein the nanovial is configured to be sorted by a fluorescence-activated cell sorter, an image-activated cell sorter, or an optofluidic microdevice and then introduced into a microfluidic droplet generator.

[0365] Embodiment 183: A method for detecting the cell-killing function of chimeric antigen receptor (CAR)-T cells or T cell receptor (TCR)-expressing T cells in response to binding of an antigen of interest, the method comprising the steps of: (a) providing or obtaining a nanovial, the nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading CAR-expressing T cells or TCR-expressing T cells within the cavity of the nanovial; (c) contacting the CAR-expressing T cells or TCR-expressing T cells with target cells that produce the antigen of interest; (d) capturing on the nanovial one or more cell-killing markers released from the target cells upon lysis or permeabilization of the target cells; and (e) detecting the one or more cell-killing markers directly or with a detection agent to obtain a cell-killing signal.

[0366] Embodiment 184: The method of embodiment 183, further comprising: (f) performing a sequencing assay on nucleic acid derived from the CAR-expressing cell or TCR-expressing cell, thereby generating a CAR or TCR sequence; (g) associating the CAR or TCR sequence with one or more cell-killing signals; and (h) identifying a CAR or TCR that functionally interacts with the antigen of interest based on the associating step of (g).

[0367] Embodiment 185: The method of embodiment 183, further comprising, after (e), sorting the nanovials based on one or more cell-killing signals.

[0368] Embodiment 186: The method of any one of embodiments 183 to 185, wherein the cavity of the nanovial further comprises one or more cell capture agents immobilized thereon.

[0369] Embodiment 187: The method of embodiment 186, wherein the one or more cell capture agents comprise an antibody or fragment thereof that binds to a protein expressed on the surface of a CAR-expressing cell or a TCR-expressing cell.

[0370] Embodiment 188: The method of embodiment 186, wherein the one or more cell capture agents bind to a label present on the surface of the CAR-expressing or TCR-expressing cell.

[0371] Embodiment 189: The method of embodiment 188, wherein the label is streptavidin or biotin.

[0372] Embodiment 190: The method of embodiment 186, wherein the one or more cell capture agents are antigens or MHC-presented antigens to which CAR-expressing or TCR-expressing cells can bind.

[0373] Embodiment 191: The method of embodiment 186, wherein one or more cell capture agents are antigen-presenting cells that express the antigen of interest on their surface or present the antigen in an MHC complex.

[0374] Embodiment 192: The method of any one of embodiments 183 to 191, wherein the affinity agent is an antibody capture moiety.

[0375] Embodiment 193: The method of embodiment 192, wherein the antibody capture moiety has affinity for a fluorescent protein.

[0376] Embodiment 194: The method of any one of embodiments 183 to 192, wherein the detection agent is an antibody or an oligonucleotide having affinity for an intracellular biomolecule of the target cell.

[0377] Embodiment 195: The method of any one of embodiments 183 to 194, wherein the detection agent is directly or indirectly labeled with a detectable label.

[0378] Embodiment 196: The method of embodiment 195, wherein the detectable label is selected from the group consisting of a fluorescent label, an oligonucleotide tag, and a magnetic particle.

[0379] Embodiment 197: The method of embodiment 196, wherein the detectable label is a fluorescent label.

[0380] Embodiment 198: The method of embodiment 197, wherein the detecting step (e) comprises determining the amount of detection agent bound to the one or more cell killing markers.

[0381] Embodiment 199: The method of embodiment 198, wherein the amount of detection agent bound to the one or more cell killing markers corresponds to the level of fluorescence.

[0382] Embodiment 200: The method of any one of embodiments 197 to 199, further comprising, following (e), sorting the nanovials based on the level of fluorescence above background levels.

[0383] Embodiment 201: The method of embodiment 200, wherein the classifying step includes implementing a flow-based classification method.

[0384] Embodiment 202: The method described in embodiment 201, wherein the flow-based classification method is fluorescence-activated cell sorting (FACS).

[0385] Embodiment 203: The method of embodiment 196, wherein the detectable label is an oligonucleotide tag.

[0386] Embodiment 204: The method of embodiment 203, further comprising sequencing the oligonucleotide tag.

[0387] Embodiment 205: The method of embodiment 204, wherein the detecting step (e) comprises counting the number of oligonucleotide tags bound to one or more cell killing markers.

[0388] Embodiment 206: The method of any one of embodiments 183 to 205, further comprising, following (e), lysing the CAR-expressing or TCR-expressing cells.

[0389] Embodiment 207: The method of embodiment 206, further comprising reverse transcribing the mRNA released from the CAR-expressing or TCR-expressing cells after lysis to produce cDNA.

[0390] Embodiment 208: The method of embodiment 207, further comprising sequencing the cDNA.

[0391] Embodiment 209: The method described in any one of embodiments 183 to 208, wherein the sequencing assay comprises single-cell RNA sequencing.

[0392] Embodiment 210: The method of any one of embodiments 183 to 209, further comprising, prior to (d), encapsulating the nanovials within the droplet.

[0393] Embodiment 211: The method of any one of embodiments 183 to 209, wherein the cavity of the nanovial comprises an opening to the surface of the nanovial.

[0394] Embodiment 212: The method of embodiment 211, further comprising, prior to (d), adding one or more blocking particles to block the openings or reduce the size of the openings.

[0395] Embodiment 213: The method of any one of embodiments 183 to 212, further comprising, prior to (e), washing the nanovial. [Example]

[0396] The following examples are included for illustrative purposes only and are not intended to limit the scope of the disclosure.

[0397] Example 1: Nanovial Fabrication and Characterization The nanovials are fabricated using an optofluidic manufacturing setup, which includes a pump for passing a precursor polymer fluid through a microfluidic chip and a microscope configured with a focused ultraviolet light source on the microfluidic chip to initiate crosslinking of the precursor polymer fluid. Generally, the nanovials are fabricated from a precursor polymer fluid containing a crosslinked PEG solution and a crosslinker solution containing dextran or other components that phase separate upon mixing with the PEG solution. Optionally, the precursor polymer fluid contains other additives for the addition of sensing moieties (e.g., oxygen- or pH-sensitive fluorophores or dyes), affinity tags (e.g., biotin, oligonucleotides), or other chemical or biomolecular functional groups (e.g., peptides, nucleic acids, nucleophiles or electrophiles for chemical conjugation, carboxylic acids, amines, small molecule sensors, etc.). For photoinitiated polymerization or crosslinking, the precursor polymer fluid also contains a photoinitiator. These precursor polymer fluids are co-flowed into a microfluidic chip containing a droplet generator to initiate phase separation, forming at least a cross-linked PEG-rich phase, and then the precursor polymer fluids are polymerized, e.g., by exposure to UV light.

[0398] In a non-limiting example, the cross-linked PEG comprises 4-arm PEG-norbornene and the cross-linker solution comprises dithiothreitol (DTT) cross-linker. In this example, one of the precursor polymer fluids comprises 4-arm PEG-norbornene, biotin-PEG-thiol, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator, and another precursor polymer fluid comprises dextran and DTT.

