Microfluidic method for sorting biomolecules

Abstract

A continuous analysis method comprising generating microfluidic droplets comprising multiple copies of a same antigen, multiple copies of a same primary antibody potentially capable of specifically binding to said antigen, a multivalent crosslinking agent being a biomolecule or a support, and a fluorescent labeling agent, said continuous microfluidic analysis being a fluorescence-based sorting.

Description

[0001] MICROFLUIDIC BIOMOLECULE SORTING PROCESS

[0002] The invention relates to a microfluidic method for sorting and screening (screening, phenotyping) biomolecules, cells or compartments thereof.

[0003] Microfluidic chips allow the manipulation and analysis of minute volumes of fluids. Multiple functions can be implemented on a single chip.

[0004] These miniature devices enable precise control of fluids, thus offering numerous applications in the fields of life sciences, biotechnology, chemistry, medical diagnostics, drug discovery, genetic analysis, environmental monitoring and field testing.

[0005] Microfluidic experimentation involves generating fluid flow in micrometer-sized channels located within a microfluidic chip. In most situations, fluid flow in these channels results in frictional forces due to viscosity far outweighing inertial forces. This leads to laminar flow in which the fluid molecules progress while maintaining their relative positions.

[0006] When two immiscible fluid streams (e.g., water and oil) meet at a channel junction of a specific geometry within the chip, an emulsion of droplets dispersed in a continuous fluid forms due to shear forces, surface tension, the junction geometry, and flow dynamics. The emulsion droplets can be stabilized by surfactants dispersed in the aqueous and / or oily phase (also called the continuous phase). The surfactants form an interface around and / or within the droplets to prevent coalescence. These stabilizing agents reduce the interfacial tension between the immiscible phases and promote interface formation.

[0007] The ability to handle very small sample volumes is a major advantage of microfluidic analysis. This allows for analyses to be performed on small amounts of material, reduces reagent consumption, yields more rapidly detectable analyte concentrations, and provides faster results.

[0008] More generally, a microfluidic experimentation device includes, but is not limited to, a microscopy module (preferably inverted), on either side of a stage, a fluorescence detection module with light excitation comprising one or more excitation sources (e.g. lasers or photodiodes) and photosensitive sensors (e.g. photomultiplier tubes), a pneumatic module typically comprising pumps and / or pressure regulators and / or solenoid valves, a fluidic module comprising connecting tubes, tanks, an electronic module equipped with a power supply for the components, means for acquiring and processing signals, mechanical parts and supports.

[0009] US patent application 2019 / 101537 describes a microfluidic droplet system for determining the activity of immune system cells. The system co-encapsulates an immune system cell and a target cell from two different streams, along with particles decorated first with streptavidin and then with a biotinylated antibody specific for recognizing a cytokine potentially synthesized by the immune system cell. It also includes a dual detection system: one for target cell viability and the other for the presence of the cytokine. Downstream sorting is not described in detail. A sorting system combining signal intensity measurement and the application of an electric field is suggested to separate droplets with signal intensity exceeding a fixed threshold from those below it.However, the measurement of induced cell death is carried out by confocal microscopy, which does not allow for screening.

[0010] Among the possibilities offered by microfluidic droplet technology, Patent EP 3207373 describes an analysis process in which an elongated aggregate along a principal axis is formed by applying a magnetic field, which allows for good detection and even sorting of droplets, depending on whether a fluorescent signal is measured at the level of the elongated aggregate along a direction, or not.

[0011] However, this type of measurement limits the choice of the nature and size of the particles, while the inventors have noticed that the gathering of the signal due to the application of the magnetic field risks creating artifacts, "false positives".

[0012] Furthermore, the inventors noted that, paradoxically, this type of measurement on an elongated cluster is complex to implement, even though it seemed simpler. The elongated cluster must be aligned with the excitation and detection beam, it must remain intact, and it can break and become misaligned due to hydrodynamic forces within the droplets.

[0013] US 2022 / 074929 describes a sorting method for extracellular vesicles, comprising capture beads, for example 5 µm in diameter, coupled to an antibody recognizing a marker of these vesicles and a labeled antibody, for example comprising a segment with enzymatic activity. Selection can be performed either in a multi-well method or via microdroplets, which implies the presence of only one extracellular vesicle per microdroplet.

[0014] Brief description of the invention

[0015] A first aspect of the present invention relates to a method for analyzing an interaction between biomolecules comprising generating aqueous microfluidic drops 1 in an oily phase said drops 1 comprising several copies of the same capture agent 3 specifically recognizing an analyte (secreted) 4, 13; several copies of the same analyte (secreted) 4, 13 potentially capable of specifically binding to said capture agent 3; a multivalent crosslinking agent 5, 5', 6, 6', 12 and a labeling agent 5', 7, 7' (fluorescent, colored or radiolabeled), said capture agent 3 being fixed on a support 2, said support 2 having an equivalent diameter between 10 nm and 1 m and preferably being made of metal (e.g. iron), plastic, a polymer (including a hydrogel), or a resin, said multivalent crosslinking agent 5, 5', 6, 6', 12 being a biomolecule 5 having a high affinity for said capture agent 3, or said support 2, preferably made of metal,plastic, a polymer, or a resin linking several capture agents 3 (possibly via several biomolecules 5 grafted onto said support 2) or a labeled biomolecule 5' having a high affinity for a detection agent 6, 6' said detection agent 6, 6' linking an epitope of the secreted analyte 4 not involved in the binding with its capture agent 3 or a biomolecule 6, 6', 12 having a high affinity for the analyte 4, 13 or for a detection antigen 14, 14', and said labeling agent 7, 7' being linked to the crosslinking agent 5', 6, 6', 12 or to a detection agent 6, 6' specifically recognizing an epitope of the secreted analyte 4 not involved in the binding with its capture agent 3, said step of generating microfluidic droplets 1 comprising the combination of a minimum of two aqueous flow, a first aqueous flow comprising copies of the same analyte 4 or cells 8, 8' capable of secreting said analyte 4 and / or secreting subcellular structures 13,and a second stream comprising copies of the same capture agent 3 bound to the support 2 and a detection agent 6 (or 6'), said microfluidic analysis being a screening and / or sorting (at high throughput) based on the aggregation of said labeling, said drops 1 having a volume between 1 pL and 10 nL, preferably between 10 pL and 500 pL, preferably between 50 pL and 100 pL.,

[0016] Preferably, this aggregation of the marking is three-dimensional.

