Microfluidic Methods for Single-Cell Analysis

Abstract

A first aspect of the present invention relates to a method for detecting a target compound in a microfluidic system. A second aspect of the present invention relates to the use of the method according to the first aspect for monitoring a biological event. Another aspect of the present invention relates to a microfluidic system and its use for implementing the method according to the first aspect.

Description

[0001] This application is a divisional application of the invention patent application filed on April 18, 2019, with application number 201980033806.1 and invention name “Microfluidic method for single cell analysis”. Technical Field

[0002] The present invention relates to the field of cell and molecular biology and is based on a method for detecting target compounds produced by single cells in droplets. The present invention also relates to the field of microfluidics and includes microfluidic devices and the use of microfluidic devices in performing biological assays. Background Art

[0003] One of the steps in the drug discovery process involves validating drug candidates based on their intended biological effects. For this purpose, either in vivo or in vitro models can be used. In vivo experiments offer the advantage of addressing questions in whole organisms. However, animal models do not necessarily predict what will happen in humans. Furthermore, in vivo studies are expensive, and their use is limited by ethical considerations. On the other hand, in vitro systems can be performed on human cells, even if they cannot replicate the exact cellular state of the organism, and are particularly well-suited for high-throughput screening processes. These cell-based assays are typically performed on target cells. However, in some cases, such as immune cells, each is unique, leading to significant interest in functional cell-based assays at the single-cell level. Measuring immune responses in large populations increases the risk of masking the unique behavior or contribution of each individual cell, especially when the immune response is highly heterogeneous or driven by rare cell populations. Therefore, single-cell-based assays are needed to better understand potential differences between different cells, taking into account individual cell phenotypes.

[0004] Recent advances in single-cell analysis methods have improved our understanding of biology within single cells by characterizing the relationships between cells within a population. Therefore, by identifying rare cellular events or subtle changes between individual cells, it is possible to address unresolved questions in cancer, immunology, infectious diseases, stem cells, as well as developmental biology and neurology.

[0005] Immune cells protect their hosts from disease by producing antibodies, chemokines, and cytokines. The former are a group of proteins secreted by innate and adaptive immune cells as chemical messengers. They are produced by immune cells to enable the body to mount immune responses, and therefore hold high clinical diagnostic value. Therefore, studying the secretion dynamics of antibodies and cytokines can provide crucial information for disease diagnosis and personalized treatment.

[0006] However, the lack of quantitative, single-cell, high-throughput systems for analyzing individual secretory cells has limited the study of immune response dynamics.

[0007] Droplet-based microfluidic systems have recently attracted great interest due to their range of applications in cell biology and their ability to control mechanical, biological, and fluid environments at the single-cell level. This technology enables assays to be performed very rapidly (up to thousands of cells and / or droplets per second). In addition, the system provides large-scale (picoliter or nanoliter volumes of sample and reagents) cell culture experiments, where biological samples are confined in droplets, allowing for the rapid detection of high concentrations of compounds (from the pM to μM range). Furthermore, the system minimizes sample loss and cross-contamination, yet allows for rapid mixing, heat transfer, and chemical reactions. Interestingly, the technology offers the possibility of large-scale genotypic and phenotypic screening at the single-cell level.

[0008] In the past few years, different microfluidic devices and systems have been proposed for single-cell analysis (Gross et al. 2015, Int. J. Mol. Sci. 16(8): 16897-16919; Reece et al. 2016, Curr. Opin. Biotechnol. 40: 90-96).

[0009] Different methods and techniques have been proposed for cell sorting in microfluidics. The sorting principles can be divided into two main categories: methods based on the physical properties of cells (such as size, deformability, electrical properties or optical properties) and methods based on biomolecular properties (especially specific surface antigens).

[0010] High-purity cell isolation and sorting can be achieved using monoclonal antibodies that bind to cellular components. Widely used antibody-based cell analysis and / or separation techniques include cell panning, magnetic cell sorting (MACS), and fluorescence-activated cell sorting (FACS), including fluorescence-activated droplet sorting (FADS).

[0011] In cell panning, cells expressing a specific antigen are selectively attached to an antibody-coated surface. Although this technique can provide high purity, it is still subject to certain limitations, such as high cell loss or effects on cell survival.

[0012] In other cell panning techniques for single cell sorting by flow cytometry, cells that secrete specific molecules can be selectively captured by antibodies bound to the cell surface or extracellular matrix (e.g., antibody-coated surfaces) (Campbell et al., 2010 J. Immunol. 185: 28-32; Manz et al., 1995, Proc. Natl. Acad. Sci. USA 92: 1921-1925). Although this technique can detect secreted molecules, it is subject to certain limitations when coupled to a flow cytometer at the single cell level, such as high background due to cell concentration (thus affecting cell purity) and lack of quantitative separation based on secretion and lack of real-time quantitative secretion rate measurement.

[0013] MACS uses magnetic beads bound to antibodies to capture specific antigens on the cell surface. Cell populations labeled with magnetic beads can be selectively collected under a magnetic field generated by a permanent magnet. Compared to FACS, MACS allows for significantly higher yields, but cannot perform single-cell sorting and has lower purity. Another significant limitation is the difficulty in separating and removing the beads after separation, which can hinder subsequent analysis.

[0014] Another example of cell separation is the use of a microfluidic approach based on the use of magnetic bead particles as a bead string. This approach, disclosed in International Patent Application WO 2016 / 059182 A1, features a particle aggregate formed by a string of magnetic beads designed to detect the presence of secreted molecules by capturing the molecules on the bead string and detecting elements on the string. The method proposed in WO 2016 / 059182 A1 has the advantage of being able to assess secreted molecules at the single-cell level. However, the method disclosed in WO 2016 / 059182 A1 relies on the presence of particle aggregates, thus hindering complex assays requiring multiple cells in the same compartment. This assay has inherent limitations in flexibility. Furthermore, sensitivity is inherently limited by the binding capacity of the particle aggregates.