[0399] The crosslinked PEG solution is prepared by dissolving the LAP photoinitiator in a 3% w / w stock solution in PBS, followed by dissolving biotin-PEG thiol in the LAP stock solution to achieve a concentration of 0.5-10 mg / mL, preferably 10 mg / mL, and 4-arm PEG-norbornene to achieve a concentration of 28.9% w / w. The crosslinker solution is prepared by dissolving dextran and DTT in deionized (DI) water at 15% w / w and 5% w / w, respectively. The dissolved dextran and DTT solutions are then combined in DI water to achieve a dextran concentration of 11% w / w. The DTT concentration is adjusted over a range of concentrations to achieve a stoichiometric ratio of thiol to norbornene of 0.8. Free radicals are maintained for optimal crosslink density in the nanovials and for biotin-PEG-thiol conjugation. The dextran or PEG phase may further contain an adhesion peptide bearing a thiol group or a cysteine ​​amino acid (e.g., RGD, poly-L-lysine) that binds to the free norbornene groups. Preparation of these precursor solutions is preferably performed immediately prior to nanovial fabrication, although the precursors may be frozen and stored at -20°C for a week or more and thawed for use at a later date. In practice, biotin-PEG-thiol is most soluble at concentrations below 15 mg / mL. Addition of PEG-norbornene can enhance solubility, ensuring that the final layer remains homogeneous. Higher concentrations of biotin may also be incorporated by conjugation to the nanovials after fabrication.

[0400] To prepare for nanovial fabrication, the precursor polymer fluid is pipetted into a 1 mL plastic syringe preloaded with Novec™ 7500 to reduce precursor polymer fluid loss to the dead volume in the syringe and tubing. In an alternative embodiment, a glass syringe is used to reduce the volume of the syringe / tubing system. A 25-gauge luer stub is attached to the tip of the syringe, and this luer stub is connected to 0.020" ID PEEK tubing. Novec™ 7500 and a surfactant, e.g., Krytox 157 FSH, Krytox 157 Prepare a partitioning oil from a combination of FSL, Pico-Surf™, and FluoSurf. The surfactant concentration should be 0.25%-2% w / w. Mineral oil containing 1-5% Span 80 as a surfactant or hexadecane containing 1-5% Span 80 as a surfactant may also be used. Load this oil into a 10 mL or 20 mL plastic syringe and attach a 25-gauge Luer stub to the end. Connect the stub to 0.020" ID Tygon® tubing using 0.020" ID PEEK tubing. Place the syringe into a separate syringe pump and set the syringe pump to deliver a constant flow rate of 0.1-50 μL / min.

[0401] Nanovials may be fabricated according to the microfluidic device design and use procedures described in WO2020037214A1, which is incorporated herein by reference in its entirety. In one example, before inserting the tubing into the chip inlet, the syringe is primed by running the pump at an increased flow rate (10-100 μL / min) to remove air from the tubing. PEEK tubing is used to connect the tubing to the chip inlet. When initializing the chip, the oil phase is run first to prevent the PEG and dextran phases from loading into the chip and prematurely polymerizing, which could block the chip. After fully priming the chip with oil, the PEG and dextran phases are pumped into the chip to initiate droplet generation. The PEG phase flows through two channels, sandwiching the dextran phase within the microfluidic channel to form a coaxial flow. The oil phase partitions the coaxial flow into droplets containing the PEG and dextran phases. The stability of droplet generation depends on the flow rate used. The flow rate is momentarily increased or decreased to force the fluid into a stable droplet generation regime. Preferably, the flow rate is controlled so that droplet generation is below 1200 Hz, and less preferably below 2000 Hz. Stable coaxial flow may require up to 30 minutes of continuous operation to achieve.

[0402] The microfluidic device used to prepare the nanovials has a standard fluid-focusing design. The channels leading to the precursor inlet converge together to form a coaxial flow that intersects with two perpendicular channels carrying oil and surfactant. Droplets are generated immediately after this confluence and then connect to a deeper channel region downstream. The precursor droplet size is controlled by varying the flow rates of the precursor and oil solutions and adjusting the width and height of the droplet generation confluence. Using a device with a channel height of 18 microns, a confluence width of 40 microns, a flow rate of 0.5 μL / min for both precursor solutions, and a flow rate of 10 μL / min for the oil and surfactant phases, 28 μm precursor droplets are formed. Using a device with a channel height of 50 microns, a confluence width of 40 microns, a flow rate of 2.25 μL / min for both precursor solutions, and a flow rate of 27 μL / min for the oil and surfactant phases, 50 μm precursor droplets are formed. Once stability is achieved, the droplets move along a deeper downstream channel sized to >125% of the droplet size. The fluorescence microscope is configured with a collimated UV (365 nm) LED that transmits UV light through the chip. The downstream region of the chip is configured with a channel width sized to accommodate various objective lens sizes to facilitate uniform illumination. For 28 micron precursor droplets, 400-1000 mW / cm was achieved with a 20x objective. 2 A UV intensity of 0.5-1 s and an exposure time of 0.5-1 s are sufficient to crosslink the polymeric precursor, resulting in nanovials with a highly uniform morphology (CV less than 5%) and an outer diameter of 38 microns and a cavity diameter of 24 microns (Figures 1A-1C). Ideally, a wide range of UV conditions is used to determine the optimal UV exposure for a given nanovial. For 50 micron precursor droplets, a UV intensity of 400-1000 mW / cm is recommended. 2A 20x objective lens with an exposure time of 0.5–1 s sufficiently crosslinks the polymer precursor, resulting in nanovials with a highly uniform morphology (CV less than 5%), with an outer diameter of 60 microns and a cavity diameter of 38 microns (Figures 2A–2C). After UV exposure, the crosslinked particles in oil are collected in a tube and washed according to the procedure outlined in WO2020037214A1. To reduce adhesion of nanovials to instrument surfaces such as flow cytometers and FACS systems, as well as to fluidic channels, capillaries, ducts, or microfluidic channels, capillary filtration of unreacted functional groups is performed after nanovial particle fabrication and washing. For example, N-ethylmalemide is used to cap any remaining thiol groups on or within the nanovials to prevent disulfide bond formation between two or more nanovials. To do this, the concentrated nanovials are diluted approximately 5-fold in PBS supplemented with 0.05% w / w Pluronic F127 and 1.25 mg / mL N-ethylmaleimide and incubated at room temperature for at least 2 hours. The uniformity of the characterized particles is determined using a microscope, flow cytometer, cell sorter, etc. The nanovial morphology is optimized to achieve a CV uniformity within 5%, while the nanovial staining is uniform, similar to commercially available labeled beads. The precursor ratio is adjusted to provide nanovials that are fully open or partially closed. Confirmation and characterization of affinity molecules, such as biotin, are confirmed with complementary fluorescent molecules (e.g., streptavidin) using a fluorescence microscope (Figure 3B) or measured using flow cytometry (Figures 4A-4C). Flow cytometry confirms similar labeling uniformity compared to commercially available fluorescent polystyrene beads (Phosphorex Orange 10 μm, 2227) (Figures 4A-4C).

