Multiplexed analysis system

Abstract

Methods and apparatus for determining at least one characteristic of a droplet, in particular of a biological entity within the droplet, based on the outcome of multiple assays, in particular by simultaneously measuring a diffuse fluorescent signal and a localised fluorescent signal from the droplet.

Description

[0001] M&C PC934028GB

[0002] 1

[0003] Multiplexed analysis system

[0004] FIELD

[0005] The present invention relates to a method of determining at least one characteristic of a droplet, in particular of a biological entity within the droplet, based on the outcome of multiple assays, in particular by simultaneously measuring a diffuse fluorescent signal and a localised fluorescent signal from the droplet.

[0006] BACKGROUND

[0007] Droplet microfluidics offer several advantages over traditional experimental techniques, including the ability to dramatically reduce the volume requirements of samples, minimal reagent consumption and high throughput analysis. Advantageously, it has been established that microfluidics platforms are compatible with homogenous FRET assays which allow the analysis of therapeutic molecules in their native solution state. Microfluidic platforms have successfully been used to select and retrieve droplets comprising cells with desirable properties fordownstream processing. However, despite the monumental impact of microfluidics technologies, there are numerous challenges that remain. To date there is a lack of reliable microfluidics methods that can be used to detect or quantify secreted analytes from a single cell. Moreover, multiplexing remains a hurdle for these technologies.

[0008] Multiplexing has transformed biological research and development by enabling the analysis of multiple targets or parameters within a single experiment. This approach offers numerous benefits, including reduced sample requirements, increased throughput, enhanced assay accuracy, cost efficiency, and a more comprehensive understanding of complex biological systems. As this technology advances, researchers are increasingly utilizing combined assays to gain deeper insights into intricate biological processes and to drive innovation in healthcare and biotechnology. Nonetheless, multiplexing presents challenges such as the need for specialized assays and equipment, sophisticated data analysis and software tools.

[0009] The Applicant’s proprietary picodroplet technology, as described in International patent application WO2016 / 193758, hereby incorporated by reference, facilitates automated, high-throughput screening and selection of single cells based on surface markers or secreted molecules. The instrument performs high-throughput single-cell analysis using

[0010] 15267483-1M&C PC934028GB

[0011] 2

[0012] a fluorescent assay; single cells are encapsulated into picodroplets, and sorted based on detected fluorescence after excitation with an excitation laser. See, for example, Craig et al, “Cyto-Mine®: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance”, SLAS Technology, Volume 25, Issue 2, 2020, Pages 177-189, https: / / doi.org / 10.1177 / 2472630319892571. This allows for a single assay to be used as the basis for sorting droplets. Background prior art can be found in: US2020 / 0376488; US2024 / 0271226; US2022 / 0212188; US2012 / 0194805; US2021 / 0016276; US2016 / 0231324; WO2022 / 103814; and US2021 / 0107001.

[0013] SUMMARY

[0014] The invention is set out in the independent claims; further aspects are defined in the dependent claims.

[0015] In one aspect there is provided a method of determining at least one characteristic of a droplet based on the outcome of at least one assay within said droplet; the method comprising:

[0016] a. providing a droplet comprising at least one biological entity to a fluid flow; b. performing at least one assay in the droplet;

[0017] c. determining optical characteristics of the droplet by exposing the droplet to a plurality of distinct excitation light sources and detecting a diffuse fluorescent signal and a localised fluorescent signal emitted in response to the distinct excitation light sources, wherein the distinct excitation light sources are selected to stimulate distinct emission spectra.

[0018] The term “emission spectrum” can refer to a range of optical signals emitted from an entity, for example, a biological entity associated with a fluorophore, in response to an excitation light source. Such signals may include light having a range of wavelengths. The term “signal”, “detected signal”, or “emitted signal” and similar wording refers to the part of the emission spectra detected by the detector, which may include a narrower range of wavelengths selected from the full emission spectra.

[0019] In an embodiment, step a) comprises providing a fluid sample comprising at least one biological entity; and preparing a droplet from said sample wherein the droplet comprises at least one biological entity.

[0020] 15267483-1M&C PC934028GB

[0021] 3

[0022] In an embodiment, the plurality of excitation light sources comprises at least one laser, e.g. at least four lasers. In an embodiment, the emitted signal is detected using at least one optical sensor.

[0023] In an embodiment, the diffuse fluorescent signal is indicative of at least one of: i) the secretion of at least one analyte from the at least one biological entity (for example when the at least one biological entity is a cell said cell may have secreted or released proteins, cytokines or metabolites into the extracellular environment in the droplet); ii) the presence of an unbound molecule or ligand in the droplet; iii) the presence of at least one pathogen within the droplet that has not attached to or entered the at least one biological entity.

[0024] In an embodiment, the localised fluorescent signal is indicative of at least one characteristic of the at least one biological entity. For example, the localised fluorescent signal may indicate the viability of a cell, i.e. whether as cell is live and functioning or dead, or dying. Alternatively, or additionally, the localised fluorescent signal may indicate cell type, for example due to the expression of specific cell surface markers.

[0025] In an embodiment, the localised signal is indicative of metabolic activity of the at least one biological entity, and / or a ligand binding to the surface of the at least one biological entity, and / or a pathogen has bound to or entered the at least one biological entity.

[0026] In some embodiments, step c) comprises measuring the intensity and / or duration of a plurality of signals. In an embodiment, step c) takes place when the fluid flow path of the droplet passes an excitation light source and a detector. Preferably, step c) detects multiple emission signals with multiple detectors. More preferably, step c) takes place multiple times in series or simultaneously as the fluid flow path of the droplet passes multiple excitation light sources and multiple detectors.

[0027] In some embodiments, the method further comprises: (d) sorting said droplet dependent on the outcome of the determination step; and (e) dispensing the sorted droplet into a reservoir, wherein said dispensing comprises, after said sorting: selecting a fluid flow path for fluid containing the sorted droplet; and ejecting the sorted droplet from the selected path.

[0028] 15267483-1M&C PC934028GB

[0029] 4

[0030] In some embodiments, step c) and / or d) comprise sequentially comparing pairs of detected signals from different optical sensors to determine at least one characteristic of a droplet.

[0031] The method may further comprise an additional detecting step carried out as part of the dispensing step e). This allows for subsequent checking of the characteristics of the droplet. Detection information may be recorded (e.g., stored as data), optionally together with imaging data from the dispensed droplet and the location in which it is dispensed. This allows subsequent verification of the process and the characteristics of the droplet. The additional detecting step will typically be performed in the same manner as the initial detecting step; that is, with multiple excitation sources and preferably multiple detected signals.

[0032] In an embodiment the method comprises a plurality of determination steps and a single sorting step. Alternatively, the method may comprise a plurality of sequential determination and sorting steps.

[0033] In an embodiment of the method, when there are sequential determination and sorting steps, the method further comprises classifying the sorted droplets according to the intensity and / or duration of a signal.

[0034] In some embodiments, step d) comprises sorting said droplet according to the profile of said detected signals in step c). In some embodiments, the sorting step comprises routing a droplet to a specific fluid flow path based on the optical characteristics of a droplet. In some embodiments, step c) comprises determining the presence and / or location of an analyte within a droplet through the emission of a fluorescent signal.

[0035] In a preferred embodiment, at least one localised and / or dispersed fluorescent signal is detected. More preferably, at least one localised and at least one dispersed fluorescent signal are detected simultaneously.

[0036] In an embodiment, the method further comprises at least one incubation step. In some embodiments there is a first incubation step followed by at least one subsequent incubation step. Optionally, the method comprises an incubation step before, after or

[0037] 15267483-1M&C PC934028GB

[0038] 5

[0039] during step b) and / or an incubation step before, after or during step c) (for example, immediately before, after, or during steps b) and / or c)). In an embodiment, the incubation step of the method comprises at least one of growing and / or maintaining one or more biological entities, a reaction between one or more biological entities, and / or interactions between one or more biological entities.

[0040] In an embodiment, the dispensing step comprises dispensing a sorted droplet at a defined location in response to the determination of the optical characteristics of a droplet. In some embodiments the droplet may be dispensed into an external device.

[0041] In an embodiment of the method, the assay is selected from at least one of a protein detection assay, cell detection assay, secretion assay, uptake assay, cytotoxicity assay, stability assay, localisation assay, enzyme assay, binding assay, proliferation assay, cell death assay, immunoassay, or tolerance assay. Preferably, the assay is a multiplex assay wherein a plurality of excitation and emission spectra are used to detect a plurality of analytes in a droplet. More preferably, the assay is a multiplex assay wherein a plurality of excitation and emission spectra are used to detect a plurality of characteristics of at least one analyte in a droplet. Most preferably, the emission spectra are used to detect a diffuse fluorescent signal and a localised fluorescent signal simultaneously. In an embodiment the diffuse fluorescent signal is indicative of the secretion of at least one molecule from the at least one biological entity and the localised fluorescent signal is indicative to at least on characteristic of the at least one biological entity.

[0042] In an embodiment, the assay comprises determining the presence of at least one molecule on the surface of a bead, or a cell and / or inside a cell based on the detection of at least one localised fluorescent signal and / or at least one dispersed fluorescent signal.

[0043] In an embodiment, the assay comprises detecting an interaction between at least two molecules based on the detection of at least one localised fluorescent signal.

[0044] The method can involve detecting secretion of at least one target protein from a single cell based on the detection of at least one fluorescent signal.

[0045] 15267483-1M&C PC934028GB

[0046] 6

[0047] The method can detect the secretion of at least one antibody from a single cell and the binding of said antibody to an antigen based on the detection of at least three different signals.

[0048] In an embodiment, the method comprises determining whether a cell is apoptotic based on the detection of at least one fluorescent signal.

[0049] In some embodiments at least four different characteristics of at least one cell or molecule are determined based on the detection of at least four different signals.

[0050] In an embodiment, the biological entity is selected from at least one of a cell, a protein, a carbohydrate, a lipid, an antibody, an antigen, an enzyme or a nucleic acid.

[0051] In another aspect there is provided an apparatus suitable for carrying out the method of any preceding claim wherein said apparatus comprises: at least one microfluidic input channel; a plurality of fluid flows; a plurality of distinct excitation light sources; a plurality of detectors for detecting at least one diffuse fluorescent signal and at least one localised fluorescent signal emitted in response to the distinct excitation light sources, wherein the distinct excitation light sources are selected to stimulate distinct emission spectra; and at least one microfluidic output channel.

[0052] In some embodiments the apparatus further comprises one or more of: a droplet formation device for preparing a droplet from a fluid which contains at least one biological entity; a droplet sorting device for sorting a said droplet dependent on an outcome of said determination; and a dispensing unit for dispensing a said sorted droplet from a fluidic flow path. In an embodiment, the dispensing unit comprises an isolation unit for isolating a said sorted droplet from said fluidic flow path and a guide for guiding a said isolated droplet into a second fluidic flow path. In an embodiment, the dispensing unit comprises a high pressure unit, e.g. a pump, for dispensing a droplet via pressurised fluid ejection. In an embodiment, the plurality of excitation light sources comprise a plurality of lasers and / or the plurality of detectors comprise a plurality of photomultiplier tubes. In an embodiment, the apparatus comprises at least one optical filter.

[0053] 15267483-1M&C PC934028GB

[0054] 7

[0055] In an embodiment, the apparatus further comprises a droplet storage unit for storing an isolated droplet. In an embodiment, the apparatus further comprises an incubator for incubating a droplet.

[0056] In an embodiment, the apparatus further comprises one or more of a fluorescence detector, a scattered light detector, an imaging detector, an acoustic wave generating and detecting unit, and a magnetic-activated cell sorting device.

[0057] BRIEF DESCRIPTION OF THE FIGURES

[0058] Figure 1- Schematic representation of surface-binding assay, for secreted antibodies. A) Antibody-secreting cells are encapsulated together with target cells expressing antigen on the cell surface, and detection (secondary) antibody. B) Upon secretion, primary antibody is recognized by detection antibody and also binds to the antigen on the surface of the cell. C) Binding of antibody complex on surface of the target cell results in translocation of some of the detection antibody from homogenously dispersed appearance into cell-surface localization, exhibited as an increase in green fluorescence on a scatter plot.

[0059] Figure 2- Isolation of subpopulation of secreting cells. A hybridoma cell line was stained with either eFluor450 (Blue) or eFluor670 (Far Red) and mixed with Mouse FRET probes. The mixture was loaded onto the droplet-based microfluidic instrument. A) Schematic representation of experimental design. B) Detection of different stained cell populations before sorting (Blue and Far-Red fluorescence). C) Cells were gated for FRET signal only, during sorting. D) Cells dispensed based on the gating strategy were analyzed for the presence of eFluor450 and eFluor670 labelled cells. E) Cells were first gated for FRET signal (left panel) and then on eFluor450 membrane dye during sorting (right panel). F) Cells dispensed based on the gating strategy were analysed for the presence of eFluor450 and eFluor670 labelled cells. G and H) Fluorescence microscopy analysis of dispensed cells from 2D or 2F. I) Statistical analysis of isolation accuracy of FRET+ or FRET+ / eFluor450 populations.