[0015] Generally, limitations affecting currently available methods for analyzing and / or isolating single cells based on secreted molecules include low efficiency or low yield / recovery in terms of single cells isolated per second, decreased cell viability / functionality during the isolation process, poor reliability, poor flexibility, and / or low throughput. Therefore, it is clear that there is a strong need for improved microfluidic methods for analyzing and isolating compound-secreting single cells that address the aforementioned issues. Summary of the Invention

[0016] A first aspect of the present invention relates to a method for detecting a target compound in a microfluidic system, the method comprising the following steps:

[0017] a. generating at least one droplet in the microfluidic system, the at least one droplet comprising:

[0018] i. at least one single cell,

[0019] ii. one or more first capture agents, wherein the one or more first capture agents are capable of binding to the single cell and the target compound,

[0020] iii. one or more second capture agents comprising a label, wherein the one or more second capture agents are capable of binding to the target compound,

[0021] b. culturing the at least one droplet capable of producing a detectable event,

[0022] c. performing direct detection on said at least one droplet,

[0023] wherein the presence or relocation of the detectable event in the at least one droplet determines the presence of the target compound.

[0024] A second aspect of the invention relates to the use of the method according to the first aspect for monitoring biological events.

[0025] A third aspect of the present invention relates to a method for detecting a target compound in a droplet, the method comprising the following steps:

[0026] a. Provide a microfluidic system, comprising:

[0027] i. at least one entrance,

[0028] ii. at least one exit,

[0029] iii. one or more channels,

[0030] b. injecting a stream of droplets into the microfluidic system, wherein at least one droplet comprises:

[0031] i. At least one single cell

[0032] ii. a plurality of first capture agents capable of binding to the single cell and the target compound, and

[0033] iii. a plurality of second capture agents, each second capture agent comprising a label, wherein the plurality of second capture agents are capable of binding to the target compound,

[0034] c. culturing the plurality of droplets under conditions that allow production of a target compound, such that if the target compound is produced by a single cell, the target compound will be captured by the plurality of first capture agents and the plurality of second capture agents,

[0035] d. Determining the presence of the target compound by detecting the presence or relocation of the label.

[0036] A fourth aspect of the present invention relates to a microfluidic system comprising:

[0037] a. At least one entrance,

[0038] b. At least one exit,

[0039] c. one or more channels,

[0040] d. A module for creating at least one droplet, the at least one droplet comprising:

[0041] i. one or more single cells,

[0042] ii. a first capture agent,

[0043] iii. Second capture agent.

[0044] e. Detection module, detecting droplets containing cells producing target compounds

[0045] f. An analysis module configured for signal analysis.

[0046] A fifth aspect of the present invention relates to the use of the microfluidic system according to the fourth aspect for implementing the method according to the first aspect or the third aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A and Figure 1B Single-cell droplet secretion assay for cytokine secretion detection.

[0048] While the examples presented here focus on cytokine secretion detection and / or antibody secretion detection using fluorescent detection reagents, the assays provided are applicable to secretion detection of any target compound and using any labeled detection reagent. PBMCs are stimulated on-chip (i.e., in droplets) or off-chip (i.e., outside the droplets, in a separate container) (specifically using antigen-presenting cells labeled with a specific antigen, or non-specifically, such as by using cross-linked antibodies or phorbol esters), and pre-labeled (on-chip or off-chip) with capture reagents and encapsulated into droplets as single cells with fluorescent detection reagents under conditions that prevent cytokine secretion. After culturing the droplets under conditions that allow cytokine secretion, secreting cells are detected by the presence or relocation of the detection reagent on the cells. Figure 1A Cytokine-secreting cells: These cells secrete the target cytokine that is bound to the capture agent. The detection reagent binds to the secreted cytokine, resulting in the presence or relocation of a fluorescent signal on the cell. Figure 1BNon-secreting cells: The cells being analyzed do not secrete the target cytokine, and the detection reagent remains homogenous within the droplet. No fluorescence is observed.

[0049] Figure 2A and Figure 2B Single-cell droplet detection of IFNγ secretion is sensitive and specific.

[0050] Figure 2A ) IFNγ secreted by activated T cells was detected specifically on a single-cell droplet basis compared to non-activated cells. or Intercalating agents can prevent cells from dying before the experiment or within the droplets before or after secretion of the target cytokine, thereby preventing any nonspecific events that could represent a substantial nonspecific binder. In droplets, IFNγ secretion was detected at 0.14% and 16.7% in droplets containing non-activated and activated cells, respectively. Figure 2B Flow-based detection of IFNγ secretion by activated T cells. In flow cytometry, IFNγ secretion was detected by 0% and 16% of non-activated and activated cells, respectively. The migration of cell populations is a serious limitation of flow-based systems due to high background from nonspecific capture of secreted molecules by nearby cells during staining.

[0051] Figure 3A 、 Figure 3B 、 Figure 3C and Figure 3D Single-cell droplet detection of IFNγ secretion was sensitive (<1 nM), efficient (>80%), and 100% specific.

[0052] Droplets containing single, non-activated CD8+ T cells, pre-labeled with capture reagent and co-flowed with detection reagent, were reinjected into the microfluidic device in the presence of varying concentrations of purified IFNγ, and the fluorescence of each droplet was analyzed using proprietary software. Figure 3A ) Select droplets with the correct width and properties for different emulsion / concentration conditions. Figure 3B ) Droplets containing CD8+ T cells were selected based on cell markers. Figure 3C ) IFNγ in the droplets was detected for each cytokine concentration. Figure 3D ) For each tested concentration of IFNγ, the percentage of positive droplets detected was determined and compared to a negative control (0 nM). Cytokine concentrations as low as 1 nM were detected in droplets, and approximately 80% of cells were detected using a droplet-based single-cell secretion assay. False positives were not selected, as 0% of cells / droplet were observed to be positive in the presence of 0 nM IFNγ.

