Fluidic device for drug testing on cells

Abstract

Disclosed herein are systems and methods for releasing controlled drug doses to cells within fluidic channels. In certain aspects, stimuli such are selectively provided to drug-containing substrates or surface-bound drug libraries to release one or more drugs in time, location, and dose-specific manners.

Description

FLUIDIC DEVICE FOR DRUG TESTING ON CELLSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 712,306 filed October 25, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Associating drug activity to cellular state and behavior is a central challenge in therapeutic research. Drugs often elicit multifaceted responses that increase in complexity through intercellular and extracellular responses to treatment regimens. High throughput screening methods are generally unable to capture phenotypic changes of individual cells, or to associate these changes to parameters of a drug dosing regimen. Accordingly, there is a need for highly multiplexed analysis methods that enable multifaceted single cell monitoring during controlled dosing assays.SUMMARY

[0003] In one embodiment, the present application provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; inputting a substrate comprising the drug into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell; and releasing the drug from the substrate, thereby allowing the drug to contact the cell.

[0004] In some aspects, the chamber co-encloses the cell with the substrate. In further aspects, the chamber is synthesized co-enclosing the cell with the substrate. In another aspect, a wall of the chamber is impermeable to the substrate. In an additional aspect, a wall of the chamber is impermeable to the drug. In a particular aspect, the inputting the substrate comprises flowing the substrate through a wall of the chamber, and the substrate is sufficiently small to pass through a pore of the wall. In certain aspects, the inputting the substrate is before, after, or concurrent with chamber synthesis.

[0005] In one aspect, the releasing comprises degrading the substrate, thereby releasing the drug from an interior portion of the substrate. In select aspects, the degrading comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof. In a certain aspect, only a portion of the substrate is degraded. In a particular aspect, the portion of the substrate is degraded by: irradiating a fraction of an interior space of the chamber with light; irradiating only the portion of thesubstrate with light; inputting only a fraction of light required to completely degrade the substrate; inputting only a fraction of light required to release all instances of the drug comprised by the substrate; inputting only a fraction of a concentration of a reagent required to completely degrade the substrate; inputting only a fraction of a concentration of a reagent to release all instances of the drug comprised by the substrate; or a combination thereof. In select aspects, the releasing comprises cleaving a bond or linker between the drug and the substrate. In certain aspects, the cleaving converts the drug from a prodrug to an active form of the drug.

[0006] In particular aspects, the substrate comprises a plurality of instances of the drug, and only a subset of the plurality of instances of the drug are released from the substrate. In some such aspects, the subset of the instances of the drug is released from the substrate by: shining a light on a fraction of an interior space of the chamber; inputting only a fraction of light required to release all instances of the drug from the substrate; inputting only a fraction of a concentration of a reagent required to release all instances of the drug from the substrate; or a combination thereof.

[0007] In certain aspects, the releasing comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof. In further aspects, the substrate comprises a particle. In some such aspects, the particle is photolytically, chemically, thermally, pH, or enzymatically degradable.

[0008] In some aspects, the chamber encloses an additional substrate comprising an additional drug. In particular aspects, the method comprises releasing the additional drug from the additional substrate. In further aspects, the releasing the additional drug from the additional substrate is performed before or after the releasing the drug from the substrate. In certain aspects, the method comprises determining whether the additional drug diminishes an activity of the drug, enhances the activity of the drug, acts synergistically with the drug, alters a side-effect of the drug, or a combination thereof. In additional aspects, the method comprises measuring a phenotypic change of the cell and then releasing either the drug from the substrate or the additional drug from the additional substrate based on the measured phenotypic change. In certain aspects, the method further comprises measuring a phenotypic change of the cell subsequent to the releasing the drug from the substrate. In some aspects, the substrate is immobilized on a surface of the fluidic channel.

[0009] A further embodiment of the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymerprecursor within the fluidic channel, the chamber co- enclosing the cell with a substrate comprising the drug; and releasing the drug from the substrate, thereby allowing the drug to contact the cell. In some cases, the substrate is coupled to a surface of the fluidic channel.

[0010] In some aspects, the method further comprises fabricating the substrate on the surface of the fluidic channel, the fabricating comprising: inputting the drug into the fluidic channel; inputting an additional polymer precursor into the fluidic channel; and photopolymerizing the additional polymer precursor in the fluidic channel, thereby generating the substrate adhered to the surface of the fluidic channel and enclosing the drug. In certain aspects, the method further comprises fabricating the substrate on the surface of the fluidic channel, the fabricating comprising: inputting the drug into the fluidic channel, wherein the drug is coupled to an additional polymer precursor; and photopolymerizing the additional polymer precursor in the fluidic channel, thereby generating the substrate adhered to the surface of the fluidic channel and coupled to the drug. In additional aspects, the method further comprises fabricating the substrate on the surface of the fluidic channel, the fabricating comprising: spotting the drug and an additional polymer precursor on the surface of the fluidic channel; or grafting the additional polymer precursor to the surface of the fluidic channel in the presence of the drug. In some aspects, the drug is coupled to the additional polymer precursor. In further aspects, the fabricating is in the fluidic channel. In additional aspects, the fabricating is performed prior to formation of the fluidic channel.

[0011] In one aspect, the releasing comprises degrading the substrate. In another aspect, only a portion of the substrate is degraded, such that only a subset of instances of the drug comprised by the substrate are released from the substrate. In some such cases, the portion of the substrate is degraded by: shining a light on a fraction of an interior space of the chamber; inputting only a fraction of light required to completely degrade the substrate; inputting only a fraction of a concentration of a reagent required to completely degrade the substrate; or a combination thereof.

[0012] In a particular aspect, the substrate comprises a polymer or a hydrogel. In one aspect, the substrate is in the shape of a disc, a pillar, or a sphere. In a further aspect, the fluidic channel comprises a plurality of instances of the substrate comprising a plurality of doses of the drug, and the method further comprises synthesizing a plurality of chambers co-enclosing a plurality of cells with at least a subset of the plurality of the instances of the substrate and releasing the drug from at least the subset of the plurality of instances of the substrate. In another aspect, the fluidic channel comprises a plurality of instances of the substrate comprising the drug, and the method further comprises synthesizing a plurality of chambersco-enclosing a plurality of cells with at least a subset of the plurality of the instances of the substrate and releasing different amounts of the drug from at least the subset of the plurality of instances of the substrate.

[0013] In an additional aspect, the fluidic channel comprises an additional substrate comprising an additional drug, and the method further comprises synthesizing an additional chamber co-enclosing an additional cell with the additional substrate, and releasing the additional drug from the additional substrate. In a further aspect, the chamber co-encloses an additional substrate comprising an additional drug with the cell and the substrate. In certain aspects, the method further comprises releasing the additional drug from the additional substrate. In some aspects, the releasing the additional drug from the additional substrate is prior to, concurrent with, or subsequent to the releasing the drug from the substrate.

[0014] In a particular aspect, the substrate is enclosed within an additional chamber that is enclosed within the chamber, and wherein the cell is enclosed within the chamber and disposed outside of the additional chamber. In another aspect, the cell is enclosed within an additional chamber that is enclosed within the chamber, and the substrate is enclosed within the chamber and disposed outside of the additional chamber. In a certain aspect, the method further comprises degrading the additional chamber after the releasing the drug from the substrate.

[0015] In a further embodiment, the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell into a fluidic channel; inputting a polymer precursor and optionally an additional polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing a substrate comprising the drug; synthesizing an additional chamber with the polymer precursor or the additional polymer precursor, the additional chamber enclosing the chamber and the cell; releasing the drug from the substrate; and degrading the chamber, thereby allowing the drug to contact the cell.

[0016] In particular aspects, the method further comprises measuring a distance between the substrate and the cell, and using the distance to determine a dose of the drug that diffuses to the cell from the substrate following the releasing.

[0017] In an additional embodiment, the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell into a fluidic channel; measuring distances between the cell and each of one or more substrates in the fluidic channel, wherein each substrate of the one or more substrates comprises the drug; selecting a subset of the one or more substrates based at least in part on the measured distances of theone or more substrates; releasing at least a portion of the drug comprised by the subset of the one or more substrates; and incubating the cell, thereby allowing the dose of the drug to diffuse to the cell.

[0018] In certain aspects, the method further comprises inputting a polymer precursor into the fluidic channel, and synthesizing a chamber that encloses the cell with the polymer precursor within the fluidic channel. In additional aspects, the releasing comprises irradiating the subset of the one or more substrates with light. In particular aspects, non-selected substrates of the one or more substrates are not irradiated. In further aspects, the irradiating cleaves a bond or a linker between the drug and the subset of the one or more substrates. In another aspect, the irradiating degrades the subset of the one or more substrates. In an additional aspect, the selecting is further based at least in part on amounts or concentrations of the drug comprised by each substrate of the one or more substrates.

[0019] In a particular aspect, the method further comprises measuring distances between the cell and each of one or more additional substrates in the fluidic channel, wherein each substrate of the one or more additional substrates comprises an additional drug: selecting a subset of the one or more additional substrates based at least in part on the measured distances of the one or more additional substrates; releasing at least a portion of the additional drug comprised by the subset of the one or more additional substrates; incubating the cell, thereby allowing the dose of the additional drug to diffuse to the cell. In a certain aspect, the selecting the subset of the one or more additional substrates is further based at least in part on amounts or concentrations of the additional drug comprised by each substrate of the one or more additional substrates.

[0020] In an additional embodiment, the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting a polymer precursor into a fluidic channel; inputting the cell into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and a spatially-addressed nucleic acid sequence coupled to a surface of the fluidic channel; wherein at least a portion of the spatially-addressed nucleic acid sequence is hybridized to a drug-nucleic acid complex, the drug-nucleic acid complex comprising the drug coupled to a nucleic acid capture sequence that is complementary to at least a portion of the spatially-addressed nucleic acid sequence; and releasing the drug from the spatially-addressed nucleic acid sequence by: dehybridizing the nucleic acid capture sequence from the spatially-addressed nucleic acid sequence, cleaving the drug from the nucleic acid capture sequence, or a combination thereof; thereby allowing the drug to contact the cell.

[0021] In some aspects, the method further comprises, before the releasing the drug, inputting the drug-nucleic acid complex into the fluidic channel and hybridizing the nucleic acid capture sequence to the spatially-addressed nucleic acid sequence or the portion of the spatially-addressed nucleic acid sequence. In certain aspects, the inputting the drug-nucleic acid complex into the fluidic channel is prior to the inputting the polymer precursor and the cell into the fluidic channel. In further aspects, the cleaving the drug from the nucleic acid capture sequence converts the drug from a prodrug form to an active form of the drug. In additional aspects, the cleaving the drug from the nucleic acid capture sequence comprises chemical cleavage, enzymatic cleavage, thermal cleavage, pH-mediated cleavage, or photolysis.

[0022] In certain aspects, the method further comprises inputting an additional drug-nucleic acid complex into the fluidic channel, wherein the additional drug-nucleic acid complex comprises an additional drug coupled to an additional nucleic acid capture sequence. In further aspects, the additional drug-nucleic acid complex hybridizes to at least a portion of an additional spatially-addressed nucleic acid sequence coupled to the surface of the fluidic channel, and: the additional spatially-addressed nucleic acid sequence is disposed outside of the chamber, or the additional spatially-addressed nucleic acid sequence is co-enclosed with the cell and the spatially-addressed nucleic acid sequence within the chamber. In some aspects, the method comprises selectively cleaving the drug from the nucleic acid capture sequence, wherein the selectively cleaving the drug from the nucleic acid capture sequence does not cleave the additional drug from the additional nucleic acid capture sequence. In further aspects, the additional nucleic acid capture sequence is complementary to the spatially-addressed nucleic acid sequence or the portion of the spatially-addressed nucleic acid sequence.

[0023] A further embodiment of the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell and into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber co-enclosing the cell and a plurality of drugs coupled to a surface of the fluidic channel through a plurality of cleavable linkers; and selectively cleaving a cleavable linker of the plurality of cleavable linkers coupled to the drug, thereby allowing the drug to contact the cell.

[0024] In one aspect, the surface is a top surface of the fluidic channel or a bottom surface of the fluidic channel. In another aspect, the selectively cleaving comprises chemical cleavage, enzymatic cleavage, thermal cleavage, pH-mediated cleavage, or photolysis. In a particularaspect, the selectively cleaving converts the drug from a prodrug form to an active form of the drug.

[0025] In certain aspects, the method further comprises selectively cleaving an additional cleavable linker of the plurality of cleavable linkers, thereby releasing an additional drug of the plurality of drugs from the surface of the fluidic channel.

[0026] In further aspects, the method further comprises coupling the plurality of drugs to the surface of the fluidic channel, the coupling comprising: hybridizing a plurality of splint oligonucleotides to a plurality of spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel, wherein each splint oligonucleotide of the plurality of splint oligonucleotides comprises: a sequence complementary to at least a portion of a spatially-addressed nucleic acid sequence of the spatially-addressed nucleic acid sequences, and a drug-capture sequence; inputting the plurality of drugs into the fluidic channel, wherein each drug of the plurality of drugs is coupled to: a cleavable linker of the plurality of cleavable linkers, and a nucleic acid capture sequence complementary to the drug-capture sequence; incubating the plurality of drugs under conditions that permit hybridization between the drugcapture sequences of the plurality of splint oligonucleotides and the nucleic acid capture sequences coupled to the plurality of drugs; and ligating the plurality of spatially-addressed nucleic acid sequences to the plurality of nucleic acid capture sequences coupled to the plurality of drugs, thereby coupling the plurality of drugs to the surface of the fluidic channel.

[0027] In additional aspects, the method further comprises coupling the plurality of drugs to the surface of the fluidic channel, the coupling comprising: inputting the plurality of drugs into the fluidic channel, wherein each drug of the plurality of drugs is coupled to: a cleavable linker of the plurality of cleavable linkers, and a nucleic acid capture sequence complementary to at least a portion of a spatially-addressed nucleic acid sequence of a plurality of spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel; and incubating the plurality of drugs under conditions that permit hybridization between the plurality of spatially-addressed nucleic acid sequences and the nucleic acid sequences coupled to the plurality of drugs, thereby coupling the plurality of drugs to the surface of the fluidic channel.

[0028] An additional embodiment of the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell into a fluidic channel; inputting a prodrug of the drug into the fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidicchannel, the chamber enclosing the cell; and converting the prodrug into an active form of the drug, thereby allowing the drug to contact the cell.

[0029] In some aspects, the chamber encloses the prodrug with the cell. In further aspects, the chamber is impermeable to the prodrug, the drug, or the prodrug and the drug. In additional aspects, the inputting the prodrug into the fluidic channel is subsequent to the synthesizing the chamber, and wherein the prodrug, the drug, or the prodrug and the drug are sufficiently small to pass through a wall of the chamber. In particular aspects, the converting comprises cleaving a promoiety from the prodrug. In additional aspects, the converting comprises chemically modifying the prodrug. In certain aspects, the converting comprises photolysis, a chemical conversion, an enzymatic conversion, or a combination thereof.

[0030] In a particular aspect, the converting comprises photolysis, and the method further comprises determining a dose of the active form of the drug delivered to the cell based on: a distance between the photolysis and the cell; an intensity of the photolysis; a duration of the photolysis; a concentration of the prodrug in the fluidic channel; a rate of conversion of the prodrug to the active form of the drug during the photolysis; or a combination thereof. In some aspects, the converting is a physiological conversion performed by the cell. In certain aspects, the converting is subsequent to uptake of the prodrug by the cell.

[0031] In one aspect, a plurality of instances of the prodrug are input into the fluidic channel, and the converting comprises converting a subset of the instances of the prodrug into the active form of the drug.

[0032] In another embodiment, the present disclosure provides a method for analyzing a capture agent, the method comprising: inputting a cell into a fluidic channel; inputting an additional cell into the fluidic channel; inputting a polymer precursor into the fluidic channel; inputting the capture agent into the fluidic channel, wherein the capture agent is configured to simultaneously bind to the cell and the additional cell; incubating the cell, the additional cell, and the capture agent in the fluidic channel under conditions that allow the capture agent to bind to the and additional cells; and synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and the additional cell.

[0033] In a further embodiment, the present disclosure provides a method for analyzing a capture agent, the method comprising: inputting a cell into a fluidic channel; inputting an additional cell into the fluidic channel; inputting the capture agent into the fluidic channel; and determining whether the capture agent simultaneously binds to the cell and the additional cell.

[0034] In some aspects, the method further comprises inputting a polymer precursor into the fluidic channel and synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and the additional cell. In further aspects, the method further comprises capturing the capture agent.

[0035] In additional aspects, the method further comprises identifying the capture agent. In some aspects, the identifying comprises sequencing a nucleic acid barcode coupled to or cleaved from the capture agent. In particular aspects, the nucleic acid barcode encodes at least a portion of an amino acid sequence of the capture agent. In additional aspects, the identifying comprises binding a nucleic acid capture sequence coupled to or cleaved from the capture agent to a spatially-addressed nucleic acid sequence coupled to a surface of the fluidic channel, and detecting the binding of the nucleic acid capture- sequence to the spatially-addressed nucleic acid sequence. In further aspects, the detecting the binding of the nucleic acid capture-sequence to the spatially-addressed nucleic acid sequence comprises detecting a location of the capture agent in the fluidic channel. In another aspect, the detecting the binding of the nucleic acid capture-sequence to the spatially-addressed nucleic acid sequence comprises detecting a location of the capture agent in the fluidic channel. In a particular aspect, the binding the nucleic acid capture sequence to the spatially-addressed nucleic acid sequence occurs before the synthesizing the chamber. In a further aspect, the cell is input into the fluidic channel bound to the capture agent.

[0036] In another aspect, the method further comprises determining a binding affinity of the capture agent for the cell, the additional cell, or the cell and the additional cell. In an additional aspect, the method further comprises inputting a third cell into the fluidic channel, and determining a specificity of the capture agent for the cell and the additional cell relative to the third cell.

[0037] In another aspect, the method further comprises inputting a plurality of additional cells into the fluidic channel, and determining a specificity of the capture agent for the cell and the additional cell relative to the plurality of additional cells. In a certain aspect, the cell, the additional cell, and the plurality of additional cells are derived from a blood sample, a peripheral blood mononuclear cell (PBMC) sample, a buffy coat sample, a solid biopsy, a liquid biopsy, or a tumor section.

[0038] In a further aspect, the cell comprises an effector cell and the additional cell comprises a target cell of the effector cell. In a particular aspect, the method further comprises measuring cytotoxicity of the effector cell against the target cell. In one aspect, the cell comprises an immune cell. In another such aspect, the cell comprises a B cell, a basophil, acytokine induced killer cell, a cytotoxic T cell, a dendritic cell, an eosinophil, a granulocyte, a hypersegmented neutrophil, a lymphoid cell, a macrophage, a mast cell, a megakaryocyte, a monocyte, a myeloid cell, a natural killer cell, a neutrophil, a regulatory T cell, a T cell, a T helper cell, or a thrombocyte. In a particular aspect, the target cell comprises a cancer cell. In a specific aspect, the cell is a T cell and the capture agent is configured to bind to a PD-1 antigen on the T cell. In an additional aspect, the capture agent is configured to bind to an immune checkpoint molecule on the immune cell. In one such aspect, the immune checkpoint molecule is PD-1, CTLA-4, BTLA, LAG-3, TIM-3, TIGIT, CD27, CD28, CD112R, CD137, ICOS, 4-1BB, OX-40, or VISTA. In another aspect, the immune checkpoint molecule is PD-1.

[0039] In one aspect, the method further comprises determining whether the capture agent modulates cytotoxicity of the effector cell against the target cell.

[0040] In some aspects, the inputting the capture agent into the fluidic channel comprises inputting a particle comprising the capture agent into the fluidic channel. In one such aspect, the method further comprises releasing the capture agent from the particle. In particular aspects, the releasing comprises cleaving a bond or linker that couples the capture agent to a surface of the particle; the releasing comprises cleaving a bond or a linker that confines the capture agent within the particle; the capture agent is coupled to a surface of the particle, and the releasing comprises lysing the particle or disrupting the surface of the particle to release the capture agent from the surface of the particle; the capture agent is contained in an interior space within the particle, and the releasing comprises lysing or opening the particle to release the capture agent from the interior space within the particle; or the capture agent is noncovalently coupled to a surface of the particle, and the releasing comprises changing a condition in the fluidic channel to desorb the capture agent from the surface of the particle. In a specific aspect, the method comprises coupling the particle to a surface of the fluidic channel. In one such aspect, the particle comprises a nucleic acid capture sequence that hybridizes to a spatially-addressed nucleic acid sequence coupled to the surface of the fluidic channel. In certain aspects, the method comprises enclosing the particle in the chamber. In some aspects, the particle comprises a nanoparticle, a microparticle, a lipid particle, a vesicle, a ceramic particle, a metal particle, a metal oxide particle, a polymeric particle, a polysaccharide particle, a magnetic particle, a hydrogel particle, or a combination thereof.

[0041] In further aspects, the capture agent comprises an antibody, an antibody fragment, a nanobody, a minibody, an affibody, a diabody, a triabody, a tetrabody, an intrabody, a bis-scFv, a single domain antibody, a DARPin, an anticalin, a knottin, a receptor ligand, anaptamer, an adheron, or a combination thereof. In some such aspects, the capture agent comprises an antibody. In particular aspects, the antibody comprises a fragment crystallizable (Fc) region configured to bind to the cell and a fragment antigen-binding (Fab) region configured to bind to the additional cell. In further aspects, the antibody comprises a fragment antigen-binding (Fab) region configured to bind to the cell and an additional fragment antigen-binding (Fab) region configured to bind to the additional cell.

[0042] In a particular aspect, the capture agent is input into the fluidic channel after the cell and the additional cell are input into the fluidic channel.

[0043] In a further embodiment, the present disclosure provides a method for analyzing a capture agent, the method comprising: inputting a cell into a fluidic channel; inputting an additional cell into the fluidic channel; inputting a third cell into the fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell, the additional cell, and the third cell; incubating the cell, the additional cell, and the third cell under conditions in which the third cell expresses the capture agent; and measuring binding of the capture agent to the cell and the additional cell. In a particular aspect, the third cell comprises a B cell.

[0044] In an additional embodiment, the present disclosure provides a method for modulating intercellular interactions, the method comprising: inputting a cell into a fluidic channel comprising a surface comprising a capture agent configured to bind to the cell and an additional capture agent configured to bind to an additional cell; inputting the additional cell into the fluidic channel; incubating the cell and the additional cell in the fluidic channel under conditions that allow the capture agent to bind to the cell and the additional capture agent to bind to the additional cell; inputting a polymer precursor into the fluidic channel; and synthesizing a chamber with the polymer precursor that co-encloses the cell with the additional cell; wherein the cell is able to contact the additional cell when the cell is bound to the capture agent and the additional cell is bound to the additional capture agent.

[0045] In some aspects, the capture agent and the additional capture agent comprise nucleic acid capture sequences that are hybridized to spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel. In additional aspects, the capture agent is coupled to the surface by a linker and the additional capture agent is coupled to the surface by an additional linker. In one such aspect, the method further comprises cleaving the linker, the additional linker, or the linker and the additional linker.

[0046] In certain aspects, the capture agent and the additional capture agent are independently selected from an antibody, an antibody fragment, a nanobody, a minibody, anaffibody, a diabody, a triabody, a tetrabody, an intrabody, a bis-scFv, a single domain antibody, a DARPin, an anticalin, a knottin, a receptor ligand, an aptamer, an adheron, or a combination thereof.

[0047] In a particular aspect, the method further comprises determining whether the cell binds to the additional cell. In an additional aspect, the inputting comprises inputting a biological sample comprising the cell, the additional cell, and a plurality of additional cells into the fluidic channel. In a specific aspect, the capture agent and the additional capture agent do not bind to the plurality of additional cells.

[0048] An additional embodiment of the present disclosure provides a method for releasing a dose of a drug to a cell, the method comprising: inputting a plurality of cells into a fluidic channel; inputting a polymer precursor into the fluidic channel; inputting a substrate comprising the drug into the fluidic channel; synthesizing a plurality of chambers with the polymer precursor within the fluidic channel, wherein each chamber encloses a cell from the plurality of cells; measuring a characteristic of the enclosed cell in each chamber; and selectively releasing the drug in one or more of the plurality of chambers based on the measured characteristic of the enclosed cell in each chamber.

[0049] In certain aspects, the plurality of cells comprises a library of gene edited cells, wherein each of the gene edited cells comprises a guide RNA associated with the edited gene, the method further comprising: subsequent to the selectively releasing the drug from one or more of the plurality of chambers, determining a sequence of the guide RNA. In another aspect, the method further comprises measuring another characteristic of the enclosed cell in each chamber subsequent to the selectively releasing the drug; and correlating the sequence with the measured characteristic or the measured another characteristic.

[0050] In another embodiment, the present disclosure provides a method for releasing a dose of a drug to at least one cell of a plurality of cells, the method comprising: inputting the plurality of cells into a fluidic channel; inputting a polymer precursor into the fluidic channel; inputting a plurality of substrates into the fluidic channel, wherein each of the substrates comprise the drug; synthesizing a plurality of chambers with the polymer precursor within the fluidic channel, wherein each of the chambers encloses a cell of the plurality of cells; and releasing the drug from the plurality of substrates, thereby allowing the drug to contact the cell.