[0060] Figure 3- Detection of antibody binding to its target surface antigen. A431 readout cell line was labelled with eFluor670 (eFL670) and mixed with Cetuximab and DL488 labelled anti-hlgG antibody. The mixture was loaded onto the droplet-based microfluidic instrument. Droplets were gated during sorting foreFluor670 and increased DL488 peak.

[0061] 15267483-1M&C PC934028GB

[0062] 8

[0063] Dot plots show data accumulated during sorting of droplets in the droplet-based microfluidic instrument in the absence (A) or presence (B) of Cetuximab.

[0064] Figure 4- Validation of performance of an example instrument in cell isolation based on multiplexing of cell staining and surface-binding secretion assay. A) Schematic representation of surface binding assay design. B) Detection and gating strategy of 3 different colors in surface-binding assay. C) Microscopy validation of instrument-based cell isolation.

[0065] Figure 5- Validation of droplet-based microfluidic instrument performance in cell isolation based on multiplexing of cell staining and surface-binding secretion assay. A) Schematic representation of advanced surface binding assay design. B) Gating strategy for sorting of anti-EGFR secreting cells from sample with ratio 1:1 of EGFR / TNFa cells. C) Gating strategy for sorting of anti-EGFR secreting cells from sample with ratio 1:100 of EGFR / TNFa cells. D) FlowJo analysis of binding events in the population of irrelevant secreting cells (anti-TNFa, stained CT- Orange). E) Microscopy validation of cells dispensed following 4-color surface binding secretion assay. F) Isolation accuracy of antibody secreting cells, based on surface-antigen binding assay. Droplets from experiments conducted using different ratios of anti-EGFR vs anti-TNFa antibody secreting cells (Figures 4 and 5) were dispensed into 96-well plates (2 plates / experiment). Isolation accuracy was calculated as specified in the Methods section. Mean value of 2-3 experiments is presented.

[0066] Figure 6- Schematics of an embodiment of the steps used to sort and dispense droplets.

[0067] Figure 7- Differentiating FRET+ dead / dying cells from live cells in picodroplets.

[0068] CHO cells secreting hlgG4 antibody were mixed with Cyto-Cellect® probes and viability dye. Cells were loaded onto the droplet-based microfluidic instrument and incubated for 1 hour. Cells were gated for apoptosis and FRET signal, (a) Schematic diagram for assay reaction and gating strategy during sorting, (b) List of fluorochromes used in the assay, (c) Gating of cells during sorting, (d) Analysis of FRET response in live versus apoptotic cell population, (e) Examples of localized and diffuse fluorescence spectra used for differentiating live from apoptotic cells, (f) An example of an apoptotic cell indicating exposed phospholipid phosphatidylserine.

[0069] 15267483-1M&C PC934028GB

[0070] 9

[0071] Figure 8- Image of an example cartridge for use with the described techniques.

[0072] Figure 9- Schematics of example detection of markers in a picodroplet, a) detection of a secreted marker, b) detection of an internal marker, c) detection of a surface marker.

[0073] Figure 10- Schematics of detection of different cell types, antigen binding and cell viability, (a) Illustrates examples assays, (b) illustrates identifying which cell line is in a droplet based on localized fluorescent colour (two signals) and simultaneously identifying antibody production by the cell line (two diffuse, fluorescent signals from FRET), (c) illustrates assays (a multiplexed assay) that simultaneously identify specific cell lines and verify the functional binding of their secreted antibodies. The localized signals identify the antibody secreting cell (ASC) line, and the (green) “contrast” signal verifies that the secreted antibody actually binds to its target, a reporter cell (or bead) that is encapsulated within the droplet and displays a surface receptor (antigen). The localized spike of the contrast signal is at the location of the reporter cell (or bead).

[0074] Figure 11- Schematics of fluorescence detection modes

[0075] Figure 12- Schematics of an example optical configuration with four distinct excitation light sources at 405, 488, 561 and 638 nm and four distinct detectors in the form of photomultiplier tubes (PMTs).

[0076] Figure 13- Schematics of example selection of one or more polygon regions on a 2D plot with fluorescence signals on the axes, (a) illustrates classification of combinations of fluorescence signal based on selected polygon region(s). (b) illustrates logical combination of regions (classifications), where AND requires multiple regions, OR requires any one region, and EXCLUDED defines everything except the selected region.

[0077] DETAILED DESCRIPTION

[0078] The following description details an example method of determining at least one characteristic of a biological entity contained within a droplet based on the outcome of a plurality of assays performed simultaneously within the droplet.

[0079] 15267483-1M&C PC934028GB

[0080] 10

[0081] The method comprises providing a droplet comprising at least one biological entity, e.g. a cell, to a fluid flow and performing the plurality of assays on the biological entity in the droplet. This involves determining optical characteristics of the droplet by exposing the droplet to a plurality of distinct excitation light sources and detecting signals emitted in response to the distinct excitation light sources. The distinct excitation light sources are configured to stimulate the emission of at least one diffuse fluorescent signal and at least one localised fluorescent signal. The plurality of assays can alternatively be referred to as a single “multiplexed” assay.

[0082] For example the system can use four distinct lasers to excite multiple fluorophores simultaneously — for example, a 405 nm (Violet) laser, a 488 nm (Blue) laser, a 561 nm (Yellow-Green) laser, and a 638 nm (Red) laser. Consequently, the at least one diffuse fluorescent signal may comprise a fluorescent signal that is excited by a first of these distinct excitation light sources at a first wavelength, while the at least one localised fluorescent signal comprises a fluorescent signal that is excited by a second of the distinct excitation light sources at a second, different wavelength. (As used herein, “wavelength” and “colour” are used broadly synonymously).

[0083] In general the fluorescence signals are of different colours (wavelengths) to excitation colours; they can be detected e.g. by applying different filters, e.g. bandpass filters, to one or more detectors, e.g. one or more photomultiplier tubes. In general the droplet is a droplet of water, carried in a fluid flow of oil e.g. through a microfluidic channel. The fluorescence signals are detected as the droplet flows past the one or more detectors. This can result in a localised signal or spike, as the entity passes the detector(s); or a diffuse signal, as the droplet passes the detector(s); or both i.e. a spike superimposed on a diffuse signal. As used herein a spike superimposed on a diffuse signal is a form of localised signal, also referred to as a “contrast” signal. That is, the localized signal results from just a localized part of the droplet (i.e., the part containing the biological entity), whilst the diffuse signal can be generated throughout the droplet.

[0084] In these assays, the at least one diffuse fluorescent signal can comprises a fluorescent signal that results from secretion of a molecule from the biological entity and that is contained within the droplet. Examples of such secreted molecules include an antibody (IgG), an Fc fusion protein, or a viral capsid protein such as p24; some further examples include cytokines, hormones, and growth factors. The at least one localised fluorescent

[0085] 15267483-1M&C PC934028GB

[0086] 11

[0087] signal generally comprises a fluorescent signal that is localised on a surface of the biological entity or on a surface of a bead within the droplet.

[0088] The method can distinguish the diffuse and localized signals in various ways, e.g. based on duration. In some implementations the at least one diffuse fluorescent signal can be determined by integrating the area under a fluorescence curve of fluorescence signal (of a particular colour) against time as the droplet flows past a fluorescence detector, e.g. to determine an "average" or "area" signal, e.g. as curve area divided by curve based width. In some implementations a different form of averaging can be used. The at least one localised fluorescent signal can be determined by determining a peak of a fluorescence signal against time as the droplet flows past a fluorescence detector, e.g. to determine a "peak" or "height" signal.

[0089] The at least one diffuse fluorescent signal can comprise a fluorescent signal, e.g. a FRET signal, that results from secretion of a molecule from the biological entity and that is contained within the droplet. Unlike flow cytometry a droplet-based approach contains secreted molecules facilitating their detection. The at least one localised fluorescent signal can comprises a fluorescent signal that is localised on a surface of the biological entity or on a surface of a bead within the droplet. A combination of the at least one diffuse fluorescent signal and the at least one localised fluorescent signal can be used to determine the at least one characteristic of the biological entity.

[0090] The fluorescent signals may be combined in many different ways, as described later, e.g. using AND, OR, or EXCLUDING “X”, and / or by defining regions on one or more graphs in two (or more) dimensions that has different fluorescence signals on different axes, all according to the desired assay / selection criteria.

[0091] For example implementations of the techniques can be used to determine both whether a cell producing a molecule of interest and also something else about the cell, e.g. a cell type or whether the cell has particular surface binding marker or some “stain” or other characteristic.

[0092] In some implementations to analyse secretion of a molecule, such as an antibody, from an entity such as a cell, the method can use the distinct excitation light sources to stimulate the emission of two diffuse fluorescent signals of different colours. The

[0093] 15267483-1M&C PC934028GB

[0094] 12

[0095] characteristic of the biological entity is then determined from a combination of the two diffuse fluorescent signals and the at least one localised fluorescent signal. For example this can involve calculating a ratio between a green FRET donor signal and an orange FRET acceptor signal. This can facilitate robustly identify secreting cells based on the spectral shift caused by the secretion event, e.g. between a non-secreting cell (e.g. high donor, low acceptor) and a secreting cell (e.g. low donor, high acceptor).

[0096] Where the secreted molecule comprises an antibody, the method can use the at least one localised fluorescent signal to determine whether the antibody binds to a surface receptor on the surface of the cell or bead. This facilitates determining the characteristic of the biological entity from a combination of whether the cell secretes the antibody and whether the antibody binds to a surface receptor on the surface of the cell or bead.

[0097] To confirm that the antibody binds to the surface receptor, the method can include an antibody stain in the droplet, the antibody stain comprising a secondary antibody with a fluorescent tag that provides a further fluorescent signal. The method can then detect binding of the secondary antibody to the antibody bound to the surface receptor by detecting localization of the secondary antibody at the surface of the cell or bead. In particular detecting the localization can involve detecting a contrast between the localized secondary antibody and the background of the droplet (this may create a halo or ring around the entity). Such approaches can help to detect cells that i) secrete an antibody and ii) for which the antibody binds to a specific target.

[0098] In implementations involving multiple cell populations, the biological entity can comprise a cell from one of at least two antibody secreting cell lines. Here, the method can use the distinct excitation light sources to stimulate the emission of one of two localized fluorescent signals of different colours, each associated with a different surface receptor or cell identification marker. For example, Cell Line 1 may be identified by a yellow localized signal while Cell Line 2 may be identified by a blue localized signal.

[0099] The method can distinguish between the antibody secreting cell lines by detecting a distinctive localised fluorescent signal associated with each cell line using the localized fluorescent signal from the droplet. Furthermore, the method may involve determining whether an antibody secreted by the cell binds to the surface receptor based on detection of a third localised fluorescent signal localised on a surface of the reporter entity. This

[0100] 15267483-1M&C PC934028GB

[0101] 13

[0102] allows for the simultaneous identification of the cell line and verification of the specific binding of its secreted antibody.

[0103] For example such techniques can simultaneously identify and distinguish between two different populations of antibody-secreting cells in the same run. The cell lines can be distinguished by their own surface labels, whilst, e.g., they both screen against the same reporter target.

[0104] In some embodiments where it is necessary to confirm that the secreted antibody functionally binds to the surface receptor, the method includes an antibody stain within the droplet. This antibody stain typically comprises a secondary antibody conjugated with a fluorescent tag that provides a further fluorescent signal, distinct from the signals used for secretion detection or cell line identification.

[0105] The method then involves detecting the binding of this secondary antibody to the secreted antibody, but only where the secreted antibody has successfully bound to the surface receptor on the cell or bead. This specific binding event is identified by detecting the localization of the secondary antibody at the surface of the reporter entity.

[0106] Detecting this localization can be done by identifying a "contrast" between the localized secondary antibody and the background of the droplet. Physically, this represents at least a partial transition of the further fluorescent signal from a diffuse state (where the secondary antibody is unbound and floating in the droplet) to a localized state (where it concentrates on the surface).

[0107] As the droplet travels past the detector, this "contrast" signal manifests as a specific optical profile: the baseline diffuse signal is overlaid by a distinct spike or peak at the location of the cell or bead. This signal mode, distinct from purely diffuse or purely localized modes, is referred to as "contrast" and serves as a positive confirmation of antigen-specific binding (Figure 11).

[0108] In further embodiments, such as those illustrated in Figures 10b and 10c, the method is adapted to simultaneously distinguish between multiple cell populations while verifying antigen specificity. In this configuration, the biological entity comprises a cell selected from a first antibody-secreting cell line and a second antibody-secreting cell line.

[0109] 15267483-1M&C PC934028GB

[0110] 14

[0111] To facilitate this multiplexing, the droplet can include a reporter entity, e.g., a bead or reporter cell, presenting a surface receptor, along with a labelled detection antibody.

[0112] The at least one diffuse fluorescent signal comprises a fluorescent signal emitted by the labeled detection antibody (in the droplet background). This background signal serves as a baseline for the contrast measurement.

[0113] Implementations of the method use distinct localized signals to identify the specific cell line present in the droplet, in particular a first localised fluorescent signal that identifies the first antibody-secreting cell line (e.g., a yellow / orange signal), and a second localised fluorescent signal (which is spectrally distinct from the first) that identifies the second antibody-secreting cell line (e.g., a blue signal).