[0053] Figure 4A 、 Figure 4B 、 Figure 4C and Figure 4D Single-cell droplet antigen-specific activation of T cells by antigen-presenting cells.

[0054] Figure 4A Antigen-presenting cells (APCs) pulsed with a specific peptide library and primary CD8+ T cells (pre-labeled with a capture reagent) were co-encapsulated in droplets. The droplets were incubated overnight under conditions that allowed APCs to activate T cells, as detected by cytokine secretion. The next day, the droplets were reinjected into the microfluidic device, and the fluorescence signal was analyzed to detect activated T cells that had secreted and were secreting IFNγ. Figure 4B The target droplets consist of a co-encapsulated T cell and an antigen-presenting cell. Both cells can be fluorescently labeled with different colors to efficiently select droplets containing both cells. Figure 4C ) Before or during the experiment / activation, a fluorescent dead cell marker was used to control the viability of the cells in the droplets and exclude any false positives due to cell death. After overnight culture, the cells encapsulated in the droplets showed high activity, as 94% of them were detected as viable. Figure 4D The droplet secretion assay was used to detect antigen-specific T cell activation of APCs in droplets. As expected based on the frequency of responding T cells, 1.2% of droplets containing both live T cells and live APCs were detected to be secreting IFNγ, demonstrating successful, high viability, antigen-specific activation, and detection of IFNγ secretion by single T cells in droplets.

[0055] Figure 5A 、 Figure 5B 、 Figure 5C 、 Figure 5D and Figure 5E Single-cell droplet endocrine assay suitable for detecting any secreted molecule.

[0056] The methods according to the present invention are highly scalable and can be adapted to detect a variety of biological events. Although the examples presented here focus on the detection of cytokine and / or antibody secretion using fluorescent detection reagents, the provided assays are applicable to the detection of secretion of any target compound and using any labeled detection reagent. Figure 5A) Examples of detecting the secretion of different target compounds in the droplets of interrogated cells, including the possibility of performing multiple determinations. Here, multiple determination methods for antibody and cytokine secretion are proposed, but the present invention can be applied to any target compound mentioned. PBMCs stimulated outside or on the chip are pre-labeled with cytokine-specific capture reagents, and B cells are pre-labeled with antibody-specific capture reagents. Under conditions that prevent the secretion of cytokines and antibodies before encapsulation into single cells, the two cell populations are co-encapsulated as droplets together with cytokine-specific fluorescent detection reagents and antibody-specific fluorescent detection reagents as single cells. The markers of the two detection reagents are wisely selected according to the assay (fluorescent in this example, but can be by any means). After culturing the droplets under conditions that allow cytokine and antibody secretion, the secreting cells are detected by the presence or repositioning of the detection reagent on the cells. The secreted cytokines are combined with the capture reagents that specifically bind to the cytokine-secreting cells and are detected by the presence or repositioning of fluorescent anti-cytokine detection reagents. The secreted antibodies are combined with the capture reagents that bind to the antibody-secreting cells and are detected by the presence or repositioning of fluorescent anti-antibody detection reagents on the cells. Antibody-specific capture reagents can be specific for all immunoglobulins, allowing detection of global antibody responses, or can be composed of the target antigen, allowing detection of antigen-specific antibody responses. Figure 5B ) Example of using co-flow capture and detection reagents for in-droplet cytokine secretion detection. Under conditions that prevent cytokine secretion, PBMCs stimulated off-chip or on-chip are encapsulated into droplets as single cells together with capture reagents and detection reagents (which can be fluorescent as exemplified here or any other means). After culturing the droplets under conditions that allow cytokine secretion, secreting cells are detected by the presence or relocation of the detection reagent on the cells. The concentrations of both capture and detection reagents are suitable for producing the highest signal / background ratio and for maximizing the fluorescent signal on the cells being interrogated. ( Figure 5C ) Example of in-droplet cytokine secretion detection by a first capture reagent bound to a cell composed of two or more molecules. PBMCs stimulated off-chip or on-chip are pre-labeled with a cytokine-specific capture reagent composed of two or more molecules. The two or more molecules consist of an antibody specific for the target cell membrane conjugated to ligand A and an antibody specific for the target cytokine conjugated to ligand B, wherein ligands A and B can interact and form a stable association. Under conditions that prevent cytokine secretion, the cells are encapsulated into droplets as single cells together with a fluorescent detection reagent. After culturing the droplets under conditions that allow cytokine secretion, the secreting cells are detected by the presence or relocation of the detection reagent on the cells. ( Figure 5D) An example of cytokine secretion detection in a droplet in which a first capture agent is co-flowed and consists of two or more molecules. Under conditions that prevent cytokine secretion, PBMCs stimulated off-chip or on-chip are encapsulated into droplets as single cells together with a capture agent and a fluorescent detector. The co-flowing cytokine-specific capture reagent consists of two or more molecules. The two or more molecules consist of an antibody specific for the target cell membrane conjugated to ligand A and an antibody specific for the target cytokine conjugated to ligand B; wherein ligands A and B can interact and form a stable association. After culturing the droplets under conditions that allow cytokine secretion, the secreting cells are detected by the presence or relocation of the detection reagent on the cells. The concentrations of both the capture and detection reagents are suitable for producing the highest signal / background ratio and for imparting the maximum fluorescent signal to the cells being interrogated. ( Figure 5E ) An example of cytokine secretion detection in droplets, wherein the first capture reagent consists of two molecules, one part binds to the cell and the other part co-flows. PBMCs stimulated off-chip or on-chip are pre-labeled with the first part of the capture reagent, which contains a cell membrane-specific antibody conjugated to ligand A. These cells are encapsulated into droplets as single cells together with the second part of monocytes. Under conditions that prevent cytokine secretion, the capture reagent consists of an antibody specific for the target cytokine that binds to ligand B, and is also composed of a fluorescent detection reagent. Ligands A and B can interact and form a stable association. After culturing the droplets under conditions that allow cytokine secretion, secreting cells are detected by the presence or relocation of the detection reagent on the cells. The concentrations of the second part of the capture reagent and the detection reagent are suitable for producing the highest signal / background ratio and for directing the maximum fluorescent signal to the cells being interrogated.