[0403] After fabrication, the nanovials are used in a variety of instruments to analyze particles and cells, including, but not limited to, flow cytometers, fluorescence-activated cell sorters (FACS), imaging flow cytometers, image-activated cell sorters, Coulter counters, particle counters, microscopes, microfluidic droplet generators, microwell arrays, microfluidic chips with microvalves, microfluidic SlipChips, optofluidic microdevices, microfluidic channels, and the like, as further described herein. Preferably, the nanovials are sized to be less than about 100 micrometers in diameter for use in these systems, more preferably less than about 60 micrometers in diameter. Typically, the nanovials are flowed through glass capillaries, cuvettes, microfluidic channels, and the like, and analyzed by optical, electrical, or magnetic excitation. The channel or capillary through which the nanovials flow is sized to allow the nanovials to flow rapidly without clogging. Preferably, the size of the channel is 1.5 times larger than the size of the nanovials. For example, for a 60-micrometer diameter nanovial, the channel is preferably sized to be greater than 90 micrometers in diameter (e.g., a 100- or 130-micrometer diameter channel). The nanovials are flowed through these capillaries and channels at high speeds (e.g., greater than 10 cm / sec, or greater than 1 m / sec). Excitation of the nanovials and potential cells therein is achieved using, for example, a focused laser, a focused LED, an arc lamp, a halogen lamp, light emitted by fiber optics, or other visible, ultraviolet, infrared, or other light source. In some cases, excitation is provided by applying an electric field using electrodes configured to apply the electric field or electric field gradient across the capillary or channel. In some cases, excitation is provided by an electric field or electric field gradient from a permanent magnet or electromagnet proximate to or embedded in the capillary or channel.Following excitation, signals from the nanovials or cells therein include emitted or scattered light, dielectric properties resulting in a dielectric spectrum, an impedance spectrum, or capacitance, molecular weight distribution of components, or magnetic properties resulting in fluctuations in the current of a coil or magnetic sensor, or deflection or movement of the nanovials, e.g., overall movement of fluid streamlines. This signal is used to generate an analysis of one or more nanovials. In some cases, the analysis of the nanovials is used to trigger a classification decision. The classification decision is used to direct or deflect the nanovials and potential cells contained therein into separate channels, containers, or vessels.

[0404] In some cases, the nanovials are flowed through a microfluidic channel or flow cell of an imaging flow cytometer, such as with a Luminex ImageStreamX® MKII Imaging Flow Cytometer, which analyzes over 10,000 objects per experiment. For example, 40- or 60-micrometer diameter nanovials containing cells are flowed through a microfluidic channel or flow cell of an imaging flow cytometer, such as with a Luminex ImageStreamX® MKII Imaging Flow Cytometer, which analyzes over 10,000 objects per experiment. 7The nanovials were introduced into the ImageStreamX® MKII at a concentration of 1000 nanovials / mL. The nanovials and cells were simultaneously imaged across multiple channels / stains, including brightfield to observe nanovial and cell morphology, AF488 to observe nanovial fluorescence, side-scattered light to observe granularity, and Draq5 to stain intracellular DNA. In this example, fixed Jurkat T cells and hybridoma cells were analyzed. Gating was performed to select focused nanovials and nanovials containing cells. For example, gating using a high-gradient RMS or aspect ratio / area graphs can be used to identify focused nanovials. A high aspect ratio (0.4-1) and low area correspond to a single nanovial. Cell-bearing nanovials were gated based on high AF488 and high Draq5 signals. Nanovials containing cells within the nanovial cavity can also be gated based on a high internalization score, indicating the presence of a cellular fluorescent signal within the brightfield signal from the nanovial. Spot counting of Draq5 fluorescent images can also be used to identify single or multiple cells within the nanovial and to gate on single, two, three, multiple, or any number of cells within the nanovial (Figure 20A). Other fluorescent cell trackers, viability dyes, cell surface stains, or nucleic acid stains can also be used to count cells within nanovials during imaging cytometry. Nucleic acid stains are preferred because they provide greater separation between cell nuclei and are easier to distinguish. Due to the non-spherical structure of nanovials, the fluorescent centroid of the nanovial can also be used to determine nanovials imaged at a specific orientation. To improve analytical uniformity, gating can be performed to analyze only nanovials with a specific orientation.

[0405] In a related example, a single-cell secretion assay is performed on nanovials using an ImageStreamX® MKII. In this experiment, B cells are isolated from human PBMCs and loaded onto nanovials functionalized with B cell capture antibodies and IgG capture antibodies. IgG is secreted by B cells for 1 hour, captured in nanovials, and then stained with a fluorescent antibody against IgG (anti-IgG APC). B cell surface markers are also stained with fluorescent antibodies (anti-CD138 PE, anti-CD38 PerCP Cy5.5, anti-IgM Pacific Blue). The nanovials are then loaded onto an ImageStreamX® MKII, which allows over 7,000 objects to be imaged and analyzed using 12 images per object. Using laser excitation at 405 nm, 488 nm, 642 nm, and 785 nm, the following wavelength ranges were analyzed: (430–470 nm, brightfield), (595–660, PE), (660–745, PerCP Cy5.5), (745–785, side scatter), (430–505, Pacific Blue), (575–595, brightfield), (660–720, APC). Images were taken using surface marker staining to show cells within the nanovial cavity, and the cavity of the nanovial was stained with anti-IgG APC staining if secretory cells were also present in the cavity (Figure 20B). An aspect ratio of 0.6–1.0 and an area of ​​2e3–5e3 were used to gate on single nanovials containing cells. Imaged nanovials containing cells were gated based on CD138, CD38, or a combination of both. These markers are then correlated with IgG secretion. By masking the location of cells within the nanovial using a threshold value in another fluorescence channel, e.g., the CD38 channel, and identifying the masked region, secreted IgG around the cells on the nanovial can be analyzed (Figure 20C). Only the fluorescence intensity on the nanovial associated with IgG (APC channel) on the nanovial and outside the masked region can then be quantified.The highest signal for masked IgG secretion was found to be associated with high levels of CD38 and CD138 signals on cells associated with the nanovials in the collected images (Figure 20D). Similar approaches to analyzing and gating nanovial samples using imaging flow cytometry as described herein may be used with an image-activated cell sorter, such as the BD CellView™ image-activated cell sorter.

[0406] In some cases, nanovials are introduced into the microwell array for imaging-based analysis and sorting. The microwell array may contain over 5,000 or even over 20,000 cylindrical wells with diameters of 50-100 micrometers. By adjusting the size of the wells in the microwell array to be similar to the size of the nanovials (e.g., 50-micrometer wells for 35-micrometer nanovials), each well can carry a single vial (Figure 21). Nanovials or nanovials containing cells can be imaged within the microwell array and, optionally, sorted based on fluorescent and / or brightfield imaging information. Image analysis techniques similar to those described herein for imaging flow cytometry are used. Sorting is achieved using focused sound waves and acoustic forces to eject target nanovials, for example, from the microwells into microchannels located on the microwell array for downstream collection.