[0051] In certain aspects, each of the chambers co-encloses the cell of the plurality of cells with the substrate. In further aspects, each of the chambers is synthesized co-enclosing the cell of the plurality of cells with the substrate. In particular aspects, a wall of the chamber isimpermeable to the substrate. In another aspect, a wall of the chamber is impermeable to the drug. In certain aspects, the inputting the plurality of substrates comprises flowing the substrate through a wall of the chamber, wherein the substrate is sufficiently small to pass through a pore of the wall. In additional aspects, the inputting the plurality of substrates is before, after, or concurrent with chamber synthesis. In further aspects, the releasing comprises degrading the substrate, thereby releasing the drug from an interior portion of the substrate.

[0052] In certain aspects, a plurality of fluorophore complexes are coupled to the surface of the fluidic channel through an additional cleavable linker, wherein the selectively cleaving the cleavable linker also cleaves the additional cleavable linker to release at least a portion of the plurality of fluorophores from the surface of the fluidic channel, and wherein the method further comprises measuring a change in fluorescence intensity from at least a portion of the surface of the fluidic channel following the selectively cleaving. In an additional aspect, a plurality of fluorophore-quencher complex are coupled to the surface of the fluidic channel, wherein the fluorophore-quencher complex comprises a fluorophore coupled to a quencher through an additional cleavable linker, wherein the selectively cleaving the cleavable linker also cleaves the additional cleavable linker to release the quencher from the surface of the fluidic channel, wherein the fluorophore remains coupled to the surface of the fluidic channel following the selectively cleaving, and wherein the method further comprises measuring a change in fluorescence intensity from at least a portion of the surface of the fluidic channel following the selectively cleaving. In a specific aspect, the method further comprises determining an amount of the drug released from the surface of the fluidic channel based on the change in fluorescence intensity.

[0053] In some aspects, the substrate comprises a cleavable linker coupled to the drug. In additional aspects, the substrate is coupled to a surface of the fluidic channel. In further aspects, the substrate comprises a nucleic acid barcode. In particular aspects, the substrate comprises a capture probe. In specific aspects, the releasing comprises cleaving the cleavable linker by photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof.

[0054] In certain aspects, the method further comprises inputting an antibody or a lipid into the fluidic channel, wherein the antibody or the lipid is configured to bind to cells within the fluidic channel, and wherein the antibody or the lipid is coupled to a nucleic acid that comprises a nucleic acid sequence specific to the fluidic channel.

[0055] In additional aspects, the method further comprises determining a characteristic of the cell. In particular aspects, the characteristic is an mRNA expressed by the cell. In some such aspects, the determining comprises lysing the cell or at least a subset of the aggregate of the cells, capturing the mRNA on a capture element coupled to a surface of the flow cell, reverse transcribing the mRNA to generate a cDNA molecule that optionally comprises a barcode sequence derived from the capture element, and sequencing the cDNA molecule.

[0056] In certain aspects, the characteristic comprises a soluble factor secreted by the cell. In some such aspects, said detecting said soluble factor comprises disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell, and detecting the soluble factor bound to the capture surface. In further aspects, said detecting said soluble factor bound to said capture surface comprises contacting said soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody. In particular aspects, the capture surface is at least partially enclosed in the chamber.

[0057] In further aspects, the cell comprises an effector cell, and the characteristic comprises cytotoxicity. In particular aspects, the determining the cytotoxicity comprises measuring a rate or an occurrence of the effector cell killing additional cells. In additional aspects, the measuring comprises contacting the additional cells with a dye that indicates whether a cell of the additional cells is dead.

[0058] In certain aspects, the characteristic comprises activation. In some such aspects, the cell is activated by an additional cell. In further aspects, the additional cell is located outside of the chamber. In other aspects, the additional cell is co-enclosed in the chamber with the cell. In some aspects, the determining the activation comprises detecting expression of a surface marker, a morphology change, a cytotoxicity increase, a proliferation rate change, a soluble factor, a genomic sequence, an mRNA, or a combination thereof of the cell.

[0059] In particular aspects, the characteristic comprises proliferation. In one aspect, the cell comprises an edited gene and a guide RNA associated with the edited gene, the method further comprising determining a sequence of the guide RNA and correlating the sequence with the determined characteristic of the at least one cell.

[0060] In particular aspects, the polymer precursor comprises a crosslinker and a photoinitiator. In additional aspects, the polymer precursor comprises a porogen.

[0061] In certain aspects, the method further comprises mixing contents of the fluidic channel. In particular aspects, the mixing comprises: flowing liquid back and forth within the fluidic channel; applying an alternating magnetic field to the fluidic channel, thereby movingmagnetic species within the fluidic channel; applying an alternating electric field to the fluidic channel, thereby moving charged species within the fluidic channel; shaking the fluidic channel; or a combination thereof.

[0062] In some aspects, the method further comprises, after the synthesizing, inputting a water-immiscible fluid into the fluidic channel. In one such aspect, the water-immiscible fluid does not displace aqueous medium from the chamber. In another such aspect, the water-immiscible fluid does not enter the chamber. In a further aspect, the water-immiscible fluid encloses the chamber. In a particular aspect, the water-immiscible fluid surrounds the chamber. In an additional aspect, the water-immiscible fluid fills a space within the fluidic channel, wherein the space is outside of the chamber. In certain aspects, the chamber comprises a drug, and wherein the water-immiscible fluid inhibits diffusion of the drug from the chamber to an additional chamber. In further aspects, the chamber comprises a drug, and wherein the water-immiscible fluid inhibits diffusion of the drug through a wall of the chamber. In select aspects, the diffusion through the wall comprises diffusion from an inner surface of the wall to the outer surface of the wall. In some aspects, the chamber comprises a cell secretion, and wherein the water-immiscible fluid inhibits diffusion of the cell secretion from the chamber to an additional chamber. In additional aspects, the method further comprises replacing at least a portion of the water-immiscible fluid within the fluidic channel with an aqueous medium. In particular aspects, the water-immiscible fluid comprises a mineral oil, a silicone oil, a fluorocarbon oil, a perfluorinated oil, a vegetable oil, a bean oil, a PEG-perfluoroalkyl block copolymer, or a combination thereof. In certain aspects, a polymeric wall of the chamber comprises pores, and wherein the pores are about 0.5 nm to about 25 nm.

[0063] A further embodiment of the present disclosure provides a method for analyzing a cell, comprising: inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, wherein the chamber encloses the cell; inputting a water-immiscible fluid into the fluidic channel, wherein: i) the water-immiscible fluid does not displace aqueous medium from the chamber; ii) the water-immiscible fluid does not enter the chamber; iii) the water-immiscible fluid encloses the chamber; iv) the water-immiscible fluid surrounds the chamber; v) the water-immiscible fluid fills a space within the fluidic channel, wherein the space is outside of the chamber; or vi) a combination thereof; and analyzing the cell.

[0064] In some aspects, the method further comprises removing the water-immiscible fluid from the fluidic channel. In further aspects, the chamber comprises a cell secretion, and thewater-immiscible fluid inhibits diffusion of the cell secretion from the chamber to an additional chamber in the fluidic channel.

[0065] In some aspects, the water-immiscible fluid comprises a mineral oil, a silicone oil, a fluorocarbon oil, a perfluorinated oil, a vegetable oil, a bean oil, a PEG-perfluoroalkyl block copolymer, or a combination thereof.

[0066] Further embodiments of the present disclosure provide a method comprising: a) forming a chamber in a fluidic device, wherein the chamber comprises one or more walls, wherein the one or more walls extend from a top surface of the fluidic device to a bottom surface of the fluidic device, wherein the one or more walls comprise a plurality of pores; and b) forming a layer on the one or more walls, wherein the layer at least partially blocks at least a pore of the plurality of pores on the one or more walls.

[0067] In some aspects, the chamber comprises a cell. In further aspects, comprises polymerizing one or more polymer precursors, thereby forming the one or more walls of the chamber. In certain aspects, the polymerizing comprises applying light to the one or more polymer precursors. In additional aspects, a) comprises selectively encapsulating at least a subset of cells of a plurality of cells within the chamber. In select aspects, the subset of cells comprises at least one cell. In particular aspects, the chamber comprises aqueous medium. In particular aspects, the layer inhibits a secretion of the cell from passing through at least a pore of the plurality of pores. In further aspects, the method further comprises removing the layer, thereby unblocking at least a pore of the plurality of pores. In certain aspects, the layer comprises a water-immiscible fluid. In one aspect, the water-immiscible fluid comprises a mineral oil, a silicone oil, a fluorocarbon oil, a perfluorinated oil, a vegetable oil, a bean oil, a PEG-perfluoroalkyl block copolymer, or a combination thereof.

[0068] In another aspect, the method further comprises releasing a dose of a drug to the cell. In a certain aspect, the releasing comprises: inputting a substrate comprising the drug into the fluidic device, and releasing the drug from the substrate, thereby allowing the drug to contact the cell. In one aspect, the chamber comprises (i) the cell or the subset of cells and (ii) the substrate. In a further aspect, a) comprises selectively encapsulating (i) the cell or the subset of cells and (ii) the substrate within the chamber. In some aspects, the one or more walls of the chamber are impermeable to the substrate or the drug. In particular aspects, the substrate or the drug are sufficiently small to pass through at least a pore of the plurality of pores. In select aspects, the releasing comprises degrading the substrate, thereby releasing the drug from an interior portion of the substrate. In certain aspects, the degrading comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymaticdegradation, or a combination thereof. In another aspect, only a portion of the substrate is degraded.BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The novel features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0070] FIG. 1 is a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments.

[0071] FIG. 2A is an illustration of a portion of a system as provided herein including an energy source, according to some embodiments.

[0072] FIG. 2B is an illustration of a polymer matrix being formed around a biological component in a portion of a system as provided herein, according to some embodiments;

[0073] FIG. 2C is an illustration of a method of forming a polymer matrix around a biological component in a system as provided herein, according to some embodiments

[0074] FIG. 3A is an illustration of a top view of the bottom layer of a flow cell without any spatial barcoded oligonucleotides.

[0075] FIG. 3B is an illustration of a top view of the spacer layer with a cut-out region suitable for use as part of the flow cell.

[0076] FIG. 3C is an illustration of a top view of the top layer of the flow cell where the top layer has an inlet and outlet opening.

[0077] FIG. 3D is an illustration of a cross-sectional side view of a top layer and a bottom layer sandwiching a spacer layer to form multiple channels of a flow cell.

[0078] FIG. 4A is an illustration of a system with a flow cell and an imaging apparatus that are capable of implementing methods of the present disclosure.

[0079] FIG. 4B is an illustration of a flow cell, including a blown-up view of a portion of a channel containing cells disposed in hydrogel chambers.

[0080] FIG. 5 is a schematic of a computer system that may be programmed or otherwise configured to perform methods described herein.

[0081] FIG. 6A is an illustration of a method for releasing a dose of a drug from a substrate to a cell.

[0082] FIG. 6B is an illustration of a method for releasing doses of drugs from multiple substrates to a cell.

[0083] FIG. 6C is an illustration of a method for releasing a dose of a drug within a chamber and then removing the chamber to release the dose of the drug to a nearby cell.

[0084] FIG. 7 is an illustration of a method of releasing a dose of a drug from a substrate to a cell.

[0085] FIG. 8 is an illustration of a method for localizing multiple drugs to selected regions within a fluidic channel and then releasing the drugs to cells within the fluidic channel.

[0086] FIG. 9 is an illustration of a method for delivering a dose of an active form of a drug to a cell by converting a prodrug into the active form of the drug in proximity to the cell.

[0087] FIG. 10 is an illustration of a method for simultaneously coupling two cells to a bispecific capture agent.

[0088] FIG. 11 is an illustration of a method for expressing a multispecific capture agent in proximity to cells targeted by the multispecific capture agent.

[0089] FIG. 12 is an illustration of a method for sequentially releasing individual drugs to a cell within a fluidic channel.

[0090] FIG. 13 is an illustration of a method for binding multiple cells to a capture agent and binding the capture agent to a surface of a fluidic channel.

[0091] FIG. 14 is an illustration of a method for binding multiple cells to a capture agent-functionalized particle coupled to a surface of a fluidic channel.

[0092] FIG. 15 is an illustration of a method for releasing a capture agent from a particle coenclosed with multiple cells that are targeted by the capture agent.

[0093] FIG. 16 is an illustration of a method for calculating a dose of a drug released to multiple cells in proximity to a degraded drug-containing substrate.

[0094] FIG. 17 is an illustration of a method for co-localizing two cells within a fluidic channel.

[0095] FIG. 18A is a fluorescence image of a fluidic channel surface with multiple fluorophore-containing regions.

[0096] FIG. 18B is a fluorescence image of the fluidic channel surface shown in FIG. 18A following fluorophore photobleaching along portions of the fluidic channel surface.

[0097] FIG. 18C is a plot of fluorescence intensities of the fluidic channel surface shown in FIGS. 18A-B

[0098] FIG. 19A is a schematic of a method for spatially-controlled fluorophore-quencher conjugate delivery and photoactivation through cleavage of the quencher from the fluorophore-quencher conjugate.

[0099] FIG. 19B is a plot of fluorophore-quencher conjugate fluorescence intensity as a function of illumination time, wherein the illumination cleaved the quencher group from the fluorophore-quencher conjugate.

[0100] FIG. 19C is a pair of fluorescence images of a fluidic device surface with fluorophore-quencher conjugates following 30 seconds (left) and 300 seconds (right) of illumination to cleave the quencher from the fluorophore-quencher conjugates.

[0101] FIG. 20A is a brightfield image of a 50 pm cell-containing, aqueous media-filled chamber following 24 hours of incubation within oil.

[0102] FIG. 20B is a brightfield image of a 100 pm cell-containing, aqueous media-filled chamber following 24 hours of incubation within oil.

[0103] FIG. 20C is a plot of cell viabilities following 48 hour incubations within chambers of varying sizes and surrounded by oil or media.

[0104] FIG. 21A is a fluorescence image of a doxorubicin and cell-containing chamber following enclosure of the chamber within an oil phase.

[0105] FIG. 21B is a fluorescence image of a cell-containing chamber following enclosure of the chamber within an oil phase.DETAILED DESCRIPTION

[0106] Disclosed herein are systems and methods for titrating drugs to cells within fluidic channels. Drug doses may be selectively released from drug-containing “drug depots” in proximity to individual cells or collections of cells. As used herein, a drug depot may be a structure or a portion of a fluidic channel that contains a plurality of instances of a drug. Examples of drug depots include degradable (e.g., photodegradable) substrates such as photocleavable gels in which one or more drugs is encapsulated, solid structures to which a drug is bound, such as a particle coupled to a plurality of instances of a drug through one or more cleavable bonds or linkers, and a portion of a fluidic device surface that contains a plurality of oligonucleotide capture probes coupled to a plurality of instances of one or more drugs. A drug may be inaccessible or inactive (e.g., in prodrug form) while it is bound to or contained within a drug depot, such as a substrate. The drug may be selectively released from the drug depot, for example by degrading at least a portion of a drug depot to release a drug contained within the drug depot, or by cleaving a linker that couples a drug to the drug depot.In this context, “selectively” can denote that the time and optionally the degree of substrate release is controlled. For example, when drug release is mediated through photocleavage, the timing of the drug release can be selected by controlling the duration, location, intensity, wavelength, and timing of substrate irradiation. Similarly, when drug release is mediated by a catalyst, the timing and concentration of catalyst input into the fluidic device that contains the substrate can dictate the timing, rate, and degree of drug release from a substrate.

[0107] For example, in some aspects, the present application provides a method for releasing a dose of a drug to a cell, the method comprising: inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber co-enclosing the cell and a drug depot comprising a drug; and releasing the drug from the drug depot, thereby allowing the drug to contact the cell. In some cases, the chamber co-encloses the cell with a plurality of instances of the drug depot comprising the drug. In such cases, the method may comprise releasing the drug from a subset of the plurality of instances of the drug depot, or may comprise releasing the drug from all instances of the drug depot. In some cases, the chamber co-encloses the cell with a plurality of drug depots comprising a plurality of drugs. In such cases, the method may comprise releasing a subset of the plurality of drugs from a subset of the plurality of drug depots, or may comprise releasing the plurality of drugs from the plurality of drug depots. In general, all instances, a subset of instances, or no instances of a drug may be released from a drug depot. For example, if a chamber encloses three drug depots, all instances of drugs comprised by the drug depot may be released, 50% of instances of drugs comprised by the additional drug depot may be released, and no drug may be released from the third drug depot. Such selective release may be mediated, for example, by selectively heating (e.g., by selectively modulating an array of heaters below the fluidic channel) or irradiating (e.g., by generating a spatially-defined light intensity pattern with a digital micromirror device (DMD)) portions of a fluidic channel. A drug depot may comprise a single type of drug or multiple types of drugs.

[0108] As used herein, a substrate can denote a structure that contains or is coupled to a plurality of instances of one or more drugs. Examples of substrates consistent with the present disclosure include microparticles, nanoparticles, lipid particles, gels, polymer matrices, nanomaterials (e.g., carbon nanotubes), and the like. In some examples, the substrate may include a nucleic acid polymer. The substrate may contain the drug. For example, the drug may be enclosed within an interior portion of the substrate, such as a cavity within a particle or space between neighboring polymer chains within a polymer matrix. Thepolymer matrix may be cleavable. Examples of polymers utilizable in such substrates include polymers that have hydrolytically degradable groups such as lactic acid, glycolic acid, and caprolactone; polymers that have enzymatically degradable groups or peptides such as gelatin, collagen, and fibrin; polysaccharides such as dextran, hyaluronic acid, and alginate; polymers that contain one or more disulfide groups; polymers that contain one or more diselenide groups; polymers that contain one or more vicinal diol pairs; polymers that contain one or more silyl ether group; and polymers that contain one or more carbamate groups. The substrate may also be coupled to a drug through a cleavable bond or linker. For example, the substrate may be a nanoparticle or microparticle coupled to drug molecules through cleavable linkers or a polymer matrix that contains a drug within its polymer backbone or is coupled to drug molecules through polymer side chains. For example, the substrate may include a capture probe bound to a surface of the fluidic channel, such as an oligonucleotide or other nucleic acid polymer, and the drug molecule is released by cleaving a linker coupling the drug molecule to the capture probe.

[0109] As used herein, a drug can be a species that alters the physiology of a cell or organism. It will be understood that a drug, in the context of the present disclosure, can refer to a known drug or to a potential drug or drug candidate. A drug can also be a species that alters the activity of another drug. For example, a method may present varying dosages of a drug and a drug blocker or drug sensitizer to cells. The term “drug” may thus be used interchangeably herein with terms such as “therapeutic agent”, “medication”, “pharmacological agent”, and “active agent”.

[0110] In one aspect, the present application provides a method for releasing a dose of a drug to a cell that includes inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; inputting a substrate comprising the drug into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell; and releasing the drug from the substrate, thereby allowing the drug to contact the cell.[oni] In many cases, the chamber co-encloses the cell with the substrate. The co-enclosure may force the cell and substrate into proximity, thereby imposing a maximum distance between the cell and substrate at the time of drug release. Thus, when drugs are simultaneously or successively released from a large number of substrates - for example 500 to 5xl06substrates- to chamber-enclosed cells within a fluidic channel, rate, timing, and doses of the drugs may be controlled and optionally may be standardized for separate cells in distinct chambers.

[0112] The substrate may be input into the fluidic channel before, after, or concurrently with chamber synthesis. In some cases, the chamber is synthesized around (that is co-enclosing) the cell and substrate. In such cases, the chamber may be impermeable to the substrate.Alternatively, the substrate may diffuse into the chamber subsequent to chamber synthesis around the cell. For example, inputting the substrate into the fluidic channel can comprise flowing the substrate through a wall of the chamber, wherein the substrate is sufficiently small to pass through a pore of the wall. In certain cases, the chamber is impermeable to the drug. However, even when the chamber is permeable to the drug, the chamber may slow the rate at which the drug diffuses away from the cell.

[0113] The drug may be released once or multiple times to a cell. In some cases, the drug is released at defined time intervals spanning minutes, hours, or days. For example, a fluidic channel may comprise a plurality of chambers that separately enclose a plurality of cells with a plurality of instances of the substrate, and the drug can be released in separate doses and intervals (e.g., every 15 minutes to every 7 days) in separate chambers to identify an optimal dosing regimen for the drug. As the present invention enables simultaneous enclosure and analysis of thousands, tens of thousands, or hundreds of thousands of cells, complex dosing parameters can be rapidly analyzed and associated with specific cellular responses.

[0114] Drug release may comprise degrading the substrate to release the drug from an interior portion of the substrate. For example, the drug may be released from an interior portion of a particle or hydrogel substrate through degradation of that substrate. In some cases, the drug is contained within a cavity or chamber of the substrate, such that substrate degradation releases all instances of the drug from the substrate. Alternatively, the drug may be dispersed throughout interior portions of the substrate, such that the amount of drug released from the substrate is proportional to substrate degradation. For example, the substrate may comprise a polymer matrix within which the drug is noncovalently dispersed, such that partial degradation of the substrate releases instances of the drug only from degraded portions of the substrate. In such cases, a dose of the drug released to the cell may be controlled by degrading only a selected portion of the substrate. As non-limiting examples, the substrate may be partially degraded by irradiating a fraction of an interior space of the chamber with light; irradiating only the portion of the substrate with light; inputting only a fraction of light required to completely degrade the substrate; inputting only a fraction of light required to release all instances of the drug comprised by the substrate; inputting only a fraction of a concentration of a reagent required to completely degrade the substrate;inputting only a fraction of a concentration of a reagent to release all instances of the drug comprised by the substrate; or a combination thereof.

[0115] Drug release can also comprise cleaving a bond or a linker that couples the drug to the substrate. The drug may be coupled to an exterior portion of the substrate by the bond or the linker, such as the surface of particle. Alternatively or in addition thereto, the drug may be coupled to an internal surface or structure (e.g., a polymer chain) within the substrate, and may diffuse out of the substrate following bond or linker cleavage. For example, the drug may be coupled to the backbone of a polymer matrix substrate through photocleavable bonds, such that cleavage of the drug from the substrate leaves the backbone of the polymer matrix intact.

[0116] In some cases, drug release (through, for example, substrate degradation or bond or linker cleavage) comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof.

[0117] An example of drug release from a substrate co-enclosed with a cell within a chamber is depicted in FIG. 7. In this example, the substrate (702) is a particle that is coupled to a drug (704) through a cleavable linker (703). The substrate (702) may be flowed into proximity with a cell (701) and then co-enclosed (710) with the cell (701) within a chamber (705), for example by synthesizing a wall or walls of the chamber (705) surrounding the substrate (702) and cell (701). In such cases, the substrate (702), either on its own or in combination with the linker (703) and drug (704), may be prohibitively large to flow through pores in a wall or walls of the chamber (705). The substrate (702) may also be flowed into the chamber (705). In such cases, the chamber (705) may be synthesized around the cell (701), and, as depicted by arrow (720), the substrate (702) can be flowed through pores in a wall or walls of the chamber (705). The substrate (702) may optionally be coupled to a surface. For example, the substrate may be coupled to a nucleic acid capture sequence that hybridizes to a spatially-addressed nucleic acid sequence coupled to a surface of a fluidic device. The drug (704) can then be released (730) from the substrate (702) by cleaving the cleavable linker (703). The chamber (705) may comprise a low permeability to the drug (704), and may thereby localize the drug within the chamber (705) and in proximity to the cell (701).

[0118] A chamber may enclose multiple substrates. The substrates may be identical, or may contain different drugs and / or concentrations of drugs. In some cases, a chamber encloses multiple instances of the substrate that comprise the same amount of the drug. As a further example, a chamber may enclose multiple instances of the substrate that contain different amounts of the drug. When a chamber encloses two or more instances of the substrate, thedrug may be selectively released from individual instances of the substrate, for example by selectively photodegrading or photocleaving all instances or a subset of instances of the drug from individual instances of the substrate. For example, if a chamber encloses three instances of the substrate, the first instance of the substrate may be fully photodegraded, half of the second instance of the substrate may be photodegraded, and the third instance of the substrate may be left undegraded.

[0119] Similarly, a fluidic channel may comprise multiple instances of the substrate that are enclosed in separate chambers and contain different or identical amounts of the drug.Following chamber synthesis, a controlled dose of the drug can be generated by releasing the drug from a selected subset of the instances of the substrate. Similarly, multiple substrates may be configured for different forms of drug release. For example, a fluidic channel may comprise a substrate configured for photodegradation and an additional substrate configured for hydrolase-mediated cleavage.

[0120] A chamber may also enclose a plurality of substrates that contain different drugs. The drugs may be simultaneously or sequentially released to enclosed cells to identify possible synergistic or inhibitory behavior between two or more drugs. In particular, the method can include determining whether an additional drug diminishes an activity of the drug, enhances the activity of the drug, acts synergistically with the drug, alters a side-effect of the drug, or a combination thereof. For example, the method can include measuring a phenotypic change of the cell following the release of the drug and / or additional drug such as a change in cell surface marker expression, mRNA expression, morphology, activation, cytotoxicity, or proliferation.