[0017] Preferably, a plurality of molecules of the same capture agent 3 are coupled on support 2.

[0018] Preferably, the support 2 is a plurality of spherical or substantially spherical particles. Advantageously, the support 2 directly comprises one or more copies of the fixing agent 3, or one or more chemical functionalities enabling stable association (for several hours or more) of one or more copies of the fixing agent.

[0019] Advantageously, the capture agent 3 is biotinylated and the support 2 comprises one or more avidin 5 groups, preferably the capture agent 3 comprises one or more biotin residues and / or the support 2 comprises several avidin 5 groups, for example the capture agent 3 comprises one (and only one) biotin residue and the support 2 comprises a plurality of avidin 5 groups.

[0020] Thus, advantageously, the support 2 comprises one or more avidin groups 5 and the capture agent 3 is a mono- or multi-biotinylated binding antibody 3 (IgG or VhH), said binding antibody 3 specifically recognizing a constant region of a secreted analyte 4 being an antibody to be tested for its ability to bind an antigen 14, 14' (detection antigen), said antigen 14 being labeled or said antigen 14' not being labeled and a labeled detection antibody 6' 7' recognizing said unlabeled antigen 14' being applied or said antigen 14 being fluorescently labeled and further comprising a fluorescent detection antibody 6 7, the fluorescence of antigen 14 being different from the fluorescence 7 of the detection antibody 6 and the detection antibody specifically recognizing a constant region of the secreted analyte (antibody) 4,or said antigen 14' not being labeled and a detection antibody 6' labeled 7' recognizing said unlabeled antigen 14' being applied and in addition, a detection antibody 6 fluorescent 7 recognizing the antibody 4 to be tested (secreted analyte), the fluorescence 7 of the second labeling being different from the fluorescence 7' of the detection antibody 6'.,

[0021] According to an advantageous variant, the capture agent 3 is an antigen fixed to the support 2, for example, a biotinylated antigen fixed to beads decorated with avidin. This process is particularly advantageous when the analyte 4 is an antibody secreted into the microfluidic droplet 1 by an antibody-producing cell 8, said process preferably comprising a final step of sorting and / or selecting (at high throughput) the microfluidic droplets 1 in which the secreted antibody 4 has been bound to the antigen 3, 14, 14', preferably followed by a step of isolating the antibody-producing cell 8 and / or characterizing the sequence of antibody 4.

[0022] In the context of the present invention, when cellular material is encapsulated in a microfluidic droplet, only one antibody-producing cell (or cell of any other biomolecule) 8 or one effector cell 8 is present in a given microfluidic droplet, but there is no encapsulation of multiple antibody-producing cells (or cells of any other biomolecule) or effector cells 8 in a single microfluidic droplet. Conversely, when a target cell 9 is encapsulated, multiple cells can be encapsulated in a single microfluidic droplet.

[0023] Preferably, the (bivalent or) multivalent crosslinking agent 5, 5', 6, 12 is: a secondary antibody 12, preferably tetrameric, recognizing the secreted antibody 4, said secondary antibody 12 preferably being present in the second stream, a multivalent aptamer recognizing the secreted antibody 4, said aptamer preferably being present in the second stream, a biotinylated secondary antibody 6 recognizing the secreted antibody 4, or an epitope present on the portion of the secreted analyte 4 not involved in the recognition of the capture agent 3, and, preferably, an avidin 5', said biotinylated secondary antibody 6 preferably being present in the second stream and said avidin 5' preferably being present in the first stream,

[0024] - a secondary antibody recognizing the capture agent 3, the capture agent 3 preferably being a binding antibody (or an antibody fragment or a VHH). Advantageously, the labeling agent 7,7' is covalently bound to the multivalent crosslinking agent 5',6,6',12, preferably a 6,6' secondary antibody derivatized with a fluorescent molecule, or an avidin derivatized with a 5' fluorescent molecule.

[0025] Advantageously, the capture agent 3 is linked to a biotin and the crosslinking agent 5 is an avidin, said avidin being linked to support 2.

[0026] Preferably, according to an alternative, the capture agent 3 is an antibody recognizing an analyte 4, 13 and the first stream includes a cell 8 potentially secreting this analyte 4, 13 recognized by said capture antibody 3; this allows selection of microfluidic drops 1 including a cell 8 y secreting the analyte recognized by the capture antibody 3.

[0027] Preferably, a detection antibody 6, preferably fluorescent 7, is then added, so as to highlight and / or potentiate the possible crosslinking of the analyte 4, 13.

[0028] For example, advantageously, the fluorescent detection antibody 6, 7 recognizes an epitope of the analyte 4, 13, different from the epitope recognized by the capture antibody 3.

[0029] Another aspect of the present invention relates to a method for high-throughput analysis and / or screening of a metabolic pathway in which a substrate 9 is modified by an effector 8', comprising generating microfluidic droplets 1 comprising said substrate 9, said effector 8' and a reporter probe 10 capable of emitting fluorescence or absorbing light at a determined wavelength and interacting differently with said substrate 9 compared to said substrate modified by said effector 8', said step of generating microfluidic droplets 1 comprising the combination of at least two streams, the first stream comprising said substrate 9 and the second stream comprising said effector 8', said effector interacting with said substrate 9 so as to modify the fluorescence or absorbance of the reporter probe 10, and, preferably, a third stream comprising the reporter probe 10, a support 2 binding a capture agent 3,a crosslinking agent 5, and / or a labeling agent 6,7. Advantageously, in this process, the effector and secretory cell 8' is a cytotoxic immune cell and the substrate 9 is a cancer cell.