[0407] In some cases, nanovials are analyzed in an automated cell counter by introducing 35-micrometer nanovials or 35-micrometer nanovials containing cells. In an exemplary embodiment, the nanovial solution is stained 1:1 with the viability dye trypan blue and introduced into a CytoSmart™ automated cell counter. It has been found that nanovials take up trypan blue and therefore appear as "dead cells" in automated counting algorithms. The number or concentration of nanovials in a sample may be counted automatically by a "dead cell count" number. To accurately count nanovials, a cell size gate may be set to include a larger range, such as 4-33 micrometers in diameter or objects greater than 33 micrometers. Nanovials containing cells may also be counted. Cells are counted as viable if they do not take up trypan blue stain. A count of nanovials containing live cells may also be obtained by locating overlapping coordinates of live and dead cells in the automated counting process. Hundreds to thousands of nanovials and the cells loaded in the nanovials are counted in an automated fashion.

[0408] In some cases, nanovials are also flowed through microfluidic channels, capillaries, conduits, 3D printed channels, electrowetting in dielectric (e.g., digital microfluidic) devices, etc., to encapsulate them into droplets or other compartments. For example, to encapsulate the nanovials (and potential cells therein) within aqueous droplets to form a water-in-oil emulsion, the nanovials are flowed as part of an aqueous sample fluid through a droplet generator microfluidic device, such as a fluid-focusing droplet generator, a T-junction droplet generator, a step-emulsion droplet generator, or a coaxial capillary droplet generator. The droplets of the water-in-oil emulsion are substantially uniform in volume. The droplet volume is preferably less than 100 nL, more preferably less than 10 nL. The droplet volume is also preferably greater than 1 pL, more preferably greater than 50 pL. Nanovials are introduced at a concentration such that the majority of droplets containing nanovials contain only a single nanovial, with the following Poisson loading statistics: average lambda greater than 0.20, preferably lambda approximately 0.1. A device containing multiple inlets and inlet channels is used to introduce the nanovials along with other reagents to form droplets containing the nanovials and additional reagents or buffers. These additional reagent streams are used to perform other operations on the nanovials (and potential cells therein) in the droplets, such as cell lysis, enzymatic reactions, polymerization reactions, staining reactions, etc.

[0409] In some cases, nanovials are introduced into a microwell, an array of microwells, a microchannel with a valved chamber, or a microchannel with a separate chamber or reservoir capable of holding cells, particles, and other microobjects at a slower flow rate than the main channel or open fluid reservoir, incorporated into the substrate. Preferably, the well, chamber, or reservoir provides a volume that reduces the flow rate by at least five times compared to the velocity at the inlet to the chamber, adjacent channel, or feed channel. The nanovials (and potentially cells therein) are incubated in the chamber or reservoir for a period of time under a low-flow environment that minimizes convective transport. The volume of the chamber or microwell is preferably less than 100 nL, more preferably less than 10 nL. The volume of the chamber or microwell is also preferably greater than 1 pL, more preferably greater than 50 pL. Nanovials are introduced to a concentration such that the majority of chambers or microwells containing nanovials contain only a single nanovial, with the following Poisson loading statistics: mean lambda greater than 0.20, preferably lambda = about 0.1. In some embodiments, the nanovials (and potential cells therein) are manipulated or moved within a channel, chamber, or reservoir using dielectrophoresis or light-induced dielectric forces, which can be used to ensure a single nanovial is present within the chamber. In some cases, the nanovials (and potential cells therein) are moved using magnetic forces. The nanovials or cells therein contain some magnetic material, such as iron oxide microparticles or nanoparticles. The nanovials (and potential cells therein) are also analyzed in wells, chambers, or reservoirs by optical, electrical, or magnetic excitation. Optical excitation is preferably used, using a light source such as a laser, LED, or arc lamp to image the nanovials by detecting emitted or scattered light from the nanovials (and potential cells therein) using a CCD, CMOS, or other 2D detector array. Following excitation, analysis is performed to identify nanovials for sorting or selection.The nanovials (and potential cells therein) are then sorted for collection, for example, by using dielectrophoresis or light-induced dielectric forces to move the nanovials from the chamber into free flow. Alternatively, a high-power laser pulse is used to push the nanovials (and potential cells therein) from the microwell in which they are located into free flow for collection. Alternatively, focused sound waves are used to push the nanovials (or potential cells therein). Alternatively, a micropipette or automated picker is used to pick the nanovials (and potential cells therein) contained in the microwell, or, if the nanovials contain magnetic material, a magnetic rod or tip is used to attract and select the nanovials.

[0410] Example 2: Method of using nanovials with a flow cytometer. In a non-limiting example, it may be desirable to use a flow cytometer and flow sorter to analyze and / or sort nanovials and their associated cells and biomolecules at high throughput. To perform analysis and sorting with improved results (e.g., yield, purity, reduced clogging, enhanced singlet detection, improved limit detection, absolute quantification), consideration is given to sample solution preparation (e.g., concentration of nanovials in the sample vessel, choice of buffer) and specific instrument and associated software settings. This example describes considerations and principles for running nanovial samples on a flow cytometer and flow sorter instrument.

[0411] Optimal concentration of nanovials for use with FACS When running nanovial samples on flow cytometers and flow sorters, the concentration of the nanovials is adjusted to improve singlet events and reduce clogging, reduce abort events, improve yield, and improve purity. Many commercially available FACS instruments use Poisson statistics to dictate the interval between analytical events. Increasing sample concentration increases the likelihood of having multiple nanovials within the same event window. Decreasing concentration reduces the likelihood of doublet or multiplet events. The ideal concentration depends on the size of the nanovials and the instrument and size of the flow cytometry nozzle or tip used. Generally, a wide range of concentrations is run for each configuration (e.g., any combination of instrument, nozzle / tip size, and nanovial size) to identify an appropriate concentration range for the intended application. An exemplary workflow includes labeling a fraction of the nanovials with a fluorescent or otherwise distinguishable label or stain, running the nanovials through a flow sorter, sorting based on the label signal, and measuring downstream purity, recovery, and / or enrichment by microscopy or by re-running the sorted sample through the instrument. Generally, increasing nanovial size decreases the concentration (defined here as the number of nanovials / volume) and allows for greater spacing between events. As nanovial size decreases, a higher concentration of nanovials can be analyzed and sorted with higher yield and purity. Increasing the size (e.g., cross-sectional dimension) of the sorting nozzle decreases the concentration of nanovials, reducing the likelihood of doublet events.

[0412] Considerations for various applications For applications where single-cell or clonal colony formation is important (e.g., detection of B-cell monoclonal antibodies, development of monoclonal cell lines for master and working cell banks), the nanovial is further diluted with sample solution to reduce doublet events and improve single-cell purity. For applications where enrichment is a priority (e.g., increasing concentration of rare events) and lower purity can be tolerated (e.g., enrichment of more productive subpopulations of producer cells), increasing concentration increases throughput at the expense of single-cell purity.