[0114] The method can distinguish between the first and second antibody-secreting cell lines based on the detection of either the first or the second localised fluorescent signal. Simultaneously, the method can determine whether an antibody secreted by the identified cell binds to the surface receptor. This determination can be based on the detection of a third localised fluorescent signal (i.e. a contrast signal) localised on the surface of the reporter entity. This allows for the simultaneous assessment of which cell line is present and whether its secreted product is functional against the target.

[0115] Implementations of this technique may be combined, i.e. in a combined assay, with those previously described (and also the cell viability check described later). For example such a combination can be used to identify a molecular, e.g. Ab, secretion from a cell, and also that the molecule binds to a particular target.

[0116] In some implementations of the techniques, but not necessarily, the method uses the detected signals to determine the characteristic of the biological entity by defining one or

[0117] 15267483-1M&C PC934028GB

[0118] 15

[0119] more regions on a graph that has first and second axes corresponding to fluorescence signals of respective first and second colours. The characteristic is determined according to whether the at least one diffuse fluorescent signal and the at least one localised fluorescent signal fall within the one or more regions.

[0120] This can involve providing a graphical user interface to enable a user to define multiple said regions, and selection logic to enable the user to define one or more logical combinations of the regions, such as AND, OR, and EXCLUDED (i.e. fluorescence in a particular region should not be present), to determine the characteristic. Then, for a plurality of the droplets, the method can select droplets containing particular biological entities dependent on the characteristic. For example the method can selectively perform one or more of incubating, sorting, and dispensing a droplet, dependent on the determined at least one characteristic of the biological entity.

[0121] Some implementations of the described techniques can include a viability analysis of the biological entity, e.g. cell, within the droplet, as well as or instead of the other assay(s) described above. Then the droplet may be processed, e.g. incubated, sorted, or dispensed accordingly, e.g. to retain only viable entities.

[0122] That is, the characteristic of the biological entity may include a viability of the cell. The method can use the at least one localised fluorescent signal to perform a surface receptor binding assay to identify cell apoptosis. For example, the system may detect the binding of a phospholipid-binding protein, such as Annexin V, with a fluorescent tag, that is conjugate to phosphatidylserine exposed on the outer surface of the cell, exposed phosphatidylserine indicating an apoptotic cell. The method can determine whether or not the cell is viable dependent on whether or not cell apoptosis is identified.

[0123] Further examples

[0124] More generally, described herein are methods and systems, e.g. an instrument, for determining at least one characteristic of a droplet, more particularly of a biological entity, such as a cell or (bio)molecule, within a droplet. A method is described of determining at least one characteristic of a droplet, more particularly of a biological entity, based on the outcome of at least one assay, in general multiple assays, performed in the droplet.

[0125] 15267483-1M&C PC934028GB

[0126] 16

[0127] More particularly, and as previously described, a method of determining at least one characteristic of a droplet based on the outcome at least one assay within said droplet can involve providing a droplet comprising at least one biological entity to a fluid flow; performing at least one assay in the droplet; and determining optical characteristics of the droplet by exposing the droplet to a plurality of distinct excitation light sources and detecting at least one diffuse fluorescent signal and at least one localised fluorescent signal emitted in response to the distinct excitation light sources, wherein the distinct excitation light sources are selected to stimulate distinct emission spectra.

[0128] Determining at least one characteristic of a droplet can for example refer to one or more of: presence of a single cell, presence of multiple cells, presence of specific cell types such as cancer cells or stem cells, presence of cells secreting antibodies, presence of at least one apoptotic cell, presence of molecular interactions, cell viability, cell proliferation, gene expression, metabolic activity, presence of pathogens, responses to stimuli, presence of biomarkers or any other characteristic of interest that can be determined using the described methods. As will be explained herein, such characteristics can be determined based on the outcome of various assays; the assays can be selected so as to relate to a particular characteristic of the droplet. Further, as explained herein, the characteristics may be determined in a multiplex manner by use of multiple distinct excitation light sources.

[0129] Assays refer to experimental methods for assessing the presence, localisation, and / or biological activity of a substance in living cells and / or biological matrices (including within droplets). Examples of assays include but are not limited to: cell viability assays, protein binding assays, apoptosis assays, cell proliferation assays, enzyme activity assays and cytotoxicity assays. In the context of the described methods at least one assay is carried out in a droplet on a microliter, nanoliter, or picoliter scale; preferably, the droplet is a picodroplet (the volume of which is below approximately one thousand or a few thousand picolitres). At least one, two, three, four, five, six, seven, eight, nine, ten or more assays are carried out in a single droplet.

[0130] In some embodiments a droplet is provided wherein said droplet comprises at least one biological entity, such as: a cell, nucleic acid, protein, carbohydrate or lipid. It should be appreciated that any type and any number of entities may be within a droplet.

[0131] 15267483-1M&C PC934028GB

[0132] 17

[0133] In an embodiment, the at least one entity in the droplet emits fluorescence; for example, said molecule may be fluorescently dyed or labelled. Non-limiting examples of fluorescent dye known in the art include: DAPI (4',6-diamidino-2-phenylindole), fluorescein isothionacynate and tetramethylrhodamine dyes. Non-limiting examples of fluorescent labels known in the art include: GFP, YFP, RFP and nitrobenzoxadiazole. It should be appreciated that any type and any number of available fluorescent labels and / or dyes can be used in the described methods and the skilled person will be able to select appropriate fluorescent labels and / or dyes to meet their specific requirements.

[0134] It will be understood that “determining at least one optical characteristic of a droplet” may refer to various specific embodiments. For example, detection of fluorescence in a droplet may include detecting one or more point sources of fluorescence (for example, from biological molecules localised within the droplet), and / or detecting one or more diffuse sources of fluorescence (for example, from biological molecules distributed throughout the droplet). As will be described herein, these different approaches permit a number of different types of assays to be conducted, and provide great flexibility in the uses and applications of the methods described herein.

[0135] In some embodiments the method further comprises preparing a droplet from a sample, preferably a fluid sample, the droplet comprising at least one biological entity. Various methods and devices for preparing a droplet from a fluid may be used. Examples include, but are not limited to, T-junctions, Y-junctions, flow focusing devices, and others.

[0136] There is also described a microfluidic system for providing a droplet containing one or more entities, the system comprising: a droplet formation device for preparing a droplet from a fluid which contains a plurality of entities.

[0137] In the context of this specification a droplet generally comprises an aqueous fluid in an oil carrier fluid or an aqueous core surrounded by an oil shell in an aqueous carrier fluid.

[0138] In an embodiment, the droplet is provided to a fluid flow path. A fluid flow path refers to a defined channel or network of channels through which fluid can travel.

[0139] 15267483-1M&C PC934028GB

[0140] 18

[0141] In an embodiment, an assay is carried out in the droplet after said droplet enters a fluid flow path. In alternative embodiments, an assay is carried out in the droplet before said droplet enters a fluid flow path.

[0142] The droplet is exposed to a plurality of distinct excitation light sources in order to determine the optical characteristics of the droplet. In some embodiments the plurality of distinct excitation light sources are at a single location, although preferably the plurality of distinct excitation light sources are at multiple different locations.

[0143] An excitation light source refers to a device which emits light at a wavelength that will excite or induce fluorescence in a molecule. Examples of excitation light sources include lasers, halogen lamps and LEDs. Preferably, the excitation light source is a laser.

[0144] A distinct excitation light source refers to an excitation light source that can emit light at a different wavelength to the other light sources used in the method. Distinct excitation light sources may be obtained by filtering a single white light source.

[0145] Any number of distinct excitation light sources may be used such as one, two, three, four, five, six, seven, eight, nine or ten distinct excitation light sources. In an embodiment between one and four distinct excitation light sources are used, and preferably four distinct excitation light sources are used. However, in some embodiments more than four excitation light sources may be used.

[0146] In a preferred embodiment the distinct excitation light sources comprise up to four lasers that each emit a different wavelength. However, in alternative embodiments, the distinct excitation light sources comprise more than four lasers, for example, five, six, seven, eight, nine or ten lasers that each emit a different wavelength.

[0147] Any wavelength of light can be used an excitation light source and the skilled person would be able to select excitation light sources to meet their specific needs; based on, for example, the properties of the desired fluorophores to be used.

[0148] In a preferred and non-limiting embodiment there are four excitation light sources that emit light at 405 nm (violet), 488 nm (blue), 561 nm (yellow-green) and 638 nm (red).

[0149] 15267483-1M&C PC934028GB

[0150] 19

[0151] Each excitation light source will excite a different emission spectra in response to the excitation light source. Put another way, each distinct excitation light source will stimulate a distinct emission spectra (for example, from a number of distinct fluorophores).

[0152] In a non-limiting example, when the excitation light sources emit light at 405 nm (violet), 488 nm (blue), 561 nm (yellow-green) and 638 nm (red) distinct fluorophores are selected that when activated or excited emit a spectra of over a range of wavelength longer than that of the excitation light source. For example, the detection ranges of emission spectra of various dyes may be 429-463 nm (blue), 511-529 nm (green), 595-615 nm (orange) and 663-693 nm (far red) respectively, as depicted in table 1. Optical filters may be used to selectively detect signals emitted from the excited fluorophore, for example, around the peak of the emission spectra (e.g., 429-463 nm for eFluor 450). Excitation and emission peaks will be known to the skilled person for a given dye.

[0153] In general there is at least one optical filter which is configured to selectively transmit light of a specific wavelength or a specific range of wavelengths, Multiple types of optical filter are known in the art such as: absorptive filters, dichroic filters, notch filters, bandpass filters, short-pass filters and long-pass filters. Any optical filter may be used that is appropriate to meet the needs of the user.

[0154] In embodiments a plurality of optical filters are used to select a plurality of optical signals at specific wavelengths. The skilled person can choose an appropriate optical filter to meet their specific needs, taking into account the excitation light sources being used and expected emission spectra.

[0155] Preferably, the emission spectra emitted by the excited fluorophore pass an optical filter which screens for specific optical signals (e.g., light of a given wavelength or a range of wavelength) and said optical signal then reaches a detector.

[0156] The emitted optical signals are detected by a detector. In general the detector is an optical sensor, that is a device which is sensitive to light and measures various properties such as light intensity and wavelength. In embodiments the detector will detect light at a single wavelength (or narrow band of wavelengths), particularly where an optical filter is present. In preferred embodiments, the detector determines light intensity over time at a

[0157] 15267483-1M&C PC934028GB

[0158] 20

[0159] given wavelength (or narrow band of wavelengths). The light wavelength is not directly determined by the detector.

[0160] In some embodiments the detector is a photomultiplier. Examples of photomultipliers include photomultiplier tubes (PMTs) and silicon photomultipliers. However, any alternative optical detector may be used that can detect an emission spectra.

[0161] Table 1: Excitation light sources, fluorophores and detectors used in a preferred and non-limiting embodiment.

[0162]

[0163] It should be understood that the emission spectra emitted in response will vary according the specific excitation light sources and fluorophores used. The skilled person would be able to select appropriate excitation light sources and corresponding emission spectra to meet their specific requirements.

[0164] The method can involve the additional steps of sorting said droplet dependent on the outcome of the determination step; and dispensing the sorted droplet into a reservoir, wherein said dispensing comprises, after said sorting: selecting a fluid flow path for fluid containing the sorted droplet; and ejecting the sorted droplet from the selected path.

[0165] Sorting refers to separating droplets based on one or more characteristics. Generally droplets are sorted according to their optical characteristics.

[0166] Droplets are sorted based on the optical signal emitted in response to the plurality of distinct excitation light sources. A droplet may be sorted based on any number of the following characteristics: number of detection wavelength windows detected, intensity of

[0167] 15267483-1M&C PC934028GB

[0168] 21

[0169] one or more signal, duration of a signal for one or more detection wavelength windows or the profile of a signal.

[0170] In some embodiments droplets are sorted when (after) the fluid flow path of said droplet passes an excitation light source and a detector. Preferably, there are multiple sorting steps in series as the fluid flow path of a droplet passes multiple excitation light sources and multiple detectors. In some embodiments the droplet is exposed to multiple excitation light sources at a single point and multiple detectors that can detect a plurality of optical signals simultaneously.

[0171] The sequential determination and sorting steps can further comprise classifying or grouping sorted droplets according to the intensity and / or duration of at least one optical signal emitted in response to at least one excitation light source. Preferably, droplets that are grouped together due to the outcome of the determination step will enter the same fluid flow path which is distinct from the fluid flow path of droplets that are in different groups as a result of their optical characteristics.

[0172] In an embodiment, the determination step comprises gating droplets according to the detected signal of the emission spectra emitted from a droplet whereby droplets are selected based on their optical characteristics.

[0173] In some embodiments gating may be carried out for a single fluorescent signal whereby droplets are selected based on the presence or absence of a single colour or wavelength range.

[0174] In some embodiments, gating may be carried out based on a plurality of fluorescent signals to identify droplets with specific combinations of characteristics.

[0175] In some embodiments, gating may be carried out sequentially whereby pairs of fluorescent signals are compared to identify specific subpopulations.