[0121] A chamber may be synthesized co-enclosing a plurality of substrates that comprise a plurality of drugs. Alternatively or additionally, one or more substrates of the plurality of substrates may be added to the chamber following its synthesis. In some cases, the chamber encloses the substrate comprising the drug and an additional substrate comprising an additional drug. The additional drug can be partially or completely released from the additional substrate. For example, a fluidic channel may comprise a plurality of chambers that each enclose a cell, an instance of the substrate comprising the drug, and an instance of the additional substrate comprising the additional drug. In such cases, variable amounts of the first and additional drugs can be released in separate chambers to ascertain the effects, individually and in combination, of first and additional drug dosing.

[0122] The first and additional drugs can be released simultaneously or sequentially. The additional drug may be released at the same time as the drug, or may be released following apredetermined time delay subsequent to drug release from the substrate. When a fluidic channel comprises a plurality of chambers that separately enclose a plurality of cells with a plurality of instances of the first and additional substrates, the delay between first and additional drug release may be varied between chambers to assess the effects of first and additional drug dose timing on the cells. Dose size and timing can be assessed in a similar, multiplexed fashion for 3, 4, 5, or more drugs.

[0123] In some cases, a substrate is disposed on and optionally adhered to a surface of a fluidic device. The substrate can contain a controlled amount of one or more drugs that may be released by degrading the substrate or cleaving a bond or linker between the substrate and the drug. As an example, a method for releasing a dose of a drug to a cell may include inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber co-enclosing the cell with a substrate comprising the drug; and releasing the drug from the substrate, thereby allowing the drug to contact the cell. The substrate may be coupled to a surface of the fluidic channel. For example, the substrate may comprise a polymer matrix that is attached to the fluidic channel surface.

[0124] In some cases, the releasing comprises degrading the substrate. In such cases, a controlled dose of the drug may be released by degrading only a portion of the substrate, such that only a subset of instances of the drug comprised by the substrate are released from the substrate. The portion of the substrate may be selectively degraded by, for example, shining a light on a fraction of an interior space of the chamber (e.g., irradiating only a portion of the substrate); inputting only a fraction of light required to completely degrade the substrate; inputting only a fraction of a concentration of a reagent required to completely degrade the substrate; or a combination thereof.

[0125] An example of this method is provided in FIG. 6A, which depicts a cell (601) in a fluidic device channel that includes a substrate (602) that contains a drug (603). The substrate (602) may be a degradable polymeric structure in which the drug (603) is embedded or covalently attached. The cell (601) and the substrate (602) can be co-enclosed within a chamber (611) such as a chamber defined by a cylindrical hydrogel wall that is synthesized (610) around the cell (601) and substrate (602) and extends between top and bottom surfaces of the fluidic device channel. The chamber (611) may be impermeable to the drug (e.g., contain walls with pores that are smaller than the drug) such that drug (603) released from the substrate remains co-enclosed with the cell (601). The substrate (602) may be degraded (620) to release the drug (603) into the chamber (611). The substrate (602) may be fully degraded,or may be partially degraded, leaving a residual portion of substrate (621) in the chamber. The degree to which the substrate (602) is degraded (620) can be controlled so that a predetermined amount of the drug (603) is released. For example, when the degrading (620) comprises photolysis, the intensity, duration, and wavelength of light may be selected to degrade (620) a predetermined amount of the substrate (602) to thereby release a controlled dose of the drug (603). Similarly, when the degrading (620) comprises photolysis, light can be applied to only a portion of the substrate (602), such as 25%, 50%, or 75% of the cross-sectional area of the substrate (602) to degrade only a fraction of the substrate (602), and commensurately to release only a portion of the drug (603) from the substrate (602).Following drug (603) release, the cell (601) may be profiled for transcriptomic, surfaceomic, secretomic, viability, or other phenotypic characteristics.

[0126] A fluidic device channel may comprise hundreds or thousands of identically prepared chambers (611) with cells (601) and substrates (602). By varying the amount of substrate degradation within each chamber, this type of system may be used to study drug (603) dosing on individual cells or consortias of cells (e.g., each chamber may contain 2 cells, 3 cells, or a variable number of cells such as 2-20 cells). For example, a fluidic device channel can comprise about 1000 chambers (611) that each enclose a single substrate (602) and a single cell (601). The degree of substrate (602) degradation (620), and thus drug release, in each chamber may vary from between about 1% and 100%. Similarly, instances of the substrate (602) in different chambers (611) may include different amounts of the drug (603). In such cases, each instance of the substrate (602) that is disposed within a chamber (611) may be degraded to release varying amounts of the drug (603) to different cells (601). Following drug (603) release, the cells (601) can be stained with a vital dye to determine whether they survived drug (603) exposure. Cell (601) survival can then be correlated to drug (603) dose. Alternatively or in addition thereto, one or more characteristics of the cells (601) such as a surfaceome, secretome, activation status, or morphology can be monitored before, during, and / or after drug (603) exposure.

[0127] The method can be performed sequentially or in parallel on a plurality of cells in the fluidic channel. For example, in some cases, the fluidic channel comprises a plurality of instances of the substrate comprising the drug, and the method further comprises synthesizing a plurality of chambers co-enclosing a plurality of cells with at least a subset of the plurality of the instances of the substrate and releasing varying amounts of the drug from at least the subset of the plurality of instances of the substrate.

[0128] Furthermore, a fluidic device can also include multiple substrates that contain different drugs. In some cases, the fluidic channel comprises an additional substrate comprising an additional drug. In such cases, the method can further comprise synthesizing a second chamber co-enclosing an additional cell with the additional substrate, and releasing the additional drug from the additional substrate. Similarly, the chamber can co-enclose an additional substrate comprising an additional drug with the cell and the substrate, and the method can optionally include releasing the additional drug from the additional substrate. The releasing the additional drug from the additional substrate can be prior to, concurrent with, or subsequent to the releasing the drug from the substrate.

[0129] For example, as depicted in FIG. 6B, a fluidic device can contain two or more substrates (602, 604) that contain different drugs (603, 605). The two or more substrates (602, 604) can be in close proximity such that these substrates (602, 604) can be co-enclosed in a single chamber (611), and can be selectively released (both in terms of timing and dosage) to facilitate controlled dosing assays on individual cells or consortias of cells. While FIG. 6B depicts a chamber (611) that encloses two substrates (602, 604), a chamber may contain 3, 4, 5, 6, 7, 8, 9, 10, or more substrates that may each be selectively degraded (620, 630) to release a drug (603, 605).

[0130] In the multi-substrate drug-release assay depicted in FIG. 6B, a substrate (602) that contains a drug (603) and an additional substrate (604) that contains an additional drug (605) are attached to a surface of a channel of a fluidic device. The two substrates (602, 604) can be in close proximity within the fluidic channel to facilitate co-encapsulation within a chamber (611). For example, the chamber can be synthesized surrounding the substrate (602), the additional substrate (604), and a cell (601). The channel can include a large number of instances of the two substrates (602, 604), such that a plurality of cells can be individually encapsulated with an instance of the substrate (602) and an instance of the additional substrate (604).

[0131] The substrate (602) and the additional substrate (604) can then be degraded (620, 630) within the chamber (611), either simultaneously or at separate times. For example, when the substrate (602) and the additional substrate (604) are photodegradable, they may be separately or collectively irradiated to release drugs (603, 605) therefrom. The substrate (602) and the additional substrate (604) can be partially degraded (620, 630), leaving nondegraded portions of these substrates (621, 631). The degree to which the substrate (602) and the additional substrate (604) are degraded (620, 630) can be controlled to release predetermined amounts of the first and additional drugs (603, 605) within the chamber (611). Alternatively,the substrate (602) and / or the additional substrate (604) can be fully degraded (620, 630). For example, the substrate (602) and the additional substrate (604) can be synthesized with predetermined amounts of the first and additional drugs (603, 605), such that complete degradation (620, 630) of the substrate (602) and the additional substrate (604) releases the predetermined amounts of the first and additional drugs (603, 605) within the chamber (611). The cell (601) may then be analyzed. In some cases, the cell (601) is contacted with a vital dye that indicates whether the cell is alive or dead. One or more additional characteristics of the cell (601) such as surfaceome, secretome, activation status, or morphology can also be measured and correlated with doses of the first and additional drugs (603, 605).

[0132] The substrate, the cell, and optionally further cells and / or substrates may be partitioned within a subspace of a first chamber by a second chamber that is enclosed within the first chamber. In some cases, the substrate is enclosed within an additional chamber that is enclosed within the chamber, and the cell is enclosed within the chamber and disposed outside of the additional chamber. In other cases, the cell is enclosed within an additional chamber that is enclosed within the chamber, and the substrate is enclosed within the chamber and disposed outside of the additional chamber. The additional chamber may be degraded after the drug is released from the substrate, for example to release a chamber-impermeable drug to the cell. For example, method for releasing a dose of a drug to a cell can include inputting the cell into a fluidic channel; inputting a first polymer precursor and optionally a second polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber enclosing a substrate comprising the drug; synthesizing a second chamber with the first polymer precursor or the second polymer precursor, the second chamber enclosing the first chamber and the cell; releasing the drug from the substrate; and degrading the first chamber, thereby allowing the drug to contact the cell.

[0133] An example of this method is depicted in FIG. 6C. In this figure, a substrate (802) that contains a drug (803) is enclosed in a first chamber (804) and a cell (801) is enclosed in a second chamber (805) that circumscribes the first chamber (804). The first chamber (804) is impermeable to the drug (803). Accordingly, when the substrate (802) is degraded or partially degraded (810), releasing the drug (803) into the first chamber (804) and optionally leaving a residual undegraded portion of the substrate (811), the drug (803) remains in the first chamber. The first chamber (804) can be degraded (820), releasing the drug to the cell (801).

[0134] In certain designs, the substrate (and optionally additional substrates) is fabricated on the surface of the fluidic channel. The fabricating may be prior or subsequent to fluidicchannel formation. For example, the substrate may be fabricated on a glass slide that is later incorporated into a fluidic device as an upper or lower surface of the fluidic channel. In some cases, fabricating the substrate on the surface of the fluidic channel comprises inputting the drug into the fluidic channel; inputting an additional polymer precursor into the fluidic channel; and photopolymerizing the additional polymer precursor in the fluidic channel, thereby generating the substrate adhered to the surface of the fluidic channel and enclosing the drug. In some cases, fabricating the substrate on the surface of the fluidic channel comprises inputting the drug into the fluidic channel, wherein the drug is coupled to an additional polymer precursor; and photopolymerizing the additional polymer precursor in the fluidic channel, thereby generating the substrate adhered to the surface of the fluidic channel and coupled to the drug. In some cases, fabricating the substrate on the surface of the fluidic channel comprises spotting the drug and an additional polymer precursor on the surface of the fluidic channel. In some cases, fabricating the substrate on the surface of the fluidic channel comprises grafting the additional polymer precursor to the surface of the fluidic channel in the presence of the drug. The drug can be coupled to the additional polymer precursor, for example within the backbone or a side chain of a crosslinker through one or more cleavable bonds or linkers, or noncovalently contained within the substrate. Such fabrication methods may be particularly suitable for generating polymeric, and in particular hydrogel substrates. The substrate may comprise any geometry, for example a disc, pillar, or sphere. In particular cases, the substrate is a portion of a sphere or spheroid coupled to a surface of the fluidic channel. Any of the foregoing fabrication methods may be repeated with additional (e.g., second, third, fourth, etc.) drugs and the same additional polymer precursor or with a different additional polymer precursor.Calculate Drug Dose

[0135] The dose of a drug delivered to a cell may be determined by measuring a distance between a substrate and the cell. Under quiescent conditions, drug diffusion between the cell and substrate can be modeled according to the Stokes-Einstein equation as detailed in Miyamoto and Shimono, Molecules, 2020; 25(22):5340, using the drug size, solution viscosity, and solution temperature to determine its diffusion coefficient. The instantaneous concentration of the drug at the cell can then be calculated based on its diffusion coefficient, initial distance from the cell (i.e., the distance between the cell and the substrate from which the cell is released), concentration (i.e., the amount of drug released from the substrate), the dimensions of the fluidic device, and diffusion time. The aggregate dose of a drug thatreaches the cell can similarly be calculated by summing instantaneous drug doses at the cell over time.

[0136] This concept is illustrated in FIG. 16, which depicts a top-down view of a fluidic channel (1700) that comprises cells (1701) and drug-containing substrates (1702). Drug may be released to the cells (1701) by degrading one or more of the substrates (1702). Upon substrate degradation and concomitant drug release (1710), the concentration of the drug at one or more cells can be calculated as a function of their distances from the substrate (1702) and the amount of drug released from the substrate (1702). Doses of the drug that diffuse to individual cells can be calculated using measured distances (1712, 1713, 1714) between each cell (1701) and the degraded substrate (1711).

[0137] Similarly, a subset of drug-containing substrates within a fluidic channel may be selected for drug-release to achieve a desired drug concentration at one or more cells. Such a method can include inputting the cell into a fluidic channel; measuring distances between the cell and each of one or more substrates in the fluidic channel, wherein each of the one or more substrates comprises the drug; selecting a subset of the one or more substrates based at least in part on the measured distances of the one or more substrates; releasing at least a portion of the drug comprised by the subset of the one or more substrates; and incubating the cell, thereby allowing the dose of the drug to diffuse to the cell. The method can further include inputting a polymer precursor into the fluidic channel, and synthesizing a chamber that encloses the cell with the polymer precursor within the fluidic channel. Chamber synthesis may be performed before, after, or concurrently with drug release. In many cases, drug release is performed by irradiating the subset of the one or more substrates, for example to photodegrade the substrate or cleave linkers between the drug from the subset of the one or more substrates. Light may be selectively input into the fluidic channel to irradiate the subset of the one or more substrates and not irradiate non-selected substrates of the one or more substrates.

[0138] Doses may be calculated or selected for multiple drugs. For example, the disclosed methods can further include measuring distances between the cell and each of one or more additional substrates in the fluidic channel, wherein each substrate of the one or more additional substrates comprises an additional drug; selecting a subset of the one or more additional substrates based at least in part on the measured distances of the one or more additional substrates; releasing at least a portion of the additional drug comprised by the subset of the one or more additional substrates; and incubating the cell, thereby allowing the dose of the additional drug to diffuse to the cell. As with the drug, the selecting the subset ofthe one or more additional substrates can be further based at least in part on amounts or concentrations of the additional drug comprised by each substrate of the one or more additional substrates.

[0139] A method can also include enclosing multiple cells in separate chambers and then selectively releasing one or more drugs to a subset of the cells. The subset of the cells may be selected based on a characteristic, such as cell type. As an example, a method for releasing a dose of a drug to a cell, the method comprising: inputting a plurality of cells into a fluidic channel; inputting a polymer precursor into the fluidic channel; inputting a substrate comprising the drug into the fluidic channel; synthesizing a plurality of chambers with the polymer precursor within the fluidic channel, wherein each chamber encloses a cell from the plurality of cells; measuring a characteristic of the enclosed cell in each chamber; and selectively releasing the drug in one or more of the plurality of chambers based on the measured characteristic of the enclosed cell in each chamber. In some cases, the plurality of cells comprises a library of gene edited cells, each of the gene edited cells comprises a guide RNA associated with the edited gene, the method further comprises determining a sequence of the guide RNA subsequent to the selectively releasing the drug from one or more of the plurality of chambers. In some cases, the method comprises measuring another characteristic of the enclosed cell in each chamber subsequent to the selectively releasing the drug; and correlating the sequence with the measured characteristic or the measured another characteristic.Deliver Drugs From Spatially-Addressed Nucleic Acid Barcodes

[0140] Further disclosed herein are methods of localizing drugs to specific locations within fluidic devices. A drug can be coupled to a nucleic acid capture sequence that localizes the drug to a particular location along a surface of a fluidic channel, allowing the identity of the drug to be ascertained based on its location. The identities of one or more drugs released to a cell in a particular region of a fluidic channel can similarly be determined based on the location of the cell. As an example, a method for releasing a dose of a drug to a cell can include inputting a polymer precursor into a fluidic channel; inputting the cell into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and a spatially-addressed nucleic acid sequence coupled to a surface of the fluidic channel; wherein at least a portion of the spatially-addressed nucleic acid sequence is hybridized to a drug-nucleic acid complex, the drug-nucleic acid complex comprising the drug coupled to a nucleic acid capture sequence that is complementary to at least a portion of the spatially-addressed nucleic acid sequence; releasing the drug from thespatially-addressed nucleic acid sequence by: a) dehybridizing the nucleic acid capture sequence from the spatially-addressed nucleic acid sequence, b) cleaving the drug from the nucleic acid capture sequence, or c) a combination thereof; thereby allowing the drug to contact the cell. While this method is amenable to multiplexing, wherein multiple different drugs are localized to discrete locations within the fluidic channel, this method may also be used to localize a single drug to multiple locations within the fluidic channel. The drug-nucleic acid complex may be input into the fluidic channel prior to drug release, thereby allowing the nucleic acid capture sequence to hybridize to the spatially-addressed nucleic acid sequence or the portion of the spatially-addressed nucleic acid sequence. In some cases, the drug-nucleic acid complex is input into the fluidic channel before the polymer precursor and the cell.

[0141] The drug may be cleaved from the nucleic acid capture sequence using, for example, chemical cleavage, enzymatic cleavage, thermal cleavage, pH-mediated cleavage, or photolysis. In many cases, the drug is photolytically cleaved from the nucleic acid capture sequence. In such cases, the drug may be coupled to the nucleic acid capture sequence through a photocleavable group or through a linker that contains a photocleavable group. Examples of photocleavable groups consistent with the present disclosure include o-nitrobenzyl, coumarin, o-nitroanilide, disulfide, and ruthenium -based photoremovable protecting groups. In some cases, the drug is activated through cleavage from the nucleic acid capture sequence. In particular cases, the cleaving the drug from the nucleic acid capture sequence converts the drug from a prodrug form to an active form of the drug wherein, for example, a cleavable linker that couples the drug to the nucleic acid capture sequence acts as a promoiety that limits activity of the drug.

[0142] In some cases, the method further comprises inputting an additional drug-nucleic acid complex into the fluidic channel, wherein the additional drug-nucleic acid complex comprises an additional drug coupled to an additional nucleic acid capture sequence. In such cases, the additional drug-nucleic acid complex can hybridize to at least a portion of an additional spatially-addressed nucleic acid sequence coupled to the surface of the fluidic channel. In some cases, the additional spatially-addressed nucleic acid sequence is disposed outside of the chamber. However, the additional spatially-addressed nucleic acid sequence may also be co-enclosed with the cell and the spatially-addressed nucleic acid sequence within the chamber.

[0143] Alternatively, multiple (e.g., 2, 3, 4, 5, 6, 8, 10, or more) drug-nucleic acid complexes may bind to a common spatially-addressed nucleic acid sequence in the fluidic channel. Forexample, an additional nucleic acid capture sequence of an additional drug-nucleic acid complex may be complementary to the spatially-addressed nucleic acid sequence or the portion of the spatially-addressed nucleic acid sequence to which the nucleic acid capture sequence of the drug-nucleic acid complex binds.

[0144] An example of location-specific drug delivery is depicted in FIG. 8, which illustrates a fluidic device that comprises a plurality of spatially-addressed nucleic acid sequences (901). The spatially-addressed nucleic acid sequences (901) can be coupled to one or more surfaces of the fluidic device, and can contain sequences that correspond to their locations within the fluidic device. A fluidic device may comprise a plurality of regions, for example about 102to 103, about 102to 104, about 102to 105, about 102to 106, about 102to 107, about 103to 104, about 103to 105, about 103to 106, about 103to 107, about 104to 105, about 104to 106, about 104to 107, about 105to 106, about 105to 107, or about 106to 107regions that contain uniquely identifiable spatially-addressed nucleic acid sequences (901). In such methods, drug may be selectively released from a subset of the regions, for example to selectively release one or more drugs to a single type of cell within the fluidic channel.

[0145] A plurality of drug-nucleic acid complexes can be input into the fluidic device (910). The drug-nucleic acid complexes can comprise nucleic acid capture sequences (911) that are complementary to counterpart spatially-addressed nucleic acid sequences (901) in the fluidic device and a drug (912). Following an incubation period, the drug-nucleic acid complexes hybridize to their counterpart spatially-addressed nucleic acid sequences (901), and thereby localize to desired and / or predetermined regions within the fluidic device. In this way, each region can be prepared with a unique drug (912), combination of drugs (912), and / or concentration of drugs (912).

[0146] In a subsequent step (920), cells (922) can be input into the fluidic device and chambers (921) can be synthesized surrounding the cells (922). Each chamber may enclose one or multiple cells (922) with one or more drug-nucleic acid complexes coupled to spatially-addressed nucleic acid sequences (901). The drugs may then be released to the cells (922). As non-limiting examples, the drugs (912) may be cleaved (930) from the nucleic acid capture sequences (911), the nucleic acid capture sequences (911) may be released (940), for example dehybridized, from the spatially-addressed nucleic acid sequences (901), or a combination thereof.

[0147] In some cases, multiple drugs are delivered to a single region of a fluidic device and then sequentially released to cells. For example, a method can include inputting the cell and into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing achamber with the polymer precursor within the fluidic channel, the chamber co-enclosing the cell and a plurality of drugs coupled to a surface of the fluidic channel through a plurality of cleavable linkers; selectively cleaving a first cleavable linker of the plurality of cleavable linkers coupled to the drug, thereby allowing the drug to contact the cell. The surface may be a top, bottom, or side surface of the fluidic channel. The selectively cleaving can comprise chemical cleavage, enzymatic cleavage, thermal cleavage, pH-mediated cleavage, or photolysis. In some cases, the plurality of cleavable linkers are cleaved through different methods, such as different proteases or nucleases that can be input into the fluidic channel at different times. The cleavable linkers can alternatively share a common cleavage mechanism, such as photolysis under a specific wavelength of light. In such cases, the cleavage mechanism can be selectively applied to release a specific drug or collection of drugs from the surface of the fluidic channel. For example, multiple drugs coupled to the surface of the fluidic channel may be localized within subregions that contain a single type of drug, such that a single type of drug can be released from the fluidic device surface by irradiating a selected subregion or set of subregions. Accordingly, the method may include sequentially or simultaneously releasing the drug, a additional drug, and optionally additional (third, fourth, etc) drugs from the surface of the fluidic channel. Optionally, cleavage from the surface of the fluidic device can convert a drug (e.g., the drug) from a prodrug form to its active form.

[0148] An example of this method is depicted in FIG. 12, which illustrates a portion of a fluidic device with one or more spatially-addressed nucleic acid sequences (1301). Splint oligonucleotides (1311) that are configured to bind to the spatially-addressed nucleic acid sequences (1301) can be input into the fluidic device (1310). Each splint oligonucleotide (1311) can comprise a first sequence complementary to a spatially-addressed nucleic acid sequence (1301) and a drug-capture sequence configured to bind to a nucleic acid sequence (1321) unique to a particular drug (1323). Multiple splint oligonucleotides (1311) can comprise identical first sequences that bind to the same spatially-addressed nucleic acid sequence (1301). Alternatively, a single region in the fluidic device may comprise multiple spatially-addressed nucleic acid sequences (1301) to which multiple distinct splint oligonucleotides (1311) bind. Multiple drugs can then be input into the fluidic channel (1320). Each drug can comprise a nucleic acid sequence complementary to a drug-capture sequence of a splint oligonucleotide (1321), a cleavable linker (1322), and a drug moiety (1323). The drugs can be ligated to the spatially-addressed nucleic acid sequences (1330) to affix the drugs in place within the fluidic device. Next, a cell (1342) can be input into the fluidic device and co-enclosed (1340) in a chamber (1341) with the drugs. The drugs can thenbe sequentially released (1350, 1360, 1370) to the cell (1342). Each drug can include a unique cleavable linker (1322) capable of being selectively cleaved. For example, each cleavable linker (1322) can include a unique protease or nuclease recognition sequence. Alternatively, a collection of cleavable linkers (1322) can comprise photocleavable groups that cleave under non-overlapping wavelengths.

[0149] The plurality of drugs may be covalently coupled to the surface of the fluidic channel through splint-ligation. In particular, the plurality of drugs can be coupled to the surface of the fluidic channel by hybridizing a plurality of splint oligonucleotides to a plurality of spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel, wherein each splint oligonucleotide of the plurality of splint oligonucleotides comprises: i) a first sequence complementary to at least a portion of a spatially-addressed nucleic acid sequence of the spatially-addressed nucleic acid sequences, and ii) a drug-capture sequence; inputting the plurality of drugs into the fluidic channel, wherein each drug of the plurality of drugs is coupled to: a) a cleavable linker of the plurality of cleavable linkers, and b) a nucleic acid capture sequence complementary to the drug-capture sequence; incubating the plurality of drugs under conditions that permit hybridization between the drug-capture sequences of the plurality of splint oligonucleotides and the nucleic acid capture sequences coupled to the plurality of drugs; and ligating the plurality of spatially-addressed nucleic acid sequences to the plurality of nucleic acid capture sequences coupled to the plurality of drugs, thereby coupling the plurality of drugs to the surface of the fluidic channel.