[0413] Singlet selection based on scattered light In some cases, it may be desirable to distinguish subpopulations of nanovial samples using the flow cytometer's light scatter signal readout. This includes, but is not limited to, distinguishing between empty nanovials and nanovials containing cells, between nanovial events and unbound cell events, and between nanovials containing a single cell and nanovials containing multiple cells. Light scatter measurements may include forward scatter (e.g., height, width, and / or area), side scatter (e.g., height, width, and / or area), or any combination of the aforementioned measurements. In some cases, nanovials, as described herein, may be fabricated to be larger than cells and have a high forward scatter signal (e.g., forward scatter height), while having a similar or lower side scatter signal than nanovials fabricated from smooth hydrogel materials. This unique feature enhances the ability to identify various populations (e.g., cells, nanovials, and / or cell-containing nanovials) (Figures 4A-4C). Nanovials containing cells may have both high forward and side scatter light signals and may be distinguishable from cells or nanovials alone.

[0414] From empty nanovials and free cells to cell-containing nanovials Samples containing nanovials without cells, samples containing nanovials with cells, and samples containing cells without nanovials are analyzed separately using the instrument to identify a unique scattered light signal threshold for each sample. The nanovials and cells are labeled with distinct fluorescent labels to identify scattered light signals associated with suspended cells, empty nanovials, and nanovials containing cells. Nanovials with attached cells are distinguished from empty nanovials by gating on a single side scatter component (e.g., height, width, or area) or forward scatter component (e.g., height, width, or area), for example, using a side scatter threshold associated with nanovials with attached cells and a forward scatter threshold associated with the presence of nanovials. Nanovials with attached cells are distinguished from empty nanovials by gating on 2-D axes in a 2D plane, for example, thresholds or gates, of two scattered light parameters, including a side scatter component (e.g., height, width, or area) and a forward scatter component (e.g., height, width, or area). Higher dimensional light scatter gating (e.g., 3D, 4D) or multiple combinations of 1D, 2D and higher dimensional gates are used to select a subpopulation of nanovials containing cells of interest.

[0415] In a non-limiting example, nanovials are engineered to have lower forward and / or side scatter than free cells. This unique feature allows users to distinguish cell-containing nanovial populations by increased FSC, SSC, or both compared to empty nanovials and / or free cells (Figures 4A-4C). The size of the nanovials can be adjusted to make their scatter or fluorescence signal more discernible from cells. For example, nanovials larger than cells may have similar scatter or fluorescence peak heights, but substantially larger scatter or fluorescence signal widths than cells. The exact gating parameters depend on the laser power and detector sensitivity of a particular instrument. The specific thresholds and gates described herein for a particular instrument are determined by running the control samples described above.

[0416] Doublet removal Nanovials containing single cells are distinguished from nanovials containing two or more cells based on their light scatter signals. Identification of specific gates is performed using a similar experimental strategy as outlined in the section above, "Cell-Containing Nanovials from Empty Nanovials and Free Cells." An additional strategy is used in which cells from two subpopulations are labeled with two unique fluorophores or other labels identified using a flow cytometer or flow sorter instrument. Nanovials are overloaded with cells containing one type of label and cells containing a second type of label, so that a significant fraction contains multiple cells bearing both types of labels. The sample is then analyzed using a flow cytometry instrument to identify the subpopulation containing multiple cells by searching for events containing both types of fluorescent signal, which correlates with multiple cells. Once identified, the light scatter signal corresponding to this subpopulation is compared to the light scatter signal from a sample containing nanovials with single cells (based on their lower loading density). Similar to the above section, simple 1D, 2D, 3D, 4D, etc. gating and more complex 1D, 2D, 3D, 4D, etc. gating, or a combination of light scatter gating and thresholding, are used to select the subpopulation of singlets of interest.

[0417] In some cases, the nanovials are engineered to have low side-scattered light signals, thereby making the side-scattered light of cells distinguishable. Cell doublets loaded in the nanovials are identified by defining thresholds for the side-scattered light height (SSC-H) and side-scattered light area (SSC-A) of the analyzed events. Events that exceed one or more thresholds are classified as cell doublets and excluded from further analysis. The above experimental procedures are used to further identify the exact conditions for each sample. The side-scattered light of the nanovials is further reduced, thereby ensuring that only signals from cells, not nanovials, exceed the event threshold. This can be facilitated by adjusting the composition of the nanovial itself, for example, by using a higher molecular weight precursor to reduce crosslink density, or by adding additives to the sample buffer and / or sheath fluid to match the refractive index of the nanovial to that of the solution. For example, a solution of Optiprep (ratio = 1.429 in the original solution) and water (ratio = 1.33) can be used to match the refractive index of the PEG nanovial to approximately 1.35, up to 1.40, depending on the weight of the fraction. In this case, the nanovial contains almost no scattered light signal because its refractive index is the same as that of the surrounding solution, allowing for the use of gating strategies typically used to gate free cell doublets and multiplets. For example, when gating based on side scatter width (SSC-W) and SSC-A, doublets / multiplets are expected to have a significantly higher SSC-W signal. When gating based on SSC-H and SSC-A, doublets are expected to have a slightly lower SSC-H for a given SSC-A value. In gating based on SSC-W and SSC-H, nanovials containing doublets or multiplets are expected to have a higher SSC-W for a given SSC-H signal. A similar strategy is used for discrimination based on forward scatter signal.

[0418] In some cases, the scattered light signal of cells and / or nanovials is modulated by modification with light-scattering nanoparticles or microparticles (e.g., polystyrene beads, magnetic beads) to increase the differential detected signal. For example, cells are labeled with biotinylated antibodies specific for surface markers and then conjugated with avidin-coated polystyrene particles to enhance their side-scattered light (Spherotech, Avidin Coated Polystyrene Particles).

