Methods and systems for fluidics assisted functional cell sorting

Abstract

Provided herein are methods and systems for characterizing cells using specific biomolecule detection and label-free cell sorting. These systems can include a microfluidic system directed to processing of cells producing a biomolecule of interest and subsequent detection, isolation, and recovery of such cells.

Description

Methods and Systems for Fluidics Assisted Functional Cell SortingInventors: Arum Han, Paul de Figueiredo, Jianxun Song, Fengguang Guo, Han Zhang, Jing DaiTechnical Field

[0001] This disclosure relates to systems and methods for fluidics-assisted functional cell sorting methods for characterization of cells, selection of recombinant cells for therapeutics, and other applications.Background

[0002] Cell characteristics and functional phenotypes can be analyzed through cell surface biomarkers or secreted biomolecules. Importantly, cell characterization based on secreted biomolecules can provide information that surface biomarkers may not be able to provide. In some instances, measuring both the surface biomarkers and secreted biomolecules can provide comprehensive information about the cells of interest. Analyzing cells based on their cell surface biomarkers is easily achieved using various cell labeling strategies, both in bulk as well as at single-cell resolution. Analyzing cells based on the secreted biomolecules can also be conducted when analyzing groups of cells in bulk, using well-established immunoassay or enzyme-linked immunoassay methods. However, single-cell-resolution analysis of secreted biomolecules remains technically challenging for several reasons, including (i) their presence in ultralow analyte concentrations (attomolar to picomolar range), (ii) rapid diffusion of the biomolecular analytes away from the cells that secrete them, leading to signal dilution, and (iii) the need for spatial- temporal confinement to maintain analyte-cell associations, unambiguously attributing the secreted biomolecules to their originating single cells. Finally, having to select and sort only those single cells that produce the target molecule of interest further adds to these challenges.

[0003] Secreted biomolecules, such as carbohydrates, lipids, proteins, and nucleic acids, encode special biological information that can be used to define types, lineages, functions, and statuses of different cells. Rapid detection of those biomolecules with high sensitivity and specificity has been implemented using various scientific tools (e.g., ELISA, PCR, flow cytometry, fluorescence-activated cell sorting) that are widely deployed in diagnosis, monitoring, and therapeutic development. Cellular immunotherapies, based on adoptive cell transfer of ex vivo genetically engineered and expanded immune cells to enhance their disease-fighting capabilities,have demonstrated curative potential in oncology and autoimmune diseases. However, these therapeutic cells (engineered and / or expanded) are typically heterogeneous, and thus exist in mixtures that include desired functional cells but also non-functional cells, or even undesirable cells with functions that may cause adverse effects.

[0004] Several methods have been developed for refined analyses and isolation of specific cell types for characterization and therapeutic applications. Fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are current gold standards for cell sorting, which rely on fluorescent or magnetic labeling or binding of unique proteins expressed on the surface of target cell population. However, the labeling process can alter the properties of the cells and therefore affects their cellular responses and functions. More importantly, these methods are not suitable for many cell types, in which specific surface markers either do not exist or have not been discovered yet, and thus these cells can only be defined by their intracellular and / or secreted proteins.Summary

[0005] Provided here are systems and methods to address these shortcomings of the art and provide other additional or alternative advantages. Embodiments include cells isolated or selected using the disclosed systems and methods.

[0006] Certain embodiments include methods for detecting a biomolecule in a cell sample. One such method includes the steps of contacting the sample with a reaction mixture containing one or more of pairs of single-stranded DNA (ssDNA) fragments. Each ssDNA fragment contains 3-10 nucleotides of complementary region at 3’ end and a conjugated antigen receptor at the 5’end. The reaction mixture further contains a DNA polymerase for primer extension (such as a T4 polymerase), a Type V CRISPR / Cas effector protein, a guide RNA containing a region that binds to the Type V CRISPR / Cas effector protein and a guide sequence that hybridizes with the target DNAs, and a labeled detector ssDNA that does not hybridize with the guide sequence of the guide RNA. The method further includes the step of measuring a detectable signal produced by cleavage of the labeled detector ssDNA by the Type V CRISPR / Cas effector protein, thereby detecting the biomolecule of interest. In certain embodiments, the biomolecule of interest can be a surface biomarker or a secreted biomolecule. The biomolecule of interest can be an antigen, The antigen in the sample can include one or more of a toxin, which can be of bacterial, viral, or fungal origin. The biomolecule of interest in the sample can include a peptide or a protein. In some embodiments,the biomolecule of interest in the sample can include a cytokine or a chemokine. The biomolecule of interest in the sample can include one or more of y-interferon, granzyme, perforin, TGF beta, interleukin 2, interleukin 4, interleukin 6, interleukin 7, interleukin 10, interleukin 11, interleukin 12, interleukin 17, interleukin 21, G-CSF, GM-CSF, CXCL10, or CCL5. The ssDNA fragment can be any sequence that can form a protospacer adjacent motif (PAM) for Cas protein recognition. The conjugated antigen receptor can include but is not limited to an antibody, an engineered antigen receptor, or any other ligand receptor. The effector protein can be any Type V CRISPR / Cas, such as Casl2a, Casl2b, which can promiscuously cleave non-targeted single stranded DNA (ssDNA) once activated. The detector single strand DNA (3-30 nt) can be labeled with a dual-labeled moiety with a fluorophore and a quencher. In certain examples, the quencher can be a Black Hole Quencher®-! (BHQ-1) entity and Black Hole Quencher®-2 (BHQ-2) entity. For instance, this dual-labeled moiety can be Dual-labeled BHQ™ probes, such as BHQ-1 with FAM, TET, or HEX, or BHQ-2 with TAMRA™, Texas Red®, or Cy™ 5. In certain examples, the dsDNA target is further amplified by recombinase polymerase amplification (RPA) or by Loop-mediated isothermal amplification (LAMP).