[0030] Preferably, in this process, probe 10 is a marker of cell death, preferably of apoptosis, or necrosis.

[0031] Thus, the present invention also relates to a method for analyzing an interaction between an effector cell 8' and a target cell 9, comprising generating microfluidic droplets comprising said effector cell 8' and said target cell 9, and further comprising: a capture agent 3 specifically recognizing an analyte 4 being a biomolecule potentially secreted due to contact between said target cell 9 and said effector cell 8', a multivalent crosslinking agent 5 and a fluorescent detection agent 6, 7, said multivalent crosslinking agent 5 being a biomolecule having a high affinity for said capture agent 3 and said fluorescent detection agent 6, 7 being a detection antibody 6 binding specifically to the biomolecule without interfering with said capture agent 3, said step of generating microfluidic droplets 1 comprising the combination of two flows,a first flow comprising the target cell and a second flow comprising the effector cell and, preferably, a third flow comprising the capture agent 3, the detection agent 6, the marking agent 7 and the crosslinking agent 5.

[0032] In this process, advantageously, the effector cell 8' is an antigen-presenting cell or a cytotoxic cell.

[0033] Preferably, the crosslinking agent 5 is a plurality of beads 2 to which a plurality of avidin are coupled, in association with a biotinylated capture antibody 3. Another aspect of the present invention relates to a method for sorting and / or quantifying subcellular structures (e.g., extracellular vesicles) 13, comprising generating microfluidic droplets 1 comprising said subcellular structure 13, a multivalent crosslinking agent 5, and a labeling agent 6, 7, said crosslinking agent 5 having a specific affinity for a capture agent 3 and said labeling agent 6, 7 having a specific affinity for another component, not recognized by said capture agent 3, of said subcellular structure 13.

[0034] Other embodiments of the microfluidic experimentation device with microscope are indicated in the attached claims.

[0035] Detailed description of the invention

[0036] The inventors have succeeded in developing a rapid analyte detection and sorting system that is based on the cross-linking of biomolecules when they are capable of interaction.

[0037] In the context of the present invention, "sorting" or "screening" preferably means that a stream of microfluidic droplets passes in front of a detector that orients the microfluidic droplets according to whether the signal (three-dimensional, see below) meets a predetermined selection criterion, advantageously exceeds a predetermined threshold, or does not. This allows the analysis of thousands, or even hundreds of thousands, of microfluidic droplets, which is not realistic in microscopy and / or via multiwells.

[0038] A first aspect of the present invention relates to a method for analyzing an interaction of biomolecules comprising generating aqueous microfluidic droplets 1 in an oily phase said droplets 1 comprising several copies of the same capture agent 3 several copies of the same analyte (secreted) 4, 13 potentially capable of binding specifically to said capture agent 3 a multivalent crosslinking agent 5 and a fluorescent, colored or radiolabeled labeling agent 7, 7' said capture agent 3 being fixed on a support 2 said support 2 having an equivalent diameter between 10 nm and 5 µm (preferably between 100 nm and 1 µm, preferably between 200 and 500 nm) and preferably being made of metal (e.g.iron), plastic, a polymer, or a resin, said multivalent crosslinking agent 5 being a) a biomolecule having a high affinity for said (or several of said) secreted analyte 4 or b) a biomolecule having a high affinity for several capture agents 3 (e.g., binding antigen or antibody), preferably for said several capture agents (e.g., binding antigens or antibodies) being modified by the addition of a recognition group to the biomolecule, preferably the recognition group being a biotin and the biomolecule being an avidin; c) a biomolecule having a high affinity for a detection agent 6 (secondary antibody), said detection agent (e.g., secondary antibody 6) specifically binding a constant portion of said secreted analyte 4 (e.g., antibody) or an epitope of the secreted analyte 4 (e.g.,cytokine) not involved in binding with its capture agent 3 and said labeling agent 7, 7' being bound to the detection agent 6, 6' (e.g., detection antibody), the detection antigen 14 or 14' (via 6 or 6'), or to the crosslinking agent 5' or to a crosslinking detection agent 12, said step of generating microfluidic drops 1 comprising the combination of a minimum of two (input) streams, a first (aqueous) stream comprising copies of the same analyte 4 or (secretory) cells 8 capable of secreting said analyte 4 or a subcellular structure 13 and a second (aqueous) stream comprising copies of the same capture agent 3, and advantageously of the detection agent, said microfluidic analysis being a screening and / or sorting based on the aggregation (crosslinking) of said labeling, said drops 1 having a volume between 1 pL and 10 nL, preferably between 10 pL and 500 pL, preferably between 50 pL and 100 pL.In the context of the present invention, "equivalent diameter" is preferably understood to mean the value d = 2* square root (Surface / ît).

[0039] Advantageously the drops are monodisperse, which means, preferably, in the context of the present invention, that 80% of the total droplets are within the range from half the average size to twice the average size, preferably within the range of average size + / - 20%, preferably average size + / - 10%, preferably average size + / - 5% (e.g. for an average size of 100 pL, 80% of the drops will have an average size between 50 and 200 pL).

[0040] The at least two mixed aqueous streams are then transformed into microdroplets in an oily phase, following the addition of an oily stream to the aqueous stream.

[0041] Preferably the crosslinking agent 5 is (are) present in the first stream (or in a third stream, but not in the second stream).

[0042] In the context of the present invention, "avidin" preferably means any multimeric (tetrameric) biomolecule capable of specifically binding biotin with high affinity. Typically, this refers to avidin, streptavidin, or neutravidin.