[0057] Figure 6 .Detection of T cell activation by secreted receptor-specific antibodies.

[0058] Figure 7 . Double positive ADCC test caused by secretion of antigen-specific antibodies and cytotoxic factor secretion detection.

[0059] Figure 8 Double positive detection of ADCC induced by secreted antigen-specific antibodies.

[0060] Figure 9 . Description of the microfluidic systems and methods according to the present invention. DETAILED DESCRIPTION

[0061] The method according to the present invention aims to solve the above-mentioned problems affecting current microfluidic technologies for single cell analysis. In particular, the present method provides improved performance for detecting, analyzing and / or quantifying the production of target compounds at the single cell level.

[0062] A first advantage of the methods disclosed herein is their high sensitivity. This property is attributed to the spatial confinement of individual cells producing the target compound within droplets, where the individual cells have freedom of migration, thereby allowing for high viability and, therefore, high yet physiological metabolic activity. Furthermore, the spatial confinement of individual cells producing the target compound within droplets, where the secreted product is confined to a limited volume of a few picoliters to nanoliters, allows for high concentrations to be achieved within minutes to hours of culture, depending on the molecule produced.

[0063] Therefore, a second advantage arising from the use of the method of the present invention is the possibility of kinetic analysis by real-time monitoring of the relocation and / or intensity changes of the detectable events. By extension, it is readily conceivable to extend to the detection of multiple secreted compounds by using differently labeled detection reagents.

[0064] Thus, a third advantage that arises from using the methods of the present invention is the possibility of performing complex and flexible assays by co-encapsulating two or more cells into droplets and monitoring cell-cell interactions. The interactions of the compounds are generated by one, two, or more cells. Complex assays that co-encapsulate two or more cells also enable the detection of secretion of two or more target compounds.

[0065] A fourth advantage that arises from the use of the methods of the present invention is the high specificity of detecting the production of the molecule. This property is attributed to the spatial confinement of the single cell producing the target compound in the droplet, where the secretion product is confined to a restricted volume, specifically trapped in the single cell, and is therefore captured only by the secreting cell.

[0066] In this regard, the inventors have discovered that secreting cells can be advantageously detected by monitoring the presence or relocation of detection reagents on cells within droplets, and that cell density / concentration does not affect the specificity of the detection.

[0067] Additionally, in the presence of non-secreting cells or cells that do not secrete the compound of interest, the detection reagent remains homogeneous within the droplet, minimizing false-positive hits.

[0068] In a first aspect, the present invention relates to a method for detecting a target compound in a microfluidic system, the method comprising the following steps:

[0069] A first aspect of the present invention relates to a method for detecting a target compound in a microfluidic system, the method comprising the following steps:

[0070] a. generating at least one droplet in the microfluidic system, the at least one droplet comprising:

[0071] i. at least one single cell,

[0072] ii. one or more first capture agents, wherein the one or more first capture agents are capable of binding to the single cell and the target compound,

[0073] iii. one or more second capture agents comprising a label, wherein the one or more second capture agents are capable of binding to the target compound,

[0074] b. culturing the at least one droplet capable of producing a detectable event,

[0075] c. performing direct detection on said at least one droplet,

[0076] wherein the presence or relocation of the detectable event in the at least one droplet determines the presence of the target compound.

[0077] In the context of the present invention, the term "microfluidic system" may refer to one or more integrated units or chips for performing the methods disclosed herein. The microfluidic system is typically represented in the form of a microfluidic chip comprising one or more microchannels and one or more microfluidic devices (e.g., micropumps, microvalves).

[0078] In the context of the present invention, "microfluidic chip" generally refers to a group of microchannels made by milling, etching, ablation or molding into a material (a polymeric material such as polydimethylsiloxane (PDMS) or polymethyl methacrylate (PMMA)), polycarbonate (PC), epoxy resin, COC, especially a photopolymerizable epoxy resin (such as sold by Norland Optical Adhesives (NOA)), glass, silicon, plastic. A microfluidic chip may include a substrate and a support that together define at least one channel.

[0079] As used herein, the term "droplet" refers to an isolated portion of a fluid that is immiscible with its surroundings. In the context of the present invention, the "droplet" can be spherical, substantially spherical, or non-spherical in shape. The shape can depend on various parameters, such as the external environment.

[0080] Methods for preparing, generating, and injecting droplets in microfluidic systems are known to those skilled in the art. An exemplary method is disclosed in US 2015 / 0057163 A1. Regarding the presence of a single cell in each droplet, those skilled in the art will recognize that this parameter can be controlled and / or estimated using a Poisson distribution.

[0081] In the context of the present invention, the expression "at least one single cell" refers to both viable and inviable single cells. The viability of the at least one single viable cell can be altered or changed according to the steps of the method according to the present invention. Notably, the ability to generate a detectable event in a droplet after the droplet incubation step according to the present method refers to the likelihood of having at least one viable single cell within the droplet.