[0007] Embodiments also include methods of using a droplet microfluidic system to isolate cells expressing specific biomolecules based on the disclosed detection methods. One such method includes the following steps: (a) encapsulating one or more cells into a water-in-oil emulsion droplet; (b) incubating the one or more cells within the droplet for a period of time sufficient for cells to express a biomolecule of interest (for example, this time period can be tens of seconds to tens of hours); (c) optionally merging this cell-encapsulated droplet with another droplet that contains other cell(s) of interest or similar type of cell(s) expressing additional biomolecule(s) of interest; (d) optionally incubating the merged droplet; (e) optionally contacting a detection or reaction mixture with the merged droplet or the cell-encapsulated droplet to detect the biomolecule(s) of interest; and (f) optionally incubating the merged droplet with the detection mixture to yield detection signals indicating the presence of cells expressing the biomolecule(s) of interest. The method can further optionally iteration of any one or more of steps (c) - (f) depending on whether multiple reagents are needed in sequence for the biomolecular assay to be completed. The detection or reaction mixture contains one or more reagents, such as any chemicals, biological molecules, labels, and / or buffers required for detection of the biomolecule of interest. The method can further include the steps of detecting target droplets, defined as the merged droplets detectedas containing cells expressing the biomolecule(s) of interest, via optical detection (such as fluorescence detection), sorting and collecting the target droplets based on the detection results; and isolating the target droplets to recover the target cells expressing the biomolecule(s) of interest from within the target droplets.

[0008] The droplet microfluidic system to achieve these steps includes one or more units directed to droplet generation, droplet merging, droplet incubation, droplet detection, droplet detection, and other droplet manipulation units comprising microfluidic channel(s) / chamber(s). An embodiment of such a system includes a droplet production unit with (i) a droplet cleaving unit containing two or more microfluidic channels, wherein a first continuous-phase fluid with merged droplets, containing the detection mixture and the cells expressing the biomolecule of interest, flows in a first microfluidic channel, a second fluid comprising a immiscible phase fluid, such as oil, flows in a second microfluidic channel, and wherein the continuous-phase fluid in the first microfluidic channel is cleaved into a second set of droplets by flow of the second fluid in the second microfluidic channel; (ii) a droplet incubation unit that allows for the cells to express the biomolecules of interest and the biomolecular detection reaction to proceed; (iii) a droplet microfluidic sorter unit that detects and sorts and / or isolates the cells based on presence of the biomolecule of interest; and (iv) a droplet dispenser unit that can isolate or dispense the sorted droplets containing the cells of interest for recovery. The continuous-phase fluid or the aqueous phase fluid can contain the reagents of the detection mixture as large particles or gel droplets that are relatively close in diameter to the cell-encapsulated droplet. These reagents include the chemical and biological molecules required for detection of the biomolecule of interest.

[0009] The fluidic channel architecture include linear, curved, or angled geometric configurations with cross-sectional heights ranging from 1 to 1,000 pm (preferably 20-200 pm). The microfluidic channel network can be configured in either (i) a two-dimensional (2D) singlelayer planar design or (ii) a three-dimensional (3D) multi-layer stacked architecture in any spatial direction with respect to the base plane. The fluidic system can be made of polydimethyl siloxane (PDMS), polymethyl methacrylate (PMMA), glass, silicon, or other materials commonly used in microfluidic devices. The size of droplet to be manipulated varies (but not limited to) from several micrometers to hundreds of micrometers. The droplet microfluidic system can be configured in either basic water-in-oil (W / O) or oil-in-water (O / W) emulsion droplets, or advanced multiemulsion architectures (e g., core-shell, Janus, or triple-emulsion droplets), and may operate insingle-step emulsification or sequential emulsion generation modes. The droplet production system may include, but is not limited to, one or more functional subunits directed to a droplet generation unit, a droplet transition unit, a droplet merging unit, and / or droplet sorting unit. The droplet generation unit includes, but is not limited to, a T-junction generator, a flow-focusing generator, a co-flow generator, and / or a step emulsification generator. In certain examples, the droplet transition unit can be achieved using a 2D reflow droplet array or 3D droplet chambers. The droplet merging unit can include an acoustic-based merger, a dielectrophoretic-based merger, a magnetic-based merger, or other droplet merging methods. The droplet sorting unit can include an acoustic-based merger, a dielectrophoretic-based merger, and / or pneumatic-based sorter.

[0010] Embodiments include cells isolated using the systems and methods provided herein, these cells of interest being isolated based on their expressed biomolecules. Certain embodiments include recombinant or naturally occurring immune cells. The expressed biomolecules can be one or more of an immunomodulator or a cell signaling protein. For example, CD8+T cells secreting y-interferon may be isolated using this method. In another example, the cells containing the biomolecule of interest are Tregcells and the biomolecule of interest is interleukin-10. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is a chimeric antigen receptor. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is an antigen-binding aspect of the CAR. Other examples include functional cell subsets, such as CD8+T cells producing perforin / granzyme B for cytotoxic response monitoring, CD4+T cells producing IL-1O / TGF- for immunosuppression assessment, macrophage cells releasing pro-inflammatory (e.g., IL- 12) or anti-inflammatory cytokines (e.g., IL- 10), tumor-infiltrating lymphocytes secreting perforin / granzyme B for cytotoxic response monitoring, or NK cells releasing IFN-y / GM-CSF for antitumor activity evaluation.

[0011] The foregoing summary has outlined broadly the features of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter, all of which form the subject matter of the claims.Brief Description of the Drawings

[0012] Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. The drawings are not necessarily to scale; emphasis instead generally being placed upon illustrating the principles of the various embodiments. Various dimensions may be altered. In certain instances, details that are not necessary for an understanding of the various aspects of the embodiments or that render other details difficult to perceive may have been omitted. It should be understood, of course, that the invention is not necessarily limited to the particular embodiments illustrated herein or specific dimensions.

[0013] FIG. 1A is a schematic representation of an antigen / ligand detection system using CRISPR Casl2a effector protein and specific antibodies / ligand-receptor conjugated oligonucleotides, according to an embodiment. FIG. IB is a graphical representation of the detection of a cytokine (IL-10) at different concentrations, measured in 2 min intervals over 2 hours, according to an embodiment. An antigen or a ligand can be a cell surface biomarker or a secreted biomolecule.

[0014] FIG. 2A is a schematic representation of an antigen / ligand detection system using CRISPR Casl2a effector protein and specific antibodies / ligand-receptor conjugated oligonucleotides, according to an embodiment. To increase the detection sensitivity, a recombinase polymerase reaction is applied to amplify the target dsDNA. FIG. 2B is a graphical representation of the detection of a cytokine (IL- 10) at different concentrations by the method showed in FIG. 2A, measured in 2 min interval over 2 hours, according to an embodiment. An antigen or a ligand can be a cell surface biomarker or a secreted biomolecule.