[0043] In the context of the present invention, "crosslinking" preferably means a three-dimensional (i.e., not rod-like, such as rods produced by a magnetic force) aggregation of the labeling (e.g., fluorescence and / or the labeling agent) caused by at least one bivalent biomolecule or by the interaction of several bivalent biomolecules. In the context of the present invention, the words "crosslinking" and "aggregation" are synonymous and interchangeable; in particular, in the context of the present invention, aggregation advantageously also means a three-dimensional aggregation. In the context of the present invention, crosslinking advantageously occurs only when there is interaction between the binding agent and the analyte, and preferably, this crosslinking remains stable until screening.

[0044] In the context of the present invention, "three-dimensional assembly" is preferably understood to mean a structure whose largest dimension is not 10 times greater than the smallest dimension, preferably not 5 times greater, preferably not 3 times greater (therefore different from a rod-like structure, for example obtained in the prior art following the assembly of balls due to the application of a magnetic field).

[0045] The inventors noted that this simplifies detection since there is no longer the constraint of focusing the signal in a precise plane, nor the need to align an elongated structure along an excitation / detection axis.

[0046] According to the most direct variant of the present process, a plurality of molecules of the same capture agent (e.g., antigen) 3 are coupled to the support 2. In addition, a plurality of the support 2 is present in each microfluidic droplet. Thus, advantageously, binding of the analyte 4 to the support 2, followed by recognition of the analyte by a detection antibody 6, and then the application of a second antibody or, if the detection antibody 6 is biotinylated, of an avidin 5', 7, will further enhance the crosslinking, which is advantageous.

[0047] This allows testing a large number of antibody-producing cells (hundreds of thousands to tens of millions of cells, or even more than hundreds of millions) for their ability to recognize / bind to the antigen (see above for the definition of screening).

[0048] In this process, preferably the capture agent (e.g., antigen 3) is biotinylated (one and only one biotin group, or several biotin groups) and the support 2 comprises one, or advantageously several, avidin group(s); preferably the capture agent (antigen) 3 comprises several biotin residues, advantageously provided that this does not interfere with the binding capacity of the secreted analyte (e.g., antibody).

[0049] An advantageous alternative is the use of support(s) 2 which are not covered with avidin groups, but which are capable of binding the capture agent 3, preferably a plurality of identical capture agents 3.

[0050] Advantageously, a preliminary test is performed to test the ability of a secreted analyte (e.g., antibody 4), known to bind the biotinylated capture agent (antigen) 3 fixed on supports (e.g., beads) 2 bearing avidin groups 5. Alternatively, or in addition, one (a single) or more biotin groups are grafted to areas of the capture agent (antigen) 3 that are not well suited to recognition by a secreted analyte (antibody), for example, at one or both ends of antigen 3 or via a "spacer".

[0051] This allows several capture agents (e.g., antigens) 3 to be bound to a support (e.g., a bead) 2, and even for one capture agent (antigen) 3 to bind to several supports (beads) 2. In other words, the capture agents (antigens) 3 are advantageously (partially) cross-linked and / or clustered, while retaining their ability to be recognized by the analyte (e.g., the antibody) secreted 4. When the analyte 4 is an antibody, its binding will amplify this initial cross-linking until, advantageously, a clustered, three-dimensional signal is produced. The same applies if the detection agent 6, 6' is multivalent, for example, a (secondary) antibody.

[0052] Other features, details and advantages of the invention will become apparent from the description given below, by way of non-limitation and with reference to the attached drawings.

[0053] Figure 1 is an example of a microfluidic process according to the invention (which shows only one support 2 per ball for the sake of simplification).

[0054] Figure 2 (a) shows crosslinking via avidin bound to the supports (beads) and (b) aggregation via free avidin binding to the biotinylated detection agent (detection antibody 6); here the capture agent 3 and the analyte are a ligand-detector system.

[0055] Figure 3 shows an aggregation obtained via the microfluidic process.

[0056] Figure 4 shows a signal with (b) or without aggregation (a).

[0057] Figure 5 shows aggregation via a multi-biotinylated antigen / capture agent.

[0058] Figure 6 shows aggregation via a multi-biotinylated antibody fragment.

[0059] Figure 7 shows aggregation via a multi-biotinylated capture antibody. Figure 8 is a schematic representation of a cytotoxicity assay; condition a), no cytotoxicity; condition b), cytotoxicity.

[0060] Figure 9 shows a three-input flow system with an effector cell and a target cell. Figure 10 depicts an aggregation of a factor potentially secreted by an effector cell coupled to a cytotoxicity assay.

[0061] Figure 11 schematically illustrates aggregation via biotinylated detection antibodies and avidin.

[0062] Figures 12 and 13 schematically illustrate aggregation via a tetrameric detection antibody.

[0063] Figure 14 shows aggregation of extracellular vesicles via a multi-biotinylated capture antibody.

[0064] In the figures, identical or analogous elements bear the same references and are included in the table below.

[0065] According to a more specific variant of this process (shown schematically for example in Figure 6), the support 2 comprises one or more avidin groups and the process includes the administration of a (multi-)biotinylated capture antibody 3, said capture (crosslinking) antibody 3 specifically recognizing a region of an analyte 4 to be tested / detected.

[0066] Advantageously this analyte is a secreted antibody which will be bound via a constant region to the capture antibody 3 and which will be tested for its ability to bind an antigen 14, 14', said antigen 14 being labeled, or not 14'.

[0067] Advantageously, the antigen 14 is fluorescently labeled and the system further comprises a fluorescent detection agent 6 7 (specifically recognizing a (other) specific part of the secreted agent 4), the fluorescence of the antigen 14 being different from the fluorescence 7 of the detection agent 6.

[0068] Conversely, the 14' antigen is not labeled and a 6', 7' detection agent recognizing another epitope of this 14' antigen is present.

[0069] Advantageously, as shown in Figure 6b, a dual detection system is present, one (6, 7) to highlight the secretion of antibody 4 and the second (6', 7') to highlight the specificity of the secreted antibody 4 towards antigen 14'; the two labeling systems being different, for example two types of fluorescence.