[0419] Operation of a FACS system adjusted for nanovials The flow cytometry instrument is adjusted to best operate for analyzing and sorting nanovials. For example, for airborne droplet flow cytometers such as the BD Aria II / III or Sony SH800, a sorting mask, such as a "purity" mask or a "single cell" mask, is selected to improve sorting purity and obtain single nanovials. Correct droplet formation is improved by adjusting the drop delay value for larger-sized nanovials. The drop delay is automatically adjusted using drop delay calibration nanovials stained with a fluorescent label similar to calibration beads known in the art (e.g., SPHERO™ AccuCount Ultra Rainbow Fluorescent Drop Delay Calibration Particles). Additionally, a larger size, e.g., a microfluidic chip diameter of 100 or 130 micrometers for the Sony SH800 system, is selected to accommodate nanovials with larger sizes compared to normal mammalian cells. Due to the size of larger nanovials, they also move downstream more slowly when focused by the hydrodynamic sheath flow. Therefore, sorting timing is increased to account for the slower velocity of the nanovials, improving the fraction of correctly sorted events. To achieve high purity and yield for sorting events at throughputs exceeding 500 events / s, nanovials with diameters of 35-55 μm are sorted using a Sony SH800 flow sorter and a 130 μm microfluidic sorting chip without adjusting the drop delay from the initial settings. Individual nanovials are then index-sorted to separate wells in a 96- or 384-well plate, resulting in an occupancy rate of over 60% for those with a single nanovial across individual wells.To achieve high purity for sorting events at throughputs exceeding 500 events / s, nanovials with diameters of 35–45 μm were sorted using a BD FACS Aria II flow sorter and a 100 μm nozzle with a calibrated drop delay on the nanovial before sorting, resulting in non-default values ​​for high-purity sorted events. To achieve high purity for sorting events at throughputs exceeding 300 events / s, nanovials with diameters of 35–55 μm were sorted using a BD FACS Aria II flow sorter and a 130 μm nozzle with a calibrated drop delay on the nanovial before sorting, resulting in non-default values ​​for high-purity sorted events. To achieve high purity for sorting events at throughputs exceeding 100 events / s, nanovials with diameters of 35–85 μm were sorted using an On-Chip Sort microfluidic flow sorter and a 150 μm diameter microfluidic chip. Density-matching solutions or thickening agents were added to reduce rapid settling within the sample reservoir. In some cases, a micropipette is used to agitate the sample immediately before sorting in the reservoir. In some cases, an 80-micrometer diameter microfluidic chip is used to sort nanovials with a diameter of less than 60 microns, more preferably nanovials with a diameter of less than 40 microns, with a throughput of more than 200 events per second and a purity of more than 80%. An 80-micrometer diameter chip is used to sort nanovials with a diameter of less than 40 microns at a throughput of more than 1,000 events per second, demonstrating a 4-fold enrichment compared to the starting population. Multiple enrichment cycles are used to achieve a high purity of more than 95% for rare populations that are at least 1% of the total population and less than 1%. More than 10,000 nanovials with an outer diameter of 35 microns, 50 microns, or 60 microns are analyzed and sorted using a Nanocellect WOLF G2 cell sorter, a microfluidic chip-based cell sorter that uses microchannel-based sorting.Nanovials of different diameters and free CHO or Jurkat cells are clearly distinguishable on a plot of forward scatter height versus backscatter height. As nanovial size increases from 35 microns to 60 microns, forward scatter height and backscatter height increase. For sorting, 35- and 50-micron nanovials are preferred to achieve high yields of sorted nanovials and nanovials containing cells. With 35-micrometer diameter nanovials, a sorting purity of 88% and a recovery of 85% are achieved. Over 18,000 nanovials are analyzed and sorted using the Nodexus NX One to a concentration of 120,000 / mL. Fluorescently labeled nanovials can be sorted from unlabeled nanovials at a concentration of 10%, achieving a sorting purity of 86.4% and a recovery of 75.6%.

[0420] Buffers for use in flow cytometers to prevent precipitation During manipulation of nanovials in a flow cytometer, settling occurs, causing inconsistent event rates within the instrument. To prevent or mitigate nanovial settling, agitation methods such as pulsating flow into the sample tube or mechanical agitation with a stir bar are used. Additives to the nanovial sample solution are also used to prevent or mitigate settling and maintain a uniform nanovial concentration during analysis. Typically, this is achieved by controlling the viscosity of the sample solution to control the settling rate, adjusting the density of the solution to adjust the net buoyancy of the nanovials, or a combination of both.

[0421] In some cases, the sample solution is supplemented with a cytocompatible thickening agent (e.g., polyethylene glycol, dextran, hyaluronic acid, alginate, etc.). The viscosity of the sample solution is altered by adjusting various additive properties, such as concentration, molecular weight, and chemical moiety. A 5% dextran solution is used to increase the viscosity to 16.7 mPa·s. Generally, the sedimentation rate is inversely proportional to the viscosity of the globule solution. To minimize the variability in event rates, the sedimentation rate should be sufficiently low compared to the sample runtime. For example, if the expected sample runtime is 1 hour and the sample solution depth in the tube is 10 cm, the viscosity should be adjusted so that the nanovials settle to less than 10% of their solution depth during the runtime. Further settling is acceptable, for example, less than 50% or even less than 25% of the sample solution depth. An additional consideration regarding viscosity is the increased shear forces on the nanovials and the cells contained within them due to increased viscosity. In some FACS instruments, pressure is increased to compensate for increased viscous drag and fluid resistance within the instrument's tubing or channels. In aerosol-based FACS instruments, droplet drive settings are sometimes adjusted to compensate for changes in droplet breakup due to additive-induced viscosity changes and interfacial tension changes. The viscosity enhancers used are optically transparent and do not contain light-scattering particulates.

[0422] In some cases, additives are added to the sample solution to increase its density and reduce settling of the nanovials. Exemplary density-matching agents include Ficoll, Percoll, and Optiprep, among others. If the nanovials are denser than the sample solution, increasing the solution's density with additives more closely aligns gravity and buoyancy, reducing settling. Ideally, the density of the sample solution matches or is slightly less dense than the nanovials (e.g., nanovials may be approximately 1.034 g / ml in phosphate-buffered saline (PBS)), so that the nanovials do not float to the top of the vial or container. For polyethylene glycol nanovials with a diameter of 35 to 60 microns, a solution containing 43 to 50% Ficoll-Paque™ PLUS Media in PBS is preferred to create neutrally buoyant nanovials. In some cases, a solution containing 44 to 50% Optiprep in PBS is also used.

[0423] Example 3: Buffer system and density adjustment for selective isolation of cell-containing nanovials. The difference in buoyancy between the nanovials and the cells is utilized to enrich or deplete specific subpopulations of samples containing, but not limited to, nanovials, cells, and nanovials with attached cells prior to downstream processing. It may be desirable to enrich nanovials containing cells, to enrich nanovials containing multiple cells, to enrich nanovials containing only a single cell, and to deplete cells not attached to the nanovials.

[0424] Generally, enrichment is achieved by adjusting the density of the sample solution, the density of the nanovials, the density of the cells, or any combination, such that differences in buoyancy cause the target population (e.g., cells bound to the nanovials) to settle to the bottom of the vial or other vessel, while the undesired subpopulation (e.g., empty nanovials) rise to the top. The target population is then pipetted or extracted from the bottom of the vessel and transferred to a new vessel, or the undesired population is removed from the top of the vessel, concentrating the target sample. In related embodiments, the target subpopulation instead rises to the top of the vial or other vessel, while the undesired subpopulation sinks to the bottom of the vial or other vessel. The target population is then pipetted or extracted from the top of the vessel and transferred to a new vessel, or the undesired population is removed from the bottom of the vessel, concentrating the target population. Examples of these various scenarios are shown in Figure 5. In related embodiments, the target population remains neutrally buoyant, while the undesired subpopulations rise to the top of the vessel and / or sink to the bottom of the vessel. The target population is pipetted or extracted from the middle of the vessel and transferred to a new vessel, or the undesired populations are removed from the top and / or bottom of the vessel, resulting in a concentrated target sample.