[0015] FIG. 3A is a schematic representation of an overview of the workflow of using the CRISPR Cast 2a method and droplet microfluidics for target cell sorting, according to an embodiment. FIG. 3B is a schematic representation of a microfluidic-based functional T cells sorting method based on the secretion of specific cytokines by T cells, according to an embodiment. In FIG. 3B, panel (A) illustrates a single T cell encapsulation in droplet. In FIG. 3B, panel (B) illustrates cultivation of T cells in a droplet for secretion of cytokine (IL-10). In FIG. 3B, panel (C) illustrates merging of these droplets with a detection mixture to facilitate the CRISPR Cas 12 reaction. In FIG. 3B, panel (D) illustrates cultivation for biosensing activation. In FIG. 3B,panel (E) illustrates the detection and sorting of the fluorescent droplet. In FIG. 3B, panel (F) illustrates a single-droplet-resolution dispensing of the target T cells within droplets.

[0016] FIG. 4 is a schematic representation of an overview of the workflow of CRISPR Casl2a method implemented in droplet microfluidics format for target cell sorting, according to an embodiment. To increase the sensitivity, a recombinase polymerase reaction is applied to amplify the target dsDNA.

[0017] FIGs. 5A and 5B show single regulatory T cells (Treg cells) encapsulation in a water-in- oil emulsion droplet and their viability after certain incubation period, according to an embodiment. FIG. 5A shows single-cell encapsulation of Tregcell in 120 pm diameter water-in-oil droplets at start (0 hours) and when cultured for up to 48 hours. FIG. 5B shows Treg cell viability results after cultivation in droplets as compared to that of well-plate cultivation.

[0018] FIGs. 6A - 6C present a comparison of a control (0 nM) and 20 nM of IL- 10 detection in droplets, where the water-in-oil emulsion droplets (120 pm diameter) were generated after bulk CRISPR reaction, according to an embodiment. FIG. 6A depicts green fluorescent channel imaging of a negative control (NC) after 18 hours of cultivation. FIG. 6B depicts 20 nM of IL-20 in a droplet imaged using green fluorescent channel. FIG. 6C shows a mix of NC and 20 nM IL- 10 containing droplets. The signal intensity is significantly different between the two different populations.

[0019] FIGs. 7A- 7D present detection of IL-10 in droplets using the biomolecular assay, according to an embodiment. FIG. 7A shows a droplet that does not contain any IL- 10. FIG. 7B shows a droplet having IL- 10 concentration at 20 nM. FIG. 7C shows bright-field image of a Tregcell cultured for 24 h in a 60 pm size droplet. FIG. 7D shows that green fluorescent signal from droplet in FIG. 7C is detectable.Detailed Description

[0020] The disclosure herein provides embodiments of systems and methods for fluidics assisted sorting of cells. Some embodiments are directed to systems and methods for fluidics assisted sorting of high-quality cells expressing specific biomolecules of interest. One such system and associated method is referred to as the FLASH-QC™ system and the FLASH-QC™ method. An embodiment of the method is a droplet microfluidics-based method that combines the powerof high specificity and sensitivity of CRISPR-based approaches for molecular detection with the single cell resolution and high throughput features of droplet microfluidics for the rapid and reliable sorting / enrichment of cells based on their surface or secreted molecule. In an embodiment, such as the FLASH-QC method, Type V CRISPR-associated proteins (such as Cast 2a, Cast 2b, Cast 2c) are used in the context of a threefold scheme to detect soluble biomolecules. First, the biomolecules are captured by DNA-conjugated ligand receptors. The DNA components are designed such that when these ligand receptors are brought into proximity by binding a target biomolecule, they form stable duplex DNA by ligand-induced proximity hybridization. The resulting dsDNA is extended by a T4 DNA polymerase generating a full-length target DNA sequence. The ligand receptor-linked DNA components are designed such that no stable duplex can form at 37 °C. However, when the DNA components are brought into proximity, a stable duplex is established. Second, the resultant dsDNA molecules are specifically recognized by Cast 2 protein complexes, which have been preloaded with crRNA that are complementary to the amplified dsDNA target sequence. Finally, when the Casl2a binds (and cleaves) the target DNA, a non-specific ssDNAase activity is unleashed, thereby enabling the cleavage of fluorophore- quencher (FQ) labeled ssDNA reporters and resulting in a fluorescent signal that can be readily detected by optical methods. To increase detection sensitivity, a recombinase polymerase amplification (RPA) can be applied in the system to produce high amount of target dsDNA.

[0021] Provided herein is a microfluidic device that enables integrated single-cell droplet encapsulation (alternatively trapping), analysis of the biomolecules using immunoassays or other non-destructive sensing techniques, followed by selective retrieval of target cells based on their biomolecular expression profiles. In some embodiments, the device includes a selective encapsulation (or alternatively trapping) of the desired target cells while non-target cells are released, thereby enabling recovery of only cells with the biomolecular profile of interest. When applied to a library of cells having different phenotype, with different surface and / or secretory biomolecular profiles, the systems and methods disclosed herein result in identifying and sorting only the desired cells of interest with extremely high purity.

[0022] Embodiments also include cells isolated using the systems and methods provided herein, these cells of interest being isolated based on expression of specific biomolecules. These biomolecules can be one or more of an immunomodulator or a signaling protein. For example, CD8+T cells secreting y-interferon may be isolated using this method. In another example, thecells containing the biomolecule of interest are Tregcells and the biomolecule of interest is interleukin-10. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is a chimeric antigen receptor. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is an antigen-binding aspect of the CAR.

[0023] In the following description, numerous details are set forth to provide a thorough understanding of the various embodiments. In other instances, well-known processes, devices, and systems may not been described in detail in order not to unnecessarily obscure the various embodiments. Additionally, illustrations of the various embodiments may omit certain features or details in order to avoid obscuring the various embodiments.

[0024] In the following detailed description, reference is made to the accompanying drawings that form a part of this disclosure. The drawings may provide an illustration of some of the various embodiments in which the subject matter of the present disclosure may be practiced. Other embodiments may be utilized, and logical changes may be made without departing from the scope of this disclosure.

[0025] The description may use the phrases “in some embodiments,” “in various embodiments,” “in an embodiment,” or “in embodiments,” which may each refer to one or more of the same or different embodiments. Furthermore, the terms “comprising,” “including,” “having,” and the like, as used with respect to embodiments of the present disclosure, are synonymous. The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0026] In an aspect, the systems and methods disclosed here are directed to the high-throughput identification and isolation of regulatory T cells (i.e., Tregcells) based on the cytokines they produce. These systems and methods can be broadly utilized to any cell type that produces distinct biomolecules that are indicative of the cell’s phenotype. Furthermore, the systems and methods disclosed here can be applied to detecting multiple different biomolecules of interest through multiplexed detection, followed by sorting the cells based on their expression of multiplebiomolecules of interest, allowing comprehensive functional profiling of heterogeneous cell populations.