[0070] The first option (Figure 6a) offers another way of modifying antigen 14, which allows more flexibility, for example in case of suspected adverse interaction between capture agent 3 (biotinylated antigen 3 described above; see Figures 2a, 3, 5) and secreted antibody 4 (secreted analyte).

[0071] The second option (Figure 6b) of this variant also allows for the rapid sorting of analytes (e.g., antibodies, cytokines) or cells secreting analyte 4. Preferably, the detection agent (6,7) specifically recognizes the secreted analyte 4, and thus masks a known epitope of this analyte. This is advantageous, for example, when sorting primary antibodies for their ability to recognize a specific epitope of an antigen (therefore different from and / or distant from that recognized by the detection agent 6,7).

[0072] The third option (with a second detection agent - secondary antibody) allows for a more specific signal.

[0073] Preferably, said capture agent 3 is a "single domain antibody"

[0074] (a VHH). Advantageously, in the above processes, the antibody (the analyte) 4 is secreted into the microfluidic droplet 1 by an antibody-producing cell 8, and this / these process(es) includes a final (or additional) step of sorting the microfluidic droplets 1 into which the secreted antibody 4 has been bound to the antigen

[0075] 3, 14, 14', preferably followed by a step of isolation of cell 8 producing antibody 4 and / or characterization of the sequence (nucleotide and / or peptide) of antibody 4.

[0076] The invention further enables a system in which the analyte (secreted) 4 to be tested is a biomolecule that will be bound by a capture antibody 3 and revealed by a labeling agent 6, 7 (recognizing another epitope of the secreted analyte 4; or, by analogy, the antibody 6, 7 of Figure 7). This makes it possible, for example, to measure the capacity of a secretory cell 8 to produce a known biomolecule.

[0077] Preferably, in the above processes, the multivalent crosslinking agent 5, 5' is: a dimeric detection antibody 6, preferably tetrameric (see Figure 12, 13) 12, recognizing the secreted analyte (e.g., the antibody

[0078] 4, Figure 12 or another analyte 4, Figure 13), said dimeric detection antibody 6, or tetrameric antibody 12 being preferably present in the second input stream of reagents upon encapsulation of the reagents in the microfluidic drops, a multivalent aptamer recognizing the analyte (the secreted antibody 4), said aptamer being preferably present in the second stream, a biotinylated detection antibody 6 (see for example Figure 11) recognizing (i) the analyte (the antibody) secreted 4, and (ii) an avidin, said detection antibody 6 being preferably present in the second stream and said avidin being preferably present in the first stream.

[0079] - an avidin molecule recognizing the binding agent 3, preferably a biotinylated antibody or antigen. In this case, preferably, the binding agent is attached to the support 2 via a mechanism independent of the one that will lead to crosslinking. Thus, for example, a biotinylated binding antibody 3 will not be both attached to a support decorated with avidin and then crosslinked by the addition of exogenous avidin. On the contrary, a biotinylated antibody will preferably be grafted to the support 2 via other types of interactions. Preferably, in the above processes, the labeling agent 7 is covalently attached to the multivalent crosslinking agent 5' or to the detection agent (e.g., a secondary antibody or an antigen). Advantageously, this labeling agent 7 is linked to this crosslinking agent 5', which is an avidin (see, for example, Figure 11).

[0080] According to a variant of this method, advantageous when testing the presence (secretion) of a biomolecule of interest (see for example Figure 7), recognized by a capture antibody 3 of known structure, the biotinylated capture antibody 3 and the crosslinking agent 5 is an avidin, said avidin being preferably present in the second stream, advantageously carried by the support 2. This variant is not advantageous when testing the ability of an antibody of unknown structure to bind a biomolecule.

[0081] Preferably, in this advantageous variant of this process, the antibody 3 is for the capture of a biomolecule 4 and the first stream comprises a cell 8 secreting a biomolecule 4 potentially recognized by said antibody 3, said process further comprising the step of selecting microfluidic microdroplets 1 comprising a cell 8 y secreting the biomolecule recognized by the capture antibody 3.

[0082] This allows for easy sorting of cells producing a biomolecule of interest under specific conditions, including immune system cells such as T lymphocytes, modified T lymphocytes (CAR-T), and / or cytotoxic cells.

[0083] Advantageously, this method can further include the application of a fluorescent detection antibody 6,7 (see Figure 6). Preferably, this detection antibody 6 binds to the biomolecule of interest 4 and to an epitope other than the capture antibody 3. The fluorescence clustering is therefore measured when the biomolecule of interest 4 binds to the capture antibody 3, which is advantageously itself cross-linked (e.g., several capture antibodies 3 on a support 2, and / or several supports 2 binding an antibody 3).

[0084] Another aspect of the present invention relates to a method for (continuous) analyzing a metabolic pathway in which a substrate is modified by an effector, comprising generating microfluidic droplets comprising said substrate, said effector and a reporter probe (specifically and exclusively binding (i) either to the substrate, (ii) or to the substrate modified by the effector) capable of emitting fluorescence or absorbing light at a determined wavelength and interacting differently with said substrate compared to said substrate modified by said effector, said step of generating microfluidic droplets comprising the combination of at least two streams, the first stream comprising said substrate and the second stream comprising said effector, said effector interacting with said substrate so that the fluorescence or absorbance of the reporter probe is modified;preferably a third stream is applied, comprising the detection agent 6, 6' and / or the labeling agent 7, 7' and the support 2, or even the detection antigen 14, 14'; capture agent 3 either the substrate and / or the modified substrate, or the effector; advantageously the support 2 comprises one or more avidin(s) and the substrate (modified), or a capture agent 3, comprises a biotin.;

[0085] Advantageously, according to an alternative of this process, the effector is a cytotoxic immune cell 8' and the substrate is a target cell, for example a cancer cell 9.

[0086] Preferably, in this method, the probe is a 10-labeled (fluorescent) marker of cell death, preferably of apoptosis or necrosis.