[0176] For example, if there are four detectors which can detect optical signals within wavelength ranges of 429-463 nm (blue 466 / 34), 511-529 nm (green 520 / 18), 595-615 nm (orange 605 / 20) and 663-693 nm (far-red 678 / 30) producing optical signals, the following pairs of signals may be compared sequentially to identify 6 different pairs of

[0177] 15267483-1M&C PC934028GB

[0178] 22

[0179] characteristics (as illustrated below) wherein the strength and width of each signal defines a subpopulation as depicted in figures 7 and 10.

[0180] • 429-463 nm (blue 466 / 34) and 511-529 nm (green 520 / 18)

[0181] • 429-463 nm (blue 466 / 34) and 595-615 nm (orange 605 / 20)

[0182] • 429-463 nm (blue 466 / 34) and 663-693 nm (far-red 678 / 30)

[0183] • 511-529 nm (green 520 / 18) and (orange 605 / 20)

[0184] • 511 -529 nm (green 520 / 18) and (far-red 678 / 30)

[0185] • 595-615 nm (orange 605 / 20) and 663-693 nm (far-red 678 / 30)

[0186] In some embodiments, logic gating may be used to combine multiple parameters, for example using Boolean logic to define complex populations of droplets.

[0187] In some embodiments the gating criteria of droplet sorting may be set to only sort droplets that emit blue; and / or green; and / or orange; and / or red fluorescence within a certain range of signal intensity. It will be appreciated that size and profile of the intensity that defines the gating set for droplet sorting may be chosen according to specific properties (e.g. fluorescence properties) the droplets (and their entities) should have. For example, the fluorescence may be diffuse or localised within the droplet, or multiple wavelengths may be co-located. In one embodiment the droplet is sorted according to the ratio between the intensity of a diffuse fluorescent signal and a localised fluorescent signal.

[0188] In an embodiment the gating procedure for droplet sorting has an accuracy of at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, Preferably, the procedure has an accuracy of 90-100%, more preferably 95-100%.

[0189] In an embodiment, the determination of the optical characteristics of a droplet comprises determining the presence and / or location of at least one analyte in the droplet through the detection of at least one fluorescent signal.

[0190] The fluorescent signal detected may be localised or dispersed; embodiments may also or instead use contrast imaging (for example, phase contrast microscopy) in determining optical characteristics.

[0191] 15267483-1M&C PC934028GB

[0192] 23

[0193] A localised or point fluorescent signal refers to a localised, concentrated source of fluorescence emitted from a single point. Localised fluorescent signals have a narrow profile (figure 11, right). Examples include single fluorescent molecules, and small clusters of fluorescent molecules such as fluorescent protein complexes within a cell or on a cell surface or a bead surface.

[0194] A dispersed or diffuse fluorescent signal refers to a diffuse and ‘spread-out’ source of fluorescence which is not confined to a single point. Generally, dispersed fluorescent signals have a lower intensity than localised fluorescent signals due to a wider distribution of fluorophores (figure 11, left). Examples include homogeneous FRET assay signals, such as donor fluorophore labelled Protein A / G and acceptor fluorophore labelled secondary Protein L binding to secreted antibodies leading to the generation of FRET fluorescence occupying the whole droplet.

[0195] A contrast signal (in, for example, contrast imaging) refers to there being differences in signal intensity between two regions of a sample. Generally, contrast signals are used to detect between different areas, for example, areas where a molecule is localising in high quantities relative to the rest of a sample. Generally the contrast signal may refer to a fluorescence possessing both localized and dispersed fluorescence. Examples include localized fluorescence from stained cellular structures such as the cell membrane or nucleus and dispersed background fluorescence from the medium of a sample occupying the whole droplet (figure 11 , middle).

[0196] In an embodiment diffuse, contrast and localised signal profiles can be detected, as depicted in figure 11.

[0197] In some embodiments a diffuse and localised signal are detected simultaneously.

[0198] In one embodiment, the diffuse signal is indicative of a molecule being secreted from a biological entity. Examples of molecules that may be secreted from the biological entity include, proteins, cytokines, antibodies, carbohydrates, lipids, nucleic acids and metabolites.

[0199] In an embodiment, the localised signal is indicative of a characteristic of said biological entity such as viability and / or cell type.

[0200] 15267483-1M&C PC934028GB

[0201] 24

[0202] In a specific non-limiting embodiment, the diffuse signal may indicate antibody secretion, while concurrently, the localised signal may indicate viability. In this example, the skilled person would be able to select the highest performing cells that are both viable and are highly productive, i.e. that secrete the most antibodies.

[0203] In some embodiments the diffuse signal is indicative of at least one unbound molecule or ligand in the droplet and the localised signal is indicative of the at least one molecule or ligand binding to at least one biological entity. In the example, the skilled person would be able to measure ligand binding.

[0204] In some embodiments the diffuse signal is indicative of at least one pathogen in the droplet and the localised signal is indicative of at least one pathogen binding to or entering the at least one biological entity.

[0205] In an embodiment, a FRET (Fluorescence Resonance Energy Transfer) signal is detected. A FRET signal occurs when there is energy transfer between two fluorophores that are in close proximity, wherein there is a transfer of energy from a donor fluorophore to an acceptor fluorophore.

[0206] In an embodiment, a FRET signal is generated by the secretion of a molecule from the at least one biological entity. Preferably, a FRET signal resulting from the secretion of a molecule is concurrent to a localised fluorescent signal that indicates a characteristic of the at least one biological entity, said characteristic may be selected from at least one of viability, the presence of a cell surface marker, or another characteristic that can be measured via a localised fluorescent signal.

[0207] In a non-limiting example, a localised signal may be measured to indicate that a cell is live or viable, for example, by measuring the localised fluorescence signal emitted by Calcein, once the cell permeant dye Calcein-AM is hydrolysed by intracellular esterases. A diffuse signal may be detected concurrently, which is generated by secretion by a fluorescently labelled molecule such as an antibody from said cell. By measuring the localised viability fluorescent signal and the diffuse secretion signal simultaneously, viable cells can be sorted based upon their secretion rate to isolate the most productive viable cells.

[0208] 15267483-1M&C PC934028GB

[0209] 25

[0210] An analyte refers to a molecule or biological entity of interest; examples of analytes include but are not limited to: cells, proteins, carbohydrates, lipids, antibodies, antigens, enzymes and nucleic acids.

[0211] Embodiments of the method described herein are particularly suitable for increasing a monoclonality assurance of, for example, viable cells in a droplet,

[0212] In some embodiments a droplet is routed to a specific fluid flow based on its optical characteristics. Optionally, droplets emitting different wavelengths; and / or wavelength intensities; and / or signal durations may be routed to different fluid flows.

[0213] The method can further comprise identifying the sorted droplet for dispensing; extracting the sorted droplet from a first fluidic flow path of said fluid by transferring said sorted droplet from the first fluidic flow path into a second fluidic flow path; and ejecting the sorted droplet from the second fluidic flow path into the reservoir by applying pressure to the second fluidic flow path.

[0214] The transferring can comprise applying pressure to the sorted droplet for dispensing whilst in a portion of the first fluidic flow path and thereafter to transfer the sorted droplet from the first fluidic flow path into the second fluidic flow path and eject the sorted droplet. In embodiments the (water in oil) emulsion flows along the first fluidic flow path, through a shared region of flow path, and may then continue along the first flow path or be diverted into the second flow path by positive and / or negative (suction) pressure applied to either flow path or to the shared flow path. Thus in embodiments the dispensing may employ a branched or T-junction channel arrangement with a shared region of flow, optionally but preferably in conjunction with a droplet selection / sorting mechanism for selecting a path.

[0215] This approach may, in embodiments, allow for one or more further sorting steps of a droplet which was already sorted previously. A droplet may thereby be detected, and optionally its constituents analysed, prior to the second sorting step. The detection and / or analysis of the sorted droplet may thereby be performed at a shared flow path of the first fluidic flow path and a path at which pressure may be applied. Alternatively, or additionally, the droplet may be detected and / or analysed prior to the shared flow path.

[0216] 15267483-1M&C PC934028GB

[0217] 26

[0218] In a preferred embodiment, the second fluidic flow path from which a droplet may be ejected may comprise first and second (or further) output channels. The pressure in the second output channel may be decreased (for example, by using a pump connected to the second output channel). A droplet may then be dispensed via the first output channel by applying pressure to the sorted droplet, as outlined above, whilst in the first fluidic flow path in response to a detection and / or analysis of the sorted droplet. This may ensure that the sorted droplet is not extracted into the second output channel using the pump. If a droplet is to be dispensed from the second output channel, e.g. if the droplet is not of interest, no pressure may be applied, such that the pump sucks the droplet into the second output channel. The analysis of a droplet to determine whether it is of interest may comprise an analysis as to a defined number of target entities in the droplet, and / or a property (e.g. fluorescence level) of the entity or entities in the droplet.

[0219] In some embodiments of the method, the dispensing comprises transferring the sorted droplet from the first fluidic flow path into the second fluidic flow path, then applying pressure to the second fluidic flow path to eject the droplet. This may be preferable as the flow of other droplets in the first fluidic flow path may not be disrupted due to the application of pressure in the second fluidic flow path only. Alternatively, however, dispensing of this type may employ a “decoupling unit” as described later to transfer an unwanted droplet from the first fluidic flow path to a second, waste path when the droplet is unwanted, a droplet being dispensed from the first flow path by, for example, closing a valve in the first flow path and applying pressure to the emulsion to eject the droplet from a nozzle or the like at the end of the first flow path.

[0220] In some embodiments of the method, the ejecting comprises heating a transport fluid (such as, but not limited to, oil) in which a said sorted droplet is transported. This allows for ejecting a droplet similar to the way in which a fluid is ejected from an ink-jet printer. In a further preferred embodiment, the ejecting comprises ejecting the identified droplet via pressurised fluid ejection.

[0221] Examples of devices used to transfer a droplet from the first fluidic flow path to the second fluidic flow path include: a rotational unit, a translational unit or a decoupling unit. Whether a droplet is to be transferred from the first fluidic flow path to the second fluidic flow path may be determined prior to the droplet entering the transfer unit, and / or while a droplet is in the transfer unit. The determination as to whether a droplet is to be

[0222] 15267483-1M&C PC934028GB

[0223] 27

[0224] transferred from the first to the second fluidic flow path may further be dependent on a defined number of target entities, and / or a property of the entity or entities in a droplet (e.g. fluorescence level).

[0225] In some embodiments, the sorted droplet is dispensed into a reservoir, in alternative embodiments the sorted droplet is dispensed into an external device; examples of such external devices include mass spectrometers and sequencing devices.

[0226] The dispensing can involve ejecting a sorted droplet at a defined location in response to said determination of at least one characteristic of a droplet. A sorted droplet may thereby be ejected, for example, into a well of a microtitre plate. The specific well of the microtitre plate into which a sorted droplet is to be dispensed is thereby chosen by the previously determined optical characteristics of the droplet (which may be a defined number of entities of one type or each of a plurality of types of entities), and / or by a property, for example a physical property (e.g. fluorescence level) of the entity or entities.

[0227] In some embodiments, the method includes an incubation step. There may be any number of incubation steps depending on the specific requirements of the skilled person. For example, there may be one, two, three, four, five, six, seven, eight, nine, ten or more incubation steps.

[0228] Incubation refers to a period of time during which a sample (in this case a droplet) is kept under controlled conditions. Incubation may allow a reaction, process or growth to occur in a droplet. Conditions that are controlled during incubation can include one or more of temperature, light exposure, pH, gas exposure and humidity. It is known in the art that the duration of an incubation period varies considerably based on the specific assay being carried out and the requirements of the skilled person; typically incubation periods can range from a few minutes to several hours or multiple days.

[0229] The at least one incubation step may have a duration of between 1-60 minutes, 1-24 hours, 1-7 days or more. The duration of the incubation step should be chosen by the skilled person to account for their specific needs.

[0230] Incubation may be carried out at around 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

[0231] 15267483-1M&C PC934028GB

[0232] 28

[0233] 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100°C, however both higher and lower temperatures may be used depending on the specific requirements of the user. Incubation may be carried out between 10-80°C, 20-70°C, 30-60°C, 30-50°C or30-40°C. Preferably incubation is carried out between 35-40°C, more preferably around 37°C. The skilled person would be able to choose any incubation temperature to meet their specific needs, without being limited to these examples.

[0234] A plurality of incubation steps may be carried out under the same conditions, or a plurality of incubation steps may be carried out under different conditions, depending on the specific needs of the skilled person.

[0235] The at least one incubation step may take place before or after at least one assay is performed in the droplet (step b); and / or an incubation step may take place before or after determining the optical characteristics of the droplet (step c).

[0236] In some embodiments the optical characteristics of a droplet may be determined and said droplet may subsequently incubated at least once before having its optical characteristics determined at least once more to determine a change in at least one characteristic of the droplet.

[0237] In some embodiments the incubation step comprises at least one of growing and / or maintaining one or more biological entities, a reaction between one or more biological entities, and / or interactions between one or more biological entities.