[0082] As used herein, the term "direct detection" refers to the possibility of detecting a compound of interest produced by a single cell in the absence of a solid support within the droplet, wherein the solid support would be used to capture the compound of interest. In the context of the present invention, the term "solid support" refers to any non-biological matrix, such as magnetic beads, gel matrix or affinity matrix, which has a given specificity for the target molecule, so that the target molecule can be immobilized on the support, which allows the target molecule to be isolated from the contents contained in the droplet.

[0083] According to an embodiment of the first aspect of the present invention, a single cell exhibits freedom of migration within the droplet.

[0084] In the context of the present invention, the detection of the target compound is independent of the orientation of the cells producing said target compound in the droplet.

[0085] According to another embodiment of the first aspect of the present invention, the single cells are not captured on the solid support.

[0086] The inventors have discovered that the presence of single cells with high mobility, which are not constrained on a solid support, can be detected with superior sensitivity in detecting the presence of target compounds secreted by the cells due to the improved distribution of the first capture agent on the cell surface.

[0087] As used herein, the term "capture agent" refers to an agent, nucleic acid, protein or peptide that has an affinity for a target compound. In the context of the present invention, the method requires the presence of a first capture agent and a second capture agent.

[0088] In the context of the present invention, the terms "first capture agent" and / or "second capture agent" can refer to a single bifunctional compound or a complex comprising two or more different compounds, each having a specific functionality. Examples of first and second capture agents contemplated by the methods of the present invention can be compounds or complexes formed from antibodies, antigens, cytokines, chemokines, hormones, or growth factors, or combinations thereof.

[0089] As used herein, the term "repositioning" refers to a change in the spatial arrangement within a droplet of the density and / or concentration of detectable events. As used herein, the term "presence" refers to the occurrence or change in the intensity of a detectable event.

[0090] An important aspect of the method according to the present invention relates to the relocalization of detectable events within the droplet. In this respect, methods known in the art do not achieve the desired "relocalization" but rather simply flush out excess localized concentrations prior to flow cytometric analysis. This characteristic, therefore, results in a method according to the present invention having a higher efficiency than existing methods.

[0091] Another important step in droplet-based microfluidic analysis is the cultivation of droplets and the generation, subcutaneous injection, merging and sorting of droplets. In the context of the present invention, cultivation can be performed off-chip or on-chip. The cultivation step can also occur in a delay line necessary to cultivate the droplets for a precise time to allow the cells to survive and produce the target compound. An exemplary method of cultivation in a delay line is disclosed in US2012 / 0121480A1. Typical culture temperatures before encapsulation range from 0°C to 16°C, typical culture temperatures after encapsulation range from 16°C to 38°C, and re-injection after cultivation to analyze secreted molecules ranges from 0°C to 38°C. Typical culture times range from milliseconds (for kinetic analysis) to over 24h (for analysis of compound production regulation mediated by cell-cell interactions).

[0092] In another embodiment of the first aspect of the invention, the method further comprises the step of measuring cell survival in the droplets after incubation. In the context of the present invention, a preferred method for measuring cell survival is accomplished by using an intercalating dye that emits fluorescence only when dead cells are detected in the droplets, e.g. Dead647Ready

[0093] According to another embodiment of the first aspect of the present invention, the one or more first capture agents are bound to the surface of the at least one single cell before or after generating the at least one single droplet.

[0094] In one embodiment of the first aspect of the present invention, one or more first capture agents are present in an amount of 10 1 to 10 8 The density of molecules / cell binds to the single cell.

[0095] According to another embodiment of the first aspect of the present invention, the target compound is produced in the droplet at a concentration of 10 pM to 100 μM.

[0096] In another embodiment of the first aspect of the invention, the volume of the droplet is from 2 pL to 10 nL.

[0097] In another embodiment of the first aspect of the invention, the label is selected from a fluorescent label, a polymer, a protein, a peptide, a hapten, a chemical, a nucleic acid, or a barcode label. As used herein, the term "barcode" refers to a label that can be attached to an analyte to convey information about the analyte. In the context of the present invention, the barcode label can be a mixture of a label, a polymer, a fluorescent label, a peptide, a hapten, a protein, a chemical, a nucleic acid.

[0098] In another embodiment of the first aspect of the present invention, the first capture agent and the second capture agent are independently selected from proteins, peptides, oligonucleotides, haptens, nucleic acids, fluorescent conjugates, enzyme conjugates, synthetic polymers, or barcodes, or combinations thereof. The barcode tag can be a mixture of a tag, a polymer, a fluorescent tag, a peptide, a heptene, a protein, a chemical, or a nucleic acid.

[0099] In another embodiment of the first aspect of the invention, the first capture agent is an antibody and the second capture agent is a fluorescent anti-compound to the antibody of interest.

[0100] According to another embodiment of the first aspect of the present invention, the first capture agent is a bifunctional antibody.