[0087] Another aspect of the present invention (schematized for example in Figure 10), which can be understood as an advantageous variant of the above, relates to a method for analyzing an interaction between an effector cell 8' and a target cell, for example a cancer cell 9, comprising generating microfluidic droplets comprising said effector cell 8' and said target cell 9, and further comprising: a capture agent 3 specifically recognizing a biomolecule 4 potentially secreted due to the contact of said target cell 9 with said effector cell 8', a multivalent crosslinking agent 5 and a detection agent 6, advantageously fluorescent 7, said multivalent crosslinking agent 5 being a biomolecule having a high affinity for said capture antibody 3 (for example biotinylated;the crosslinking agent being then an avidin) and said detection agent 6 binding a fluorescent labeling agent (7) being a detection antibody binding specifically to the biomolecule 4 without interference with said capture agent (capture antibody) 3, said step of generating microfluidic drops 1 comprising the combination of two streams, a first stream comprising the target cell 9 and a second stream comprising the effector cell 8' and, preferably (Figure 9), a third stream comprising the capture antibody 3, the labeling agent 7, the detection agent 6, the crosslinking agent 5 and the supports 2.;

[0088] Advantageously, in this process, the effector cell 8' is an antigen-presenting cell or a cytotoxic cell (T, NK, CAR-T, ...).

[0089] Preferably the biomolecule is produced by the effector cell 8' following its interaction with the target cell 9 (see for example the diagram in Figure 10).

[0090] Preferably, in this process, the crosslinking agent 5 is a plurality of avidin, carried by a plurality of beads 2, in association with a (multi-)biotinylated capture antibody 3.

[0091] This process preferably includes the addition of a probe 10, capable of labeling the potentially affected target cell 9 (e.g., cell death) by the effector and secretory cell 8. This allows for the rapid provision of information on the activity and underlying mechanism.

[0092] Another aspect of the present invention relates to a method for sorting and / or quantifying subcellular or extracellular structures (or extracellular vesicles) 13, comprising generating microfluidic droplets 1 comprising said subcellular structure 13, a multivalent crosslinking agent 5 and a detection agent (fluorescent) 6,7 said detection agent 6,7 having a specific affinity for a component of said subcellular structure 13 (Figure 14).

[0093] This process advantageously allows the concentration of any subcellular structures 13 present, provided they expose the desired component (recognized by the detection agent 6 and the capture agent 3). Preferably, the subcellular structure 13 is an extracellular vesicle, such as an exosome.

[0094] This allows us to verify the possible production of these extracellular vesicles (concentration of secreted vesicles, phenotypes, specificity, for example for the validation of a batch (clinical, or preclinical research): presence, quantification, specificity of vesicles.

[0095] Preferably the component of the subcellular structure 13 (extracellular vesicle) recognized by the capture agent 3 and / or by the detection agent 6 is more abundant (more concentrated, e.g. per unit area) on the subcellular structure 13 than on the cell that produced it 8.

[0096] Alternatively, the producing cell 8 and the subcellular structure 13 are separated, either physically before labeling, or (in silico) during labeling analysis, for example due to size differences.

[0097] Examples

[0098] Example 1

[0099] The inventors sought to develop a microfluidic system for measuring the interaction between two biomolecules without having to resort to agglutination by using magnetic forces.

[0100] Figure 1 illustrates the general principle, partly known, of generating aqueous microdroplets in an oily phase, in which an aqueous stream, here, an aqueous stream being the combination of two aqueous streams, one comprising cells 8, here cells producing an antibody to be tested, and the other stream comprising a support 2, crosslinking agents 5, an antigen 3, here fixed on the support 2 and a fluorescent detection antibody 6, 7. This stream is then combined with an oil stream, here shown by the two vertical arrows, so as to provide the aqueous droplets 1 in an oily phase.

[0101] Some of the ways of creating crosslinking are illustrated in Figure 2, via a 7-labeled 6-sensing agent which is here a bivalent antibody, or via a 7-labeled 5' avidin.

[0102] The first "proof of concept" test is shown in Figures 3 and 4. A stream containing a multi-biotinylated capture agent (an antigen) 3, fixed to a support (2) (beads with a diameter of 300 nm coupled to their surface with streptavidin molecules 5), was brought into contact with a second stream containing a fluorescent detection agent 6, 7 recognizing the antigen (capture agent) 3 (Figure 3 a, b). Agglomeration is directly visible under a microscope (Figure 3, right panel). The agglomeration is also easily shown or tracked by continuous flow analysis (Figure 4), which allows for simple sorting based on whether there has been an interaction (condition b) or not (condition a) between the detection agent 6, 7 and the capture agent 3, here an antigen. The capture of the fluorescent detection agent 6, 7 by the capture agent 3, bound to the support 2 via a streptavidin 5, results in a relocalization and concentration of fluorescence 7 on the aggregate (Figure 3b).This signal concentration is easily detectable by conventional microscopy (Figure 3, right panel) or by continuous flow microscopy in a microfluidic chip (Figure 4). The droplets of interest (Figure 4b) exhibit a distinct signal peak (illustrated in green in Figure 4c1) due to the concentration of the fluorescent signal on the aggregate. The signals at the bottom, illustrated in Figure 4 (right panel), correspond to dyes (barcodes) used to distinguish the droplets of interest (b, low-intensity fluorescent signal at the bottom) from the droplets not containing the aggregate (Figure 4a, bottom, more intense signal).

[0103] Example 2

[0104] With reference to Figure 2a and Figure 1, an aqueous stream comprising a multi-biotinylated antigen 3 bound to support 2 and a fluorescent detection antibody 6, 7 and a second aqueous stream comprising a cell 8 producing an antibody 4 to be tested, capable of recognizing the antigen 3.