[0238] In a preferred embodiment the method comprises use of a microfluidic cartridge, for example, as depicted in Figure 8. An example microfluidic cartridge can comprise the following:

[0239] • A sample loading chamber where a fluid sample can be loaded.

[0240] • An encapsulation chamber for encapsulation of the sample into a droplet.

[0241] • An incubation region or chamber where droplets can be held, preferably at a controlled temperature to allow incubation of the contents of the droplet. In embodiments this region is configured to hold the droplets at a greater density than when dispersed within the emulsion; this may be achieved by holding the

[0242] 15267483-1M&C PC934028GB

[0243] 29

[0244] droplets (which typically ‘float’) in an upper portion of the chamber and providing a valve or similar at the top of the chamber for controlled release of the droplets - in use the cartridge is orientated with the top of the incubation chamber upwards. Excess carrier fluid (oil) may be released to waste. Preferably, but not essentially, the incubation region is upstream of the sorting region.

[0245] • A sorting region comprising two or more sorting output channels coupled to said input channel and a droplet director to selectively direct a droplet from said sorting input channel to a selected one of said sorting output channels.

[0246] • A dispensing region where the droplet ejection mechanism ejects selected droplets into reservoirs based on their optical characteristics.

[0247] The microfluidic cartridge described above provides a non-limiting example of one possible apparatus to work the described methods, however other appropriate apparatus may be used.

[0248] The cartridge can be configured to interface with a microdroplet processing system -that is in embodiments the cartridge can be attached into and released from the microdroplet processing system, which provides sensing and control functions. In embodiments the microdroplet processing system provides temperature control for the incubation chamber and a droplet property sensing means, i.e. at least one detector to determine the optical characteristics of the droplet for droplet sorting. Optionally, the microdroplet processing system may also provide relative motion between the dispenser output channel and reservoir wells to collect the droplets.

[0249] In an example embodiment the cartridge generally has the form of a flat plate bearing microfluidic channels, one or more holding regions, valves and the like. It is preferably substantially optically transparent, and may be fabricated from a range of plastic materials, for example polydimethylsiloxane (PDMS) or cyclic olefin polymer or copolymer (COP or COC). The cartridge may then be mounted vertically or at a suitable angle in the system / instrument with the dispenser output channel directed downwards towards a multi-well or microtitre plate. The instrument may then move the cartridge and / or plate to direct the output channel into a selected well.

[0250] In some embodiments the cartridge is provided with a plurality of fluidic connections, which may be made automatically when the cartridge is inserted into the instrument.

[0251] 15267483-1M&C PC934028GB

[0252] 30

[0253] Generation of the emulsion may be performed on-cartridge or off-cartridge. For example, in embodiments the cartridge may be provided with reservoirs along one edge (so that these are in the correct orientation when the cartridge is vertical), to hold oil and aqueous medium (such as water and growth medium) for droplet-on-chip droplet generation.

[0254] An example workflow using the described method of comprises the steps of:

[0255] • Add cells and reagents into the sample loading portion of a cartridge or vessel.

[0256] • Generate picodroplets to encapsulate cells.

[0257] • Incubate picodroplets and optionally allow the cells to secrete biologies.

[0258] • Measure fluorescence in at least four channels and sort each picodroplet independently according to its optical characteristics.

[0259] • Measure fluorescence in at least four channels and image selected picodroplets for monoclonality and dispense into a chosen vessel ready for any downstream experimentation. The fluorescent signals measured at the dispensing step may be stored together with images and the location of well of microtiter plate. This allows offline checking for monoclonality and the characteristics of dispensed cells.

[0260] IThe determined optical characteristics of a droplet can indicate the outcome of at least one assay; examples of such assays include a protein detection assay, cell detection assay, secretion assay, uptake assay, cytotoxicity assay, stability assay, localisation assay, enzyme assay, binding assay, proliferation assay, cell death assay, immunoassay, or a tolerance assay.

[0261] The method can be used to determine at least one characteristic of at least one cell, including but not limited to any one of or any combination of: protein localisation, protein internalisation, cell-cell interactions, cell surface marker expression, protein-protein interactions, proliferation, molecule secretion, molecule uptake, changes in membrane potential and intracellular calcium levels amongst others.

[0262] In an embodiment the method can be used in a molecular biology assay, for example PGR and nucleic acid sequencing,

[0263] The described methods can be used to detect the outcome and / or at least one characteristic of any assay that uses fluorescence.

[0264] 15267483-1M&C PC934028GB

[0265] 31

[0266] In embodiments the assay is a multiplex assay. A multiplex assay simultaneously measures multiple analytes and / or characteristics of a single sample. Multiplex assays offer several advantages such as increased throughput and low sample volume requirements, and more information or combination of information for targeted or precision selection of cells with desired characteristics.

[0267] Implementations of the method provide a multimodal means to multiplex an assay whereby determination of characteristics and optional sorting based on the determination step may comprise any of the following: fluorescence analysis, imaging, scatter analysis, phase analysis and impedance analysis.

[0268] In some embodiments the assay comprises determining the presence of at least one molecule on the surface of a bead, or a cell and / or inside a cell based on the emission of at least one localised fluorescent signal and / or at least one dispersed fluorescent signal and / or at least one contrast fluorescent signal.

[0269] In some embodiments a fluorescent signal indicates the binding of at least one molecule to a bead. In alternative embodiments a fluorescent signal indicates the binding of at least one molecule to the surface of a cell. The fluorescent signal may be a localised fluorescent signal, dispersed fluorescent signal or a contrast fluorescent signal.

[0270] In some embodiments the interaction of at least two molecules is detected based on the emission of at least one fluorescent signal, preferably a localised and / or diffuse fluorescent signal.

[0271] In a non-limiting example, the method can be used to detect uptake and / or secretion of molecules by a cell.

[0272] In an embodiment, the method comprises detecting secretion of at least one target protein from a single cell based on the emission of at least one fluorescent signal. Examples of target proteins that may be secreted from a cell include but are not limited to: antibodies, hormones, enzymes and cytokines. In a non-limiting example, a fluorescently labelled single cell is encapsulated in a droplet with at least one stimulus to initiate secretion of a target molecule; the secreted molecule may be fluorescently

[0273] 15267483-1M&C PC934028GB

[0274] 32

[0275] labelled itself whereby increased fluorescence intensity of the droplet indicates secretion; or alternatively, the droplet may comprise a reagent for detection of the secreted molecule such as a fluorophore conjugated antibody.

[0276] In a non-limiting example a fluorescently labelled or dyed cell of interest that secretes antibodies may be provided in a droplet with a fluorescently labelled or dyed target cell and a fluorescently labelled secondary antibody. In this example, when the cell of interest secretes antibodies, said antibodies bind to antigens on the target cell, this interaction can then be identified with the fluorescently labelled secondary antibody that binds to the antibody-antigen complex and emits a fluorescent signal on the condition of the formation of this complex. The method can be used to detect the distinct emission spectra of the cell of interest, the target cell, and the secondary antibody bound to the antibody-antigen complex to determine whether the cell of interest exhibits the characteristic of antibody secretion.

[0277] The method can be used to sort cells according to their productivity, for example, by level of antibody secretion (and hence intensity of fluorescence).

[0278] In some embodiments the method involves determining whether a cell is apoptotic based on the emission of at least one fluorescent signal. In a non-limiting example, the method can provide discrimination between live and dead cells based on at least one of membrane integrity, esterase activity, metabolic activity and structural segmentation. For example, plasma membrane integrity can be determined by ethidium homodimer-1 which enters apoptotic cells with compromised plasma membranes to bind DNA and emit a red fluorescence; meanwhile live cells can be identified by Calcein AM, a fluorogenic cell-permeant dye that is converted to a green fluorescence after interaction with intracellular esterases.

[0279] In a further non-limiting example C12 Resazurin / SYTOX Green can be used in viability assays to identify metabolically active cells from dead and damaged cells. In metabolically active cells, Ci2-resazurin is reduced to Ci2-resorufin to emit a red fluorescence. Ci2-resazurin is the lipophilic version of resazurin that is more permeable to live cells and the Ci2-resorufin product is better retained in cells. The SYTOX Green dye enters the compromised plasma membrane of dead and dying cells, and binds DNA, resulting in a green fluorescence. Cells that have undergone damage, will display a

[0280] 15267483-1M&C PC934028GB

[0281] 33

[0282] reduced red and green fluorescence. The method can determine all of these characteristics in a single droplet.

[0283] In a non-limiting example, the method can be used to isolate cell populations based on antibody-antigen interactions. For example, a starting population of cells may be subjected to screening based on a single conserved region (e.g. human FC receptor expression). Cells exhibiting this conserved feature may then be further screened for a single target such as an antigen of interest, or alternatively multiple targets such a plurality of antigens in a multiplex assay. In a non-limiting example can be used for cellline development, for example according to the specificity of antibody antigen interactions.

[0284] In embodiments the method can be used to sort cells according to the specificity of antibody antigen interactions.

[0285] In embodiments the method can be used to detect molecules secreted by a cell and / or molecules on the cell surface, and / or intracellular molecules. In embodiments the method can be used to detect combinations of each of these molecules (for example, intracellular molecules and cell surface molecules). This can be based on different fluorescent labels, and / or localisation of the fluorescent signals within a droplet.

[0286] In an embodiment, the method can be used to detect one or more molecule such as a protein, lipid or carbohydrate in a droplet whereby a plurality of molecules are fluorescently labelled and can all be detected in a single droplet according to a method described herein.

[0287] In an embodiment, the method can be used to detect protein modifications such as phosphorylation, glycosylation, nitrosylation, methylation, acetylation and lipidation, for example, by using fluorescent dyes that specifically stain post translational modifications. For example a plurality of post translational modifications can be identified in a single droplet.

[0288] In an embodiment, the method can be used to detect interactions between multiple cellular components such as proteins, lipids and carbohydrates and / or association of molecules at the cell surface. For example, if one cellular component is labelled with a

[0289] 15267483-1M&C PC934028GB

[0290] 34

[0291] donor fluorophore and another cellular component is labelled with an acceptor fluorophore when these two cellular components interact energy transfer will occur between the donor and acceptor fluorophores leading to changes in fluorescence emission. Using the method a plurality of interactions between different components can be detected in a single droplet.

[0292] In an embodiment, the method can be used to distinguish multiple different cells. For example, multiple cells may be contained in the same droplet whereby different fluorophores are employed to label specific cell surface markers and / or intracellular molecules that are unique to each cell type.

[0293] In a non-limiting example, the method can be used to detect presence of disease and characteristics of said disease that may inform treatment plans. For examples, a breast cancer cell may be identified by presence of a tumour marker such as CA 15-3, while oestrogen receptor status, progesterone receptor status and HER2 status may also be detected in a single assay facilitated by the use of different fluorescent markers to detect each characteristic in a single droplet.

[0294] In a non-limiting example, the method can be used to detect a cell’s progression through the cell cycle. For example, the fluorescence ubiquitination cell cycle indicator (FUCCI) system, based on the expression of the cell cycle oscillators Cdt1 and geminin tagged with different fluorescent proteins, marks cells in G1 or S / G2 / M phases, respectively. In this example, cells at different stages of the cell cycle can be detected in the same single droplet by using the method.

[0295] In an embodiment, the method can be used to detect intracellular and / or extracellular signalling.

[0296] In a non-limiting example, the method can be used to monitor enzyme assays. For example, the formation of a fluorescent product from a substrate that does not fluoresce or fluoresces at a different wavelength to the product; and vice versa. The formation of enzyme-substrate complexes may also be detected as well as the action of fluorescently labelled competitive and non-competitive enzyme inhibitors. Using the method the turnover of a substrate and action of inhibitors can be monitored simultaneously in a single droplet.

[0297] 15267483-1M&C PC934028GB

[0298] 35

[0299] In a non-limiting example, the method can be used to monitor tolerance assays whereby at least one type of molecule is alongside at least one type of cell in a droplet, preferably the tolerance of multiple cells or cell types may be assayed in a single droplet.

[0300] It should be understood that due to the multiplexing ability of the method the above assays may be combined for analysis in a single droplet.

[0301] Implementations of the described method apply multiplexing capability to multifluidics technology. The method can be implemented using an instrument, in particular droplet microfluidic platform, equipped with multiple lasers and detectors for automated singlecell analysis and isolation with multiplexing capabilities. Expanding the number of excitation lasers and detection channels facilitates fluorescence multiplexing, enhancing assay specificity and enabling more targeted selection and high-throughput screening. The instrument can perform spectral multiplexing within droplets, useful for applications such as antibody discovery (AbD) and cell line development (CLD).

[0302] As exemplified below, the instrument can perform several multiplexed biological assays utilising simultaneous analysis of multiple parameters. Here it is shown that a droplet microfluidic platform equipped with multiple excitation light sources and detectors is capable of identifying multiple characteristics of droplets (including cells within those droplets) in multiplexed assays.

[0303] While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods as well as the best mode of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0304] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is

[0305] 15267483-1M&C PC934028GB

[0306] 36

[0307] to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0308] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments that are described.

[0309] Further aspects of the techniques are now described in the following non-limiting examples.