[0150] Alternatively, the plurality of drugs may be hybridized to spatially-addressed nucleic acid sequences coupled to the surface of the fluidic device. Such a method can include inputting the plurality of drugs into the fluidic channel, wherein each drug of the plurality of drugs is coupled to: a) a cleavable linker of the plurality of cleavable linkers; and b) a nucleic acid capture sequence complementary to at least a portion of a spatially-addressed nucleic acid sequence of a plurality of spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel; and incubating the plurality of drugs under conditions that permit hybridization between the plurality of spatially-addressed nucleic acid sequences and the nucleic acid sequences coupled to the plurality of drugs, thereby coupling the plurality of drugs to the surface of the fluidic channel.

[0151] A drug may also be covalently coupled to the surface of the fluidic device through a linker. An example of such a method is outlined in EXAMPLE 7, wherein a drug-linker conjugate is coupled to a reactive functional group on the surface of the fluidic device. The reactive functional group can include a nucleophile such as a thiol, an amine, or an azide; anelectrophile such as an acyl chloride, a N-hydroxysuccinimide (NHS) ester, a maleimide, or an alkyne; or a combination thereof. The drug-linker conjugate can include a reactive functional group that is configured to couple to the reactive functional group on the surface of the fluidic device. As non-limiting examples, when the surface includes a thiol or amine, the drug-linker conjugate can include an acyl chloride, an NHS ester, or a maleimide; when the surface includes an azide, the drug-linker conjugate can include an alkyne; when the surface includes an acyl chloride, NHS ester, or maleimide, the drug-linker conjugate can include an amine or a thiol; when the surface includes an alkyne, the drug-linker conjugate can include an azide; or a combination thereof. The drug-linker conjugate may also noncovalently couple to a functional group on the fluidic device surface, for example through a streptavidin-biotin interaction, an antibody (or antibody fragmentj-antigen pair interaction, a streptactin-strep tag interaction, a ubiquitin-ubiquitin binding domain interaction, or a combination thereof. In some cases, the surface may contain or couple to a linker, and the linker can subsequently couple to the drug. The drug or the drug-linker conjugate may be spotted onto the surface to control the type(s) and amount(s) of the drug at discrete locations along the surface.Prodrug Conversion

[0152] In some cases, a drug is generated from a prodrug within a fluidic channel. As used herein, a prodrug can be a modified form of a drug that has lowered or altered activity relative to the drug and is chemically convertible to the drug (i.e., an “active form” of the drug). In many cases, prodrugs comprise a promoiety coupled to a drug, wherein cleavage of the promoiety from the drug restores or otherwise modifies the drug’s activity (e.g., affinity for a target protein or uptake into a target cell). However, a prodrug can be related to a drug through other chemical transformations, including reduction, oxidation, elimination, alkylation, dealkylation, decarboxylation, esterification, hydrolysis, isomerization, cyclization, and ring opening. Examples of prodrug conversions include the photocleavage of a nitrobezyl protecting group from the amine of doxorubicin (e.g., as disclosed in Ibsen et al., Pharm Res., 2010; 27(9): 1848-1860), photocleavage of a carbamate-linked coumaryl protecting group from the amine of isotaxel followed by isotaxel conversion to paclitaxel (e.g., as disclosed in Klan et al., Chem. Rev., 2012; 773(1): 119-191), and cleavage of PEG-based polymers from therapeutic proteins (e.g., as disclosed in Mishra et al., Asian Journal of Pharmaceutical Sciences, 2016; 77(30):337-348, Fang Y et al., Cleavable PEGylation: a strategy for overcoming the "PEG dilemma" in efficient drug delivery, Drug Deliv. 2017 Dec;24 (supl):22-32.). For example, PEG-based polymers can be attached with a cleavable disulfide group to the therapeutic protein and then cleaved with a reducing agent such asglutathione. Further examples of prodrug to drug conversions are provided in Table 1. The prodrug can be converted into an active drug form by a cell (e.g., a cell enclosed in a chamber with the prodrug), by a chemical, by an enzyme or other catalyst, by a condition (e.g., temperature and / or pH), by light, or a combination thereof. The prodrug to drug conversion can include cleaving a promoiety from the prodrug, chemically modifying the prodrug, or a combination thereof. In particular cases, the cell may convert or partially convert a prodrug into an active drug form. For example, the cell may express an extracellular enzyme that converts the prodrug into an active form, or may internalize the prodrug to perform one or more intracellular conversions that convert the prodrug into an active form.Table 1Prodrug Drug ConversionH ' - H :Dihydro— C? I 'l Artemether bOxz _ o j,. / Artemisinin H*1 i • H d H O vHCLI Y OHChloramphenicol Vsxs / \ > - 0. X::::Z )>.. — Chi orampheni col MN 0 — Z011Palmitate \sssa oCl - (Ci- ( C) a 0_ JI ~ ,NHSL-Dopa DopamineOHi oOH ** OHDiacetylMorphine — 5 Morphined--d d ■■ dPrednisone Prednisolone r-T T / - ► d j \Xu "XXJ

[0153] In some cases, a method for releasing a dose of a drug to a cell includes inputting the cell into a fluidic channel; inputting a prodrug of the drug into the fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell; and converting the prodrug into an active form of the drug, thereby allowing the drug to contact the cell. The chamber may enclose the prodrug with the cell, and may optionally be impermeable to the prodrug, the drug, or the prodrug and the drug. In some cases, the prodrug is input into the fluidic channel subsequently to chamber synthesis. In such cases, the prodrug, the drug, or the prodrug and the drug are sufficiently small to pass through a wall of the chamber.

[0154] In many cases, the prodrug is converted into an active form of the drug with light, for example by photocleaving a promoiety from the prodrug. A dose of a drug active form can be determined based on a distance between the photolysis and the cell, an intensity of the photolysis, a duration of the photolysis, a volume of the fluidic channel irradiated during the photolysis, a concentration of the prodrug in the fluidic channel, a rate of conversion of the prodrug to the active form of the drug during the photolysis, or a combination of such factors. The dose of the prodrug delivered to the cell may be similarly controlled by selecting a location, intensity, and wavelength of light for prodrug to drug conversion. When a plurality of instances of the prodrug are input into the fluidic channel, a selected subset of the instances of the prodrug may be converted into the active form of the drug.

[0155] An example of a prodrug-based assay is provided in FIG. 9. In this example, a cell (1001) is input into a fluidic device channel with a prodrug (1003) comprised of a drug (1003 A) coupled to a promoiety (1003B). The cell (1001) and prodrug (1003) can be enclosed within a chamber (1002). For example, the chamber (1002) may be synthesized (1010) surrounding the cell (1001) and prodrug (1003), or the cell (1001) can be enclosed in the chamber (1002) before the prodrug (1003) is input into the fluidic device channel, and the prodrug (1003) can diffuse (1020) into the chamber (1002). The promoiety (1003B) can then be cleaved (1030) from the drug (1003A), thereby converting the prodrug (1003) into anactive form of the drug (1003A). The cleaving (1030) can be performed, for example, by inputting light, a chemical, or a catalyst into the fluidic device channel that is capable of cleaving (1030) the promoiety (1003B) from the drug (1003 A). The cleaving (1030) can be performed to completion. That is, all instances of the prodrug (1003) can be converted into the active form of the drug by cleaving the promoiety (1003B) from the drug (1003A).Alternatively, only a subset of instances of the prodrug (1003) may be converted by cleaving the promoiety (1003B) from the drug (1003A). The degree of prodrug (1003) conversion can be controlled, for example by inputting only a portion of the amount of light required to cleave the promoiety (1003B) from all instances of the prodrug (1003). When the method of FIG. 9 is performed on hundreds, thousands, tens of thousands, or more cells (1001) in parallel, the degree of prodrug (1003) conversion may be varied to determine the effects of its conversion or concentration on a cellular response.Multispecific Binding Agents

[0156] In some cases, the drug comprises multispecific binding affinity for two or more cells. In such cases, the activity of the drug may, at least in part, be an ability to co-localize cells, and the drug can be referred to as a multispecific capture agent or capture agent. In particular, the drug may be configured to simultaneously bind to an effector cell and a target cell of the effector cell to thereby induce cytotoxic activity of the effector cell against the target cell. Alternatively, the drug may be configured to simultaneously bind to a non-activated cell and a cell capable of activating the non-activated cell to induce activation of the non-activated cell.

[0157] In one aspect, the present disclosure provides a method for analyzing a capture agent that includes inputting a cell into a fluidic channel; inputting an additional cell into the fluidic channel; inputting the capture agent into the fluidic channel; and determining whether the capture agent simultaneously binds to the cell and the additional cell. The method can include synthesizing a chamber around multiple cells bound to or capable of binding to the capture agent. For example, the method can include inputting a polymer precursor into the fluidic channel and synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and the additional cell. Similarly, a method for analyzing a capture agent can include inputting a cell into a fluidic channel; inputting an additional cell into the fluidic channel; inputting a polymer precursor into the fluidic channel; inputting the capture agent into the fluidic channel, wherein the capture agent is configured to simultaneously bind to the cell and the additional cell; incubating the cell, the additional cell, and the capture agent in the fluidic channel under conditions that allow the capture agent tobind to the first and additional cells; and synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and the additional cell.

[0158] As non-limiting examples, the capture agent can comprise an antibody, an antibody fragment, a nanobody, a minibody, an affibody, a diabody, a triabody, a tetrabody, an intrabody, a bis-scFv, a single domain antibody, a DARPin, an anticalin, a knottin, a receptor ligand, an aptamer, an adheron, or a combination thereof. When the capture agent comprises an antibody, the antibody can comprise a fragment crystallizable (Fc) region configured to bind to the cell and a fragment antigen-binding (Fab) region configured to bind to the additional cell. Alternatively, the antibody can comprise a first fragment antigen-binding (Fab) region configured to bind to the cell and a second fragment antigen-binding (Fab) region configured to bind to the additional cell.

[0159] The first and / or additional cells can be input into the fluidic channel bound to the capture agent, or the capture agent can bind to the first and / or additional cells after the capture agent is input into the fluidic channel. In particular, the capture agent can be input into the fluidic channel after the cell and the additional cell are input into the fluidic channel to force the first and additional cells to co-localize within the fluidic channel.

[0160] The capture agent may be captured and / or identified. For example, the capture agent may be coupled to a nucleic acid barcode that uniquely identifies the capture agent, and the capture agent can be identified by sequencing the nucleic acid barcode coupled to or cleaved from the capture agent. For example, the nucleic acid barcode may be reverse transcribed with a spatially-addressed nucleic acid sequence to associate the barcode with a particular location, chamber, cell, or collection of cells in the fluidic channel. In particular cases, the nucleic acid barcode encodes at least a portion of an amino acid sequence (such as an antibody Fab region or a portion of an antibody Fab region) of the capture agent. Similarly, when the capture agent is coupled to a nucleic acid barcode, the capture agent can be identified by detecting binding of the nucleic acid barcode to a spatially-addressed nucleic acid sequence coupled to a surface of the fluidic device, and in particular by detecting a location of the capture agent in the fluidic channel. When the method includes chamber synthesis, the nucleic acid barcode of the capture agent may bind to the spatially-addressed nucleic acid sequence before, concurrently with, or after chamber synthesis.

[0161] The method may also include determining a binding affinity of the capture agent for the cell, the additional cell, or the cell and the additional cell. For example, the method may include inputting a plurality of instances of the cell, the additional cell, and the capture agent into the fluidic channel, and measuring the frequency of capture agent binding to the first andadditional cells. The method may also include measuring capture agent binding to the first and additional cells under multiple conditions, such as multiple temperatures, under which the affinity of the capture agent for the first and additional cells varies.

[0162] The method can also include determining the specificity of the capture agent for the first and additional cells. In such cases, the method may further include inputting a third cell into the fluidic channel, and determining a specificity of the capture agent for the cell and the additional cell relative to the third cell, or inputting a plurality of additional cells into the fluidic channel, and determining a specificity of the capture agent for the cell and the additional cell relative to the plurality of additional cells. For example, the first and additional cells may be present among a consortia of cells from a complex biological sample, such as a blood sample, peripheral blood mononuclear cell (PBMC) sample, a buffy coat sample, a solid biopsy, a liquid biopsy, or a tumor section. When the capture agent is analyzed in the presence of multiple types of cells, the cells bound to the capture agent may be identified using one or more techniques disclosed herein, including brightfield imaging or fluorescencebased surfaceome profiling.

[0163] In some cases, the method includes measuring cytotoxicity of the cell against the additional cell, and may further optionally include determining whether the capture agent modulates cytotoxicity of the cell against the additional cell. The capture agent may enhance effector cell cytotoxicity by localizing the effector cell (e.g., the cell) to its target cell (e.g., the additional cell). This cytotoxicity enhancement may be measured and correlated to the sequence and / or structure of the capture agent. In some cases, the cell comprises an immune cell such as a B cell, a basophil, a cytokine induced killer cell, a cytotoxic T cell, a dendritic cell, an eosinophil, a granulocyte, a hypersegmented neutrophil, a lymphoid cell, a macrophage, a mast cell, a megakaryocyte, a monocyte, a myeloid cell, a natural killer cell, a neutrophil, a regulatory T cell, a T cell, a T helper cell, or a thrombocyte. The capture agent may be configured to bind an immune checkpoint molecule on the immune cell. In such cases, the capture agent may prevent or limit target cell inhibition of the immune cell. For example, the capture agent may bind to an extracellular region of an immune checkpoint molecule that does not activate the immune checkpoint molecule, but which blocks access to a receptor region of the immune checkpoint molecule by one or more immune checkpoint inhibitors expressed by the target cell. In some cases, the immune checkpoint molecule is selected from PD-1, CTLA-4, BTLA, LAG-3, TIM-3, TIGIT, CD27, CD28, CD112R, CD137, ICOS, 4-1BB, OX-40, or VISTA. In particular cases, the cell is a T cell and the capture agent is configured to bind to a PD-1 antigen on the T cell. The additional cell maycomprise a cancer cell from a subject (e.g., from a liquid biopsy or tumor resection) or from a cell line. The cancer cell may be, for example, an adenocortical carcinoma cell, an agnogenic myeloid metaplasia cell, a basal cell carcinoma cell, a bile duct cancer cell, a bladder cancer cell, an osteosarcoma cell, an astrocytoma cell, a glioma cell, an ependymoma cell, an oligodenglioma cell, a meningioma cell, a craniopharyngioma cell, a haemangioblastoma cell, a medulloblastoma cell, a breast cancer cell, a lung cancer cell, a carcinoid tumor cell, a carcinoma cell, a lymphoma cell, a cervical cancer cell, a colon cancer cell, a colorectal cancer cell, an endometrial cancer cell, an esophageal cancer cell, a gallbladder cancer cell, a gastric cancer cell, an extracranial cancer cell, an ovarian cancer cell, a hepatic cancer cell, a hypopharyngeal cancer cell , an endocrine cancer cell, a laryngeal cancer cell, a leukemic cell, oropharyngeal cancer cell, a pancreatic cancer cell, a parathyroid cancer cell, a pharyngeal cancer cell, a renal cancer cell, a skin cancer cell, or a thyroid cancer cell.

[0164] An example of a capture agent analysis method is illustrated in FIG. 10. This figure depicts a capture agent (1103) that is simultaneously bound to a cell (1101) and a additional cell (1102). In this figure, the capture agent (1103) is an antibody that includes a Fab region bound to its cognate antigen (1101 A) on the surface of the cell (1101) and an Fc region bound to an Fc receptor (1102A, e.g., an FcyR) of the additional cell (1102). However, alternate capture agents are utilizable in the depicted method, including bispecific antibodies with distinct Fab regions, tandem scFv constructs, diabodies, triabodies, conjugated surface receptor ligands, and the like. After the cell (1101) and additional cell (1102) are coupled by the capture agent (1103), the cells may optionally be enclosed in a chamber (1111), for example by synthesizing (1110) a wall or walls of the chamber around the cells (1101, 1102).

[0165] The capture agent (1103) may be identified by sequencing a nucleic acid barcode coupled to the capture agent (1103) that uniquely identifies the capture agent (1103). Such barcode-based methods may utilize a spatially-addressed nucleic acid sequence coupled to a fluidic device surface to capture and optionally to encode barcode information onto the nucleic acid barcode. This method may be analogous to mRNA capture, barcoding, and sequencing processes disclosed herein. A characteristic of the capture agent (1103), such as its sequence or structure, can be correlated to its binding affinity to the first and additional cells (1101, 1102) or characteristics of the first and additional cells (1101, 1102) measured prior to, concurrently with, or subsequent to capture agent (1103) binding.

[0166] A capture agent can be localized to a specific location within a fluidic device. For example, as depicted in FIG. 13, a multispecific capture agent (1401) can be coupled to nucleic acid barcode sequence (1402) that is complementary to at least a portion of spatially-addressed nucleic acid sequence (1403) coupled to a surface of the fluidic device. The nucleic acid barcode sequence (1402) can thus localize the capture agent (1401) to the spatially-addressed nucleic acid sequence (1403) in the flow cell. A plurality of cells can be input into the fluidic device (1410), and binding of the capture agent (1401) to a cell (1411) and a additional cell (1412) of the plurality of cells can be measured. The plurality of cells input into the fluidic device can consist of the first and additional cells (1411, 1412) or can comprise additional cell types. For example, the plurality of cells can include a peripheral blood mononuclear cell (PBMC) population with numerous types of T cells, B cells, NK cells, monocytes, and dendritic cells. The types of cells bound to the capture agent (1401) can be determined with one or more techniques disclosed herein, including brightfield imaging, surfaceome profiling (e.g., to identify cell-type specific markers), transcriptome profiling, and the like.

[0167] The capture agent (1401) may be uniquely identifiable based on its nucleic acid barcode sequence (1402) and / or position in the fluidic device. In such cases, the fluidic device may comprise a plurality of spots with unique spatially-addressed nucleic acid barcodes (1403), and each capture agent (1401) can be coupled to a uniquely identifiable nucleic acid barcode sequence (1402) that binds to a single spatially-addressed nucleic acid barcode (1403). Accordingly, when the capture agent (1401) binds to a specific spatially-addressed nucleic acid barcode (1403) in the fluidic device, the capture agent (1401) can be identified based on its position in the fluidic device.

[0168] A multispecific capture agent can be coupled to a particle, such that inputting the capture agent into the fluidic channel comprises inputting a particle comprising the capture agent into the fluidic channel. The particle may comprise a nanoparticle, a microparticle, a lipid particle, a vesicle, a ceramic particle, a metal particle, a metal oxide particle, a polymeric particle, a polysaccharide particle, a magnetic particle, a hydrogel particle, or a combination thereof. The capture agent can then optionally be released from the particle, for example by cleaving a bond or linker that couples the capture agent to a surface of the particle; cleaving a bond or a linker that confines the capture agent within the particle; lysing the particle or disrupting the surface of the particle to release the capture agent from the surface of the particle; lysing or opening the particle to release the capture agent from the interior space within the particle; or changing a condition in the fluidic channel to desorb the capture agent from the surface of the particle. The particle can be coupled to a surface of the fluidic channel. For example, the particle can comprise a nucleic acid capture sequence that isconfigured to hybridize to a spatially-addressed nucleic acid sequence coupled to the surface of the fluidic channel. The particle can also be co-enclosed with a cell within a chamber.

[0169] An example of particle-based capture agent delivery is depicted in FIG. 14. In this figure, a particle (1501) is coupled to a capture agent (1502) and a nucleic acid barcode sequence (1503) that is complementary to at least a portion of spatially-addressed nucleic acid sequence (1504) coupled to a surface of the fluidic device. By hybridizing to the spatially-addressed nucleic acid sequence (1504), the particle can localize to a specific location within the fluidic device. A plurality of cells can be input into the fluidic device (1510). Binding of a cell (1511) and a additional cell (1512) from among the plurality of cells to the capture agent (1502) can then be detected. The particle can be identified based on its barcode sequence or location within the fluidic device. For example, a subset of particles (1502) within a fluidic device can be collected and their nucleic acid barcode sequences (1503) can be sequenced.

[0170] A multispecific capture agent can also be contained within a degradable particle such as a liposome. After the particle is co-localized with potential target cells, the particle can be degraded to release the capture agent in proximity to the target cells. An example of this method is depicted in FIG. 15, which shows a particle (1601) co-enclosed with first and additional cells (1604, 1605) within a chamber (1606). In this figure, the particle (1601) is coupled to a nucleic acid barcode sequence (1602) that is complementary to at least a portion of spatially-addressed nucleic acid sequence (1603) coupled to a surface of the fluidic device. However, the particle (1601) alternatively may be suspended in the fluidic device. In a step not shown in FIG. 15, the chamber (1606) may be synthesized co-enclosing the particle (1601) and the first and additional cells (1604, 1605). The particle (1601) may then be degraded (1610), thereby releasing capture agents (1611) contained within the particle (1601). The chamber (1606) may be impermeable to the first and additional cells (1604, 1605) and capture agent (1611), such that the chamber co-localizes the first and additional cells (1604, 1605) with the capture agent (1611). Each particle (1601) in the fluidic device may contain a single type of capture agent (1611), thereby facilitating identification of the capture agent (1611) based on, for example, the location of the particle (1601) in the fluidic device or its nucleic acid barcode sequence (1602). Simultaneous binding of the capture agent (1611) to the first and additional cells (1604, 1605) can then be detected (1620). The chamber (1606) can optionally be degraded to collect the first and additional cells (1604, 1605), the capture agent (1611), a complex comprised of the first and additional cells (1604, 1605) and capture agent (1611), the nucleic acid barcode sequence (1602), a nucleic acid generated byamplifying or extending over the nucleic acid barcode sequence (1602), or a combination thereof.

[0171] In certain cases, the capture agent is expressed by an additional cell in the fluidic device which may optionally be co-enclosed with the cell and the additional cell within a chamber. For example, a method for analyzing a capture agent can include inputting a cell into a fluidic channel; inputting a additional cell into the fluidic channel; inputting a third cell into the fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell, the additional cell, and the third cell; incubating the cell, the additional cell, and the third cell under conditions in which the third cell expresses the capture agent; and measuring binding of the capture agent to the cell and the additional cell. As an example, the third cell may comprise a B cell that expresses a bispecific antibody as disclosed herein.

[0172] An example of this method is provided in FIG. 11, which depicts a B cell (1201, a “third cell”) co-enclosed within a chamber (1204) with a cell (1202) and additional cell (1203). A fluidic device may comprise hundreds, thousands, tens of thousands, or more chambers (1204), each containing aB cell (1201), cell (1202), and additional cell (1203). For example, as described further herein, a detector may be used to identify regions of the fluidic device that include all three cells (1201, 1202, 1203) to direct chamber (1204) synthesis around these cells. The B cell (1201) may express (1210) an antibody (1211) while enclosed within the chamber (1204). Simultaneous binding (1220) of the antibody (1211) to the cell (1202) and the additional cell (1203) may be detected. mRNA (1231) from the B cell can be analyzed by lysing (1230) the B cell (1201), capturing and sequencing its mRNA (1231) with a method described herein. Alternatively, the antibody (1211) may be captured and sequenced, for example with mass spectrometry or by sequencing a nucleic acid barcode coupled to the antibody (1211). While this example covers antibody expression by B cells, other capture agents and capture agent-expressing cells are readily applicable to this method.Cell Co-Localization

[0173] Aspects of the present disclosure provide methods for co-localizing cells within a fluidic channel. As an example, a method can include inputting a cell into a fluidic channel comprising a surface comprising a first capture agent configured to bind to the cell and a second capture agent configured to bind to a additional cell, inputting the additional cell into the fluidic channel; incubating the cell and the additional cell in the fluidic channel under conditions that allow the first capture agent to bind to the cell and the second capture agent to bind to the additional cell; inputting a polymer precursor into the fluidic channel; andsynthesizing a chamber with the polymer precursor that co-encloses the cell with the additional cell; wherein the cell is able to contact the additional cell when the cell is bound to the first capture agent and the additional cell is bound to the second capture agent. The first capture agent and the second capture agent can be localized to specific portions of a fluidic channel by nucleic acid capture sequences that hybridize to spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel. In such cases, the first and second capture agents can be coupled to the surface by separate linkers. Accordingly, the method can include selectively cleaving the first capture agent, the second capture agent, or the first and second capture agents from the surface of the fluidic channel. As non -limiting examples, the first capture agent and the second capture agent can be independently selected from an antibody, an antibody fragment, a nanobody, a minibody, an affibody, a diabody, a triabody, a tetrabody, an intrabody, a bis-scFv, a single domain antibody, a DARPin, an anticalin, a knottin, a receptor ligand, an aptamer, an adheron, or a combination thereof. In some cases, the method comprises determining whether the cell binds to the additional cell, or determining an affinity of the cell for the additional cell. In some cases, the inputting comprises inputting a biological sample comprising the cell, the additional cell, and a plurality of additional cells into the fluidic channel. For example, the first and additional cells can be present in a PBMC or tumor biopsy sample input into the fluidic channel.