[0105] In this microfluidic process, the supports (beads) 2 bind a plurality of antigens 3, and the antigens 3 potentially bind to several beads 2. Antibodies secreted 4 by cells 8 bind to the antigen 3 if their affinity allows, and labeled detection antibodies 6,7 bind to the secreted antibodies 4, which aggregates the fluorescence. The droplets 1 for which aggregation is measured are sorted, allowing the sorting / selection of promising cells 8. This sorting can be done at high throughput, advantageously for identifying rare / very infrequent secreting cells. Example 3

[0106] This is a variant, shown in Figure 6, also intended to rapidly identify cells 8 capable of secreting a biomolecule of interest, here also an antibody 4.

[0107] An aqueous stream comprising VHH-type capture antibodies 3 recognizing a constant region of immunoglobulin G of antibody 4, the analyte to be tested secreted by cells 8, the VHH-type capture antibodies 3 being biotinylated and attached to beads 2, here coupled to streptavidin residues 5, is combined with an aqueous stream comprising secretory cells 8, here secreting an antibody 4 to be tested. The first stream further comprises a fluorescent detection antibody 6,7 and, here, a fluorescent antigen 14. The fluorescence of antigen 14 is agglomerated if the antibody 4 secreted by cells 8 binds on the one hand the antigen 14 and on the other hand the capture agent (here VhH), itself being bound to the crosslinking agents, whereas the fluorescence 7 of the detection antibody is agglomerated if cell 8 produces an IgG (4).

[0108] Example 4

[0109] In this variant illustrated in Figure 7, an aqueous stream comprising a cell 8 to be tested capable of secreting the analyte 4 is combined with an aqueous stream comprising beads 2 coated with a capture antibody 3 for the binding of this analyte 4 to be tested and this stream comprising a second antibody, which is a detection antibody 6, capable of binding to another epitope of the analyte, and fluorescent 7,

[0110] The presence of analyte 4 will agglomerate the fluorescence of the detection antibody onto the agglomerate formed by the support beads coupled to streptavidin and the capture antibody.

[0111] The involvement of two antibodies recognizing two distinct sites of analyte 4 increases the specificity of detection, somewhat analogous to ELISA tests.

[0112] Here, the analyte is a cytokine.

[0113] Example 5

[0114] The inventors also developed a test for the interaction between two cell types (Figures 8, 9, and 10). Figure 9 illustrates the encapsulation of the reagents in droplets: A first aqueous stream contains the target cells, here a cancer cell 9. A second aqueous stream contains the effector cells 8', and a third aqueous stream contains beads 2 coated with a capture antibody 3 and a fluorescent detection antibody 6 7, and a dead cell detection probe 10. Figure 10: The interaction between the effector cell 8' and the target cell 9 produces a molecule 4, here a cytokine, which is bound by the capture antibody 3, according to its affinity, and which in turn binds the fluorescent detection antibody 6 7. The aggregation of fluorescence indicates that the interaction between the two cells has resulted in the secretion of the cytokine of interest.The presence of probe 10 assembled at cell 9 (intracellular labeling, a marker expressed on the cell surface or secreted) confirms that the interaction with the effector cell 8' resulted in the death of this cell 9. Verification of the labeling of cell 9 by probe 10 and of the assembly / aggregation of the label 7 allows us to conclude that the interaction between the effector cell 8' resulted in the death of the target cell 9 and the secretion of a biomolecule 4 recognized by the capture antibody 3 and the detection antibody 6.

[0115] This system can easily be multiplexed by incorporating other 6 detection antibodies, coupled to different labeling agents and recognizing other potentially secreted biomolecules.

[0116] Example 6

[0117] Figures 11 and 12-13 respectively show variants in which the crosslinking agent 5 is a fluorescent streptavidin 5',7, or a modified tetrameric fluorescent antibody 12,7.

[0118] Example 7

[0119] Figure 14 shows the adaptation of this system for extracellular vesicle-producing cells, where the extracellular vesicles 13 are the secreted analyte and are aggregated with the beads 2.

Claims

24 DEMANDS 1. A method for analyzing an interaction between biomolecules comprising generating aqueous microfluidic droplets (1) in an oily phase said droplets (1) comprising several copies of the same capture agent (3) specifically recognizing an analyte (4, 13) several copies of the same analyte (4, 13) potentially capable of specifically binding to said capture agent (3) a multivalent crosslinking agent (5, 5', 6, 6', 12) and a fluorescent, colored or radiolabeled labeling agent (5', 7, 7') said capture agent (3) being fixed on a support (2) or on a plurality of supports (2), said support (2) having an equivalent diameter between 10 nm and 5 m, preferably between 10 nm and 1 m and preferably being made of metal, plastic, a polymer, or a resin.said multivalent crosslinking agent (5, 5', 6, 12) being a biomolecule (5) having a high affinity for said capture agent (3) or a labeled biomolecule (5') having a high affinity for a detection agent (6), said detection agent (6) binding an epitope of the analyte (4, 13) not involved in binding with its capture agent (3) or a biomolecule (6, 12) having a high affinity for the analyte (4) or a biomolecule (6') having a high affinity for an antigen (14') recognized by the secreted antibody (4), and said labeling agent (7, 7') being bound to the crosslinking agent (5', 12) or to a detection agent (6, 6') specifically recognizing an epitope of the analyte (4, 13) not involved in binding with its capture agent (3) or a detection antigen (14, 14'), said step of generating microfluidic drops (1) comprising the combination of a minimum of two aqueous streams, a first aqueous stream comprising copies of the same. analyte (4, 13) or cells (8, 8') capable of secreting said analyte (4, 13), and a second stream comprising copies of the same capture agent (3) bound on the support (2) and a detection agent (6, 7) said microfluidic analysis being a screening and / or sorting based on the aggregation of said labeling, said drops (1) having a volume between 1 pL and 10 nL, preferably between 10 pL and 500 pL, preferably between 50 pL and 100 pL.