[0101] In another embodiment of the first aspect of the invention, the compound of interest is a compound secreted by a cell, which is selected from, but not limited to, antibodies (IgG (IgG1, IgG2, IgG3, IgG4), IgE, IgA (IgA1, IgA2), IgM, cytokines (IL-1-like, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6-like, IL-6, IL-7, IL-9, IL-10-like, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, common b chain (CD131), LIF, OSM, interferons (IFN-α, IFN-β, IFN-γ), TNF, TNF -α, TNF-β, CD153, CD154, LT-β, 4-1BBL, APRIL, CD70, CD132, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, Tpo, Flt-3L, SCF, M-CSF, MSP), chemokines (CCL 1. CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL1 4. CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL 26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, X1L1, XCL2, CX3CL1), hormones (estrogen, progesterone, thyroxine, steroids, insulin, epinephrine, melatonin, triiodothyronine, thyroxine, prostaglandins, leukotrienes, prostacyclin, Therocis, adipose tissue hormone, adrenocorticotropic hormone (or adrenocorticotropic hormone), amylin (or islet amyloid polypeptide), angiotensin and angiotensin, islet amyloidosis anti-Müllerian hormone (or Müllerian inhibitory factor or hormone), antidiuretic hormone (or vasopressin, arginine vasopressin), atrial peptide (or atrial natriuretic peptide), calcitonin, cholecystokinin, adrenocorticotropic hormone-releasing hormone, cortistatin, endothelin, enkephalin, erythropoietin, follicle-stimulating hormone, galanin, gastric inhibitory polypeptide, gastrin, glucagon, glucagon-like peptide-1, gonadotropin-releasing hormone, guanosine, heparin, human chorionic gonadotropin, inhibin, insulin,Insulin-like growth factor (or growth hormone), leptin, lipoprotein, melanocyte-stimulating hormone, motilin, orexin, osteocalcin, oxytocin, relaxin, renin, secretin, somatostatin, thrombopoietin, uroguanosine, vasoactive intestinal peptide, steroids, estrogen, glucocorticoids, progesterone, steroids), growth factors (G-CSF, GM-CSF, Fas-ligand, adrenomedullin (AM), angiogenin (Ang), autotaxin, bone morphogenetic protein (BMP), ciliary neurotrophic factor family, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), leukemia Interleukin-6 (IL-6), colony stimulating factor, macrophage colony stimulating factor (m-CSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), E-factor (A1-A5, B1-B3), erythropoietin (EPO), fibroblast growth factor (FGF1-FGF23), fetal bovine growth hormone (FBS), GDNF ligand family, glial cell line-derived neurotrophic factor (GDNF), neuraminic acid, persephin, artemin, growth differentiation factor 9 (GDF 9)), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor, insulin-like growth factor-1 (IGF-1 and IGF-2), interleukins; IL-1 cofactor of IL-3 and IL-6, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, keratinocyte growth factor (KGF), migration stimulating factor (MSF), macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like protein (HGFLP), myostatin (GDF-8), neuregulin (NRG1-NRG 4), neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), placental growth factor (PGF), platelet-derived growth factor (PDGF), nephrotic enzyme (RNLS) – anti-apoptotic survival factor, T cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factor α (TGF-α, TGF-β (TGF-β1, TGF-β2, TGF-β3)), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF)).

[0102] A second aspect of the present invention includes the purposes of the method according to the first aspect of the invention in monitoring one or more biological events that may occur simultaneously. As used herein, the term "biological event" refers to a physiological process and / or a change in state that describes the physiological state that occurs in a subject and affects living cells. Typical examples are that the secretion of a compound is linked to the death of induction (non-limiting examples are ADCC, CDC, ADCP, cytokine-induced cell dissolution, apoptosis, chromium release), and another example is that the compound of secretion activates and / or inhibits cell pathways (as non-limiting examples, G protein coupled receptor activation, B-arrestin, caspase activation, PKC / NFKB pathway, MAP kinase, Pi3K, AKT pathway, Ras / Mek / Erk, PLC / Ca++).

[0103] In an embodiment of the second aspect of the invention, the biological event is an immune response. Typical examples include detecting antigen recognition by a T cell-induced compound secretion, including antigen recognition by a B cell-induced compound secretion, and monitoring T cell activation induced by a compound secreted by a T cell and induced by a second secreted compound (this example includes detecting secreted compounds by two different cell types and distinguishing them using barcodes specific to each), and including monitoring B cell activation induced by a compound secreted by a B cell and induced by a second secreted compound.

[0104] A third aspect of the present invention relates to a method for detecting a target compound in a droplet, the method comprising the following steps:

[0105] a. Provide a microfluidic system, comprising:

[0106] i. at least one entrance,

[0107] ii. at least one exit,

[0108] iii. one or more channels,

[0109] b. injecting a stream of droplets into the microfluidic system, wherein at least one droplet comprises:

[0110] i. At least one single cell

[0111] ii. a plurality of first capture agents capable of binding to the single cell and the target compound, and

[0112] iii. a plurality of second capture agents, each second capture agent comprising a label, wherein the plurality of second capture agents are capable of binding to the target compound,

[0113] c. culturing the plurality of droplets under conditions that allow production of a target compound, such that if the target compound is produced by a single cell, the target compound will be captured by the plurality of first capture agents and the plurality of second capture agents,

[0114] d. Determining the presence of the target compound by detecting the presence or relocation of the label.

[0115] A fourth aspect of the present invention relates to a microfluidic system comprising:

[0116] a. At least one entrance,

[0117] b. At least one exit,

[0118] c. one or more channels,

[0119] d. A module for creating at least one droplet, the at least one droplet comprising:

[0120] i. one or more single cells,

[0121] ii. a first capture agent,

[0122] iii. Second capture agent.

[0123] e. Detection module, detecting droplets containing cells producing target compounds

[0124] f. An analysis module configured for signal analysis.

[0125] According to an embodiment of the fourth aspect of the present invention, the microfluidic system is characterized by the presence of at least two modules in communication with each other, the modules being selected from the group consisting of: a module for droplet generation, a module for droplet detection, a module for droplet analysis, a module for sorting droplets, a module for labeling droplets, and a module for recovering droplets. In the context of the present invention, the module for recovering droplets is intended to be used to perform additional procedures (e.g., genotyping, further functional analysis).

[0126] exist Figure 9 An idealized embodiment of the microfluidic system and method according to the present invention is depicted in FIG.