[0174] An example of a cell co-localization assay is provided in FIG. 17, which depicts a cell (1801A) coupled to a first capture agent (1802A) that is coupled to a surface of a fluidic channel through a first linker (1803A), and an additional cell (1801B) coupled to a second capture agent (1802B) that is coupled to the surface of the fluidic channel through a second linker (1803B). The first and second capture agents are in sufficient proximity to allow the first and additional cells (1801A, 1801B) to bind (1810). The first and additional cells (1801A, 1801B) may then optionally be enclosed (1820) in a chamber (1821) and or analyzed. In some cases, the first and / or second linker (1803 A, 1803B) comprises a nucleic acid barcode that may be sequenced to identify the first and / or second capture agent (1802A, 1802B).

[0175] A method can further include subjecting a cell to a condition and determining the effect of the condition on the cell’s response to one or more drugs. For example, the temperature, osmolarity, viscosity, salinity, pH, pressure, or oxygen level may be varied spatially or temporally within a fluidic channel or between separate fluidic channels during a drug release assay disclosed herein.

[0176] A method may also include mixing the contents of a fluidic channel. The mixing may comprise flowing liquid back and forth within the fluidic channel; applying an alternating magnetic field to the fluidic channel to move magnetic species within the fluidic channel; applying an alternating electric field to the fluidic channel to move charged species within the fluidic channel; shaking the fluidic channel; or a combination thereof.Adherent Supports

[0177] In some of the methods disclosed herein, a cell is adhered to a surface of the fluidic channel. The surface can include an adherent substrate, that is, a species to which the cell comprises a binding affinity. Examples of adherent substrates utilizable in the presently disclosed methods include fibronectin, poly-l-omithine, an RGD peptide, actinin, collagen, fibrinogen, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, talin, VCAM-1, vinculin, and vitronectin, as well as combinations and chemical derivatives of these species. As detailed further herein, an adherent substrate may improve cell survival within the fluidic device, and can also alter cell drug responses and phenotype (e.g., promote cell spreading or growth). Cell Analysis

[0178] A cell can be subjected to further analyses prior to, during, or following contact to one or more drugs to associate one or more cellular characteristics or changes to doses of the one or more drugs. As used herein, the phrase “determining a characteristic of a cell” and the term “assay” refer to processes for detecting or measuring a cellular characteristic or property of a cell. The assays disclosed herein can comprise a chemical, biochemical or molecular reaction (such as a cleavage of a bond, specific binding of complementary components, enzymatic reactions, dissolution of complementary components, or the like) or a change of physical state (such as an increase or decrease in temperature, change in energy level, or the like), along with a measurement of such reaction or change in physical state. The signals generated during an assay can include an electrical signal, an optical signal, a chemical signal, or a material output. A material signal comprises the production of a material that comprises information that can be extracted. For example, a material signal may be the amplification of a polynucleotide whose length, quantity, composition, or nucleotide sequence is indicative of a cellular characteristic. For example, a barcode oligonucleotide may be a material signal. Characteristics or properties of cells that are detected or measured in the disclosed methods may include, but are not limited to, cytotoxicity, viability, proliferation capacity under selected conditions, size, shape, motility, types and profiles of cell surface, or cell membrane proteins, types and profiles of secreted proteins, production of metabolites, transcriptome, gene copy numbers, gene or allele identity, genetic modification, chromatinaccessibility profiles, morphology, vector copy numbers for engineered or infected cells, and the like. These characteristics can then be associated with a drug dose or regimen. In some cases, determining a characteristic of a cell includes lysing the cell to release analytes for capture and further analysis. In particular, the cell can be lysed while it is enclosed within a chamber to prevent lysate from diffusing away from the lysed cell. Additional assays may include culture contamination assays including, but not limited to, viral, bacterial, yeast, mold, or mycoplasma assays, endotoxin assays, and cellular morphology assays. In specific embodiments disclosed herein, an assay may measure cell viability or survival prior to, during, or in response to a drug dose or regimen.

[0179] A method can also include determining one or more characteristics of a cell. As nonlimiting examples, the one or more cellular characteristics can be selected from cytotoxicity, proliferative capacity or proliferation rate, activation status, guide RNA expression, cellular identity, purity, gene expression profile, transcriptome, surface marker expression, soluble factor secretion, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, or gene copy number), morphology, survival (e.g., following exposure to a drug or condition), cell type, or a combination thereof. The one or more characteristics of the cell can then be related to a drug dose or regimen, a condition (e.g., dissolved oxygen concentration, pH, or temperature), or a combination thereof. Alternatively or additionally, the one or more characteristics can be used to select a cell for drug exposure, select a dose or regimen of a drug for the cell, to select a cell for enclosure within a chamber, to select a cell for further analysis (e.g., lysis and transcriptome profiling), or a combination thereof. For example, when a consortia of cells are input into a fluidic channel, hundreds, thousands, tens of thousands, or hundreds of thousands of instances of a single type of cell (e.g., CD4+T cells) may be enclosed in chambers and exposed to one or more drugs, while all other cells may be removed from the fluidic channel in one or more wash steps. The cell type may be determined, for example, by measuring cell morphology, surface marker expression, secretome, or one or more additional characteristics as disclosed herein.

[0180] A method disclosed herein can include detecting a guide ribonucleic acid (RNA) associated with a genetic modification of the cell, and then optionally associating that genetic modification with one or more responses of the cell to a drug dose or regimen. The cell can be transiently or stably transfected with a sequence encoding a guide RNA specific for a particular genomic sequence. The guide RNA can be coupled to a barcode, an exogenous messenger RNA (e.g., a selection marker), a capture sequence (e.g., a polyA tail), or acombination thereof. The cell can express a Cas protein that can utilize the guide RNA. Alternatively, a Cas protein can be delivered to the cell (e.g., in a liposome). Cellular response to a drug can then be correlated with a genomic edit imparted by a particular guide RNA sequence. For example, the cell can be lysed to release guide RNA. The guide RNA can optionally be captured, and then be used as a template for generating a cDNA molecule comprising a complement of the guide RNA sequence, and optionally additional sequences coupled to the guide RNA such as the exogenous mRNA, the barcode, or a combination thereof. The cDNA molecule can be coupled to a spatial location tag corresponding to a unique location within the flow cell. For example, the spatial location tag can be present in a capture probe that hybridizes to the guide RNA and serves as a reverse transcription primer, and the method of generating the cDNA molecule can include capturing the guide RNA on the capture probe comprising the spatial location tag or the complement thereof, and reverse transcribing the guide RNA on the capture probe, thereby generating the cDNA molecule. The method can include sequencing the cDNA, thereby detecting the guide RNA associated with the genetic modification of the cell. The genetic modification can then be associated with one or more responses of the cell to a drug or drug regimen.

[0181] In some cases, a method includes detecting one or more mRNA molecules expressed by the cell. The one or more mRNA molecules can be detected and optionally are sequenced by lysing the cell, capturing the mRNA on a capture element coupled to a surface of the flow cell, reverse transcribing the mRNA to generate a cDNA molecule that optionally comprises a barcode sequence derived from the capture element, and sequencing the cDNA molecule. In many cases, mRNA analysis comprises capturing mRNAs from a cell exposed to one or more drugs as disclosed herein. The one or more capture elements can be coupled to a surface of the fluidic device. The fluidic device containing the cell may be loaded with a lysing reagent so that messenger RNAs of the cell are released and captured by the capture elements, and with reverse transcription reagents to copy the captured oligonucleotide labels to produce complementary DNAs thereof. The complementary DNAs may then be eluted from the fluidic device (e.g., cleaved from a surface of the top or bottom layer) and sequenced. It is understood that a sequencing step may comprise additional steps in particular embodiments including, but not limited to, tagmentation, adding adaptors, cleaving the cDNA to form appropriate lengths for sequencing, and the like. In some embodiments, an additional step may be implemented for depolymerizing or degrading the polymer matrix walls of the chambers after mRNA capture. Reverse transcription reagents comprise conventional reagents for reverse transcription; namely, a reverse transcriptase (such as, a Moloney murineleukemia virus (MMLV)), dNTPs, optional RNase inhibitor, buffer. The sequencing step may be carried out at the sites of the captured mRNAs (in situ) or cDNAs may include a spatial barcode and be eluted and sequenced on a separate sequencing instrument (“external” sequencing). For in situ sequencing, further steps may include (i) amplifying the complementary DNAs, e.g. by bridge amplification, or like method, (ii) sequencing the amplified complementary DNAs, e.g. by a sequencing-by-synthesis technique, and (iii) determining relative expression of the mRNAs for the cells of each of the chambers. For external sequencing, further steps may include (i) providing capture elements comprising spatial barcodes, (ii) synthesizing cDNAs comprising spatial barcodes, and (iii) eluting and sequencing the cDNAs and correlating each cDNA with a chamber location by its spatial barcode.

[0182] The characteristic can also be a soluble factor secreted by a cell. The soluble factor may be detected by disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell, and detecting the soluble factor bound to the capture surface. Such a method can include disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell and detecting the soluble factor bound to the capture surface. Disposing the capture surface adjacent to the cell can denote enclosing or at least partially enclosing the capture surface with the cell within a chamber, and optionally removing non-enclosed capture surfaces from the fluidic system. The capture surface can be loaded into the fluidic channel at a controlled density, for example one capture surface per about 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 mm2of the fluidic channel.

[0183] In an exemplary embodiment, the capture surface comprises a particle. As used herein, the term “particle” can denote a microparticle or a nanoparticle, such as a ceramic, metal, metal oxide, polymer, or saccharide-based 30 to 10000 nm particle or bead. However, further capture surfaces, including nanotubes, nucleic acid nanostructures, and antibody Fc domains. The capture surface affinity reagent can, as non-limiting examples, include antibodies, antibody fragments, aptamers, affimers, or a combination thereof.

[0184] In a further exemplary embodiment, the soluble factor comprises a cytokine such as interferon -y (IFN-y) and interferon-a (IFN-a), an interleukins such as interleukin- 1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin- 10 (IL-10), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin-21 (IL-21), or interleukin-23 (IL-23), a colony stimulating factor (CSFs) such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), or a tumor necrosis factors (TNF) such as TNF-a or TNF-p. In another embodiment, the secreted factor comprises an effector molecule such as a granzyme.

[0185] A soluble factor bound to a capture surface (e.g., an affinity reagent of a capture surface) can be detected by contacting the soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody. Multiple soluble factors can be detected in a single assay by providing a capture surface or plurality of capture surfaces that comprise a plurality of affinity reagents configured to bind the plurality of soluble factors, contacting the plurality of soluble factors bound to the capture surface or plurality of capture surfaces with a plurality of labeled antibodies configured to bind to the plurality of soluble factors, and detecting a plurality of labels coupled to the plurality of antibodies. In this way, 2, 3, 4, 5, 6, or more soluble factors can be detected in a single assay. For example, an assay may utilize 2, 3, 4, 5, 6, or more particle that each include a different affinity reagent configured to bind to a different soluble factor and a commensurate number of antibodies configured to bind to the soluble factors and optionally containing distinguishable detectable labels (e.g., different fluorophores or oligonucleotide barcodes). In many cases, the soluble factor is a protein or a metabolite, such as a cytokine, immune active protein, hormone, or neurotransmitter.

[0186] In some cases, the cell comprises an effector cell, and the characteristic comprises cytotoxicity. In such cases, determining the cytotoxicity of the cell comprises measuring a rate or an occurrence of the effector cell killing one or more target cells. For example, an assay may measure the tumor-infiltration ability of a lymphocyte by measuring its ability to pass through a semipermeable polymer matrix, and then measure the lymphocyte’s ability to measure cancer cells trapped within or behind the semipermeable polymer matrix. As nonlimiting examples, the target cells may comprise a sample of tumor cells of a patient or a cancer cell line such as the hepatic tumor cell line SK-HEP-1, Chava et al, J. Vis. Exp., 2020 Feb 22: (156): 10.3791 / 60714. Examples of effector cells include Tel cells, Tc2 cells, Tc9 cells, Tcl7 cells, Tc22 cells, natural killer cells. In some embodiments, the effector cell is engineered for a therapeutic purpose, such as expression of a chimeric antigen receptor that confers cytotoxicity against a particular cancer cell.

[0187] The characteristic can also include activation. In some aspects, the characteristic of the one or more cells comprises activation. Cellular activation can be measured with numerous techniques disclosed herein, including surface marker expression, soluble factor secretion, transcriptomic analysis, proliferation, morphology, or a combination thereof. As one example, the disclosed methods are broadly amenable to detecting activation caused bycontact between two cells or by a soluble factor secreted by a cell and received by a additional cell. In a particular aspect of the present disclosure, determining activation comprises detecting a cell surface marker. For example, the method can comprise contacting a cell with a binding agent configured to bind to the surface marker and detecting the binding agent. As examples, activation can be determined by detecting expression of a surface marker, a morphology change, a cytotoxicity increase, a proliferation rate change, a soluble factor, a genomic sequence, an mRNA, or a combination thereof of the cell.

[0188] In some aspects, determining a characteristic of a cell includes determining a proliferation rate. It is understood that the term “proliferation rate” may include a measure of a lack of proliferation. For example, chambers enclosing one or more cells may be exposed to an agent, e.g. a drug candidate, after which such cells may be returned to normal growth conditions. In some cases, the agent may kill or retard the growth of the cells, e.g. in comparison to controls not exposed to the agent. Thus, in the case of the treated cells, a negative “proliferation rate” may be possible because the final numbers of cells counted in the chambers may be less than the original numbers; or a signal monotonically related to cell number may decline in value. In some embodiments, cells may be stained with a membrane or intracellular dye for determining proliferation by dye dilution so that an independent measure of cell proliferation may be obtained. Exemplary intracellular dyes for dye dilution include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. The desired number of single cells enclosed by chambers depends on statistical confidence desired in the measured values. If a subpopulation of interest is present as only a small fraction of a total population then a larger number chambers is required.

[0189] In some aspects, determining a characteristic of the one or more cells includes detecting a surface marker of the one or more cells. As used herein, the term “surface marker” denotes species that are expressed on the surface of a cell. Exemplary surface markers include surface proteins such as G protein-coupled receptors (GPCRs), ion channels, engineered receptors (e.g., chimeric antigen receptors), and cluster of differentiation (CD) molecules (e.g., CD3, CD4, CD5, CD6, CD7, CD8, etc.), as well as carbohydrates (e.g., sialic acids), glycolipids, and the like. However, in particular aspects of the present disclosure, the surface markers are proteins.

[0190] Surface marker detection can include contacting a cell with a binding agent configured to bind to the surface marker and detecting the binding agent, thereby detecting the surface marker. A single surface marker or plurality of surface markers can be detected in a single assay. More precisely, the binding agent can include a single binding agentconfigured to bind to a single surface marker or a plurality of binding agents configured to bind to a plurality of surface markers. Multiple surface markers can be detected simultaneously, for example by contacting the cell with multiple antibodies that bind different surface markers and are coupled to distinct detectable labels such as fluorophores that are simultaneously detectable on separate imaging channels. Alternatively or in addition thereto, two or more surface markers can be detected sequentially, for example by contacting a cell with a first antibody that binds to a surface marker, detecting the first antibody, binding the cell with a second antibody that binds to a additional surface marker, and detecting the second antibody. In particular cases, detecting a plurality of surface markers includes detecting relative expression levels of the plurality of surface markers. Exemplary binding agents include antibodies; antibody fragments such as single-chain antibody molecules, scFvs, Fab domains, diabodies, nanobodies, minibodies, linear antibodies, and cross-Fab fragments; aptamers; and affimers. A binding agent may be coupled to a detectable label such as a fluorescent label, an oligonucleotide label, a colorimetric label, an enzymatic label (e.g., pyrophosphatase), or a combination thereof. It is noted that a binding agent such as an antibody can be coupled to a cell prior to or following the cell’s introduction into a channel and / or encapsulation within a chamber.Flow Cell Design

[0191] A channel or chamber of a fluidic device (also sometimes referred to as a “fluidic channel,” “flow chamber,” “flow channel,” or “reaction chamber”) may receive or be configured to receive a biological sample. FIG. 1 shows a simplified schematic cross-sectional side view illustration of a portion of the channel 100 of the fluidic device disclosed herein. The channel 100 may comprise a surface 101 and a additional surface 102. In some embodiments, the surface 101 and the additional surface 102 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 1). For example, the surface can be provided by the bottom layer and the additional surface can be provided by the top layer that at least partially define the channel. In some embodiments, a middle spacer layer of double-sided adhesive with a cut-out portion can be used to position the surface 101 and additional surface 102 in a facing relationship to at least partly form the flow channel. As a non-limiting example, the cut-out portion can have a dimension of about 0.7 cm width and about a 10 cm length. In some embodiments, the surface and additional surface are substantially parallel, so that the perpendicular distance between them is substantially the same throughout the channel. In some embodiments, the perpendicular distance between a surface and a additional surface depends in part on the nature and size of the biologicalcomponents to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a surface and a additional surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a surface and a additional surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a surface and a additional surface may be in the range of from twice the average size of the largest biological component in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the surface 101 may be a lower surface. In certain embodiments, the additional surface 102 may be an upper surface. The channel 100 may receive a biological sample comprising one or more biological components 50, 51. The channel 100 may receive one or more polymer precursors. As illustrated in FIG. 1, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the surface 101, the additional surface 102, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 50, 51. In some cases, the surface 101 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In certain cases, the additional surface, 102 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In some embodiments, the surface and / or additional surface can be optically transmissive so that visible and UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the flow cell, and the measurement of the analyte and biological components.

[0192] In certain cases, a channel may have a cross-sectional area that is rectangular, circular, semi-circular, or oval. Accordingly, the channel may have a single, internal surface. In some cases, a channel may have a triangular, square, rectangular, polygonal, or other cross-section. Accordingly, the channel may have three or more internal surfaces. One or more of the internal surfaces may be couple or receive, or be configured to couple or receive, the one or more biological components.

[0193] The surface 101, the additional surface 102, or both surfaces 101, 102 may be functionalized, for example with a coating. As a non-limiting example, a surface coating may be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), a functional group to capture one or moremoi eties (e.g., a chemical moiety), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, an aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group to capture one or more moieties. For example, the acrylamide, the agarose, etc. may include such a functional group. In certain embodiments, the surface polymer may comprise polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or combinations thereof. In various embodiments, the surface polymer may comprise a silane polymer. In some embodiments, the surface polymer may be functionalized with at least one of an oligonucleotide, an antibody, a cytokine, a chemokine, a protein, an antibody derivative, an antibody fragment, a carbohydrate, a toxin, or an aptamer. In particular embodiments, the surface coating comprises a material for which adherent cells have a binding affinity, such as fibronectin or laminin.

[0194] In some cases, the surface 101, the additional surface 102, or both surfaces 101, 102 may comprise one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the surface 101, the additional surface 102, or both surfaces 101, 102 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein. The barcodes may cover an area of about 500 nm2to about 5000 mm2of the first or additional surface. In some embodiments, the surface 101, the additional surface 102, or both surfaces 101, 102 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodes between any of the two numbers mentioned herein. In some cases, a barcode (or a portion / subset of barcodes) may be associated with the location of the barcode on a surface (location coordinates (e.g., x-, y-coordinates) on a surface of a channel). In particular, a barcode may comprise a sequence that is unique (“spatially-addressed”) to a spot or region along a surface of the fluidic device. Each spot may comprise a plurality of nucleic acid barcodes that share this “spatially-addressed” sequence. Accordingly, the spot or region of origin of a barcode may be determined based on its spatially-addressed sequence. A spot or region that contains barcodes with common “spatially-addressed” sequences may comprisean area of about 10 to 105pm2along the first or additional surface of the fluidic device. The first or additional surface of the fluidic device may comprise about 50 to 500, about 50 to 1000, about 50 to 5000, about 50 to 104, about 50 to 5xl04, about 50 to 105, about 50 to 5xl05, about 50 to 106, about 50 to 5xl06, about 50 to 107, about 100 to 500, about 100 to 1000, about 100 to 5000, about 100 to 104, about 100 to 5xl04, about 100 to 105, about 100 to 5xl05, about 100 to 106, about 100 to 5xl06, about 100 to 107, about 500 to 1000, about 500 to 5000, about 500 to 104, about 500 to 5xl04, about 500 to 105, about 500 to 5xl05, about 500 to 106, about 500 to 5xl06, about 500 to 107, about 1000 to 5000, about 1000 to 104, about 1000 to 5xl04, about 1000 to 105, about 1000 to 5xl05, about 1000 to 106, about 1000 to 5xl06, about 1000 to 107, about 5000 to 104, about 5000 to 5xl04, about 5000 to 105, about 5000 to 5xl05, about 5000 to 106, about 5000 to 5xl06, about 5000 to 107, about 104to 5xl04, about 104to 105, about 104to 5xl05, about 104to 106, about 104to 5xl06, about 104to 107, about 5xl04to 105, about 5xl04to 5xl05, about 5xl04to 106, about 5xl04to 5xl06, about 5xl04to 107, about 105to 5xl05, about 105to 106, about 105to 5xl06, about 105to 107, about 5xl05to 106, about 5xl05to 5xl06, about 5xl05to 107, about 106to 5xl06, about 106to 107, or about 5xl06to 107spots or regions that contain barcodes with unique “spatially-addressed” sequences along one or more surfaces. Similarly, the fluidic device may comprise one or more fluidic channels, each of which may comprise about 50 to 107spots or regions that contain barcodes with unique “spatially-addressed” sequences along one or more surfaces.

[0195] A barcode may be attached to or coupled to the captured biological component. In some embodiments, the barcode may be a unique identifier that distinguishes a biological component from other biological components (e.g., that identifies a first biological component versus a second biological component). In some embodiments, a barcode may comprise a nucleic acid sequence (e.g., common sequence) to capture a biological component, or used in amplification. In some embodiments, a barcode may comprise a unique identifier comprising a unique nucleic acid sequence (e.g., DNA sequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. In some embodiments the barcode may comprise a fluorescent molecule. In some embodiments, a location of the captured biological component may be associated with the unique identifier to, for example, retain spatial information of a biological component.

[0196] In some embodiments, the fluidic device that comprises one or more fluidic channels may be a flow cell. For example, the fluidic device may be used for sequencing (e.g., DNA orRNA sequencing). In certain embodiments, the fluidic device may be a microfluidic device. In particular embodiments, the fluidic device may be a nanofluidic device.

[0197] FIGS. 3A-D illustrate an example of a fluidic device consistent with the present disclosure. The channel 100 of FIG. 1 may correspond to any one of the cut-out regions (405 A, 405B, 405C) of FIG. 3D. FIGS. 3A-D figures depict the components of a flow cell comprised of a bottom layer 400 (FIG. 3A), a spacer layer 402 (FIG. 3B), and top layer 404 (FIG. 3C). The bottom layer (400) and the top layer (404) can both independently be a glass or plastic material. In some cases, the top layer (404) is optically transparent or translucent. In some cases, the bottom layer (400) is optically transparent or translucent. In many cases, the top layer (404) and the bottom layer (400) are optically transparent or translucent. The spacer layer (402) can be a double-sided pressure sensitive adhesive with one or more cut-out regions (405A, 405B, 405C). In this design, the one or more cut-out regions of the spacer layer are sandwiched between the bottom layer and the top layer to form one or more channels. In various embodiments, the spacer layer (402) includes a core plastic (e.g., PET) layer with pressure sensitive adhesive coating on its top and / or bottom sides that contact the top (404) and bottom (400) layers. Examples of adhesive coatings consistent with the present disclosure include siloxane, silicone, acrylate, acrylamide, polyvinyl, polyurethane, epoxy, polyphenol, polyester, polyamide, polyimide, polytetrafluoroethylene, polyethylene, polypropylene, polycarbamate, polycarbonate, polyacrylic acid, sulfonated polyester, and combinations thereof.

[0198] Referring to FIG. 3B, a peripheral portion (407) of the spacer layer (402) provides a boundary for one or more cut-out regions (406) with defined widths and lengths. In many designs disclosed herein, the spacer layer (402) adheres to the top (404) and bottom (400) layers, such that a cut-out region or plurality of cut-out regions define a channel or a plurality of channels. The spacer layer (402), bottom layer (400), and / or top layer (404) can be water (or optionally more generally liquid) impermeable such that a first aqueous sample in a first cut-out region is isolated from a second aqueous sample in a second cut-out region.Biological components and reagents in a first cut-out region may similarly be isolated from other cut-regions. The one or more cut-out regions can thus define flow cell channels with defined dimensions.

[0199] A channel defined by a cut-out region can be operably coupled to an inlet (408) and / or an outlet (410) in the top layer (404), through which, for example, a gas, liquid, sample, or reagent may flow. In various embodiments, the inlet (408) and outlet (410) can both be represented as through holes in the top layer (404). However, the inlet (408) and / or outlet(410) can be disposed within other components of the flow cell, such as the bottom layer (400) or spacer layer (402).