[0310] EXAMPLES

[0311] Example 1

[0312] Introduction and Aims

[0313] The Applicant has developed a microfluidic instrument using droplet technology, equipped with multiple lasers and detectors for automated single cell analysis and isolation with advanced multiplexing capabilities. This example demonstrates the potential of spectral multiplexing within droplets, highlighting its advantages for applications such as antibody discovery and cell line development.

[0314] To validate the performance and capabilities of the droplet-based microfluidic instrument multiplexed biological assays were designed utilising simultaneous analysis of multicolour parameters. This example shows that the instrument is capable of identifying both secreted and cellular markers in fluorescence-based multiplexed assays. Cells were identified and isolated based on their productivity (IgG secretion) as well as antigen specificity (antibody-antigen binding), while maintaining accuracy. In-line with the antibody discovery workflow, the instrument is capable of highly accurate detection, analysis and isolation of rare, antibody secreting, single cells from a heterogenous starting population.

[0315] Some example implementations of the described techniques demonstrate the detection and isolation of subpopulation of cells based on a fluorescent cell label, and their secretion of IgG measured by Forster Resonance Energy Transfer (FRET) signal. They also demonstrate isolation of rare antibody-secreting cells based on the binding of a secreted antibody to a surface-expressed antigen in a multiplexed assay.

[0316] 15267483-1M&C PC934028GB

[0317] 37

[0318] Droplet-based microfluidic instrument

[0319] An example of the instrument a single cell analysis system that leverages picodroplet technology to functionally analyze encapsulated single cells follows. Further details of an example instrument that can be adapted for use with the presently described techniques can be found in WO2016 / 193758. Briefly, the droplet-based microfluidic instrument uses microfluidics to encapsulate single cells in picodroplets and transport picodroplets through a series of channels on a microfluidic cartridge. Cells are first encapsulated in picodroplets, incubated, and then transported to the analysis / sorting area of the cartridge. Analysis of picodroplets is conducted using 4 independently controllable lasers and 4 PMT detectors with excitation at 405, 488, 561, and 638 nm and detection at 446 / 34, 520 / 15, 605 / 20, 678 / 30 nm. Design of the optic channel generates a precise laser line which, can profile picodroplet contents to characterize localized, diffused, or contrasting fluorescent signals. Fluorescent signal detection is visualized using the instrument control software, and picodroplets can be sorted by users. Notably, different modes are used for optimal detection of either localized signal (surface-binding or cell staining, detected by fluorescence peak) or dispersed fluorescence (FRET signal, detected by fluorescence average). Sorted picodroplets are then transported through the cartridge to the dispensing area. Fluorescent analysis is conducted again using the same 4 lasers and detectors, and data is visualized using the instrument control software. Specific populations of picodroplets can be selected for dispensing. Selected picodroplets are then individually dispensed into a microtitre plate and the instrument captures 5 images of each dispensed picodroplet for monoclonality assurance.

[0320] Cell Staining

[0321] Three different cell dyes were used for staining: eBioscience™ Cell Proliferation Dye eFluor™ 450, CellTracker™Orange CMRA (CT- Orange), eBioscience™Cell Proliferation Dye eFluor™670. These dyes were obtained from Thermo Fisher Scientific (Leicestershire, UK). Briefly, the dyes were reconstituted in DMSO to a stock concentration of 10 mM, 2.5 mM and 5 mM, respectively. The dyes were diluted in basic media (in the absence of FBS) to a final concentration and each dye incubated at 37°C with 1.0X106of pre-washed cells. The staining was stopped by diluting the reaction with x10 volumes of cold complete media and centrifugation. The cells were then resuspended in 1 mL of encapsulation media (Complete culture media, 16% Opti-prep

[0322] 15267483-1M&C PC934028GB

[0323] 38

[0324] (Merck Life Sciences, Dorset, UK), 10% PF68 (Thermo Fisher) in desired cells concentration.

[0325] FRET assay detection of pre-stained cells

[0326] Two different hybridoma cell lines, each at 0.5x106cells / mL were stained with either eBioscience™ Cell Proliferation Dye eFluor™450 (eFL450) or eBioscience™ Cell Proliferation Dye eFluor™670 (eFL670). Stained cells were then mixed with FRET probes (Donor: anti-mouse IgG Dylight488 (DL488), Acceptor: anti-mouse IgG Dylight594 (DL594)) before being loaded into droplet-based microfluidic instrument. Cells were encapsulated and the picodroplets incubated at 37°C for 2 hours in the instrument, to allow antibody secretion. Picodroplets were gated during sorting for cellular dye and / or FRET signal. Picodroplets were dispensed into 96-well plates and scanned for the presence and fluorescence of cells on an NYONE® scanner (Synentec GmbH, Germany).

[0327] Surface antigen-binding of recombinant and secreted antibodies

[0328] A binding assay was performed to detect the binding of antibodies to their specific target antigen expressed on the surface of cells, as described in Figure 1. Accordingly, two types of cells were stained and prepared for co-encapsulation in picodroplets: the target A431 cells (over-expressing EGFR) were stained with eBioscience™Cell Proliferation Dye eFluor™670, and hybridoma cells (secreting anti-human EGFR antibodies; ECCAC) were stained with eBioscience™Cell Proliferation Dye eFluor™450. Next, 3x106target cells were mixed with antibody secreting cells (0.01x106 / mL - 0.3x106 / mL) in 1 mL of encapsulation media (Complete culture media, 16% Opti-prep, 10% PF68) together with anti-mouse DL488-conjugated antibodies (Abeam). Where stated, the binding of Cetuximab (Absolute Antibody) to eFluor™670 -stained target cells was detected using anti-human DL488-conjugated antibody (Abeam). For spike-in experiments, a different hybridoma cell-line, secreting anti-human TNF-a antibodies (ECCAC) was stained with Cell Tracker Orange CMRA, and 0.3x106cells (for 1:1 ratio) or 1x106cells (for 1:100 ratio) were mixed with the sample. The sample was then loaded onto the instrument where cells were encapsulated into picodroplets and incubated for 2 hours at 37°C. Positive droplets were sorted and dispensed into 96-well plates and analysed on an NYONE® scanner for validation.

[0329] Validation of cell isolation using microscopy

[0330] 15267483-1M&C PC934028GB

[0331] 39

[0332] NYONE® scanner was used to validate correct detection and isolation of the desired populations of picodroplets, whereby all dispensed 96-well plates were scanned at 4 different wavelengths to detect the fluorescence of the dispensed cells. The settings of NYONE® scanner use are summarised below:

[0333]

[0334] Statistical analysis

[0335] Statistical analysis was performed on images obtained as described, from dispensing of single picodroplets into 96-well plates (two plates generated per assay). All assays were repeated at least twice. For analysis of secreting cells (Figure 2) the following formula was used :

[0336] wells with eFluor450 + cells

[0337] Isolation accuracy % = - - - - - - - — X 100

[0338] total No. cell occupied wells

[0339] For the surface binding assay (Figure 4) the analysis was based on:

[0340] , , , . wells with eFluor450 AND (eFluor670 cells stained with DL488) Isolation accuracy % = - total No.cell occupied wells X 100

[0341] OR for surface-binding assay with spiking-in (Figure 5) the analysis was based on: , , , . wells with eFluor450 AND (eFluor670 cells stained with DL488) Isolation accuracy % = - total No.cell occupied wells X 100

[0342] Isolation of rare antibody secreting cells from a mixed cell population requires accuracy in detection, sorting and dispensing. Antibody secreting cells may be isolated based on the presence of secreted IgG, or the specific binding of secreted IgG to its antigen. To

[0343] 15267483-1M&C PC934028GB

[0344] 40

[0345] evaluate the ability of the droplet-based microfluidic instrument in successfully isolating these cells, we determined:

[0346] • Isolation of single cells when droplets were sorted using single or sequential gating. In this assessment both secretion and cell staining were used to successfully gate on the population of interest.

[0347] • Accuracy in detecting antibody binding to its surface-expressed antigen. Using recombinant antibody, we show the detection specificity of antibody binding to target cells.

[0348] • Accuracy in isolation of rare cells of interest by multiplexing antigen-binding detection with labelling of ASC and target cells. We show that when two different antibody secreting cells (ASC) are present in the same culture, it is possible to isolate the cells of interest with high accuracy based on the binding of the secreted antibody to its target cell.

[0349] Detection and isolation of cells based on antibody secretion and cell-based staining

[0350] To determine the isolation accuracy of ASC in the droplet-based microfluidic instrument, based on the presence of secreted antibody, we used mouse IgG 1 - secreting hybridoma cells together with FRET probes (Figure 2A). The transfer of energy from Donor probe to Acceptor probe in the presence of their target molecule (mouse IgG) will result in an increase in Acceptor fluorescence signal and reduced Donor signal (10). As shown in Figure 2A, the cells were stained with either eFluor™450 oreFluor™670 membrane dye and mixed with mouse-specific FRET probes (Donor labelled with DL488 and Acceptor with DL594). The resulting population of cells was loaded onto the droplet-based microfluidic instrument and IgG secretion was detected using the FRET signal. As shown in Figure 2B, hybridoma cells stained with either eFluor™450 or eFluor™670 were detectable and easily differentiated. Following a 2 hour incubation to allow IgG secretion, the FRET-positive population, recognized by a shift in Donor (reduced) and Acceptor (increased) fluorescence signal, was identifiable (Figure 2C). A single gate on the FRET+ population during sorting, resulted in droplets containing either eFluor™450-stained, eFluor™670- stained or a combination of the two stained cells being dispensed, as expected (Figure 2D). To show that sequential gating could be used to isolate a specific ASC population, droplets were initially gated for FRET signal followed by gating on the eFluor™450 stained cells (Figure 2E). The combination of these selection criteria, resulted in isolation of a single population of cells, the eFluor™450 stained cells only (Figure 2F). Single droplets were dispensed into 96-well plates and the fluorescence

[0351] 15267483-1M&C PC934028GB

[0352] 41

[0353] signal of the isolated cells was validated by fluorescence microscopy. As shown in Figure 2G, droplets gated solely on FRET positive population (Figure 2C), showed an expected distribution of either eFluor™450, eFluor™670 stained cells, or both (for polyclonal droplets) (Figure 2G and Figure 2I). Droplets sequentially gated for FRET and eFluor™450 however (Figure 2E), contained eFluor™450 stained cells only (100%) (Figure2G and Figure 2I). These data demonstrate accuracy of isolating cell populations using single or sequential gating during sorting (Figure 2I).

[0354] Detection of antibody binding in target cell

[0355] Next we wished to determine whether the binding of antibody to its antigen, expressed on the surface of a target cell, can be accurately detected in the droplet-based microfluidic instrument. To this end the target cells (A431 cell line), expressing EGFR, were stained with eFluor™670 and mixed with Cetuximab (anti-EGFR hlgG) and detection antibody (DL488- labelled anti-hlgG antibody).

[0356] Droplets were gated for dual expression of eFluor™670 (target cells) as well as the increase in DL488 peak signal. Data show that in the absence of Cetuximab, the background binding of the detection antibody remains low (Figure 3A). The presence of Cetuximab, however shifts the DL488 signal in 60% of total droplets (Figure 3B, equivalent to 80% of droplets containing cells).

[0357] Data shown here demonstrates the detection specificity of antibody binding to its surface antigen on a target cell.

[0358] Detection and isolation of ASC based on antigen specificity, in the presence of target cells

[0359] To determine whether presence of ASC could be detected in droplets based on binding of secreted antibody to its surface- expressed antigen, we used a hybridoma cell line that secrets anti-human EGFR mouse antibody. To demonstrate antibody binding to its target antigen, the hybridoma cells were stained with eFluor™450 and mixed with eFluor™670- stained A431 target cells (Figure 4A). Using the mouse- specific FRET probes, we confirmed that more than 50% of hybridoma cells secreted detectable levels of antibody following a 2 hour incubation in the droplet-based microfluidic instrument. Notably, staining hybridoma cells with eFluor™450 did not impair their secretion

[0360] 15267483-1M&C PC934028GB

[0361] 42

[0362] capability, as they exhibited a similar FRET profile to their unstained counterparts (data not shown).

[0363] The binding of the secreted antibody to the target cells was detected using an anti-mouse lgG-DL488 antibody. The hybridoma cells, target cells and detection antibody were loaded into the droplet-based microfluidic instrument and incubated for 2 hours to allow secretion and binding of the antibody to its target antigen. Following the incubation, the droplet population was gated during sorting for the presence of hybridoma cells (eFluor™450+) and increase in detection antibody peak signal (DL488). A second gating step identified droplets containing target cells (eFluor™670+, Figure 4B). Using this sequential gating strategy, single droplets were isolated and dispensed into 96-well plates, and the accuracy of isolation was validated with fluorescence microscopy. Representative images from a well are shown in Figure 4C.

[0364] As expected, the dispensed droplet showed the presence of secreting cells, target cells and DL488 signal. It is worth noting that the DL488 signal is only seen on the target cells, to which the secreted anti-EGFR antibody would specifically bind (Figure 4C).

[0365] These data clearly demonstrate the droplet-based microfluidic instrument’s capability to isolate secreting cells of interest by accurately detecting antigen-specific binding of the secreted antibody.