[0425] In some cases, the density of the sample solution, nanovial, cells, or a combination is adjusted to control the enrichment of the target subpopulation. For example, biocompatible water-soluble additives such as Ficoll, Percoll, Optiprep, glycerol, PEG, Dextran, and hyaluronic acid, among others, are used to adjust the density of the sample solution. To affect cell density and enhance enrichment, the osmolarity of the solution is adjusted. For example, the use of a hypotonic solution (compared to normal physiological phosphate-buffered saline) causes cells to swell and decrease their density. The density of the nanovial can be adjusted in a number of ways to enhance enrichment. For example, density can be increased by increasing the concentration of PEG used in the precursor solution, increasing crosslinking density by using lower molecular weight PEG molecules (e.g., 5 kD 4-arm PEG), or embedding or attaching higher density microparticles or nanoparticles in or on the nanovial matrix (e.g., SiO2 microparticles / nanoparticles, iron oxide microparticles / nanoparticles). Similarly, the density of nanovials can be decreased by decreasing the concentration of the PEG precursor solution, increasing the molecular weight of the precursor polymer, or embedding or attaching lower-density microparticles / nanoparticles (e.g., glass hollow particles for buoyancy-activated cell sorting - BACS particles). Alternatively, cells attached to nanovials can be labeled with particles such as glass, iron oxide, or metal particles (100 nm to approximately 3 μm in diameter) containing cell-specific affinity tags to adjust the effective density of cell-containing nanovials compared to cell-free nanovials. The effective density of nanovials relative to cells can be adjusted by using higher (or lower) density additives that are impermeable to cell membranes but transport or diffuse into the nanovial matrix.

[0426] Concentration of cell-containing nanovials at the bottom In one non-limiting exemplary embodiment, a high molecular weight, biocompatible, density-altering additive is added (10) to a vessel (e.g., a centrifuge tube) containing nanovials (fabricated using processes described elsewhere herein) and nanovials with attached cells. The solution has a density equal to or slightly greater than that of the nanovials (e.g., 1.034 g / ml) and contains density-altering molecules that are too large for transport into the nanovials to prevent them from diffusing into the nanovial matrix and altering the nanovial's density. Under these conditions, nanovials without attached cells are buoyant and rise to the top of the vessel. Nanovials with attached cells (e.g., cells with a diameter of 6-20 microns and a density of 1.05-1.08 g / ml) are not buoyant because the overall mass of the cells and nanovials is greater than the mass of the displaced solution. For example, a 40% Ficoll-Paque™ PLUS Media solution is used as the density-modifying agent to achieve a density slightly greater than 1.034 g / ml. The nanovials in the vessel are incubated with the density-modifying agent, causing the nanovials with attached cells to settle to the bottom of the vessel, or are centrifuged at 180 g to accelerate the transport of the nanovials with attached cells to the bottom of the vessel, resulting in an enriched fraction of the nanovials with attached cells at the bottom of the vessel. The nanovials from the bottom of the vessel are then removed, e.g., by pipetting, to create a solution containing an enriched fraction of the nanovials with attached cells. Alternatively, the cell-free nanovials in the supernatant at the top of the vessel are removed, e.g., by pipetting, to create an enriched set of nanovials with attached cells remaining in the vessel. After this density-based enrichment, the density-modifying additive is then replaced with a buffer, medium, or other density-matching solution, as described above, for downstream flow cytometry.

[0427] Concentration of cell-containing nanovials at the top In one alternative, non-limiting example, a low-molecular-weight biocompatible density-altering additive is added to the nanovials and the vessel (e.g., centrifuge tube) containing the nanovials with attached cells. The additive has a molecular weight small enough to diffuse freely into the hydrogel matrix of the nanovials, thereby increasing their effective density. For example, the molecular weight is less than 40 kD or less than 10 kD. For example, nanovials fabricated using the method described in the Preparative Examples (Example 1) section have an approximate density of 1.03 g / ml in water or PBS. When Optiprep is added to the solution, the density of the nanovials increases with increasing concentration. These nanovials are approximately neutrally buoyant in a solution consisting of 44% Optiprep in PBS, which has an approximate density of 1.14 g / ml. Above this concentration, the nanovials begin to float to the top of the vessel. To separate nanovials containing cells from empty nanovials, the density of the solution is adjusted to be just below the neutrally buoyant density of the empty nanovials. The sample solution is incubated to allow the empty nanovials to settle to the bottom of the vessel and the cell-containing nanovials to float to the top of the vessel. (12) In some cases, centrifugation is used to increase the transport rate of the empty nanovials to the bottom of the vessel and the cell-containing nanovials to the top of the vessel, resulting in a concentrated fraction of the cell-containing nanovials at the top of the vessel.

[0428] Concentration of nanovial-bound cells separate from free cells at the bottom In another non-limiting example, differences in buoyancy are used to separate unbound cells from cells bound to nanovials (14). For example, if the density of the cells is less than that of the sample solution and the effective density of the nanovial to which the cells are bound is greater than that of the sample solution, the cells will rise to the top and the nanovial containing the cells will sink to the bottom. In one embodiment, Optiprep is added to a vessel containing nanovials and cells so that the density of the solution is slightly greater than the neutrally buoyant density of the cells but remains below the neutrally buoyant density of the nanovials. For example, a sample solution composed of 33% v / v Optiprep in PBS or medium has an approximate density of 1.1 g / ml. When incubated with cells and cells bound to the nanovials, the free cells will rise to the top and can be removed by pipetting or aspiration, while the nanovial containing the cells will sink to the bottom of the vessel and can be transferred by pipetting. In some cases, centrifugation is used to speed up the process, as described above.

[0429] Concentration of cells bound to the nanovial at the apex, separate from free cells In another non-limiting example, differences in buoyancy are used to separate unbound cells from cells bound to the nanovial (16). For example, if the density of the cells is greater than that of the sample solution and the effective density of the nanovial to which the cells are bound is less than that of the sample solution, the cells will sink to the bottom of the vessel and the nanovial containing the cells will float to the top. In one exemplary embodiment, Ficoll is added to the vessel containing the nanovial and cells so that the density of the solution is slightly below the neutrally buoyant density of the cells but remains above the neutrally buoyant density of the nanovial. For example, a sample solution composed of 80% v / v Ficoll-Paque™ PLUS Media in PBS or culture medium has an approximate density of 1.06 g / ml. Upon incubation with cells and nanovials bound to the cells, the free cells sink to the bottom of the vessel and are removed by pipetting or aspiration, while the nanovials containing the cells rise to the top of the vessel and are transferred by pipetting to create a concentrated fraction. In some cases, centrifugation is used as described above to increase the rate of transport of the cells and nanovials to the top or bottom.