[0200] With respect to FIG. 3D, which provides an end-on view of the fluidic device, the top layer (404) can have a height of about 0.7 mm and the bottom layer can have a height of about 0.5 mm. The cut-out region (406) can have a height defined by the distance between opposing faces of the top layer (404) and the bottom layer (400). The fluidic device can include multiple cut-out regions. For example, cut-out regions 405A and 405B may be disposed on opposite sides of cut out region 406. The cut-out regions may be identical or may have different shapes and / or dimensions. The spacer layer can have a height ranging from about 50 microns to about 200 microns, and preferably, from about 70 microns to about 130 microns. The cut-out region can have dimensions of about 10.3 cm length x about 0.7 cm width.Hydrogel Chambers

[0201] FIGS. 2A-C illustrate an exemplary method for forming a chamber enclosing one or more biological components in a fluidic channel as disclosed herein, for example in a drug titration or cell capture assay. FIG. 2A shows a portion of a system as provided herein (e.g., comprising a fluidic channel as disclosed herein) including an energy source. FIG. 2B shows a polymer matrix being formed around a biological component in a portion of a system as provided herein. FIG. 2C shows a method of forming a polymer matrix around a biological component in a system as provided herein. As illustrated in FIGS. 2A-2C, in some embodiments, the one or more chambers extend from the bottom layer to the top layer of a fluidic channel.

[0202] With continued reference to FIG. 2A, the channel 200 of the system may include a surface 201 provided by the bottom layer and a additional surface 202 provided by the top layer of the fluidic channel. The energy source 203 may comprise one or more energy emitting portions (e.g., an energy emitting portion 205). In some embodiments, the energy source 203 may comprise one or more non-emitting portions (e.g., a non-emitting portion 204). The non-emitting portion 204 may not emit, or be configured to emit, energy. In some embodiments, the emitting portion 205 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic channel. For example, the energy source may comprise an LED array in which individual LEDs can be selectively activated (e.g., act as an energy emitting portion 205) to create light projections with specified patterns. In some embodiments, the fluidic channel may be coupled to or disposed on a movable stage. In other embodiments, light may be projected to or onto at least a portionof the first fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the first fluidic channel. The energy source (e.g., light source) may be coupled to the fluidic channel via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and / or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on at least a portion of a surface of the fluidic channel. The polymer matrix may be formed similarly or complementary to the pattern of energy emission.

[0203] A fluidic channel can comprise one or more polymer precursors for forming chambers. In some embodiments, the one or more polymer precursors are added to the fluidic channel along with one or more cells. In various embodiments, the one or more cells and the one or more polymer precursors can be pre-mixed and then added to the fluidic device at the same time; the one or more cells and the one or more polymer precursors can be added to the fluidic device at the same time; the one or more cells can be added to the fluidic device first and then the one or more polymer precursors can be added to the fluidic device second; or the one or more polymer precursors can be added to the fluidic device first and then the one or more cells can be added to the fluidic device second. Such precursors may be selected from a wide variety of compounds including, but not limited to, polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N’-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof. In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEGthiol / PEG-acrylate, acrylamide / N,N’-bis(acryloyl)cystamine (BACy), orPEG / PPO. In some embodiments, the following precursors and crosslinker may be used to form chambers with degradable polymer matrix (hydrogel) walls. Polymer precursors may be formed by using any precursor and crosslinker of Table 2A (columns 1 and 3, respectively) and / or a crosslinker of Table 2B. The resulting polymer matrices may be degraded with the indicateddegradation agents in Table 2A (column 4). Representative crosslinkers useful for polymer synthesis are listed in Table 2B.Table 2APrecursors Hydrogels Crosslinkers Degradation Agents Acrylamide Polyacrylamide Bis-acryloyl cystamine DTT / TCEP / THP (Structure 1)PEG-based PEG Bis(2- DTT / TCEP / THP acryloyl methacryloly)oxy ethyldisulfide (Structure 2)Dextran-based Dextran N,N’-(1,2- NaI04acryloyl Dihydroxylethylene)bis- acrylamide (Structure 3)Polysacchride-base Polysaccharide Structure 4 NaOH, acryloyl ethanolamine,DTT / TCEP / THP Gelatin-base Gelatin Structure 5 NaOH, acryloyl ethanolamine, nucleophilic bases - - Structure 6 NaOH, alkali, organic bases - - Structure 7 Acid%7^ OxcccX I A X .W}Kai . f . S- U^xx: _<:. i:j « . J¥. ., .. v . . . ».In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.wherein R1is a Ci-Ce linear or C3-C6 cyclic saturated alkyl group substituted with q instances of, the remaining substituents on R1are hydrogen, and q is an integer from 3 to 10.In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.In some cases, m is an integer selected from 0-100.In some cases, m is an integer selected from 5-50.In some cases, m is an integer from 0-10.y’’' \.. ....j - •’ / ".v-A x 's-M . .r. / i- "rv -. Z'v 'V ■ In some cases, m is an integer selected from 0-100.In some cases, m is an integer selected from 5-50.In some cases, m is an integer from 0-10..< ? % k. ••• -< / v.1V ' :-.x / 'X ”>lK t-S x--\« < A;, o' rIn some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.cZ"\„y; WO, -P' -f- ■ t- -X .In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.T wlI *F i r 4, i r o. "i•: :•: s^s: > .•.•£■%•:•v- :■ .«»>.> -XJ-- •>.;. ::In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.In some cases, m is an integer selected from 0-100.In some cases, m is an integer selected from 5-50.In some cases, m is an integer from 0-10.In some cases, p is an integer selected from 0-100.In some cases, p is an integer selected from 5-50.In some cases, p is an integer from 0-10.0-7prY- OH ,OHa< MO NH io< o-Na+HO - i tiIn some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.; 'ft"0->30 Z ; ** ■«; '^P. / 1 ( -<w- / L °^;i.In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.In some cases, m is an integer selected from 0-100.In some cases, m is an integer selected from 5-50.In some cases, m is an integer from 0-10.(A|j•: :<x-xX-- Z '- x-' < - . I- .lS '~ ; < < . >'z' « .In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.\ i. / Z A I W! T M8 o 0In some cases, n is an integer selected from 0-100.In some cases, n is an integer selected from 5-50.In some cases, n is an integer from 0-10.

[0204] A polymer precursor can further comprise additional reagents that affect polymerization and polymer matrix properties. As examples, a polymer precursor can include a crosslinker, a porogen, a viscosity-modifying agent, an acid, a base, a catalyst, a salt, a photoinitiator, or a combination thereof.

[0205] As used herein, the term “crosslinker” denotes a species with a greater number of polymerizable groups than are required to generate a linear polymer chain. For example, a crosslinker can contain two or more ethylenically unsaturated groups or three or more reactive centers for bifunctional polymer synthesis (e.g., the three methoxy groups of trimethoxybenzene in the context of polyester synthesis). Crosslinkers are thus generally configured to generate a nonlinear polymer network upon polymerization.

[0206] As used herein, the term “porogen” can denote a species that modulates the porosity of a polymer matrix but does not incorporate into the polymer matrix during polymerization. Porogens typically diffuse out of polymer matrices following polymerization, leaving pores in the regions that they occupied. Porogen size, concentration, hydrophobicity, and hydrophilicity can thus influence pore density and pore size in polymer matrices. Examples of porogens consistent with the present disclosure include particles (e.g., polymeric, ceramic, metal, metal oxide, or hydrogel particles), polymers such as polyethylene glycol and alginate, and vesicles such as liposomes or micelles.

[0207] As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In many cases, a photoinitiator included in a polymer precursor formulation is a type I photoinitiator, that is a molecule that generates radicals throughintramolecular cleavage (e.g., homolysis) upon photoexcitation, or a type II photoinitiator, that is a molecule that abstracts an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of photoinitiators utilizable in the present methods include acetophenone, anisoin, anthraquinone, anthraquinone-2-sulfonic acid, benzil, benzoin, benzophenone, 3,3’,4,4’-benzophenonetetracarboxylic dianydride, 4-benzoylbiphenyl, 2-benzyl-2-(dimethylamino)-4’-morpholinobutyrophenone, dibenzosuberenone, 2,2-diethoxyacetophenone, 2-ethylanthraquinone, ferrocene, 2-isopropylthioxanthone, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, methyl-2-benzoylbenzoate, and thiooxanthen-9-one.

[0208] In some embodiments, the generation of a polymer matrix within said fluidic channel comprises exposing the one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light at 350 nm to 800 nm. In some embodiments, the light generating device generates light at 350 nm to 600 nm. In some embodiments, the light generating device generates light at 350 nm to 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of the polymer matrix comprises between about 1 and 3 seconds of illumination, between about 1 and 5 seconds of illumination, between about 1 and 10 seconds of illumination, between about 1 and 15 seconds of illumination, between about 1 and 20 seconds of illumination, between about 1 and 30 seconds of illumination, between about 1 and 50 seconds of illumination, between about 3 and 5 seconds of illumination, between about 3 and 10 seconds of illumination, between about 3 and 15 seconds of illumination, between about 3 and 20 seconds of illumination, between about 3 and 30 seconds of illumination, between about 3 and 50 seconds of illumination, between about 5 and 10 seconds of illumination, between about 5 and 15 seconds of illumination, between about 5 and 20 seconds of illumination, between about 5 and 30 seconds of illumination, between about 5 and 50 seconds of illumination, between about 10 and 20 seconds of illumination, between about 10 and 30 seconds of illumination, between about 10 and 50 seconds of illumination, between about 20 and 30 seconds of illumination, or between about 20 and 50 seconds of illumination. In some embodiments, the generation of a polymer matrix within said fluidic channel is performed using a spatial light modulator (SLM) (i.e. a spatial energy modulation element that is capable of generating desired light intensity pattern spatially). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid crystal based. AnSLM such as a DMD may similarly be used to direct light to one or more drugs or cells during an assay.

[0209] Optionally, a first chamber of the one or more chambers can be disposed inside of a second chamber of the one or more chambers. This design can be utilized to separately partition two species (e.g., a cell and a reagent or two cells) within close proximity. This design can also be used to control the timing with which two species are contacted. For example, a method can include forming a first chamber around a cell, flowing a particle (or other assay reagent incapable of diffusing into the first chamber) adjacent to the cell, forming a second chamber surrounding the first chamber and enclosing the particle, and selectively degrading the first chamber to allow the cell and particle to come into contact within the second chamber.

[0210] In some embodiments, a functional group can be coupled to one or more chambers. Some non-limiting examples of functional group may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an aldehyde dithiolane, PEG, a thiol, an alkene, an alkyne, an azide, or a combination thereof. In some cases, the functionalized chamber may be used to capture a biomolecule enclosed therein, thereby trapping the biomolecule in proximity to a biological component (e.g., a cell) enclosed within the chamber. The biomolecule may be produced by the biological component (e.g., secretome from a cell). The functionalized surface of the polymer matrix inside the compartment may be used to capture reagents or molecules from outside the compartment. The functionalized surface may increase surface area covered by a reagent, a molecular sensor, or any molecule of interest (e.g., an antibody).

[0211] With continued reference to FIG. 2A, the polymer matrix 208, 209, or at least a portion of the polymer matrix 208, 209, may be coupled to the surface 201, the additional surface 202, or both surfaces 201, 202. In certain embodiments, the polymer matrix, or at least a portion of the polymer matrix, may be coupled to a third surface, a fourth surface, a fifth surface, etc. as appropriate. In various embodiments, the polymer matrix 208, 209 may extend from the surface 201 to the additional surface 202 (e.g., through at least a portion of a lumen of the channel 200 or a cavity of a chamber) such that the polymer matrix surrounds, or substantially surrounds, the biological component 50. In some embodiments, two or more biological components (e.g., biological components 50, 51 of FIG. 2C) that are in close physical proximity may be separated (e.g., by agitating or shaking a fluidic device that comprises the fluidic channel). The fluidic device may be agitated or shaken by physicalmovement, use of a sonic pulse, changing a flow in the channel, or any other suitable method of agitation. A polymer matrix may then be formed that surrounds (or partially surrounds) the biological components that are separated. FIG. 2B shows polymer matrices 218, 219 formed surrounding the biological component 52 after being separated from the biological component 53. FIG. 2C shows a process, according to various embodiments, of separating the two biological components 50, 51, which are in close proximity. That is, by agitating or shaking the fluidic device the biological components 50, 51 can be separated. In some embodiments, separation of the biological components is achieved through fluidic pressure, flow pulsation, dielectrophoresis, optothermal flow, or some combination thereof. In some cases, separation of the biological components is achieved through acoustic vibration. FIG.2C also shows a polymer matrix being formed to generate a compartment 222 surrounding the biological component 50 after the separation of the biological components 50, 51.

[0212] A fluidic device disclosed herein can include a detector that is configured to detect one or more locations of one or more biological components contained within a channel. In certain embodiments, the energy source 203 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In various embodiments, a mask may be generated using an image obtained from at least a portion of the fluidic device. The mask may allow or permit the energy source 203 to emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the surface 201. The mask may inhibit or prevent the energy source 203 from emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the surface 201. In some embodiments, the image may be obtained from a camera (e.g., a digital camera, fluorescent imaging camera, etc.). In some embodiments, the camera may be coupled to, connected to, or in communication with the energy source 203. For example, the camera (not shown) may be in electrical communication with the energy source 203. In some embodiments, the energy source 203 may comprise the camera. In various embodiments, the energy source 203 may comprise a microscope (e.g., a fluorescence microscope, a confocal microscope, lens-free imaging system, a transmission electron microscopy (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used to detect one or more positions of one or more biological components (e.g., in combination with the detector).

[0213] In some embodiments, one or more chambers has sufficiently large pores to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule,an antibody, etc.) therethrough. Simultaneously, one or more chambers can have sufficiently small pores to allow movement or transfer of a reagent and / or biological component (e.g., DNA, RNA, a protein, a cell, etc.). In some embodiments, the pores have a diameter from 5 nm to 100 nm. In some embodiments, the pores have a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores have a diameter smaller than 5 nm.

[0214] In particular embodiments, pores of the one or more chambers are formulated to encapsulate sufficiently large genetic material, nucleic acids with greater than 300 base pairs, but to allow smaller materials, such as reverse transcriptases and 50 base pair nucleic acid primers to pass through the pores, thereby passing in and out of the hydrogel structures. In some embodiments, the pore size of the hydrogel structures is tuned by varying the ratio of the concentrations of polymer precursors to the concentration of crosslinkers, varying pH, salt concentrations, temperature, light intensity, and the like. In some embodiments, the average diameter of pores of a chamber prevent passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater; or having a molecular weight of 50 kDa or greater; or having a molecular weight of 75 kDa or greater; or having a molecular weight of 100 kDa or greater; or having a molecular weight of 150 kDa or greater. In some embodiments, DNA or RNA retained have lengths that are sequencable using conventional sequencing-by-synthesis techniques. For example, such DNA or RNA comprise at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the pores may have an average diameter from 5 nm to 100 nm.

[0215] The pore sizes of the one or more chambers may be modulated using a chemical reagent, or by applying heat, electrical field, light, or another suitable stimulus. In other words, a chamber may comprise a tunable property (e.g., the pore size). In some cases, one or more chambers comprises a thermoresponsive or temperature-responsive polymer. A thermoresponsive polymer (e.g., poly(N-isopropylacrylamide) (NIPAAM)) may phase separate from a solution upon heating or upon cooling (e.g., polymer showing lower critical solution temperature (LCST) or upper critical solution temperature (UCST)). The polymer matrix may comprise polymer which may collapse at high temperature in order to, for example, control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that may be used to form hydrogel / polymer matrix with tunable properties may include Poly(N-vinyl caprolactam), Poly(N-ethyl oxazoline), Poly(methyl vinyl ether), Poly(acrylic acid- coacrylamide), or a combination thereof. A change intemperature may enlarge or contract average pore size in the polymer matrix to allow selected molecules, such as a nucleic acid molecule, a protein, or any biomolecule or molecule smaller than the adjusted pore size to be released from a hydrogel chamber.Cell Culture and Incubation Protocols

[0216] The cells or cell aggregates and polymer precursors can optionally be mixed outside of the fluidic system before they are loaded into the channel. In some embodiments, cells are delivered to the channel, where they are dispersed over a surface of the channel. For example, the one or more cells may be randomly disposed on the surface. Alternatively, the one or more cells may be randomly disposed across or between structures or regions for which the one or more cells comprise a binding affinity. As an example, the one or more cells may localize to regions that comprise an adherent support such as fibronectin, poly-1-omithine, or laminin, and partition away from portions of the surface that comprise exposed glass.

[0217] In various embodiments, a method of incubating one or more cells can include loading a fluidic channel (e.g., (100) in FIG. 1) with the one or more cells and one or more polymer precursors. The one or more polymer precursors can be polymerized to form one or more chambers within the channel that at least partially enclose the one or more cells. For example, after the location of the one or more cells are identified with a detector, a spatial energy modulation element may project light into the channel such that the projected light causes cross-linking of the one or more polymer precursors to form polymer matrix walls of the one or more chambers. Exemplary chamber designs include circular, elliptical, and polygonal shapes that fully enclose inner spaces (e.g., do not include a discontinuity or break). Furthermore, in many aspects, the one or more chambers extend from the bottom layer (400) to the top layer (404) of a flow cell, such that species enclosed within a chamber of the one or more chambers is isolated or partially isolated from species that are extrinsic to the one or more chambers or enclosed within other chambers. As used herein, “partially isolated” can denote that a chamber prevents a subset of species (e.g., cells and large nucleic acids) from entering or leaving a chamber enclosing a species but allows other species (e.g., small molecule analytes) to enter and leave the chamber.

[0218] An assay can include one or more assay components, which may be provided with or as part of a channel include, but are not limited to, capture elements such as capture oligonucleotides, primers for captured nucleic acid amplification, antibodies, detectable labels, and drugs. In some embodiments, such assay components may be attached to any one of a surface, a additional surface or a polymer matrix wall exclusively, or on combinations ofsuch surfaces, either exclusively, or in combination with other reagents. In some embodiments, assay components that may be provided after synthesizing gel chambers include, but are not limited to, lysing reagents, transcription reagents, reverse transcription reagents, antibodies, polymerases, primers, particles, and the like. In some embodiments, cellular or assay components may be attached or captured by capture elements on a polymer matrix wall.

[0219] In some embodiments, incubating under assay conditions may comprise only a single step of an assay comprising more than one step, such as, for example, a step of detecting a signal (or in the case of a material signal, generating a sequencing-ready nucleic acid), or such step of incubating under assay conditions may comprise a plurality of steps of a multi-step assay. In some embodiments, cells may be treated or subjected to assay steps prior to loading into a channel of a fluidic device, so that the step of incubating may comprise only a single step of a multi-step assay, such as signal generation and / or signal collection. In other embodiments, the step of incubating may comprise the implementation of an assay step or part of an assay, such as, cDNA synthesis, second strand synthesis, capture of an assay component or a cellular component, or the like. In some embodiments, assay conditions may comprise a series of steps each with different conditions (e.g. temperature, pH, presence or absence of particular reagents, such as, primers or an enzyme, e.g., a ligase, a polymerase, a transposase, or the like). For example, such steps may comprise loading primers so that they diffuse across chamber walls and anneal to target sites on a template strand in a sample in the chamber, extending the annealed primers, heating the channel so that the extended primers melt from its template strand, capturing the melted extended primers by capture agents attached to the surface, and so on.Immiscible Phases

[0220] An aqueous-filled chamber may be surrounded by a water-immiscible fluid such as oil. It was surprisingly discovered herein that polymeric chambers can retain aqueous contents upon immersion within a water-immiscible fluid, for example upon oil input into a fluidic device that contains the chambers. As overviewed in EXAMPLE 5, the chamber may prevent water-immiscible fluid ingress even when the chamber includes nanoscale pores. The chamber may also be stable in the presence of the water-immiscible fluid for the timespan of an assay (e.g., days, weeks, months, or longer). Accordingly, cells may be incubated within chambers surrounded by the water-immiscible fluid without suffering from exposure to the water-immiscible fluid. In the context of a water-immiscible fluid, ‘surrounding’ a chamber may denote that the water immiscible-fluid encompasses or encircles the chamber andoptionally further encompasses or encircles an aqueous phase immediately outside of the chamber. In this context, the water-immiscible fluid may directly contact the chamber, for example when the water-immiscible fluid forms an interface with an outer surface of the chamber, and is not separated from the outer surface of the chamber by an intervening aqueous phase. The water-immiscible fluid can be periodically replaced with nutrientcontaining buffer or media to refresh aqueous media within chambers and facilitate longitudinal (e.g., week or monthlong) cellular analyses.

[0221] It was further demonstrated herein, for example in EXAMPLE 6, that surrounding chambers with a water-immiscible fluid may prevent reagents or cellular products from diffusing between chambers. As, in some embodiments, the disclosed chambers can have nanometer-sized pores, reagents such as drugs, small molecules, and proteins, as well as cell lysate or cellular secretions such as cytokines, vesicles, and CO2 may flow between chambers with different cells and / or drug treatment regimens. Surrounding chambers with a water-immiscible fluid such as oil can prevent water soluble species from diffusing between chambers, and keep drugs and other reagents confined with chambers. Water-immiscible fluid can be in a liquid form and have water solubility of less than about 1%, 0.1%, 0.01%, 0.001%, or 0.0001% [(weight of dissolved water / weight of water-immiscible fluid) x 100%]. In addition, water-immiscible fluid can be in a liquid form and have dissolved oxygen content similar to or higher than water that can be beneficial for maintaining cell viability. In various embodiments, the water-immiscible liquid can have a dissolved oxygen content of about 1%, 5%, 10%, or 40% by volume of water-immiscible liquid (at 37 C and 1 atmosphere of pressure).

[0222] As an example, a method for cellular analysis may include inputting the cell into a fluidic channel; inputting a polymer precursor into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell; inputting a water-immiscible fluid into the fluidic device, wherein the water-immiscible fluid surrounds the chamber; and analyzing the cell using a method disclosed herein. The method can further include removing the water-immiscible fluid from the fluidic device, for example by inputting buffer or media that displaces the water-immiscible fluid. In embodiments that utilize drugs, the method can include releasing the drug within the chamber before the chamber is surrounded by the water-immiscible fluid or while the chamber is surrounded by the water-immiscible fluid. Examples of water-immiscible fluids consistent with the present disclosure include mineral oils, silicone oils, perfluorinated oils, vegetable oils, bean oils, and PEG-perfluoroalkyl block copolymers.Systems

[0223] FIG. 4A is an example system for carrying out the above method. Flow cell (500) is a component of a fluidic device that provides channels for carrying out a variety of assays and liquid handling components under programmable control for delivering samples and reagents to the channels. In this illustration, four channels (502, 504, 506, and 508) are shown. However, as detailed elsewhere herein, systems of the present disclosure can utilize flow cells with fewer or greater numbers of channels.

[0224] The system of FIG. 4A includes an optical system (521) for photosynthesizing chambers at locations of cells or other analytes in the channels (502, 504, 506, and 508) of the flow cell (500) and for collecting images and other optical signals. The optical system (521) includes a light source (522) that generates a light beam (523) of appropriate wavelength light (e.g. UV light) for synthesizing chambers (e.g., hydrogel chambers) in the flow cell (500). The light beam (523) that passes through an appropriate photo-mask or beam-shaping or beam steering (Galvo) system (524) for shaping a beam to synthesize a desired structure or structures in a channel. In some embodiments, this beam shaping system (524) includes a digital micromirror device (DMD). In other embodiments, a physical photomask may be employed. Reflected light from DMD (524) is shaped using conventional optics, e.g. collimating optics (528), and is directed through objective lens system (534) into channel 2 segment (510). In exemplary embodiments, the light is directed by one or more dichroic mirrors (530 and 531).

[0225] Chamber position, shape and polymer matrix wall thickness is determined at least in part from cell position information determined from images collected by detector (532). Objective (534) and flow cell (500) move relative to one another in the xy-directions (536) to photosynthesize chambers at any position in any of the channels. In some embodiments, the flow cell (500) moves and optical system (521) is stationary. The system may utilize light from a light source (599), such as a homogenized light condenser, that is positioned on an opposite side of the flow cell as the optical system and directs light through the flow cell (500) to the objective (534). To achieve this functionality, the light source positioned on the opposite side of the flow cell (599) can be configured to move in tandem with the optical system (521), or the light source (599) and optical system can be stationary and the flow cell (500) can be moved to the region illuminated by the light source (599) and from which light is collected by the objective (534). In some embodiments, objective (534) may also direct light beam (527) from light source (529) to targets, such as cells, on surface (514) and collect optical signals, such as fluorescent signals, from assays taking place on surface (514).Optical signal collection can also be carried out with a separate objective. Information collected by detector (532), particularly cellular positions in their respective channels, is employed by computer (538) and / or subsidiary controllers to direct DMD (524) and translation devices controlling the relative positions of objective (534) and flow cell (500) to synthesize hydrogel chambers of the appropriate shape and size at the appropriate locations.

[0226] FIG. 4B provides a blown-up view of the exemplary channel segment (510) of the flow cell of FIG. 4A. On surface (514) of channel 2 (504) a plurality of cells, e.g. (518), are each enclosed by a hydrogel chamber, e.g. (516). In some embodiments, the porosity of polymer matrix walls of the hydrogel chambers is selected to be impermeable to the cells, but permeable to assay reagents. Thus, reagents may be introduced to, and removed from, the interiors of the hydrogel chambers by flowing (520) them through the channels, but cells are retained in the hydrogel chamber (516).