2. The method according to claim 1, wherein the marking aggregation is three-dimensional.

3. The process according to claim 1 or 2 wherein a plurality of molecules of the same capture agent (3) is coupled to the support (2) and / or wherein the capture agent (3) is biotinylated and the support (2) comprises one or more avidin groups (5), preferably the capture agent (3) comprising one or more biotin residues and / or the support (2) comprising several avidin groups (5).

4. The method according to any one of the preceding claims, wherein the support (2) comprises one or more avidin groups (5) and comprising the administration of a capture agent (3) being a multi-biotinylated or mono-biotinylated binding antibody (3), said binding antibody (3) specifically recognizing a constant region of an analyte (4) being an antibody to be tested for its ability to bind an antigen (14, 14'), said antigen (14) being labeled or said antigen (14') not being labeled and a labeled (7') detection antibody (6') recognizing said unlabeled antigen (14') being applied or said antigen (14) being fluorescently labeled and further comprising a fluorescent (7) detection antibody (6), the fluorescence of the antigen (14) being different from the fluorescence (7) of the detection antibody (6) and the detection antibody specifically recognizing a constant region of the antibody (4).

5. The method according to any one of the preceding claims, wherein the crosslinking agent is present in the first stream.

6. The method according to any one of the preceding claims, wherein the analyte (4) is an antibody secreted into the microfluidic droplet (1) by an antibody-producing cell (8), said method comprising a final step of sorting and / or selecting the microfluidic droplets (1) into which 16 the secreted antibody (4) was linked to the antigen (3, 14, 14'), preferably followed by a step of isolation of the cell (8) producing the antibody (4) and / or characterization of the sequence of the antibody (4).

7. The method according to any one of the preceding claims, wherein the multivalent crosslinking agent (5, 5', 6, 12) is: a secondary antibody (12), preferably tetrameric, recognizing the secreted analyte (4), said secondary antibody (12) preferably being present in the second stream, a multivalent aptamer recognizing the analyte (4), said aptamer preferably being present in the second stream, a biotinylated secondary antibody (6) recognizing the secreted analyte (4), or an epitope present on the portion of the analyte (4) not involved in the recognition of the capture agent (3), and an avidin (5'), said biotinylated secondary antibody (6) preferably being present in the second stream and said avidin (5') preferably being present in the first stream.

8. The method according to any one of the preceding claims wherein the labeling agent (7, 7') is covalently fixed to the multivalent crosslinking agent (5', 6, 6', 12), preferably a secondary antibody (6, 6') derivatized with a fluorescent molecule, or an avidin derivatized with a fluorescent molecule (5').

9. The method according to claim 1 or 2 wherein the capture agent (3) is linked to a biotin and the crosslinking agent (5) is an avidin, said avidin being linked to the support (2).

10. The method according to claim 1, 2 or 9, wherein the capture agent (3) is an antibody recognizing an analyte (4, 13) and wherein the first stream comprises a cell (8) potentially secreting an analyte (4, 13) recognized by said capture antibody (3), said method comprising the step of selecting microfluidic drops (1) comprising a cell (8) secreting therein the analyte recognized by the capture antibody (3). 1 1. The method according to claim 10 comprising the application of a detection antibody (6), preferably fluorescent (7). 27 12. The method according to claim 10 comprising the application of a fluorescent detection antibody (6, 7) recognizing an epitope of the analyte (4, 13), different from the epitope recognized by the capture antibody (3).

13. A high-throughput analysis and / or screening method for a metabolic pathway in which a substrate (9) is modified by an effector (8'), comprising generating microfluidic droplets (1) comprising said substrate (9), said effector (8'), and a reporter probe (10) capable of emitting fluorescence or absorbing light at a predetermined wavelength and interacting differently with said substrate (9) compared to said substrate modified by said effector (8'), said step of generating microfluidic droplets (1) comprising the combination of at least two streams, the first stream comprising said substrate (9) and the second stream comprising said effector (8'), said effector (8') interacting with said substrate (9) so as to modify the fluorescence or absorbance of the reporter probe (10), and, preferably, a third stream comprising the reporter probe (10), a support (2) binding a capture agent (3), and a crosslinking agent. (5),and / or a detection and marking agent (6,7)., 14. The method according to claim 13 wherein the effector and secretory cell (8') is a cytotoxic immune cell and the substrate (9) is a cancer cell.

15. The method according to claim 13 or 14 wherein the probe is a marker of cell death, preferably apoptosis, or necrosis.

16. A method for high-throughput analysis and / or screening of an interaction between an effector cell (8') and a target cell (9) comprising generating microfluidic droplets comprising said effector cell (8') and said target cell (9), and further comprising: a capture agent (3) specifically recognizing an analyte (4) being a biomolecule potentially secreted due to contact between said target cell (9) and said effector cell (8'), a multivalent crosslinking agent (5, 5', 6, 12) and a fluorescent detection agent (6, 6', 7, 7'), said multivalent crosslinking agent (5) being a biomolecule having a high affinity for said capture agent (3) and said fluorescent detection agent (6, 7) being a detection antibody (6) binding specifically to the biomolecule without interfering with said capture agent (3), 28 said step of generating microfluidic drops (1) comprising the combination of two flows, a first flow comprising the target cell and a second flow comprising the effector cell and, preferably, a third flow comprising the capture agent (3), the detection agent (6), the labeling agent (7) and the crosslinking agent (5) and the support (2).

17. The method according to claim 16 wherein the effector cell (8') is an antigen-presenting cell or a cytotoxic cell.

18. The method according to claim 16 or 17 wherein the crosslinking agent (5) is a plurality of beads (2) to which are coupled a plurality of avidin, in association with a biotinylated capture antibody (3).

19. A method for sorting and / or quantifying subcellular structures (13), comprising generating microfluidic drops (1) comprising said subcellular structure (13), a multivalent crosslinking agent (5) and a labeling agent (6, 7), said crosslinking agent (5) having a specific affinity for a capture agent (3) and said labeling agent (6, 7) having a specific affinity for another component, not recognized by said capture agent (3), of said subcellular structure (13).

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