[0027] In an aspect, the embodiments disclosed herein provide CRISPR-based biomolecule detection and label-free cell sorting method by combing the advantage of unspecific ssDNA cleavage function of Casl2 proteins and a droplet microfluidic system. Type V CRISPR / Cas proteins (e g., Casl2 proteins) can indiscriminately cleave non-targeted single stranded DNA (ssDNA) once activated by detection of a target DNA. Once a Type V CRISPR / Cas effector protein is activated by a guide RNA, which occurs when a sample includes a target DNA to which the guide RNA hybridizes, the protein becomes a nuclease that unselectively cleaves ssDNAs. A droplet microfluidic system provides an efficient tool to isolate and sort single cells based on signal generated from the unleashed Casl2 proteins. Embodiments disclosed herein can detect biomolecule(s) of interest with femtomolar level of sensitivity and can isolate cells with specific biomolecular profiles. These embodiments have applications in health science and medical / clinical applications including, for example, pathogen detection and isolation, contamination detection, and cell-based therapies. Cells isolated using these disclosed methods and systems include functional cells for immunotherapy.

[0028] One such method for detecting an biomolecule in a sample includes the steps of contacting the sample with a mixture of one or more of pairs of ssDNA fragments with 3-10 nucleotide of complementary region at 3’ end of each ssDNA fragment and with a conjugated biomolecule receptor at the 5 ’end of each ssDNA fragment, a DNA polymerase for primer extension (i.e. T4 polymerase), a Type V CRISPR / Cas effector protein, a guide RNA containing a region that binds to the Type V CRISPR / Cas effector protein and a guide sequence that hybridizes with the target DNAs; and a labeled detector ssDNA that does not hybridize with the guide sequence of the guide RNA. The method further includes the step of measuring a detectable signal produced by cleavage of the labeled detector ssDNA by the Type V CRISPR / Cas effector protein, thereby detecting the target biomolecule. The biomolecule can be any compound of interest produced by a cell. The biomolecule in the sample can include a peptide or a protein. The biomolecules can be a surface biomarker, an intracellular protein, or a secretary protein. The biomolecule in the sample can include one or more of a toxin, which can be of bacterial, viral, or fungal origin. In some examples, the biomolecule in the sample can include a cytokine or a chemokine. The biomolecule in the sample can include one or more of y-interferon, granzyme,perforin, TGF beta, interleukin 2, interleukin 4, interleukin 6 interleukin 10, interleukin 17, G- CSF, GM-CSF, CXCL10, or CCL5. In certain examples, the ssDNAs can be any sequence that can form a protospacer adjacent motif (PAM) for Cas protein recognition. In certain examples, the conjugated biomolecule receptor can include but is not limited to an antibody, an engineered biomolecule receptor, any other ligand receptor, or a binding agent capable of binding to the biomolecule of interest. In certain examples, the effector protein can be any Type V CRISPR / Cas, such as Cas 12a, Cas 12b, which can promiscuously cleave non-targeted ssDNA once activated. In certain examples, the detector single strand DNA (3-30 nt) can be labeled with a dual-labeled moiety of a fluorophore and a quencher. In certain examples, the quencher can be a Black Hole Quencher®-! (BHQ-1) and Black Hole Quencher®-2 (BHQ-2). For instance, this dual-labeled moiety can be Dual -labeled BHQ™ probes, such as BHQ-1 with FAM, TET, or HEX, or BHQ-2 with TAMRA™, Texas Red®, or Cy™ 5. In certain examples, the dsDNA target is further amplified by recombinase polymerase amplification (RPA) or by Loop-mediated isothermal amplification (LAMP).

[0029] Embodiments further provide methods for isolating antigen, chemokine, or cytokine secreting cells, or cells expressing any biological molecule of interest, using a microfluidic system in conjunction with the disclosed detection strategies. In one embodiment, the method includes the following steps: (a) encapsulating one or more cells within a water-in-oil emulsion droplet; (b) incubating the one or more cells within the droplet for a period of time sufficient for cells to express a biomolecule of interest (e.g., from several seconds to several hours); (c) optionally merging this cell-encapsulated droplet with another droplet that contains another cell(s) of interest or similar type of cell(s) expressing additional biomolecule(s) of interest; (d) optionally incubating the merged droplet; (e) optionally contacting a detection mixture with the merged droplet to detect the biomolecule(s) of interest; (f) optionally incubating the merged droplet or the cell-encapsulated droplet with the detection mixture to yield detection signals indicating the presence of cells expressing the biomolecule(s) of interest; (g) optionally repeating any one of steps (c) - (f) as needed if multiple reagents are needed in sequence for the biomolecular assay to be completed; (h) detecting target droplets, defined as the merged droplets detected as containing cells expressing the biomolecule(s) of interest, via optical detection (e g., fluorescence, bioluminescence, Raman- active, or surface plasmon resonance detection); (i) sorting and collecting the target droplets based on the detection results; (j) isolating the target droplets and recovering the target cells expressingthe biomolecule(s) of interest from within the target droplets. In some examples, the cells are CD8+T cells and the biomolecule of interest is y-interferon. In certain examples, the microfluidic system includes, but is not limited to, a droplet production or manipulation unit. In certain examples, the droplet production or manipulation unit includes one or more microfluidic channels or chambers.

[0030] In one aspect, the methods are directed to a biomolecule (protein, peptide, cytokine, or fragments thereof, etc.) detection in a sample using a detection mixture. Individual components of the detection mixture can be provided as a kit or as independent reagents. In some cases, the detection mixture includes some or all of the following: one or more pairs of single strand DNA fragments with 3-10 nucleotides of complementary region at 3’ end and conjugated biomolecule / ligand receptors at 5 ’end of each; a DNA polymerase (such as a T4 polymerase); a CRISPR / Casl2 effector protein; a guide RNA; and a single strand detector DNA labeled with a quencher / fluorophore pair. The method includes the step of contacting the sample with the detection mixture and measuring the fluorescence signal produced by the single stranded detector DNA cleaved by the activated effector protein (FIGS. 1A and IB).