[0366] Detection and isolation of ASC based on antigen- specificity in AbD related workflow

[0367] To determine the performance of the system in an AbD- related workflow, we set up an assay, whereby a small number of secreting cells of interest are present within a larger population of irrelevant secreting cells. To this end we mixed the anti-EGFR antibody secreting hybridoma cells (stained with eFluor™450) with an anti-TNF-aAb secreting hybridoma cell line (stained with CT- Orange) (Figure 5A). A431 target cells were stained with eFluor™670 as before and surface binding of the secreted antibody to its specific antigen was detected in the droplet-based microfluidic instrument as described in Figure 4. The two types of secreting cells were mixed in two different ratios, either in equal amounts (ratio 1:1, Figure 5B), or at 1:100 (anti-EGFR : anti-TNF-a secreting cells, Figure 5C). The latter imitates an AbD workflow, where the ASC of interest are rare.

[0368] 15267483-1M&C PC934028GB

[0369] 43

[0370] As depicted in Figure 5B and Figure 5C, two gating strategies were applied sequentially. First gating ensured the inclusion of picodroplets containing target cells (eFluor™670+) as well as an increase in detection antibody binding (DL488+). The second gating ensured the presence of the anti-EGFR antibody secreting cells (eFluor™450+) but excluded the irrelevant secreting cells (CT- Orange+). As expected, the 1:100 dilution of the relevant secreting cells resulted in a lower number of positive binding events compared to the 1:10 dilution (4% vs 0.4%, first gating, Figure 5B and Figure 5C).

[0371] However, the inclusion of a second gating, led to a similar enrichment of the relevant secreting cells (Figure 5B and Figure 5C). Importantly, the isolation accuracy, determined by presence of the expected combination of cells and fluorescent signal (antigen binding of the secreted antibodies), remained high even in the presence of increased numbers of irrelevant antibody-secreting cells (Figure 5F).

[0372] As demonstrated in Figure 5B and Figure 5C, following the first gating, a population of droplets were positive for both CT- Orange stained cells, as well as the eFluor™450+cells. To ascertain that these droplets were indeed positive for the relevant antibody secreting cells (eFluor™450 stained cells) and would therefore account for the positive binding of the detection antibody, we further analysed the sorting data. Indeed, further analyses of droplets positive for both the CT- Orange stained cells as well as detection antibody also showed the presence of eFluor™450 positive cells (Figure 5D;

[0373] 99.2%). Microscopy data of this population following dispensing, further confirmed the co-encapsulation of relevant and irrelevant ASC together with DL488- labelled target cells (Figure 5E).

[0374] The data presented above demonstrates the multiplexing in picodroplet capability of the droplet-based microfluidic instrument and its ability to detect and isolate cells of interest with high degree of accuracy. We show that this multi-laser instrument, is capable of sequential gating, accurate sorting and dispensing based on fluorescent signal detected from encapsulated cells or secreted antibody. Moreover, we show that in more complex secretion assays, where relevant cells are present only as a fraction of irrelevant cells, it is possible to accurately isolate droplets containing rare cells of interest based on antibody-antigen binding.

[0375] Detection accuracy:

[0376] 15267483-1M&C PC934028GB

[0377] 44

[0378] The platform reliably detected localised (antibody-antigen binding) as well as dispersed (IgG secretion, FRET signal) fluorescence. When combined with fluorescently stained cells, the platform accurately distinguished between different cell types based on their specific staining.

[0379] Sorting accuracy of a gated population:

[0380] The platform demonstrated remarkable sorting accuracy and enrichment of target populations following single or sequential gating.

[0381] Multiplexing capability:

[0382] The droplet-based microfluidic instrument demonstrates multiplexing capability by assessing and isolating rare secreting cells based on simultaneous detection of 4 different cellular markers.

[0383] Example 2- Differentiating FRET+ viable cells from apoptotic or dead cells, in picodroplets

[0384] Detecting and excluding dead / dying cells can increase efficiency of cell isolation, increasing success in hit selection. A Cyto-Cellect® assay kit from Sphere Fluidics (https: / / spherefluidics.com / product / cyto-cellect-fc-fusion-assay-kit / ) was used together with a viability dye (Fig 7a & Fig 7b), we show that it is possible to detect both FRET signal as well as dying / dead cells in picodroplets (Fig 7c) using the method. Although these cells are undergoing cell death, they do appear as FRET+ (Fig 7d) and exclusion of these will improve outgrowth of cells.

[0385] Bibliography

[0386] 1) Paul J. Mills and Christine T. Peterson, 2016. Multiplexing and Beyond in Biobehavioral Research.

[0387] 2) Junfang Chen and Emanuel Schwarz, 2017. Opportunities and Challenges of Multiplex Assays: A Machine Learning Perspective.

[0388] 3) Chen J. et al., 2017. The Utility of Multiplex Assays for Identification of Proteomic Signatures in Psychiatry.

[0389] 4) Edwards B.S. et al., 2007. High-throughput cytotoxicity screening by propidium iodide staining.

[0390] 15267483-1M&C PC934028GB

[0391] 45

[0392] 5) PO Krutzik and GP Nolan, 2006. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signalling profiling.

[0393] 6) Robert A.B. et al., 2021. Development and Validation of a Multiplex, Bead-based Assay to Detect Antibodies Directed Against SARS-CoV-2 Proteins.

[0394] 7) Edwards B.S. et.al., 2007. High-throughput flow cytometry for drug discovery.

[0395] 8) Brouzes E. et al., 2009. Droplet Microfluidic Technology for Single-Cell High-Throughput Screening.

[0396] 9) Dimitris Josephides et al., 2019. Cyto-Mine®: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance.

[0397] Some further aspects of the invention will now be described with reference to the following clauses:

[0398] Clause 1. A method of determining at least one characteristic of a droplet based on the outcome of at least one assay within said droplet; the method comprising:

[0399] a. providing a droplet comprising at least one biological entity to a fluid flow; b. performing at least one assay in the droplet;

[0400] c. determining optical characteristics of the droplet by exposing the droplet to a plurality of distinct excitation light sources and detecting a signal emitted in response to the distinct excitation light sources, wherein the distinct excitation light sources are selected to stimulate distinct emission spectra. Clause 2. The method of clause 1 wherein step a) comprises providing a fluid sample comprising at least one biological entity; and

[0401] preparing a droplet from said sample wherein the droplet comprises at least one biological entity.

[0402] Clause 3. The method of any preceding clause wherein the plurality of excitation light sources comprise at least one laser.

[0403] Clause 4. The method of any preceding clause wherein the signal is detected using at least one optical sensor.

[0404] Clause 5. The method of any preceding clause wherein step c) comprises measuring the intensity and / or duration of a plurality of signals.

[0405] 15267483-1M&C PC934028GB

[0406] 46

[0407] Clause 6. The method of any preceding clause wherein step c) takes place when the fluid flow path of the droplet passes an excitation light source and a detector.

[0408] Clause 7. The method of any preceding clause wherein step c) detects multiple emitted signals with multiple detectors.

[0409] Clause 8. The method of clause 7 wherein step c) takes place multiple times in series or simultaneously as the fluid flow path of the droplet passes multiple excitation light sources and multiple detectors.

[0410] Clause 9. The method of any preceding clause wherein the method further comprises d. sorting said droplet dependent on the outcome of the determination step; and e. dispensing the sorted droplet into a reservoir, wherein said dispensing comprises, after said sorting:

[0411] selecting a fluid flow path for fluid containing the sorted droplet; and ejecting the sorted droplet from the selected path

[0412] Clause 10. The method of any preceding clause wherein step c) comprises sequentially comparing pairs of detection signals from different optical sensors to determine at least one characteristic of a droplet.

[0413] Clause 11. The method of clause 9 comprising a plurality of determination steps and a single sorting step.

[0414] Clause 12. The method of clause 9 comprising a plurality of sequential determination and sorting steps.

[0415] Clause 13. The method of clause 12 wherein the sequential determination and sorting steps further comprise classifying the sorted droplets according to the intensity and / or duration of a signal.

[0416] Clause 14. The method of any of clauses 9 or 11-13 wherein step d) comprises sorting said droplet according to the profile of said detected signals in step c).

[0417] 15267483-1M&C PC934028GB

[0418] 47

[0419] Clause 15. The method of any of clauses 9 or 11-14 wherein the sorting step comprises routing a droplet to a specific fluid flow path based on the optical characteristics of said droplet.

[0420] Clause 16. The method of any preceding clause wherein step c) comprises determining the presence and / or location of an analyte within a droplet through the detection of a fluorescent signal.

[0421] Clause 17. The method of any preceding clause wherein at least one localised and / or dispersed fluorescent signal is detected.

[0422] Clause 18. The method of any preceding clause further comprising at least one incubation step.

[0423] Clause 19. The method of clause 18 comprising a first incubation step followed by at least one subsequent incubation step.

[0424] Clause 20. The method of clause 18 or 19 comprising an incubation step before, after or during step b).

[0425] Clause 21. The method of any of clauses 18-20 comprising an incubation step before, after or during step c).

[0426] Clause 22. The method of any of clauses 18-21 wherein the incubation step comprises at least one of growing and / or maintaining one or more biological entities, a reaction between one or more biological entities, and / or interactions between one or more biological entities.

[0427] Clause 23. The method of any of clauses 9 or 11-15 wherein the dispensing step comprises dispensing a sorted droplet at a defined location in response to the determination of the optical characteristics of a droplet.

[0428] Clause 24. The method of clause 23 wherein the droplet is dispensed into an external device.

[0429] 15267483-1M&C PC934028GB

[0430] 48

[0431] Clause 25. The method of any preceding clause wherein the assay is selected from at least one of a protein detection assay, cell detection assay, secretion assay, uptake assay, cytotoxicity assay, stability assay, localisation assay, enzyme assay, binding assay, proliferation assay, cell death assay, immunoassay, or tolerance assay.

[0432] Clause 26. The method of any preceding clause wherein the assay is a multiplex assay wherein a plurality of excitation and emission spectra are used to detect a plurality of analytes in a droplet.

[0433] Clause 27. The method of any preceding clause wherein the assay is a multiplex assay wherein a plurality of excitation and emission spectra are used to detect a plurality of characteristics of at least one analyte in a droplet.

[0434] Clause 28. The method of any preceding clause wherein the assay comprises determining the presence of at least one molecule on the surface of a bead, or a cell and / or inside a cell based on the emission of at least one localised fluorescent signal and / or at least one dispersed fluorescent signal.

[0435] Clause 29. The method of any preceding clause wherein the assay comprises detecting an interaction between at least two molecules based on the emission of at least one localised fluorescent signal.

[0436] Clause 30. The method of any preceding clause wherein the method comprises detecting secretion of at least one target protein from a single cell based on the emission of at least one fluorescent signal.

[0437] Clause 31. The method of any preceding clause wherein the method comprises detecting the secretion of at least one antibody from a single cell and the binding of said antibody to an antigen based on the detection of at least three different signals.

[0438] Clause 32. The method of any preceding clause wherein the method comprises determining whether a cell is apoptotic based on the emission of at least one fluorescent signal.

[0439] 15267483-1M&C PC934028GB

[0440] 49

[0441] Clause 33. The method of any preceding clause wherein at least four different characteristics of at least one cell or molecule are determined based on the emission of at least four different signals.

[0442] Clause 34. The method of any preceding clause wherein the biological entity is selected from at least one of a cell, a protein, a carbohydrate, a lipid, an antibody, an antigen, an enzyme or a nucleic acid.

[0443] Clause 35. An apparatus suitable for carrying out the method of any preceding clause wherein said apparatus comprises:

[0444] at least one microfluidic input channel;

[0445] a plurality of fluid flows;

[0446] a plurality of distinct excitation light sources;

[0447] a plurality of detectors for detecting a plurality of signals in response to the distinct excitation light sources, wherein the distinct excitation light sources are selected to stimulate distinct emission spectra;and

[0448] at least one microfluidic output channel.

[0449] Clause 36. An apparatus according to clause 35 further comprising a droplet formation device for preparing a droplet from a fluid which contains at least one biological entity.

[0450] Clause 37. An apparatus according to clause 35 or 36 further comprising a droplet sorting device for sorting said droplet dependent on an outcome of said determination.

[0451] Clause 38. An apparatus according to any of clauses 35 to 37 further comprising a dispensing unit for dispensing said sorted droplet from a fluidic flow path.

[0452] Clause 39. An apparatus according to clause 38 wherein the dispensing unit comprises an isolation unit for isolating a said sorted droplet from said fluidic flow path and a guide for guiding a said isolated droplet into a second fluidic flow path.

[0453] Clause 40. An apparatus according to any of clauses 35-39 wherein the plurality of excitation light sources comprise a plurality of lasers.

[0454] 15267483-1M&C PC934028GB

[0455] 50

[0456] Clause 41. An apparatus according to any of clauses 35-40 wherein the plurality of detectors comprise a plurality of photomultiplier tubes.

[0457] Clause 42. An apparatus according to any of clauses 35-41 wherein the apparatus comprises at least one optical filter.