[0227] One of ordinary skill in the art would recognize that optical systems with different configurations than those of FIG. 4A and 4B may be employed for carrying out these functions. In some embodiments, a plurality of DMD-objective subsystems for synthesizing hydrogel structures may be employed to increase the speed of synthesis by synthesizing multiple structures simultaneously.Computer Systems

[0228] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 5 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameter using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0229] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g.,network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and / or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1530 in some cases may be a telecommunication and / or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server.

[0230] The CPU 1505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions can be directed to the CPU 1505, which can subsequently program or otherwise configure the CPU 1505 to implement methods of the present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0231] The CPU 1505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).

[0232] The storage unit 1515 can store files, such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, e.g., user preferences and user programs. The computer system 1501 in some cases can include one or more additional data storage units that are external to the computer system 1501, such as located on a remote server that may be in communication with the computer system 1501 through an intranet or the Internet.

[0233] The computer system 1501 can communicate with one or more remote computer systems through the network 1530. For instance, the computer system 1501 can communicate with a remote computer system of a user (e.g., a laptop, a personal computer, a tablet, or a mobile phone). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones,Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1501 via the network 1530.

[0234] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software.During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations, the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.

[0235] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0236] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, termssuch as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0237] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0238] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, for example, an image of a biological component, a barcode, a signal or measurement of a local parameter.Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0239] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1505. The algorithm can, for example, identify a biological component, detect a barcode, generate a spatial modulating element (e.g., a mask), provide energy from an energy source, detect or measure a local parameter using a sensor, etc.

[0240] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled inthe art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.EXAMPLES EXAMPLE 1Spatially-Controlled Fluorophore Bleaching Along A Fluidic Channel Surface

[0241] This example covers spatially controlled fluorophore photobleaching along a fluidic channel surface. Cy5 fluorophore coupled to a polyAmRNAtag was input into a fluidic channel that contained polyT nucleic acid capture sequences coupled a surface of a flow cell. The polyA mRNA tags hybridized to the polyT capture sequences, localizing the Cy5 fluorophore to approximately 80 micron diameter circular regions along the top surface of the fluidic channel. A fluorescence image of three such regions with Cy5 fluorophore hybridized to polyT capture sequences (1901, 1902, 1903) is shown in FIG. 18A.

[0242] UV light was input into the fluidic channel in a spatially controlled manner to photobleach Cy5 fluorophore in selected portions of the fluidic channel. Referencing Cy5 fluorophore-containing regions 1901, 1902, and 1903 from FIG. 18A, a digital micromirror device (DMD) was used to fully irradiate the leftmost circle (1901), half of the middle circle (1902) with 365 nm UV light. The rightmost circle (1903) was not irradiated with UV light.

[0243] Fluorescence intensities of the three Cy5 fluorophore containing regions 1901, 1902, and 1903 were remeasured following UV irradiation. FIG. 18B is a fluorescence image of the three Cy5 fluorophore-containing regions 1901, 1902, and 1903 following UV irradiation. FIG. 18C is a plot of fluorescence intensities across the Cy5 fluorophore-containing regions 1901, 1902, and 1903 prior to (“Baseline”) and following (“Post-Illumination”) UV-irradiation as a function of horizontal position in the fluidic channel. As shown in these figures, the fluorescence intensity of the leftmost region (1901) and the left half of the middle region (1902) decreased following UV irradiation, indicating that Cy5 fluorophore photobleached in the regions subjected to UV irradiation. Fluorescence intensity decreased in Fluorescence intensity was retained in the right half of the middle region (1902) and the rightmost region (1903), confirming that the UV light was input into the fluidic channel in a spatially-controlled manner. While this example was directed to fluorophore photobleaching, the results from this example suggest that all or a portion of a drug may be photocleaved from a fluidic channel surface in a spatially-controlled manner. In addition, the DMD device was capable of applying precise amounts of UV light to relatively small predetermined areas ofthe fluidic channel surface showing the feasibility of releasing controlled amounts of drug with a photocleavable mechanism.EXAMPLE 2Spatially-Controlled Quencher Release From A Fluidic Channel Surface

[0244] This example is directed to spatially-controlled quencher release from a fluorophore-functionalized surface. Fluorophore-quencher constructs are input into a fluidic channel. Each construct contains, in order, a polyA mRNA tag, a Cy5 fluorophore, a nucleic acid linker withthe photocleavable nitrobenzyl intemucleotide linkageO' , and the quencher molecule 4'-(4-Nitro-phenyldiazo)-2'-methoxy-5'-methoxy-azobenzene-4"-(N-ethyl)-N-ethyl-2-cyanoethyl-(N,N-diisopropyl) (5BHQ-2). The top surface of the fluidic channel contains polyT capture sequences that hybridize to the polyA mRNA tags of the fluorophore-quencher constructs. Selected portions of the top surface of the fluidic channel are irradiated with UV light to cleave the nitrobenzyl-modified nucleotides of fluorophore-quencher constructs. Nitrobenzyl-modified nucleotide cleavage liberates the quencher from irradiated constructs. The fluorescence intensities of UV irradiated regions of the fluidic channel top surface increase following UV irradiation.EXAMPLE 3Fluorescence-Quantitated Drug Release

[0245] This example covers a method for quantitating drug-release from a surface of a fluidic channel. Fluorophore-quencher constructs and drug-nucleic acid complexes are input into a fluidic channel. Each fluorophore-quencher construct contains, in order, a polyA mRNA tag, a Cy5 fluorophore, a photocleavable nitrobenzyl-modified nucleic acid, and the quencher molecule 5BHQ-2. Each drug-nucleic acid complex comprises, in order, a polyA mRNA tag, a photocleavable nitrobenzyl-modified nucleic acid, and the drug molecule. The top surface of the fluidic channel contains polyT capture sequences that hybridize to the polyA mRNA tags of the constructs and drug-nucleic acid complexes. Selected portions of the top surface of the fluidic channel are irradiated with UV light to cleave the nitrobenzyl-modified nucleotides of fluorophore-quencher- constructs and drug-nucleic acid complexes.Nitrobenzyl-modified nucleotide cleavage liberates the drug and quencher from portions ofthe fluidic device surface. The fluorophore remains bound to the surface following the nitrobenzyl-modified nucleotide cleavage. Fluorescence intensities of UV irradiated regions of the fluidic channel top surface increase following UV irradiation proportional to quencher release. Fluorescence intensity increases can be used to calculate amounts of the drug released from discrete regions along the fluidic channel surface and / or to verify the release of the drug in the appropriate area where UV irradiation was directed.EXAMPLE 4Spatially-Controlled Small Molecule Release Within A Fluidic Device

[0246] This example is directed to spatially-controlled release of small molecules at defined locations within a fluidic device. An overview of the method associated with this example is provided in FIG. 19A. This method utilized a fluidic device that contained a surface (1900) with oligonucleotide capture elements (1901) arranged within discrete, circular spots. The oligonucleotide capture elements (1901) contained poly(T) capture sequences (1901A) at their free ends. Quencher-fluorophore-oligonucleotide constructs (1902) were input into the fluidic device. Each construct (1902) included, in order, a poly(A) sequence (1902 A), a fluorophore (1902B), a photocleavable group (1902C), and a quencher (1902D). The poly(A) sequences (1902D) in the constructs (1902) hybridized to the poly(T) capture sequences (1901A) of the capture elements. The proximity of the quencher (1902D) to the fluorophore (1902B) prevented the fluorophore (1902B) from fluorescing. Excess, unbound constructs (1902) were removed from the fluidic device, and a subset of the discrete, circular spots were illuminated (1903) at a wavelength capable of cleaving the photocleavable groups (1902C) within the constructs, thereby uncoupling quenchers (1902D) from the irradiated constructs (1902) and increasing the fluorescence intensities (1904) of the fluorophores (1902B) coupled to the oligonucleotide capture elements.

[0247] FIG. 19B is a plot of fluorescence intensities as a function of illumination time (for photocleavable group (1902C) cleavage). The circles and rectangles within the plot represent mean fluorescence values and standard deviation, respectively. Fluorescence increased linearly over 5 minutes of 405-430 nm irradiation with a power of about 100 to 700 mW / cm2, demonstrating that quencher release correlated with light dose.

[0248] FIG. 19C is a set of representative fluorescence images following 30 seconds (left image) and 300 seconds (right image) of illumination for photocleavable group cleavage. The rectangular illumination area covered about 21 discrete, circular spots. Minimal fluorescence intensity was observed outside of the rectangular, illuminated area, indicating thatphotocleavage was confined to the rectangular illumination area. In addition, the fluorescent intensity of the circular spots was much higher at the 300 second exposure time than the 30 second exposure time indicating more photocleavage at longer exposure times.EXAMPLE 5Cell Viability Within Oil-Surrounded Polymeric Chambers

[0249] This example is directed to cell viability during multi-day incubations within polymeric chambers surrounded by oil. Suspension-state Jurkat cells were incubated for 24 and 48 hours in polymeric chambers of varying sizes. Jurkat cells were input into fluidic devices with a photopolymerizable polymer precursor. Light was projected into the fluidic devices using a digital micromirror device to photopolymerize the polymer precursor into cylindrical polymeric chambers that (i) extended between the top and bottom surfaces of the fluidic devices, (ii) had 12 pm thick walls, and (iii) had radii ranging from 50 to 400 pm. The polymeric chambers were estimated to have pores at least 5 nm in size based on previous analyses. The fluidic devices were washed and then were filled either with fresh media or with oil and were incubated for 24 or 48 hours.

[0250] FIGS. 20A-B are brightfield images of 50 and 100 pm diameter chambers, respectfully, following 24 hours of incubation within the oil. For reference, a label is provided for a chamber (2001), oil-media boundary (2002), and cell (2003) in each figure. Phase boundaries between oil phases outside of the chambers and aqueous media inside of the chambers are visible as dark shading surrounding the chambers. Residual aqueous media (indicated with label 2004) was observed between a subset of the chambers (2001) forming an approximate shape of a bar portion of a barbell, These images indicated that the oil filled interstitial regions outside of the chambers, but did not displace aqueous media from the interiors of the chambers, and, in some regions, did not displace all aqueous media from the regions directly between adjacent chambers. Even though the chambers included pores sufficiently large for solvent molecules to traverse through, aqueous media remained within the chambers following the introduction of oil into the fluidic device.

[0251] Cell viability was measured with a Sytox dye stain (commercially available from Thermo Fisher Scientific and commonly uses Annexin V dye for an indication of cell death) following 24 and 48 hour incubations in the fluidic devices. The results of these analyses are summarized in FIG. 20C, which is a bar chart of cell viabilities (%, y-axis) for cells incubated for 48 hours within chambers of varying sizes. Cell viabilities ranged from about75% to about 90%. No difference in cell viability was observed between chambers of different sizes or between chambers surrounded by media or oil.EXAMPLE 6Direct Covalent Attachment of a Drug to a Fluidic Device Surface

[0252] This example covers a method for covalently attaching a drug to a fluidic device surface through a photocleavable linker. The fluidic device surface, a glass slide, was coated with a polyacrylamide polymer with free NHS ester functional groups. After the coating set on the surface, 100 mM diaminodiethyl disulfide in lx PBS was applied on the surface and incubated at room temperature under normal atmosphere overnight, allowing amino groups of the diaminodiethyl disulfide to couple to the NHS ester functional groups to form amide linkages to the coating . The surface was rinsed with water (200 pL x 3) and dried by vacuum suction. Athiolated surface was obtained by cleaving the disulfides (of the diaminodiethyl disulfide groups coupled to the surface) with TCEP (100 mM in H2O) for one hour at room temperature. The surface was rinsed with water and 1 x PBS.

[0253] A doxorubicin conjugate was prepared according to SCHEME 1 (wherein ‘DOXO’ represents doxorubicin, and the doxorubicin couples to the NHS ester through the amine of its amino sugar). During this step, doxorubicin (5 pL, 50mM) was mixed with TEA (10 pL, 100 mM) and was reacted with MAL-PEG4-PC-NHS (top structure in SCHEME 1, 5 pL, 50 mM) in DMSO (30 pL). MAL-PEG4-PC-NHS is a heterobifunctional polyethylene glycol linker that includes a mal eimide group (MAL), four units of an ethylene glycol group (PEG4), and a photocleavable (PC) group in the form of orthonitrobenzyl group, and an N-hydroxy succinimide group (NHS). The resulting mixture was left on a bench for one hour at room temperature. The solution was subsequently diluted with lx PBS.SCHEME 1DOXO, TEA

[0254] The doxorubicin conjugate was then applied to the thiolated surface of the fluidic device and allowed to sit overnight to affect coupling between the maleimide of the doxorubicin conjugate and thiols on the fluidic device surface. This method yielded a fluidic device surface covalently coupled to doxorubicin through a photocleavable linker.EXAMPLE 7Drug Release Within Polymeric Chambers

[0255] This example is directed to drug release and containment in chambers surrounded by an oil phase. A fluidic device surface was covalently coupled to doxorubicin through a photocleavable linker according to EXAMPLE 6. Jurkat cells were mixed with a polymer precursor and introduced into the fluidic device. Chambers were formed around cells, after which the fluidic device was rinsed three times with 200 pL of phosphate-buffered saline (PBS) to remove residual media. Subsequently, 50 pL of an oil (99% (v / v) of a polyfluoroalkane mixture and 1% (v / v) of a perfluoropoly ether-poly(propylene oxide)-poly(ethylene oxide) block copolymer with a mass of about 10 kDa) was flowed through the device. The oil displaced aqueous media outside of the chambers but did not remove aqueous media from the interiors of the chambers. A single chamber was selectively exposed to ultraviolet light (patterned with a digital micromirror device) for 5 minutes to release doxorubicin from the surface of the fluidic device within the chamber. Following UV exposure, the flow cell was imaged at 488 nm to measure doxorubicin fluorescence signal. The flow cell was then incubated at 37 °C for 1 hour. After incubation, PBS was flowed back through the device to displace the oil, and the flow cell was imaged again to evaluate postincubation fluorescence and cell behavior.

[0256] A fluorescence image of the UV-exposed chamber is shown in FIG. 21A. For reference, a fluorescence image of a non-UV-exposed chamber is shown in FIG. 21B. Only the UV-exposed chamber exhibited doxorubicin fluorescence signal. The released doxorubicin was contained within the oil encapsulated chamber and in the aqueous media trapped directly outside of the chamber. Cells inside of the UV-exposed chamber (in which doxorubicin was released from the fluidic device surface) exhibited higher fluorescence signals than cells in chambers that were not exposed to UV irradiation.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for releasing a dose of a drug to a cell, the method comprising:inputting the cell into a fluidic channel;inputting a polymer precursor into the fluidic channel;inputting a substrate comprising the drug into the fluidic channel; synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell; andreleasing the drug from the substrate, thereby allowing the drug to contact the cell.

2. The method of claim 1, wherein the chamber co-encloses the cell with the substrate.

3. The method of claim 1, wherein the chamber is synthesized co-enclosing the cell with the substrate.

4. The method of claim 3, wherein a wall of the chamber is impermeable to the substrate.

5. The method of claim 1, wherein a wall of the chamber is impermeable to the drug.

6. The method of claim 1, wherein the inputting the substrate comprises flowing the substrate through a wall of the chamber, wherein the substrate is sufficiently small to pass through a pore of the wall.

7. The method of claim 1, wherein the inputting the substrate is before, after, or concurrent with chamber synthesis.

8. The method of claim 1, wherein the releasing comprises degrading the substrate, thereby releasing the drug from an interior portion of the substrate.

9. The method of claim 8, wherein the degrading comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof.

10. The method of claim 8, wherein only a portion of the substrate is degraded.

11. The method of claim 10, wherein the portion of the substrate is degraded by:i) irradiating a fraction of an interior space of the chamber with light;ii) irradiating only the portion of the substrate with light;iii) inputting only a fraction of light required to completely degrade the substrate;iv) inputting only a fraction of light required to release all instances of the drug comprised by the substrate;v) inputting only a fraction of a concentration of a reagent required to completely degrade the substrate;vi) inputting only a fraction of a concentration of a reagent to release all instances of the drug comprised by the substrate; orvii) a combination thereof.

12. The method of claim 1, wherein the releasing comprises cleaving a bond or linker between the drug and the substrate.

13. The method of claim 12, wherein the cleaving converts the drug from a prodrug to an active form of the drug.

14. The method of claim 1, wherein the substrate comprises a plurality of instances of the drug, and wherein only a subset of the plurality of instances of the drug are released from the substrate.

15. The method of claim 14, wherein the subset of the instances of the drug is released from the substrate by:i) shining a light on a fraction of an interior space of the chamber;ii) inputting only a fraction of light required to release all instances of the drug from the substrate;iii) inputting only a fraction of a concentration of a reagent required to release all instances of the drug from the substrate; oriv) a combination thereof.

16. The method of claim 1, wherein the releasing comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof.

17. The method of claim 1, wherein the substrate comprises a particle.

18. The method of claim 17, wherein the particle is photolytically, chemically, thermally, pH, or enzymatically degradable.

19. The method of claim 1, wherein the chamber encloses an additional substrate comprising an additional drug.

20. The method of claim 19, further comprising releasing the additional drug from the additional substrate.

21. The method of claim 20, wherein the releasing the additional drug from the additional substrate is performed before or after the releasing the drug from the substrate.

22. The method of claim 20, further comprising determining whether the additional drug diminishes an activity of the drug, enhances the activity of the drug, acts synergistically with the drug, alters a side-effect of the drug, or a combination thereof.

23. The method of claim 19, further comprising measuring a phenotypic change of the cell and then releasing either the drug from the substrate or the additional drug from the additional substrate based on the measured phenotypic change.

24. The method of claim 1, further comprising measuring a phenotypic change of the cell subsequent to the releasing the drug from the substrate.

25. The method of claim 1, wherein the substrate is immobilized on a surface of the fluidic channel.

26. A method for releasing a dose of a drug to a cell, the method comprising:inputting the cell into a fluidic channel;inputting a polymer precursor into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber co- enclosing the cell with a substrate comprising the drug; and releasing the drug from the substrate, thereby allowing the drug to contact the cell.

27. The method of claim 26, wherein the substrate is coupled to a surface of the fluidic channel.

28. The method of claim 27, further comprising fabricating the substrate on the surface of the fluidic channel, the fabricating comprising:(i) inputting the drug into the fluidic channel;(ii) inputting an additional polymer precursor into the fluidic channel; and (iii) photopolymerizing the additional polymer precursor in the fluidic channel, thereby generating the substrate adhered to the surface of the fluidic channel and enclosing the drug.

29. The method of claim 27, further comprising fabricating the substrate on the surface of the fluidic channel, the fabricating comprising:(i) inputting the drug into the fluidic channel, wherein the drug is coupled to an additional polymer precursor; and(ii) photopolymerizing the additional polymer precursor in the fluidic channel, thereby generating the substrate adhered to the surface of the fluidic channel and coupled to the drug.

30. The method of claim 27, further comprising fabricating the substrate on the surface of the fluidic channel, the fabricating comprising:(i) spotting the drug and an additional polymer precursor on the surface of the fluidic channel; or(ii) grafting the additional polymer precursor to the surface of the fluidic channel in the presence of the drug.

31. The method of claim 30, wherein the drug is coupled to the additional polymer precursor.

32. The method of claim 30, wherein the fabricating is in the fluidic channel.

33. The method of claim 30, wherein the fabricating is performed prior to formation of the fluidic channel.

34. The method of claim 26, wherein the releasing comprises degrading the substrate.

35. The method of claim 26, wherein only a portion of the substrate is degraded, such that only a subset of instances of the drug comprised by the substrate are released from the substrate.

36. The method of claim 35, wherein the portion of the substrate is degraded by:i) shining a light on a fraction of an interior space of the chamber;ii) inputting only a fraction of light required to completely degrade the substrate; iii) inputting only a fraction of a concentration of a reagent required to completely degrade the substrate; oriv) a combination thereof.

37. The method of claim 26, wherein the substrate comprises a polymer or a hydrogel.

38. The method of claim 26, wherein the substrate is in the shape of a disc, a pillar, or asphere.

39. The method of claim 26, wherein the fluidic channel comprises a plurality of instances of the substrate comprising a plurality of doses of the drug, and wherein the method further comprises synthesizing a plurality of chambers co-enclosing a plurality of cells with at least a subset of the plurality of the instances of the substrate and releasing the drug from at least the subset of the plurality of instances of the substrate.

40. The method of claim 26, wherein the fluidic channel comprises a plurality of instances of the substrate comprising the drug, and wherein the method further comprises synthesizing a plurality of chambers co-enclosing a plurality of cells with at least a subset of the plurality of the instances of the substrate and releasing different amounts of the drug from at least the subset of the plurality of instances of the substrate.

41. The method of claim 26, wherein the fluidic channel comprises an additional substrate comprising an additional drug, and wherein the method further comprises synthesizing an additional chamber co-enclosing an additional cell with the additional substrate, and releasing the additional drug from the additional substrate.

42. The method of claim 26, wherein the chamber co-encloses an additional substrate comprising an additional drug with the cell and the substrate.

43. The method of claim 41, further comprising releasing the additional drug from the additional substrate.

44. The method of claim 42, wherein the releasing the additional drug from the additional substrate is prior to, concurrent with, or subsequent to the releasing the drug from the substrate.

45. The method of claim 26, wherein the substrate is enclosed within an additional chamber that is enclosed within the chamber, and wherein the cell is enclosed within the chamber and disposed outside of the additional chamber.

46. The method of claim 26, wherein the cell is enclosed within an additional chamber that is enclosed within the chamber, and wherein the substrate is enclosed within the chamber and disposed outside of the additional chamber.

47. The method of claim 45 or claim 46, further comprising degrading the additional chamber after the releasing the drug from the substrate.

48. A method for releasing a dose of a drug to a cell, the method comprising:inputting the cell into a fluidic channel;inputting a polymer precursor and optionally an additional polymer precursor into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing a substrate comprising the drug;synthesizing an additional chamber with the polymer precursor or the additional polymer precursor, the additional chamber enclosing the chamber and the cell;releasing the drug from the substrate; anddegrading the chamber, thereby allowing the drug to contact the cell.

49. The method of any one of claims 1-48, further comprising measuring a distance between the substrate and the cell, and using the distance to determine a dose of the drug that diffuses to the cell from the substrate following the releasing.

50. A method for releasing a dose of a drug to a cell, the method comprising:inputting the cell into a fluidic channel;measuring distances between the cell and each of one or more substrates in the fluidic channel, wherein each substrate of the one or more substrates comprises the drug;selecting a subset of the one or more substrates based at least in part on the measured distances of the one or more substrates;releasing at least a portion of the drug comprised by the subset of the one or more substrates; andincubating the cell, thereby allowing the dose of the drug to diffuse to the cell.

51. The method of claim 50, further comprising inputting a polymer precursor into the fluidic channel, and synthesizing a chamber that encloses the cell with the polymer precursor within the fluidic channel.

52. The method of claim 50, wherein the releasing comprises irradiating the subset of the one or more substrates with light.

53. The method of claim 52, wherein non-selected substrates of the one or more substrates are not irradiated.

54. The method of claim 52, wherein the irradiating cleaves a bond or a linker between the drug and the subset of the one or more substrates.

55. The method of claim 52, wherein the irradiating degrades the subset of the one or more substrates.

56. The method of claim 50, wherein the selecting is further based at least in part on amounts or concentrations of the drug comprised by each substrate of the one or more substrates.

57. The method of claim 50, further comprising measuring distances between the cell and each of one or more additional substrates in the fluidic channel, wherein each substrate of the one or more additional substrates comprises an additional drug;selecting a subset of the one or more additional substrates based at least in part on the measured distances of the one or more additional substrates;releasing at least a portion of the additional drug comprised by the subset of the one or more additional substrates; andincubating the cell, thereby allowing the dose of the additional drug to diffuse to the cell.

58. The method of claim 57, wherein the selecting the subset of the one or more additional substrates is further based at least in part on amounts or concentrations of the additional drug comprised by each substrate of the one or more additional substrates.

59. A method for releasing a dose of a drug to a cell, the method comprising:inputting a polymer precursor into a fluidic channel;inputting the cell into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and a spatially-addressed nucleic acid sequence coupled to a surface of the fluidic channel;wherein at least a portion of the spatially-addressed nucleic acid sequence is hybridized to a drug-nucleic acid complex, the drug-nucleic acid complex comprising the drug coupled to a nucleic acid capture sequence that is complementary to at least a portion of the spatially-addressed nucleic acid sequence; andreleasing the drug from the spatially-addressed nucleic acid sequence by:a) dehybridizing the nucleic acid capture sequence from the spatially-addressed nucleic acid sequence,b) cleaving the drug from the nucleic acid capture sequence, orc) a combination thereof;thereby allowing the drug to contact the cell.

60. The method of claim 59, further comprising before the releasing the drug, inputting the drug-nucleic acid complex into the fluidic channel and hybridizing the nucleic acid capture sequence to the spatially-addressed nucleic acid sequence or the portion of the spatially-addressed nucleic acid sequence.

61. The method of claim 60, wherein the inputting the drug-nucleic acid complex into the fluidic channel is prior to the inputting the polymer precursor and the cell into the fluidic channel.

62. The method of claim 59, wherein the cleaving the drug from the nucleic acid capture sequence converts the drug from a prodrug form to an active form of the drug.