[0031] FIG. 1A is a schematic representation of a biomolecule / ligand detection system using CRISPR Casl2a effector protein and specific antibodies / ligand-receptor conjugated oligonucleotides, according to an embodiment. FIG. IB is a graphical representation of the detection of a cytokine (IL- 10) at different concentrations, , according to an embodiment. In this example, the cytokine levels were measured in 2-minute intervals over 1, 2, or more hours. Biotinylated target strand (TS) probe and non-target strand (NTS) probes were conjugated with biotinylated capture IL 10 antibody and detection IL- 10 antibody respectively in separate reactions. After conjugation of the probe with antibodies, 100 pM of both TS-IL-10 probe and NTS-IL-10 probe were subjected to extension reaction with T4 polymerase in the presence different concentrations of IL-10 (0, 1, 2, 10, 50 pM). The CRISPR reaction was performed by incubating 5 pl of TS / NTS probe amplicon, FAM-BBQ1 reporter (400 nM), Casl2a (40 nM), and crRNA (40 nM) at appropriate temperature (i.e., 37°C). Fluorescence signal was detected in Cytation5 over a 4-hour period (BioTek with excitation / emission at 495 / 520 nm).

[0032] In certain embodiments, to increase the detection sensitivity, these methods further include amplifying the generated target double strand DNA. The amplification method can be, but not limited to, a recombinase polymerase amplification (RPA) reaction. The detection of thedetectable positive signal indicates a presence of one or more target molecules in the sample. FIG. 2A is a schematic of an example of biomolecule / ligand detection system using CRISPR Casl2a effector protein and specific antibodies / ligand-receptor conjugated oligonucleotides coupled to a RPA reaction. To increase the detection sensitivity, a recombinase polymerase reaction is applied to amplify the target dsDNA. FIG. 2B shows detection of a cytokine (IL- 10) at different concentrations by the method shown in FIG. 2A, measured in 2-minute interval over 2 hours. Biotinylated target strand (TS) probe and non-target strand (NTS) Probe were conjugated with biotinylated capture IL- 10 antibody and detection IL- 10 antibody respectively in separate reactions. After conjugation of the probe with the antibodies, 100 pM of both TS-IL-10 probe and NTS-IL-10 probe were subjected to extension reaction with T4 polymerase in the presence of different concentrations of IL-10 (0, 1, 10, 100 pM). Extended probes were subjected to RPA reaction for amplification using the recommended protocol by the kit supplier. CRISPR reaction was performed by incubating 5 pl of TS / NTS probe amplicon, FAM-BBQ1 reporter (400 nM), Casl2a (40 nM), and crRNA (40 nM) at appropriate temperature (i.e., 37°C) and fluorescence signal was detected in Cytation5 over a 4 h period (BioTek with excitation / emission at 495 / 520 nm). By introducing the DNA amplification step, the detection limit of this embodiment of the method is lower than 1 pM of IL-10 cytokine and the released fluorescence signal is much higher, and detection sensitivity is increased more than 50 times.

[0033] In an example, the droplet production unit includes (i) a droplet cleaving module containing two or more microfluidic channels, wherein a continuous-phase fluid with merged droplets, containing the detection mixture and the cells producing the biomolecule of interest, flows into a first microfluidic channel, a second fluid containing a second phase liquid, such as oil, flows into a second microfluidic channel, and wherein the continuous-phase fluid in the first microfluidic channel is cleaved into a second set of droplets by flow of the second fluid in the second microfluidic channel; (ii) a droplet incubation unit that allows for the detection reaction to proceed; and (iii) a microfluidic droplet sorter unit that sorts and / or isolates the cells containing the target cells. This continuous-phase fluid can contain the biochemical reagents of the detection mixture as large particles or gel droplets that are relatively close in diameter to the encapsulated droplet. The fluidic channel architecture characteristics include linear, curved, or angled geometric configurations with cross-sectional heights ranging from 1-1000 pm (preferably 20-200 pm). The microfluidic channel network can be configured in either (i) a two-dimensional (2D) single-layerplanar design or (ii) a three-dimensional (3D) multi-layer stacked architecture in any spatial direction with respect to the base plane. The fluidic system can be made of PDMS, PMMA, glass, silicon, or other plastic materials. The size of droplet to be manipulated varies (but is not limited to) from several micrometers to hundreds of micrometers. The droplet production system can utilize either water-in-oil or oil-in-water droplets. The droplet production system can be used with multi-emulsion droplet schemes that lead to the generation of double emulsion (or more, i.e., triple quadruple, core / shell, multicore, etc.) droplets. The droplet production system may include, but not limited to, one or more functional subunits directed to droplet generation unit; droplet transition unit; droplet merging unit; and / or droplet sorting unit. The droplet generation unit includes, but not limited to, a T-junction generator, a flow-focusing generator, a co-flow generator and / or a step emulsification generator. In certain examples, the droplet transition unit can be achieved using a 2D reflow droplet array or 3D fabricated chambers. The droplet merging unit can include an acoustic-based merger, a dielectrophoretic-based merger, and / or a magnetic-based merger. The droplet sorting unit can include an acoustic-based merger, a dielectrophoretic-based merger, and / or pneumatic-based sorter. To the extent necessary to teach specific aspects of the fluidic analysis system with the droplet production unit (and only to the extent that they are consistent with the disclosure herein), US Patent No. 11,794,188 is herein incorporated by reference.

[0034] Embodiments also include cells isolated using the systems and methods provided herein, these cells of interest being isolated based on their expression of specific biomolecules. These biomolecules can be one or more of an immunomodulator or a signaling protein. For example, CD8+T cells secreting y-interferon may be isolated using this method. In another example, the cells containing the biomolecule of interest are Tregcells and the biomolecule of interest is interleukin- 10. In another example, cells of interest are CAR T-cells, and the biomolecule of interest is a specific chimeric antigen receptor. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is an antigen-binding aspect of a specific CAR. Other examples include functional cell subsets, such as CD8+T cells producing perforin / granzyme B for cytotoxic response monitoring, CD4+T cells producing IL- 1O / TGF- for immunosuppression assessment, macrophage cells releasing pro-inflammatory (e.g., IL-12) or anti-inflammatory cytokines (e g., IL-10), tumor-infiltrating lymphocytes secreting perforin / granzyme B for cytotoxic response monitoring, or NK cells releasing IFN-y / GM-CSF for antitumor activity evaluation.