[0127] The combination of two or more of the aforementioned modules allows the microfluidic system disclosed herein to achieve improved results in high throughput (thousands of droplets can be processed per second).

[0128] An important aspect of the microfluidic system according to the present invention is that the secretion and detection steps of the method according to the first aspect of the present invention can be performed in the same module of the microfluidic system.

[0129] According to a fifth aspect, the microfluidic system according to the fourth aspect is used to perform the method disclosed in the first aspect or the third aspect of the present invention.

[0130] Example

[0131] Principle Description

[0132] Healthy donor PBMCs were pre-labeled with an excess of bifunctional antibodies, called "capture reagents," in microtubes. The capture reagents were specific for both a leukocyte-specific membrane protein (CD45) and a target cytokine. After 5 minutes of incubation under conditions that prevented cytokine secretion (i.e., at 4°C), all leukocytes were uniformly labeled with the capture reagent, and excess leukocytes were removed by extensive washing. Under conditions that prevented cytokine secretion, the pre-labeled cells were encapsulated as single cells into picoliter droplets ( ) along with a final concentration of 1% v / v of fluorescently labeled anti-cytokine antibodies. Figure 1A and Figure 1B Droplets containing single cells were incubated at 37°C in a 5% CO2-controlled incubator for 1 hour to allow cytokine secretion. The droplets were then reinjected, and each droplet was analyzed for cytokine secretion, mediated by the relocalization of the detection reagent's fluorescence signal to the cells. In droplets containing cytokine-secreting cells, the detection reagent signal relocalized to the cells, resulting in a localized increase in fluorescence within the droplet. In contrast, in droplets containing non-secreting cells, the detection reagent's fluorescence signal remained uniform throughout the droplet, and no localized increase in fluorescence was observed.

[0133] A droplet endocrine assay was used to detect secretion of IFNγ (and TNFα, not shown) by PMA / ionomycin-activated PBMCs compared to non-activated PBMCs ( Figure 1A and Figure 1B The results observed using the droplet-based microfluidics system and software were compared with flow cytometry data generated in microplates with the same cells and conditions ( Figure 2A and Figure 2B In the droplet secretion assay, 100% of secreting cells were detected as positive by flow cytometry. False-positive cells were counted in the negative control at less than 0.15%. Compared to flow cytometry, droplet detection of cytokine secretion by activated T cells is highly efficient and specific.

[0134] Quantifying the sensitivity and efficiency of cytokine secretion

[0135] Following the droplet secretion assay, non-activated and non-secreting CD8+ T cells were encapsulated in droplets with a concentration of purified IFNγ. Four emulsions were generated, each containing cells isolated as single cells and a different concentration of purified cytokine: the final concentration of IFNγ in the droplets was 0 nM, 1 nM, 5 nM, or 10 nM ( Figure 3A 、 Figure 3B 、 Figure 3C and Figure 3D Droplets from all four emulsions were refilled and the fluorescence signals analyzed. Using the droplet secretion assay, cytokine concentrations as low as 1 nM were detected with no false-positive events, demonstrating a highly sensitive and 100% specific assay. The secretion assay also demonstrated efficiency, with over 80% of positive cells detected in the droplets.

[0136] These examples show the possibility of calibrating assays to detect quantitative, real-time cytokine secretion in droplets by generating average values of standard curve sample conditions.

[0137] Antigen-specific T cell identification based on cytokine secretion by APC / T cells in co-encapsulated droplets

[0138] When co-cultured, antigen presenting cells (APCs) loaded with specific peptides can specifically activate a subset of responding T cells, leading to cytokine secretion. Droplet secretion assays are used to detect the specific activation of T cells by APCs in droplets ( Figure 4A 、 Figure 4B 、 Figure 4C and Figure 4D Under conditions that prevent cytokine secretion, APCs and T cells were co-flown into the droplets as single cells. The droplets were incubated overnight at 37°C in a 5% CO2-controlled incubator and re-injected the next day. After overnight culture in the droplets, T cell and APC activity was measured. Using this fluorescent marker for dead cells, 94% of the encapsulated cells were found to be viable. A droplet secretion assay for IFNγ secretion was used to examine T cell-specific activation by APCs. In droplets containing active T cells and APCs, 1.2% secreted IFNγ, demonstrating efficient, antigen-specific activation of T cells in the droplets.

[0139] Antigen-specific and total antibody secretion assays

[0140] By co-encapsulating B cells pre-labeled with capture reagents and detection reagents in droplets, the droplet endocrine assay can be used to assess the secretion of antibodies. The first part of the capture reagent is capable of recognizing a B cell surface marker, which can be a Pan-B marker or a specific B cell marker, a typical example being the CD138 marker for plasma cells that secrete immunoglobulins. The second part of the capture reagent is a specific capture antibody, or consists of the target antigen. In the first case, where the capture reagent consists of a portion of the capture antibody, the detection reagent consists of a detectable labeled antigen. In the second case, where the capture reagent consists of the target antigen, the detection reagent consists of a detectably labeled anti-antibody secondary antibody. Relocalization of the fluorescent signal on the B cell (here the label is fluorescent, but can be detected by any method by a person skilled in the art) indicates that the B cell present in the interrogated droplet secretes antigen-specific antibodies. The method described herein can be adapted with or without pre-incubation of the capture reagent ( Figure 5A -E). The methods described here can be applied to any of the target compounds mentioned above.