[0458] Clause 43. An apparatus according to any of clauses 35-42 wherein the apparatus further comprises a droplet storage unit for storing said isolated droplet.

[0459] Clause 44. An apparatus according to any of clauses 35-43 wherein the apparatus further comprises an incubator for incubating said droplet.

[0460] Clause 45. An apparatus according to any of clauses 35-44 wherein said dispensing unit comprises a high-pressure unit for dispensing said droplet via pressurised fluid ejection.

[0461] Clause 46. An apparatus according to any of clauses 35-45, wherein the apparatus further comprises one or more of a fluorescence detector, a scattered light detector, an imaging detector, an acoustic wave generating and detecting unit, and a magnetic-activated cell sorting device.

[0462] 15267483-1

Claims

M&C PC934028GB51CLAIMS:

1. A method of determining at least one characteristic of a biological entity contained within a droplet based on the outcome of a plurality of assays performed simultaneously on said droplet; the method comprising:providing a droplet comprising the biological entity to a fluid flow; performing the plurality of assays on the biological entity in the droplet by: determining optical characteristics of the droplet by exposing the droplet to a plurality of distinct excitation light sources and detecting a signals emitted in response to the distinct excitation light sources, wherein the distinct excitation light sources stimulate the emission of:i) at least one diffuse fluorescent signal, andii) at least one localised fluorescent signal.

2. The method of claim 1 , whereinthe at least one diffuse fluorescent signal comprises a fluorescent signal that results from secretion of a molecule from the biological entity and that is contained within the droplet; andthe at least one localised fluorescent signal comprises a fluorescent signal that is localised on a surface of the biological entity or on a surface of a bead within the droplet; and the method further comprises:using the detected signals to determine the at least one characteristic of the biological entity from a combination of the at least one diffuse fluorescent signal and the at least one localised fluorescent signal.

3. The method of claim 2, comprising:using the distinct excitation light sources to stimulate the emission of two diffuse fluorescent signals of different colours; anddetermining the at least one characteristic of the biological entity from a combination of the two diffuse fluorescent signals and the at least one localised fluorescent signal.

4. The method of claim 2 or 3, wherein the biological entity comprises a cell, wherein the secreted molecule comprises an antibody, and further comprising:using the at least one localised fluorescent signal to determine whether the antibody binds to a surface receptor on the surface of the cell or bead; and15267483-1M&C PC934028GB52determining the at least one characteristic of the biological entity from a combination of whether the cell secretes the antibody and whether the antibody binds to a surface receptor on the surface of the cell or bead.

5. The method of claim 4, wherein the biological entity comprises a cell from one of at least two antibody secreting cell lines, and further comprising:using the distinct excitation light sources to stimulate the emission of one of two localized fluorescent signals of different colours, each associated with a different surface receptor; anddistinguishing between the antibody secreting cell lines by detecting a distinctive localised fluorescent signal associated with each cell line using the localized fluorescent signal from the droplet.

6. The method of claim 4 or 5, further comprising confirming that the antibody binds to the surface receptor by:including an antibody stain in the droplet, the antibody stain comprising a secondary antibody with a fluorescent tag that provides a further fluorescent signal; and detecting binding of the secondary antibody to the antibody bound to the surface receptor by detecting localization of the secondary antibody at the surface of the cell or bead, whereindetecting the localization is detected by a contrast between the localized secondary antibody and the background of the droplet.

7. The method of any of claims 1-6, wherein the biological entity comprises a cell selected from a first antibody-secreting cell line and a second antibody-secreting cell line; wherein the droplet further comprises a reporter entity presenting a surface receptor and a labelled detection antibody; wherein the at least one diffuse fluorescent signal comprises a fluorescent signal emitted by the labelled detection antibody; wherein the at least one localised fluorescent signal comprises a first localised fluorescent signal that identifies the first antibody-secreting cell line or a second localised fluorescent signal that identifies the second antibody-secreting cell line; the method further comprising:distinguishing between the first and second antibody-secreting cell lines based on detection of the first or second localised fluorescent signal; and15267483-1M&C PC934028GB53determining whether an antibody secreted by the cell binds to the surface receptor based on detection of a third localised fluorescent signal localised on a surface of the reporter entity.

8. The method of any of claims 1-7, whereinthe at least one diffuse fluorescent signal comprises a fluorescent signal that is excited by a first of the distinct excitation light sources at a first wavelength; and the at least one localised fluorescent signal comprises a fluorescent signal that is excited by a second of the distinct excitation light sources at a second, different wavelength.

9. The method of any of claims 1-8, comprising determining the at least one diffuse fluorescent signal by integrating the area under a fluorescence curve of fluorescence signal against time as the droplet flows past a fluorescence detector.

10. The method of any of claims 1-9, comprising determining the at least one localised fluorescent signal by determining a peak of a fluorescence signal against time as the droplet flows past a fluorescence detector.

11. The method of any of claims 2-10 when dependent on claim 2, wherein using the detected signals to determine the at least one characteristic of the biological entity from a combination of the at least one diffuse fluorescent signal and the at least one localised fluorescent signal comprises:defining one or more regions on a graph that has first and second axes corresponding to fluorescence signals of respective first and second colours; and determining the at least one characteristic of the biological entity according to whether the at least one diffuse fluorescent signal and the at least one localised fluorescent signal fall within the one or more regions.

12. The method of claim 11 , further comprising:providing a graphical user interface to enable a user to define multiple said regions, and selection logic to enable the user to define one or more logical combinations of the regions to determine the at least one characteristic of the biological entity.15267483-1M&C PC934028GB5413. The method of any of claims 1-12, further comprising, for a plurality of the droplets, selecting droplets containing particular biological entities dependent on the at least one characteristic of the biological entity in the respective droplets.

14. The method of any of claims 1-13, further comprising, selectively performing one or more of incubating, sorting, and dispensing the droplet, dependent on the at least one characteristic of the biological entity.

15. The method of any of claims 1-14, wherein the biological entity comprises a cell, wherein the at least one characteristic of the biological entity includes a viability of the cell, and further comprising:using the at least one localised fluorescent signal to perform a surface receptor binding assay to identify cell apoptosis; anddetermining whether or not the cell is viable dependent on whether or not cell apoptosis is identified.

16. The method of any preceding claim, further comprising:providing a fluid sample comprising at least one biological entity; and preparing a droplet from said sample wherein the droplet comprises at least one biological entity.

17. The method of any preceding claim wherein the plurality of excitation light sources comprises at least one laser.

18. The method of any preceding claim wherein the diffuse fluorescent signal indicates at least one of:i. the secretion of at least one analyte from the at least one biological entity into the droplet, orii. an unbound molecule or ligand in the droplet.

19. The method of any preceding claim wherein the localised fluorescent signal indicates at least one characteristic of the at least one biological entity, wherein the characteristic is selected from at least one of: cell viability, metabolic activity, expression of a cell surface marker, or ligand binding to the at least one biological entity.15267483-1M&C PC934028GB5520. The method of any preceding claim wherein the signal is detected using at least one optical sensor.

21. The method of any preceding claim wherein determining optical characteristics of the droplet comprises measuring the intensity and / or duration of a plurality of signals.

22. The method of any preceding claim wherein determining optical characteristics of the droplet takes place when the fluid flow path of the droplet passes an excitation light source and a detector.

23. The method of any preceding claim wherein determining optical characteristics of the droplet detects multiple emitted signals with multiple detectors.

24. The method of any preceding claim, wherein determining optical characteristics of the droplet takes place multiple times in series or simultaneously as the fluid flow path of the droplet passes multiple excitation light sources and multiple detectors.

25. The method of any preceding claim wherein the method further comprises sorting said droplet dependent on the outcome of the determination step; and dispensing the sorted droplet into a reservoir, wherein said dispensing comprises, after said sorting:selecting a fluid flow path for fluid containing the sorted droplet; and ejecting the sorted droplet from the selected path.

26. The method of any preceding claim, wherein determining optical characteristics of the droplet comprises sequentially comparing pairs of detection signals from different optical sensors to determine at least one characteristic of a droplet.

27. The method of any preceding claim, comprising a plurality of determination steps and a single sorting step or, a plurality of sequential determination and sorting steps.15267483-1M&C PC934028GB5628. The method of any preceding claim, wherein the sequential determination and sorting steps further comprise classifying the sorted droplets according to the intensity and / or duration of a signal.

29. The method of any of claims 25-28, wherein dispensing the sorted droplet comprises sorting said droplet according to the profile of said detected signals when determining optical characteristics of the droplet.

30. The method of any of claims 25-29, wherein the droplet is sorted according to the ratio between the intensity of the diffuse fluorescent signal and the localised fluorescent signal.

31. The method of any of claims 25-30, wherein the sorting step comprises routing a droplet to a specific fluid flow path based on the optical characteristics of said droplet.

32. The method of any preceding claim wherein determining optical characteristics of the droplet comprises determining the presence and / or location of an analyte within a droplet through the detection of a fluorescent signal.

33. The method of any preceding claim further comprising at least one incubation step.

34. The method of claim 33, comprising a first incubation step followed by at least one subsequent incubation step.

35. The method of claim 33 or 34, comprising an incubation step before, after or during performing the plurality of assays on the biological entity, and / or an incubation step before, after or during determining optical characteristics of the droplet.

36. The method of any of claims 33-35, wherein the incubation step comprises at least one of growing and / or maintaining one or more biological entities, a reaction between one or more biological entities, and / or interactions between one or more biological entities.15267483-1M&C PC934028GB5737. The method of any of claims 25-36 wherein the dispensing step comprises dispensing a sorted droplet at a defined location in response to the determination of the optical characteristics of a droplet.

38. The method of claim 37, wherein the droplet is dispensed into an external device.

39. The method of any preceding claim wherein the assay is selected from at least one of a protein detection assay, cell detection assay, secretion assay, uptake assay, cytotoxicity assay, stability assay, localisation assay, enzyme assay, binding assay, proliferation assay, cell death assay, immunoassay, or tolerance assay.

40. The method of any preceding claim wherein the assay is a multiplex assay wherein a plurality of excitation and emission spectra are used to detect a plurality of analytes in a droplet.

41. The method of any preceding claim wherein the assay is a multiplex assay wherein a plurality of excitation and emission spectra are used to detect a plurality of characteristics of at least one analyte in a droplet.

42. The method of any preceding claim wherein the assay comprises determining the presence of at least one molecule on the surface of a bead, or a cell and / or inside a cell based on the emission of at least one localised fluorescent signal and / or at least one dispersed fluorescent signal.

43. The method of any preceding claim wherein the assay comprises detecting an interaction between at least two molecules based on the emission of at least one localised fluorescent signal.

44. The method of any preceding claim wherein the method comprises detecting the secretion of at least one antibody from a single cell and the binding of said antibody to an antigen based on the detection of at least three different signals.

45. The method of any preceding claim wherein the method comprises determining whether a cell is apoptotic based on the emission of at least one fluorescent signal.15267483-1M&C PC934028GB5846. The method of any preceding claim wherein at least four different characteristics of at least one cell or molecule are determined based on the emission of at least four different signals.

47. The method of any preceding claim wherein the biological entity is selected from at least one of a cell, a protein, a carbohydrate, a lipid, an antibody, an antigen, an enzyme or a nucleic acid.

48. An apparatus suitable for carrying out the method of any preceding claim wherein said apparatus comprises:at least one microfluidic input channel;a plurality of fluid flows;a plurality of distinct excitation light sources;a plurality of detectors for detecting at least one diffuse fluorescent signal and at least one localised fluorescent signal in response to the distinct excitation light sources, wherein the distinct excitation light sources are selected to stimulate distinct emission spectra; andat least one microfluidic output channel.

49. An apparatus according to claim 48, further comprising a droplet formation device for preparing a droplet from a fluid which contains at least one biological entity.

50. An apparatus according to claim 48 or 49, further comprising a droplet sorting device for sorting said droplet dependent on an outcome of said determination.

51. An apparatus according to any of claims 48-50, further comprising a dispensing unit for dispensing said sorted droplet from a fluidic flow path.

52. An apparatus according to claim 51 wherein the dispensing unit comprises an isolation unit for isolating a said sorted droplet from said fluidic flow path and a guide for guiding a said isolated droplet into a second fluidic flow path.

53. An apparatus according to any of claims 48-52, wherein the plurality of excitation light sources comprise a plurality of lasers.15267483-1M&C PC934028GB5954. An apparatus according to any of claims 48-53, wherein the plurality of detectors comprise a plurality of photomultiplier tubes.

55. An apparatus according to any of claims 48-54, wherein the apparatus comprises at least one optical filter.

56. An apparatus according to any of claims 48-55, wherein the apparatus further comprises a droplet storage unit for storing said isolated droplet.

57. An apparatus according to any of claims 48-56, wherein the apparatus further comprises an incubator for incubating said droplet.

58. An apparatus according to any of claims 51-57 wherein said dispensing unit comprises a high pressure unit for dispensing said droplet via pressurised fluid ejection.

59. An apparatus according to any of claims 48-58, wherein the apparatus further comprises one or more of a fluorescence detector, a scattered light detector, an imaging detector, an acoustic wave generating and detecting unit, and a magnetic-activated cell sorting device.15267483-1

Images