[0035] In another aspect, the embodiments disclosed herein are directed to a cell sorting method by detecting biomolecule / ligand (protein, peptide, cytokine, etc.) produced by encapsulated cells. In some cases, method includes the steps of encapsulating single cell in a droplet with one or more components of a detection mixture. The components can include one or more of (i) one or more pair of single strand DNA fragments with 3-10 nt of complementary region at 3’ end and conjugated biomolecule / ligand receptors at 5’end of each; (ii) a DNA polymerase (e.g., T4 polymerase); (iii) a CRISPR / Casl2 effector protein; (iv) a guide RNA; and (v) a single strand detector DNA labeled with quencher / fluorescence dye pair. The method includes the step of incubating the droplets and allowing for signal generation. The encapsulated cells are maintained under conditions sufficient to allow release of biomolecules and the formation of one or more target dsDNAs. The method includes the step of sorting cells based on a fluorescent signal produced when the single stranded detector DNA is cleaved by the activated effector protein.

[0036] FIG. 3 A is a schematic representation of an overview of the workflow of CRIPR Cast 2a method for target cell sorting, according to an embodiment. Single cells are encapsulated with the CRISPR-based biomolecule / ligand detection component in a droplet produced by a droplet generator. The droplet is incubated under conditions sufficient to allow production of specific biomolecule(s) and the formation of one or more target dsDNAs, which activate CRISPR / Casl2 effector protein that then cleaves the single stranded detector DNA to produce a fluorescence signal. Then, these droplets flow through a microfluidic droplet sorter such that fluorescence signal is detected. When the droplet’s signal is greater than the set threshold, the sorter is triggered to pull that droplet towards the “Hit” channel while others flow out to the “Waste” channel.

[0037] FIG. 3B is a schematic representation of a microfluidic-based functional T cells sorting method based on the secretion of specific cytokines, according to an embodiment. Panel A shows single T cell encapsulation in droplets. Panel B shows cultivation of T cells in a droplet for secretion of cytokine (IL-10). Panel C shows merging of cultivated droplets with CRISPR Cas 12 mix. Panel D shows cultivation for biosensing activation. Panel E shows detecting and sorting the fluorescent droplet. Panel F shows the single-droplet-resolution being dispensed, so that the target T cells within droplets can be recovered.

[0038] In certain cases, to increase the detection sensitivity, such methods further include amplifying the generated target dsDNA. The amplification method can be, but is not limited to, arecombinase polymerase amplification (RPA) reaction. FIG. 4 is a schematic overview of the workflow of CRIPR Casl2a method for target cell sorting. To increase the sensitivity, a recombinase polymerase reaction is applied to amplify the target dsDNA. In another aspect, the embodiments disclosed herein are directed to a cell sorting method similar as the method described above, however the reaction reagents contained in different droplets. Here, the reaction reagents are accommodated in two different droplets: Droplet #1 contains one cell and one or more pair of single strand DNA fragments with 3-10 nt of complementary region at 3’ end and conjugated biomolecule / ligand receptors at 5’end of each, and T4 polymerase, while Droplet #2 contains the amplification reagents (PRA and primers); CRISPR / Casl2 effector proteins; single strand detector DNAs labeled with quencher / fluorescence dye pair. This method includes incubating the Droplet #1 under conditions to generate target dsDNA, and then sequentially merging with Droplet #2 to generate detection signals. After the reaction, the cells are sorted based on the fluorescent signal produced by the single stranded detector DNA cleaved by the activated effector protein.

[0039] Various examples are described herein to illustrate selected aspects of the various embodiments. For example, an embodiment includes the detection of cytokine production by regulatory T cells (Treg cells). Treg cells are the basis of cell-based immunotherapeutics that can target autoimmune diseases. However, T cell therapies can suffer from relatively high toxicity and low potency, stemming from low purity Tregcells. This is due to the difficulty in identifying and isolating Tregcells, as there are many different sub-populations of T cells, and surface marker alone cannot identify these Tregcells. If such T cells are mixed into the Treg cell population, this leads to high toxicity and low potency. Thus, identifying and sorting only Treg cells from a diverse T cell subpopulation is key to a safe and effective therapeutics. Treg cells can only be identified by their cytokines they produce (such as IL-10). Thus, the systems and methods disclosed here allow the detection of the specific cytokine production at a single target cell level, and the ability to recover those target cells, obtaining a pure population of Treg cells for therapeutics.

[0040] FIGs. 5A and 5B show single Tregcell encapsulation and viability test results. FIG. 5A shows single-cell encapsulation of Tregcell in 120 pm diameter water-in-oil droplets for culture at the start and at 48 hours. FIG. 5B presents the viability test after cultivation of the Treg cells in droplets as compared to the viability of the cells in cell culture wells-plate. The Tregcells in droplets were as viable as the cells cultivated in cell culture wells, demonstrating the success of these methods in capturing and maintaining viability of the cells of interest.

[0041] FIGs. 6A - 6C provide a comparison of 0 nM and 20 nM of IL-10 detection in droplets. The water-in-oil droplets (120 pm diameter) were generated after bulk CRISPR reaction using engineering oil (Novec 7500) with oil-phase surfactant (Pi co- Surf®) and aqueous solution that contains CRISPR mix after the reaction, according to an embodiment. FIG. 6A shows EGFP channel imaging of a negative control (NC) after 18 hours of cultivation at 37 °C. This negative control condition contains only CRISPR mix, but no IL-10 in the droplet. FIG. 6B shows 20 nM of IL-20 in a droplet under EGFP channel. FIG. 6C shows a mix of NC with 20 nM IL-10 for intensity comparison. The signal intensity is significantly different.

[0042] FIGs. 7A- 7D show detection of IL- 10 in droplets using the developed Flash-QC system, according to an embodiment. Here, the Tregcells were first encapsulated in droplets containing TS & NTS probes (250 pM) and cultivated overnight to allow IL- 10 secretion and extension. Then, the droplets were merged with a second group of droplets containing primers (10 pm) and a reaction mix for a 30 min RPA amplification (37°C). After that, the RPA-amplified droplets were again merged with the third group of droplets containing Casl2a (1 pM), crRNAl pM and FAM- BHQ-1 reporter 10 pM. The fluorescence was measured after 2 hours of reaction. FIG. 7A shows IL-10 concentration in the droplet being 0 nM. FIG. 7B shows presence of IL-10 (20 nM) in the droplet containing a Treg cell. FIG. 7C is a bright-field image of a Treg cell cultured for 24 h in a 60 pm diameter droplet. FIG. 7D shows green fluorescent signal corresponding to the presence of IL- 10 from droplet (C) is detectable.Embodiments.