[0141] Secreted receptor-specific antibodies detect T cell activation

[0142] Binding of antibodies specific for a given T cell receptor can activate T cells, leading to, for example, the secretion of cytokines. Droplet secretion assays can detect the activation of T cells by T cell receptor-specific antibodies secreted by immunoglobulin-expressing cells in droplets ( Figure 6 ). A typical example includes PBMC, which is pre-labeled with a capture reagent that is encapsulated in a droplet together with cells expressing immunoglobulins. Droplets are produced in the presence of a labeled detection reagent (the label here is fluorescent, but can be produced in any way) and under conditions that prevent antibody production. After culturing the droplets under conditions that allow antibody production, T cell activation is detected by detecting, for example, cytokine secretion. The binding of T cell receptor-specific antibodies activates T cells in turn, and the T cells then secrete, for example, cytokines. The secreted cytokines are repositioned on the capture reagent bound to the T cells, and the fluorescent detection reagent is repositioned on the target cytokine. Due to the repositioning of the detection reagent on the activated T cells, droplets containing T cells activated by the secreted antibodies subsequently show a detectable signal. The method described here can be adjusted with or without pre-culturing the capture reagent. A typical example of the above method is the detection of anti-CD3 antibodies that trigger T cell activation. By extension, this system can be used to identify anti-checkpoint antibodies.

[0143] Antigen-specific antibody secretion-induced ADCC double-positive test and cytotoxic factor secretion test

[0144] In the case of secretion of antigen-specific antibodies with ADCC activity, titer endocrine assays can be used to assess induced mortality ( Figure 7 ). The double positive test introduced here can detect cytotoxic factors secreted by killer cells (e.g., primary natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells and cell culture cell lines), and target cell death induced by compounds secreted by killer cells. Under non-saturated conditions, killer cells are pre-labeled with capture reagents specific for target cytotoxic factors. The non-saturated conditions of the capture reagent are mandatory so that the secretory secretory compounds of interest can be captured and detected by killing cells, and the effect of the secreted compounds that have not yet been captured on the target cells ultimately leads to cell death. The target cells are then co-encapsulated in droplets with cells and killer cells that produce immunoglobulins. Before encapsulation, the encapsulated cells are co-flowed with a detection reagent specific for the target cytotoxic factors under conditions that prevent antibody production. After production, the droplets are cultured under conditions that allow the production of antibodies. The specific antibodies are repositioned on the target cells, and the killer cells bind to the antibodies through Fc receptors. Once bound to an antibody with ADCC activity, the killer cell releases cytotoxic factors, leading to target cell death. Some of the secreted cytotoxic factors are captured by capture reagents on the killer cells and relocate the detection reagent, enabling the detection of cytotoxic factor production. Cell death is monitored by releasing compounds from dying target cells expressing the target antigen. Alternatively, cell death can be monitored by cell surface markers or any other suitable marker known to those skilled in the art. By extension, the in-drop assay can be applied to complement-dependent cytotoxicity and opsonophagocytosis or any other assay described above.

[0145] Alternatively and / or in addition to this example, instead of detecting secreted cytotoxic factors, and / or in addition to detecting secreted cytotoxic factors, the production of antibodies may be detected, with or without detection of cell death. Figure 8 ).

Claims

1. A droplet composition comprising droplets, wherein the droplets include: a. at least one single cell, said at least one single cell exhibiting freedom of migration within said droplet, b. one or more first capture agents bound to the single cell, wherein the one or more first capture agents are capable of directly or indirectly binding to the single cell and directly or indirectly binding to a target compound, c. one or more second capture agents comprising a label, wherein the one or more second capture agents are capable of directly or indirectly binding to the target compound, wherein the label is directly or indirectly bound to the one or more second capture agents, wherein the droplet is capable of being cultured and producing a detectable event, the presence or relocation of the detectable event in the droplet determining the presence of the target compound; The target compound is a compound secreted by cells.

2. The composition of claim 1, wherein the label is selected from a fluorescent label, a polymer, a protein, a peptide, a hapten, a chemical, a nucleic acid or a barcode label.

3. The composition according to any one of claims 1 or 2, wherein the composition is used to detect a target compound produced by the at least one single cell. The composition according to claim 3 , wherein the target compound is present in the droplets at a concentration of 10 pM to 100 μM.

5. The composition of any one of claims 1 or 2, wherein one or more target compounds are detected.

6. The composition of any one of claims 1 or 2, wherein the kinetics of target compound production is monitored.

7. The composition according to any one of claims 1 or 2, wherein the production of the target compound by one or more cells is triggered by the interaction of at least two cells.

8. Use of the composition according to any one of claims 1 to 7 for monitoring biological events.

9. A microfluidic system for generating droplets, wherein at least one droplet comprises: a. at least one single cell, said at least one single cell exhibiting freedom of migration within said at least one droplet, b. one or more first capture agents bound to the single cell, wherein the one or more first capture agents are capable of directly or indirectly binding to the single cell and directly or indirectly binding to a target compound, c. one or more second capture agents comprising a label, wherein the one or more second capture agents are capable of directly or indirectly binding to the target compound, wherein the label is directly or indirectly bound to the one or more second capture agents, The target compound is a compound secreted by cells.

10. A microfluidic system capable of detecting a target compound in a droplet, wherein the droplet comprises: a. at least one single cell, said at least one single cell exhibiting freedom of migration within said droplet, b. one or more first capture agents bound to the single cell, wherein the one or more first capture agents are capable of directly or indirectly binding to the single cell and directly or indirectly binding to a target compound, c. one or more second capture agents comprising a label, wherein the one or more second capture agents are capable of directly or indirectly binding to the target compound, wherein the label is directly or indirectly bound to the one or more second capture agents, wherein the microfluidic system cultures the plurality of droplets comprising the droplets under conditions that allow production of a target compound, such that if the target compound is produced by the at least one single cell, it will be captured by the plurality of first capture agents and the second capture agents, The microfluidic system determines the presence of the target compound by detecting the presence or relocation of the label.

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