63. The method of claim 59, wherein the cleaving the drug from the nucleic acid capture sequence comprises chemical cleavage, enzymatic cleavage, thermal cleavage, pH- mediated cleavage, or photolysis.

64. The method of claim 59, further comprising inputting an additional drug-nucleic acid complex into the fluidic channel, wherein the additional drug-nucleic acid complex comprises an additional drug coupled to an additional nucleic acid capture sequence.

65. The method of claim 64, wherein the additional drug-nucleic acid complex hybridizes to at least a portion of an additional spatially-addressed nucleic acid sequence coupled to the surface of the fluidic channel, and wherein:i) the additional spatially-addressed nucleic acid sequence is disposed outside of the chamber, orii) the additional spatially-addressed nucleic acid sequence is co-enclosed with the cell and the spatially-addressed nucleic acid sequence within the chamber.

66. The method of claim 64 or claim 65, wherein the method comprises selectively cleaving the drug from the nucleic acid capture sequence, wherein the selectively cleaving the drug from the nucleic acid capture sequence does not cleave the additional drug from the additional nucleic acid capture sequence.

67. The method of claim 64, wherein the additional nucleic acid capture sequence is complementary to the spatially-addressed nucleic acid sequence or the portion of the spatially-addressed nucleic acid sequence.

68. A method for releasing a dose of a drug to a cell, the method comprising:inputting the cell and into a fluidic channel;inputting a polymer precursor into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber co-enclosing the cell and a plurality of drugs coupled to a surface of the fluidic channel through a plurality of cleavable linkers; andselectively cleaving a cleavable linker of the plurality of cleavable linkers coupled to the drug, thereby allowing the drug to contact the cell.

69. The method of claim 68, wherein the surface is a top surface of the fluidic channel or a bottom surface of the fluidic channel.

70. The method of claim 68, wherein the selectively cleaving comprises chemical cleavage, enzymatic cleavage, thermal cleavage, pH-mediated cleavage, or photolysis.

71. The method of claim 68, wherein the selectively cleaving converts the drug from a prodrug form to an active form of the drug.

72. The method of claim 68, further comprising selectively cleaving an additional cleavable linker of the plurality of cleavable linkers, thereby releasing an additional drug of the plurality of drugs from the surface of the fluidic channel.

73. The method of claim 68, further comprising coupling the plurality of drugs to the surface of the fluidic channel, the coupling comprising:hybridizing a plurality of splint oligonucleotides to a plurality of spatially- addressed nucleic acid sequences coupled to the surface of the fluidic channel, wherein each splint oligonucleotide of the plurality of splint oligonucleotides comprises:i) a sequence complementary to at least a portion of a spatially- addressed nucleic acid sequence of the spatially-addressed nucleic acid sequences, andii) a drug-capture sequence;inputting the plurality of drugs into the fluidic channel, wherein each drug of the plurality of drugs is coupled to:a) a cleavable linker of the plurality of cleavable linkers, and b) a nucleic acid capture sequence complementary to the drug-capture sequence;incubating the plurality of drugs under conditions that permit hybridization between the drug-capture sequences of the plurality of splint oligonucleotides and the nucleic acid capture sequences coupled to the plurality of drugs; andligating the plurality of spatially-addressed nucleic acid sequences to the plurality of nucleic acid capture sequences coupled to the plurality of drugs, thereby coupling the plurality of drugs to the surface of the fluidic channel.

74. The method of claim 68, further comprising coupling the plurality of drugs to the surface of the fluidic channel, the coupling comprising:inputting the plurality of drugs into the fluidic channel, wherein each drug of the plurality of drugs is coupled to:a) a cleavable linker of the plurality of cleavable linkers, and b) a nucleic acid capture sequence complementary to at least a portion of a spatially-addressed nucleic acid sequence of a plurality of spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel; andincubating the plurality of drugs under conditions that permit hybridization between the plurality of spatially-addressed nucleic acid sequences and the nucleic acid sequences coupled to the plurality of drugs, thereby coupling the plurality of drugs to the surface of the fluidic channel.

75. A method for releasing a dose of a drug to a cell, the method comprising:inputting the cell into a fluidic channel;inputting a prodrug of the drug into the fluidic channel;inputting a polymer precursor into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell; andconverting the prodrug into an active form of the drug, thereby allowing the drug to contact the cell.

76. The method of claim 75, wherein the chamber encloses the prodrug with the cell.

77. The method of claim 75, wherein the chamber is impermeable to the prodrug, the drug, or the prodrug and the drug.

78. The method of claim 75, wherein the inputting the prodrug into the fluidic channel is subsequent to the synthesizing the chamber, and wherein the prodrug, the drug, or the prodrug and the drug are sufficiently small to pass through a wall of the chamber.

79. The method of claim 75, wherein the converting comprises cleaving a promoiety from the prodrug.

80. The method of claim 75, wherein the converting comprises chemically modifying the prodrug.

81. The method of claim 75, wherein the converting comprises photolysis, a chemical conversion, an enzymatic conversion, or a combination thereof.

82. The method of claim 81, wherein the converting comprises photolysis, and the method further comprises determining a dose of the active form of the drug delivered to the cell based on:i) a distance between the photolysis and the cell;ii) an intensity of the photolysis;iii) a duration of the photolysis;iv) a concentration of the prodrug in the fluidic channel;v) a rate of conversion of the prodrug to the active form of the drug during the photolysis; orvi) a combination thereof.

83. The method of claim 75, wherein the converting is a physiological conversion performed by the cell.

84. The method of claim 75, wherein the converting is subsequent to uptake of the prodrug by the cell.

85. The method of claim 75, wherein a plurality of instances of the prodrug are input into the fluidic channel, and the converting comprises converting a subset of the instances of the prodrug into the active form of the drug.

86. A method for analyzing a capture agent, the method comprising:inputting a cell into a fluidic channel;inputting an additional cell into the fluidic channel;inputting a polymer precursor into the fluidic channel;inputting the capture agent into the fluidic channel, wherein the capture agent is configured to simultaneously bind to the cell and the additional cell;incubating the cell, the additional cell, and the capture agent in the fluidic channel under conditions that allow the capture agent to bind to the and additional cells; and synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and the additional cell.

87. A method for analyzing a capture agent, the method comprising:inputting a cell into a fluidic channel;inputting an additional cell into the fluidic channel;inputting the capture agent into the fluidic channel; anddetermining whether the capture agent simultaneously binds to the cell and the additional cell.

88. The method of claim 87, further comprising inputting a polymer precursor into the fluidic channel and synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell and the additional cell.

89. The method of claim 86 or claim 87, further comprising capturing the capture agent.

90. The method of claim 86 or claim 87, further comprising identifying the capture agent.

91. The method of claim 86 or claim 87, further comprising determining a binding affinity of the capture agent for the cell, the additional cell, or the cell and the additional cell.

92. The method of claim 86 or claim 87, further comprising inputting a third cell into the fluidic channel, and determining a specificity of the capture agent for the cell and the additional cell relative to the third cell.

93. The method of claim 86 or claim 87, further comprising inputting a plurality of additional cells into the fluidic channel, and determining a specificity of the capture agent for the cell and the additional cell relative to the plurality of additional cells.

94. The method of claim 93, wherein the cell, the additional cell, and the plurality of additional cells are derived from a blood sample, a peripheral blood mononuclear cell (PBMC) sample, a buffy coat sample, a solid biopsy, a liquid biopsy, or a tumor section.

95. The method of claim 86 or claim 87, wherein the cell comprises an effector cell and the additional cell comprises a target cell of the effector cell.

96. The method of claim 95, further comprising measuring cytotoxicity of the effector cell against the target cell.

97. The method of claim 95, wherein the cell comprises an immune cell.

98. The method of claim 97, wherein the cell comprises a B cell, a basophil, a cytokine induced killer cell, a cytotoxic T cell, a dendritic cell, an eosinophil, a granulocyte, a hypersegmented neutrophil, a lymphoid cell, a macrophage, a mast cell, a megakaryocyte, a monocyte, a myeloid cell, a natural killer cell, a neutrophil, a regulatory T cell, a T cell, a T helper cell, or a thrombocyte.

99. The method of claim 95, wherein the target cell comprises a cancer cell.

100. The method of claim 95, wherein the cell is a T cell and the capture agent is configured to bind to a PD-1 antigen on the T cell.

101. The method of claim 97, wherein the capture agent is configured to bind to an immune checkpoint molecule on the immune cell.

102. The method of claim 101, wherein the immune checkpoint molecule is PD-1, CTLA- 4, BTLA, LAG-3, TIM-3, TIGIT, CD27, CD28, CD112R, CD137, ICOS, 4-1BB, OX-40, or VISTA.

103. The method of claim 102, wherein the immune checkpoint molecule is PD-1.

104. The method of claim 96, further comprising determining whether the capture agent modulates cytotoxicity of the effector cell against the target cell.

105. The method of claim 90, wherein the identifying comprises sequencing a nucleic acid barcode coupled to or cleaved from the capture agent.

106. The method of claim 105, wherein the nucleic acid barcode encodes at least a portion of an amino acid sequence of the capture agent.

107. The method of claim 90, wherein the identifying comprises binding a nucleic acid capture sequence coupled to or cleaved from the capture agent to a spatially-addressed nucleic acid sequence coupled to a surface of the fluidic channel, and detecting the binding of the nucleic acid capture-sequence to the spatially-addressed nucleic acid sequence.

108. The method of claim 107, wherein the detecting the binding of the nucleic acid capture-sequence to the spatially-addressed nucleic acid sequence comprises detecting a location of the capture agent in the fluidic channel.

109. The method of claim 107, wherein the binding the nucleic acid capture sequence to the spatially-addressed nucleic acid sequence occurs before the synthesizing the chamber.

110. The method of claim 107, wherein the cell is input into the fluidic channel bound to the capture agent.

111. The method of claim 86 or claim 87, wherein the inputting the capture agent into the fluidic channel comprises inputting a particle comprising the capture agent into the fluidic channel.

112. The method of claim 111, wherein the method further comprises releasing the capture agent from the particle.

113. The method of claim 112, wherein:i) the releasing comprises cleaving a bond or linker that couples the capture agent to a surface of the particle;ii) the releasing comprises cleaving a bond or a linker that confines the capture agent within the particle;iii) the capture agent is coupled to a surface of the particle, and the releasing comprises lysing the particle or disrupting the surface of the particle to release the capture agent from the surface of the particle;iv) the capture agent is contained in an interior space within the particle, and the releasing comprises lysing or opening the particle to release the capture agent from the interior space within the particle; orv) the capture agent is noncovalently coupled to a surface of the particle, and the releasing comprises changing a condition in the fluidic channel to desorb the capture agent from the surface of the particle.

114. The method of claim 111, wherein the method comprises coupling the particle to a surface of the fluidic channel.

115. The method of claim 114, wherein the particle comprises a nucleic acid capture sequence that hybridizes to a spatially-addressed nucleic acid sequence coupled to the surface of the fluidic channel.

116. The method of claim 111, wherein the method comprises enclosing the particle in the chamber.

117. The method of claim 111, wherein the particle comprises a nanoparticle, a microparticle, a lipid particle, a vesicle, a ceramic particle, a metal particle, a metal oxide particle, a polymeric particle, a polysaccharide particle, a magnetic particle, a hydrogel particle, or a combination thereof.

118. The method of claim 86 or claim 87, wherein the capture agent comprises an antibody, an antibody fragment, a nanobody, a minibody, an affibody, a diabody, a triabody, a tetrabody, an intrabody, a bis-scFv, a single domain antibody, a DARPin, an anticalin, a knottin, a receptor ligand, an aptamer, an adheron, or a combination thereof.

119. The method of claim 118, wherein the capture agent comprises an antibody.

120. The method of claim 119, wherein the antibody comprises a fragment crystallizable (Fc) region configured to bind to the cell and a fragment antigen-binding (Fab) region configured to bind to the additional cell.

121. The method of claim 119, wherein the antibody comprises a fragment antigen-binding (Fab) region configured to bind to the cell and an additional fragment antigen-binding (Fab) region configured to bind to the additional cell.

122. The method of claim 86 or claim 87, wherein the capture agent is input into the fluidic channel after the cell and the additional cell are input into the fluidic channel.

123. A method for analyzing a capture agent, the method comprising:inputting a cell into a fluidic channel;inputting an additional cell into the fluidic channel;inputting a third cell into the fluidic channel;inputting a polymer precursor into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, the chamber enclosing the cell, the additional cell, and the third cell;incubating the cell, the additional cell, and the third cell under conditions in which the third cell expresses the capture agent; andmeasuring binding of the capture agent to the cell and the additional cell.

124. The method of claim 123, wherein the third cell comprises a B cell.

125. A method for modulating intercellular interactions, the method comprising: inputting a cell into a fluidic channel comprising a surface comprising a capture agent configured to bind to the cell and an additional capture agent configured to bind to an additional cell;inputting the additional cell into the fluidic channel;incubating the cell and the additional cell in the fluidic channel under conditions that allow the capture agent to bind to the cell and the additional capture agent to bind to the additional cell;inputting a polymer precursor into the fluidic channel; andsynthesizing a chamber with the polymer precursor that co-encloses the cell with the additional cell;wherein the cell is able to contact the additional cell when the cell is bound to the capture agent and the additional cell is bound to the additional capture agent.

126. The method of claim 125, wherein the capture agent and the additional capture agent comprise nucleic acid capture sequences that are hybridized to spatially-addressed nucleic acid sequences coupled to the surface of the fluidic channel.

127. The method of claim 125, wherein the capture agent is coupled to the surface by a linker and the additional capture agent is coupled to the surface by an additional linker.

128. The method of claim 127, further comprising cleaving the linker, the additional linker, or the linker and the additional linker.

129. The method of claim 125, wherein the capture agent and the additional capture agent are independently selected from an antibody, an antibody fragment, a nanobody, a minibody, an affibody, a diabody, a triabody, a tetrabody, an intrabody, a bis-scFv, a single domain antibody, a DARPin, an anticalin, a knottin, a receptor ligand, an aptamer, an adheron, or a combination thereof.

130. The method of claim 125, further comprising determining whether the cell binds to the additional cell.

131. The method of claim 125, wherein the inputting comprises inputting a biological sample comprising the cell, the additional cell, and a plurality of additional cells into the fluidic channel.

132. The method of claim 131, wherein the capture agent and the additional capture agent do not bind to the plurality of additional cells.

133. The method of any one of claims 1-132, further comprising inputting an antibody or a lipid into the fluidic channel, wherein the antibody or the lipid is configured to bind to cells within the fluidic channel, and wherein the antibody or the lipid is coupled to a nucleic acid that comprises a nucleic acid sequence specific to the fluidic channel.

134. The method of claim 1-132, further comprising determining a characteristic of the cell.

135. The method of claim 134, wherein the characteristic is an mRNA expressed by the cell.

136. The method of claim 135, wherein the determining comprises lysing the cell or at least a subset of the aggregate of the cells, capturing the mRNA on a capture element coupled to a surface of the flow cell, reverse transcribing the mRNA to generate a cDNA molecule that optionally comprises a barcode sequence derived from the capture element, and sequencing the cDNA molecule.

137. The method of claim 134, wherein the characteristic comprises a soluble factor secreted by the cell.

138. The method of claim 137, wherein said detecting said soluble factor comprises disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell, and detecting the soluble factor bound to the capture surface.

139. The method of claim 138, wherein said detecting said soluble factor bound to said capture surface comprises contacting said soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody.

140. The method of claim 138, wherein the capture surface is at least partially enclosed in the chamber.

141. The method of 134, wherein the cell comprises an effector cell, and wherein the characteristic comprises cytotoxicity.

142. The method of claim 141, wherein the determining the cytotoxicity comprises measuring a rate or an occurrence of the effector cell killing additional cells.

143. The method of claim 142, wherein the measuring comprises contacting the additional cells with a dye that indicates whether a cell of the additional cells is dead.

144. The method of claim 134, wherein the characteristic comprises activation.

145. The method of claim 144, wherein the cell is activated by an additional cell.

146. The method of claim 145, wherein the additional cell is located outside of the chamber.

147. The method of claim 145, wherein the additional cell is co-enclosed in the chamber with the cell.

148. The method of claim 144, wherein the determining the activation comprises detecting expression of a surface marker, a morphology change, a cytotoxicity increase, a proliferation rate change, a soluble factor, a genomic sequence, an mRNA, or a combination thereof of the cell.

149. The method of claim 134, wherein the characteristic comprises proliferation.

150. The method of claim 134, wherein the cell comprises an edited gene and a guide RNA associated with the edited gene, the method further comprising determining a sequence of the guide RNA and correlating the sequence with the determined characteristic of the at least one cell.

151. The method of any one of claims 1-49, 51-86, or 88-132, wherein the polymer precursor comprises a crosslinker and a photoinitiator.

152. The method of any one of claims 1-49, 51-86, or 88-132, wherein the polymer precursor comprises a porogen.

153. The method of any one of claims 1-132, further comprising mixing contents of the fluidic channel.

154. The method of claim 153, wherein the mixing comprises:(i) flowing liquid back and forth within the fluidic channel;(ii) applying an alternating magnetic field to the fluidic channel, thereby moving magnetic species within the fluidic channel;(iii) applying an alternating electric field to the fluidic channel, thereby moving charged species within the fluidic channel;(iv) shaking the fluidic channel; or(v) a combination thereof.

155. A method for releasing a dose of a drug to a cell, the method comprising:inputting a plurality of cells into a fluidic channel;inputting a polymer precursor into the fluidic channel;inputting a substrate comprising the drug into the fluidic channel; synthesizing a plurality of chambers with the polymer precursor within the fluidic channel, wherein each chamber encloses a cell from the plurality of cells; measuring a characteristic of the enclosed cell in each chamber; and selectively releasing the drug in one or more of the plurality of chambers based on the measured characteristic of the enclosed cell in each chamber.

156. The method of claim 155, wherein the plurality of cells comprises a library of gene edited cells, wherein each of the gene edited cells comprises a guide RNA associated with the edited gene, the method further comprising: subsequent to the selectively releasing the drug from one or more of the plurality of chambers, determining a sequence of the guide RNA.

157. The method of claim 156, further comprising measuring another characteristic of the enclosed cell in each chamber subsequent to the selectively releasing the drug; and correlating the sequence with the measured characteristic or the measured another characteristic.

158. The method of claim 68, wherein a plurality of fluorophore complexes are coupled to the surface of the fluidic channel through an additional cleavable linker, wherein the selectively cleaving the cleavable linker also cleaves the additional cleavable linker to release at least a portion of the plurality of fluorophores from the surface of the fluidic channel, and wherein the method further comprises measuring a change in fluorescence intensity from at least a portion of the surface of the fluidic channel following the selectively cleaving.

159. The method of claim 68, wherein a plurality of fluorophore-quencher complex are coupled to the surface of the fluidic channel, wherein the fluorophore-quencher complex comprises a fluorophore coupled to a quencher through an additional cleavable linker, wherein the selectively cleaving the cleavable linker also cleaves the additional cleavablelinker to release the quencher from the surface of the fluidic channel, wherein the fluorophore remains coupled to the surface of the fluidic channel following the selectively cleaving, and wherein the method further comprises measuring a change in fluorescence intensity from at least a portion of the surface of the fluidic channel following the selectively cleaving.

160. The method of claim 158 or claim 159, further comprising determining an amount of the drug released from the surface of the fluidic channel based on the change in fluorescence intensity.

161. The method of claim 1, wherein the substrate comprises a cleavable linker coupled to the drug.

162. The method of claim 161, wherein the substrate is coupled to a surface of the fluidic channel.

163. The method of claim 162, wherein the substrate comprises a nucleic acid barcode.

164. The method of claim 163, wherein the substrate comprises a capture probe.

165. The method of claim 164, wherein the releasing comprises cleaving the cleavable linker by photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof.

166. The method of any one of claims 1-47 51, 59-72, 75-86, 88, or 123-165, further comprising, after the synthesizing, inputting a water-immiscible fluid into the fluidic channel.

167. The method of claim 166, wherein:i) the water-immiscible fluid does not displace aqueous medium from the chamber;ii) the water-immiscible fluid does not enter the chamber;iii) the water-immiscible fluid surrounds the chamber;iv) the water-immiscible fluid fills a space within the fluidic channel, wherein the space is outside of the chamber; orv) a combination thereof.

168. The method of claim 166, wherein the chamber comprises a drug, and wherein the water-immiscible fluid inhibits diffusion of the drug from the chamber to an additional chamber.

169. The method of claim 166, wherein the chamber comprises a drug, and wherein the water-immiscible fluid inhibits diffusion of the drug through a wall of the chamber.

170. The method of claim 169, wherein the diffusion through the wall comprises diffusion from an inner surface of the wall to the outer surface of the wall.

171. The method of claim 166, wherein the chamber comprises a cell secretion, and wherein the water-immiscible fluid inhibits diffusion of the cell secretion from the chamber to an additional chamber.

172. The method of claim 166, further comprising replacing at least a portion of the water- immiscible fluid within the fluidic channel with an aqueous medium.

173. The method of claim 166, wherein the water-immiscible fluid comprises a mineral oil, a silicone oil, a fluorocarbon oil, a perfluorinated oil, a vegetable oil, a bean oil, a PEG- perfluoroalkyl block copolymer, or a combination thereof.

174. The method of claim 166, wherein a polymeric wall of the chamber comprises pores, and wherein the pores are about 0.5 nm to about 25 nm.

175. A method for analyzing a cell, comprising:inputting the cell into a fluidic channel;inputting a polymer precursor into the fluidic channel;synthesizing a chamber with the polymer precursor within the fluidic channel, wherein the chamber encloses the cell;inputting a water-immiscible fluid into the fluidic channel, wherein:i) the water-immiscible fluid does not displace aqueous medium from the chamber;ii) the water-immiscible fluid does not enter the chamber;iii) the water-immiscible fluid surrounds the chamber;iv) the water-immiscible fluid fills a space within the fluidic channel, wherein the space is outside of the chamber; or v) a combination thereof; andanalyzing the cell.

176. The method of claim 175, further comprising removing the water-immiscible fluid from the fluidic channel.

177. The method of claim 175, wherein the chamber comprises a cell secretion, and wherein the water-immiscible fluid inhibits diffusion of the cell secretion from the chamber to an additional chamber in the fluidic channel.

178. The method of claims 166-177, wherein the water-immiscible fluid comprises a mineral oil, a silicone oil, a fluorocarbon oil, a perfluorinated oil, a vegetable oil, a bean oil, a PEG-perfluoroalkyl block copolymer, or a combination thereof.

179. A method comprising:a) forming a chamber in a fluidic device,wherein the chamber comprises one or more walls,wherein the one or more walls extend from a top surface of the fluidic device to a bottom surface of the fluidic device,wherein the one or more walls comprise a plurality of pores; and b) forming a layer on the one or more walls, wherein the layer at least partially blocks at least a pore of the plurality of pores on the one or more walls.

180. The method of claim 179, wherein the chamber comprises a cell.

181. The method of claim 179 or claim 180, wherein (a) comprises polymerizing one or more polymer precursors, thereby forming the one or more walls of the chamber.

182. The method of claim 181, wherein the polymerizing comprises applying light to the one or more polymer precursors.

183. The method of any one of claims 179-182, wherein a) comprises selectively encapsulating at least a subset of cells of a plurality of cells within the chamber.

184. The method of claim 183, wherein the subset of cells comprises at least one cell.

185. The method of any one of claims 179-184, wherein the chamber comprises aqueous medium.

186. The method of any one of claims 179-185, wherein the layer inhibits a secretion of the cell from passing through at least a pore of the plurality of pores.

187. The method of any one of claims 179-186, further comprising removing the layer, thereby unblocking at least a pore of the plurality of pores.

188. The method of any one of claims 179-187, wherein the layer comprises a water-immiscible fluid.

189. The method of claim 188, wherein the water-immiscible fluid comprises a mineral oil, a silicone oil, a fluorocarbon oil, a perfluorinated oil, a vegetable oil, a bean oil, a PEG-perfluoroalkyl block copolymer, or a combination thereof.

190. The method of any one of claims 180-189, further comprising releasing a dose of a drug to the cell.

191. The method of claim 190, wherein the releasing comprises:inputting a substrate comprising the drug into the fluidic device, and releasing the drug from the substrate,thereby allowing the drug to contact the cell.

192. The method of claim 191, wherein the chamber comprises (i) the cell or the subset of cells and (ii) the substrate.

193. The method of claim 191, wherein a) comprises selectively encapsulating (i) the cell or the subset of cells and (ii) the substrate within the chamber.

194. The method of any one of claims 190-193, the one or more walls of the chamber are impermeable to the substrate or the drug.

195. The method of any one of claims 190-194, the substrate or the drug are sufficiently small to pass through at least a pore of the plurality of pores.

196. The method of any one of claims 190-195, wherein the releasing comprises degrading the substrate, thereby releasing the drug from an interior portion of the substrate.

197. The method of claim 196, wherein the degrading comprises photolysis, chemical degradation, thermal degradation, pH-mediated degradation, enzymatic degradation, or a combination thereof.

198. The method of claim 196 or claim 197, wherein only a portion of the substrate is degraded.

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