[0043] Embodiment 1: A method for detecting a biomolecule in a sample, the method comprising: (a) contacting the sample containing cells expressing a biomolecule with (i) one or more of pairs of ssDNA fragments with a complementary region of 3-10 nucleotides at 3’ end of each ssDNA fragment and with a conjugated biomolecule receptor at the 5’end of each ssDNA fragment, (ii) a DNA polymerase for primer extension to form double-stranded DNA targets, (iii) a Type V CRISPR / Cas effector protein, (iv) a guide RNA containing a region that binds to the Type V CRISPR / Cas effector protein and a guide sequence that hybridizes with the double stranded DNA targets, and (v) a labeled detector ssDNA that does not hybridize with the guide sequence of the guide RNA; and (b) detecting the target biomolecule by measuring a detectablesignal produced by cleavage of the labeled detector ssDNA by the bound Type V CRISPR / Cas effector protein.

[0044] Embodiment 2: The method of Embodiment 1, wherein the biomolecule is a surface biomarker, an intracellular biomolecule, or a secreted biomolecule. These biomolecules can be recombinant molecules or naturally occurring molecules.

[0045] Embodiment 3: The method of Embodiments 1 or 2, wherein the biomolecule in the sample is one or more of a bacterial, viral, or fungal toxin.

[0046] Embodiment 4: The method of Embodiment 1, wherein the biomolecule in the sample is a secretory peptide or a protein, including glycosylated or post-translationally modified variants of the same.

[0047] Embodiment 5: The method of Embodiment 1, wherein the biomolecule in the sample is a molecule with cytokine or chemokine activity.

[0048] Embodiment 6: The method of Embodiment 1, wherein the biomolecule in the sample is one or more of y-interferon, granzyme, perforin, TGF-P, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, G-CSF, GM-CSF, CXCL10, or CCL5, or recombinant variants thereof, or biomolecules with equivalent biological activities.

[0049] Embodiment 7: The method of any one of Embodiments 1-6, wherein the ssDNA fragments are a sequence with a protospacer adjacent motif (PAM) for Cas protein recognition.

[0050] Embodiment 8: The method of any one of Embodiments 1-7, wherein the conjugated biomolecular receptor is an antibody, an engineered biomolecule receptor, an affinity binding reagent, or any other ligand receptor.

[0051] Embodiment 9: The method of any one of Embodiments 1-8, wherein the Type V CRISPR / Cas effector protein is a Casl2a or a Casl2b.

[0052] Embodiment 10: The method of any one of Embodiments 1-9, wherein the labeled detector ssDNA is labeled with a fluorophore and a quencher.

[0053] Embodiment 11: The method of any one of Embodiments 1-10, further comprising amplifying the double stranded DNA targets by recombinase polymerase amplification or by loop- mediated isothermal amplification.

[0054] Embodiment 12: A method of isolating cells expressing a biomolecule of interest, the method comprising:(a) passing the one or more cells through a microfluidic system containing a droplet production unit that encapsulates the one or more cells with an oil / aqueous solution to form a cell-encapsulated droplet;(b) incubating the cell-encapsulated droplet for a period of time sufficient for the one or more cells to produce a biomolecule of interest;(c) optionally merging this cell-encapsulated droplet with another droplet that contains another cell(s) of interest or similar type of cell(s) expressing additional biomolecule(s) of interest;(d) optionally incubating the merged droplet;(e) optionally injecting a detection mixture into the merged droplet to detect the biomolecule(s) of interest;(f) optionally incubating the merged droplet with the detection mixture to yield detection signals indicating the presence of cells producing the biomolecule(s) of interest;(g) optionally repeating steps (c) - (f) as needed if multiple reagents are needed in sequence for the biomolecular assay to be completed;(h) detecting target droplets, defined as the merged droplets detected as containing cells expressing the biomolecule(s) of interest, via optical detection (e.g., fluorescence detection);(i) sorting and collecting the target droplets based on the detection results; and(j) isolating the target droplets and recovering the target cells producing the biomolecule(s) of interest from within the target droplets.

[0055] Embodiment 13: The method of Embodiment 12, wherein the size of the merged droplet varies from a few microns to several hundred microns.

[0056] Embodiment 14: The method of Embodiment 12 or Embodiment 13, wherein the microfluidic system contains a droplet sorting unit.

[0057] Embodiment 15: The method of any one of Embodiments 12-14, wherein the microfluidic system contains one or more of the droplet production unit, a droplet incubation unit, and a droplet sorting unit.

[0058] Embodiment 16: The method of any one of Embodiments 12-15, wherein the droplet production unit comprises a droplet generator, which can be one of a T-junction generator, a flowfocusing generator, a co-flow generator, or a step emulsification generator.

[0059] Embodiment 17: The method of any one of Embodiments 12-16, wherein the merger of the cell-encapsulated droplet with the droplet containing a detection mixture can be an acousticbased merger, a dielectrophoretic-based merger, or a magnetic-based merger.

[0060] Embodiment 18: The method of any one of Embodiments 12-24, wherein the step of merging of the cell-encapsulated droplet with the aqueous solution containing a detection mixture comprises the steps of cleaving the droplet followed by a dielectrophoretic-based merger.

[0061] Embodiment 19: The method of any one of Embodiments 12-18, wherein the step of detecting target droplets includes an optical, an electrochemical, or an impedance sensing detection.

[0062] Embodiment 20: The method of any one of Embodiments 14 or 15, wherein the droplet sorting unit contains an acoustic-based sorter, a dielectrophoretic-based sorter, or a pneumaticbased sorter.

[0063] Embodiment 21: The method of any one of Embodiments 12-20, wherein the droplet production unit contains a plurality of fluidic channels.

[0064] Embodiment 22: The method of Embodiment 21, wherein, the fluidic channel architectures include linear, curved, or angled geometric configuration.

[0065] Embodiment 23: The method of Embodiment 21 or Embodiment 22, wherein the fluidic channel architectures include a two-dimensional (2D) single-layer planar design or (ii) a three-dimensional (3D) multi-layer stacked architecture in any spatial direction with respect to the base plane.

[0066] Embodiment 24: The method of any one of Embodiments 21-23, wherein the fluidic system is made of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, silicon, or other materials commonly used in microfluidic devices.