[0430] Non-Limiting Examples of Buoyancy-Based Fractionation of Nanovials In one embodiment, buoyancy-based separation of cell-bound nanovials from free cells is achieved by first coating biotin-functionalized PEG-norbornene 35 μm diameter nanovials with streptavidin at a final concentration of 30 μg / mL. The streptavidin-conjugated nanovials are then washed three times with wash buffer using the wash procedure (described herein) and resuspended in wash buffer to a concentration of 4000 nanovials / μL. Anti-human CD45 antibody (biotinylated) is then conjugated to the nanovials by mixing 165 μL of 8 μg / mL anti-CD45 antibody with 165 μL of streptavidin-conjugated nanovial solution. The mixed solution is pipetted and incubated at room temperature for 30 minutes to allow the antibody to bind to the nanovials. The nanovial solution is washed three times using the wash procedure and resuspended to a final concentration of 330 μL. A 24-well plate is prepared with 1 mL of medium per well. Each well was filled with 3 μL of antibody-coated nanovials and allowed to sit for 30 minutes. Jurkat cells were seeded into the wells at a concentration of 122,000 cells / well. The cells were allowed to settle for 20 minutes, then transferred to an incubator for 60 minutes to allow binding of the loaded cells to the nanovial cavities. The samples were then pipetted into tubes, centrifuged at 300 g for 1 minute, and resuspended in 24%, 29%, 31%, or 33% Opti pre-treated solutions mixed with wash buffer or wash buffer control. The samples were vortexed for 10 seconds to resuspend and centrifuged at 600 g for 30 minutes ...

Claims

1. 1. A method for linking functional single-cell information with genomic, transcriptomic, and / or proteomic single-cell information, comprising: (a) performing a functional assay on a single cell located in a cavity of a nanovial, said functional assay being associated with a first barcode or a first label; (b) performing a genomic, transcriptomic, and / or proteomic assay on the single cell in the nanovial, wherein the genomic, transcriptomic, and / or proteomic assay is associated with a second barcode or a second label; (c) linking the functional single-cell information with the genomic, transcriptomic, and / or proteomic single-cell information by associating the first barcode or the first label with the second barcode or the second label; A method comprising:

2. 10. The method of claim 1, further comprising, prior to (b), sorting the nanovials based on a signal generated by the functional assay.

3. The method of claim 1 , wherein the functional assay is an antibody secretion screening assay.

4. The method of claim 1 , wherein the functional assay is an antibody affinity assay.

5. The method of claim 1 , wherein the functional assay is an antibody specificity assay.

6. The method of claim 1 , wherein the genomic assay is a single-cell RNA sequencing assay.

7. The method of claim 1 , wherein the nanovial is associated with a unique oligonucleotide tag.

8. The method of claim 7, wherein the unique oligonucleotide tag comprises a poly-dT capture region.

9. 2. The method of claim 1, wherein the first barcode or the first label comprises an oligonucleotide having a polyA region.

10. A nanovial having a size, shape, surface chemistry, density, or any combination thereof, configured to fit into and be used with a number of different types of instruments designed to analyze particles and / or cells.

11. A method for identifying an antibody or fragment thereof that binds to an antigen of interest, comprising the steps of: (a) providing or obtaining a nanovial, said nanovial comprising a cavity formed therein and an affinity agent immobilized therein; (b) loading antibody-producing cells within the cavity of the nanovial; (c) incubating the nanovial so that one or more antibodies or fragments thereof are secreted from the antibody-producing cells and bind to the affinity agent; (d) adding a detection agent to the cavity of the nanovial such that the detection agent binds to the one or more antibodies or fragments thereof; (e) detecting one or more signals related to binding of the detection agent to the one or more antibodies or fragments thereof; (f) performing a sequencing assay on nucleic acid derived from the antibody-producing cells, thereby generating sequences for the one or more antibodies or fragments thereof; (g) relating the sequence of the one or more antibodies or fragments thereof to the one or more signals related to binding of the detection agent to the one or more antibodies or fragments thereof; (h) identifying the one or more antibodies or fragments thereof that bind to the antigen of interest based on the associating step of (g); A method comprising:

12. The method of claim 11, wherein the cavity of the nanovial further comprises one or more cell capture agents fixed thereto.

13. The method of claim 11, wherein the affinity agent is an antigen-presenting cell that expresses the antigen of interest on its surface.

14. The method of claim 11, wherein the detection agent is directly or indirectly labeled with a detectable label.

15. The method of claim 14, wherein the detectable label is selected from the group consisting of a fluorescent label, a protein affinity tag, an oligonucleotide tag, and a magnetic particle.

16. The method of claim 15, wherein the detectable label is an oligonucleotide tag.

17. The method of claim 11, wherein the cavity of the nanovial includes an opening to a surface of the nanovial.

18. The method of claim 17, further comprising, prior to (c), adding one or more blocking particles to block the opening or reduce the size of the opening.

19. A method for analyzing cells, comprising: (a) providing or obtaining a plurality of nanovials, each nanovial of the plurality having an opening to a surface of the nanovial; (b) loading cells into said plurality of nanovials such that a subset of said nanovials loads at least one cell; (c) adding a plurality of blocking particles such that the blocking particles interact with the opening of the nanovial and block the opening or reduce the size of the opening; (d) releasing the nucleic acid molecule from at least one cell carried within the nanovial, thereby producing a released nucleic acid molecule; A method comprising:

20. The method of claim 19, wherein the plurality of nanovials are disposed in a fluid.

21. The method of claim 20, wherein the blocking particles inhibit the released nucleic acid molecules from being transported out of the nanovial.

22. The method of claim 19, wherein the maximum diameter of the blocking particle is greater than the diameter of the opening of the nanovial.

23. The method of claim 19, wherein the blocking particle is in contact with or surrounds the opening of the nanovial.

24. The method of claim 19, wherein the blocking particles are sized to maintain greater than 50% of the volume of the hollow portion of the nanovial.

25. The method of claim 19, wherein the blocking particles have an average diameter of about 20 micrometers to about 50 micrometers.

26. The method of claim 19, wherein the blocking particles are spherical or substantially spherical in shape.

27. The method of claim 19, wherein the blocking particles comprise a polymer hydrogel.

28. The method of claim 27, wherein the polymer hydrogel comprises polyethylene glycol (PEG).

29. The method of claim 19, wherein the blocking particles are coated with one or more affinity agents.

30. The method of claim 29, wherein the one or more affinity agents comprise a nucleic acid.

31. The method of claim 30, wherein the nucleic acid comprises an oligonucleotide tag or an oligonucleotide barcode.

32. The method of claim 30, wherein the nucleic acid comprises an oligo-dT sequence.

33. The method of claim 32, wherein the released nucleic acid molecule is an RNA molecule.

34. The method of claim 33, further comprising a step of sequencing the RNA molecule.

35. The method of claim 29, wherein the one or more affinity agents comprise an antibody capture moiety or an antigen.

36. The method of claim 35, wherein the antibody capture moiety is selected from the group consisting of an anti-IgG antibody or fragment thereof, an anti-Fc antibody or fragment thereof, protein A, and protein G.

Images