[0067] Embodiment 25: The method of any one of Embodiments 12-24, wherein the one or more cells containing the biomolecule of interest are CD8+T cells and the biomolecule of interest is y-interferon.

[0068] Embodiments also include immune cells isolated using the systems and methods provided herein, these cells of interest are isolated based on their expressed biomolecules. These biomolecules can be one or more of an immunomodulator or a signaling protein. In an example, the cells containing the biomolecule of interest are Tregcells and the biomolecule of interest is interleukin-10. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is a chimeric antigen receptor. In another example, cells containing the biomolecule of interest are CAR T-cells and the biomolecule of interest is an antigen-binding aspect of the CAR. Other examples include functional cell subsets, such as CD8+T cells producing perforin / granzyme B for cytotoxic response monitoring, CD4+T cells producing IL-1O / TGF- for immunosuppression assessment, macrophage cells releasing pro-inflammatory (e.g., IL-12) or anti-inflammatory cytokines (e.g., IL- 10), tumor-infdtrating lymphocytes producing perforin / granzyme B for cytotoxic response monitoring, or NK cells releasing IFN-y / GM-CSF for antitumor activity evaluation.

[0069] Other objects, features and advantages of the disclosure will become apparent from the foregoing figures, detailed description, and examples. It should be understood, however, that the figures, detailed description, and examples, while indicating specific embodiments of the disclosure, are given by way of illustration only and are not intended to limit the scope thereof in any way. Additional modifications and variations within the spirit and scope of the disclosure will be readily apparent to those skilled in the art in the view of the detailed description. In further embodiments, features from specific embodiments may be combined with features from other embodiments. For example, features from one embodiment may be combined with features from any of the other embodiments. In further embodiments, additional features may be added to the specific embodiments described herein, without departing from the scope of the disclosure.

Claims

ClaimsWhat is claimed is:

1. A method for detecting a biomolecule in a sample, the method comprising: contacting the sample containing cells secreting or displaying on a cell surface a biomolecule with (i) one or more of pairs of ssDNA fragments with a complementary region of 3-10 nucleotides at 3’ end of each ssDNA fragment and with a conjugated biomolecule receptor at the 5 ’end of each ssDNA fragment, (ii) a DNA polymerase for primer extension to form double-stranded DNA targets, (iii) a Type V CRISPR / Cas effector protein, (iv) a guide RNA containing a region that binds to the Type V CRISPR / Cas effector protein and a guide sequence that hybridizes with the double stranded DNA targets, and (v) a labeled detector ssDNA that does not hybridize with the guide sequence of the guide RNA; and detecting the target biomolecule by measuring a detectable signal produced by cleavage of the labeled detector ssDNA by the bound Type V CRISPR / Cas effector protein.

2. The method of Claim 1, wherein the biomolecule in the sample is one or more of abacterial, viral, or fungal toxin.

3. The method of Claim 1 , wherein the biomolecule in the sample is a secretory or cell surface displayed peptide or a protein, including glycosylated or post-translationally modified variants of the same.

4. The method of Claim 1, wherein the biomolecule in the sample is a molecule with cytokine or chemokine activity.

5. The method of Claim 4, wherein the biomolecule in the sample is selected from: y- interferon, granzyme, perforin, TGF-P, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, G-CSF, GM- CSF, CXCL10, or CCL5, or recombinant variants thereof, or biomolecules with equivalent biological activities.

6. The method of Claim 1, wherein the ssDNA fragments are a sequence with a protospacer adjacent motif (PAM) for Cas protein recognition.

7. The method of Claim 1 , wherein the conjugated biomolecular receptor is an antibody, an engineered biomolecule receptor, or any other ligand receptor.

8. The method of Claim 1, wherein the Type V CRISPR / Cas effector protein is a Casl2a or a Casl2b.

9. The method of Claim 1, wherein the labeled detector ssDNA is labeled with a fluorophore and a quencher.

10. The method of Claim 1, further comprising amplifying the double stranded DNA targets by recombinase polymerase amplification or by loop-mediated isothermal amplification.

11. A method of isolating cells producing a biomolecule of interest, the method comprising:(a) passing the one or more cells through a microfluidic system containing a droplet production unit that encapsulates the one or more cells with an oil / aqueous solution to form a cell-encapsulated droplet;(b) incubating the cell-encapsulated droplet for a period of time sufficient for the one or more cells to produce a biomolecule of interest;(c) optionally merging the cell-encapsulated droplet with another droplet that contains another cell(s) of interest or similar type of cell(s) expressing additional biomolecule(s) of interest;(d) optionally incubating the merged droplet;(e) optionally injecting a detection mixture into the merged droplet to detect the biomolecule(s) of interest;(f) optionally incubating the merged droplet with the detection mixture to yield detection signals indicating the presence of cells producing the biomolecule(s) of interest;(g) optionally repeating steps (c) - (f) as needed if multiple reagents are needed in sequence for the biomolecular assay to be completed;(h) detecting target droplets, defined as the merged droplets detected as containing cells producing the biomolecule(s) of interest, via optical;(i) sorting and collecting the target droplets based on the detection results; and(j) isolating the target droplets and recovering the target cells producing the biomolecule(s) of interest from within the target droplets.

12. The method of claim 11, wherein the microfluidic system contains a droplet sorting unit.

13. The method of claim 11, wherein the microfluidic system contains one or more of the droplet production unit, a droplet incubation unit, and a droplet sorting unit.

14. The method of claim 13, wherein the droplet sorting unit contains an acoustic-based sorter, a dielectrophoretic-based sorter, or a pneumatic-based sorter.

15. The method of claim 11, wherein the step of merging of the cell-encapsulated droplet with the droplet containing a detection mixture is carried out as an acoustic-based merger, a dielectrophoretic-based merger, or a magnetic-based merger.

16. The method of claim 11, wherein the step of merging of the cell-encapsulated droplet with the aqueous solution containing a detection mixture is carried out in a droplet cleaving and dielectrophoretic-based merger unit.

17. The method of claim 11 , wherein the step of detecting the target droplet includes an optical, an electrochemical, or an impedance sensing detection.

18. The method of claim 11, wherein the one or more cells containing the biomolecule of interest are CD8+T cells and the biomolecule of interest is y-interferon.

19. The method of claim 11, wherein the one or more cells containing the biomolecule of interest are Tregcells and the biomolecule of interest is interleukin-10.

20. A cell of interest isolated by the method of claim 11.

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