QUANTIFICATION OF SUCCESSFUL ENCAPSULATION INTO MICROFLUIDIC COMPARTMENTS

Abstract

The invention provides a method for quantifying the number of target entities that were successfully encapsulated into a microfluidic compartment such as a microfluidic droplet. The invention is predicated upon a continuous quantifiable detectable signal corresponding to the quantity of entities encapsulated in a microfluidic compartment. The present invention is useful in the context of high throughput microfluidic screening approaches which require thorough signal normalization in order to reliably improve signal to noise ratio and to reliably identify screening hits.

Claims

1. A method for the quantification of the number of target entities encapsulated in a micro compartment, the method comprising the steps of: (a) Providing a detection molecule comprising (i) a target binding site specific for the target entity, or at least capable of binding to the target entity, and (ii) an enzymatic component capable of catalysing a chemical signalling reaction; (b) Bringing into contact the detection molecule and the target entity to allow for a specific binding of the detection molecule to the target entity; (c) Optionally, removing at least any unbound detection molecules; (d) Encapsulating one or more (an unknown number of) target entities bound with detection molecules into one or more micro-compartments together with substrate molecules, wherein the substrate molecule is a substrate of the enzymatic component, and which when brought into contact with the enzymatic component renders the enzymatic component to catalyse the chemical signalling reaction; and (e) Quantifying the detectable signal for each micro compartment and thereby quantifying the number of target entities encapsulated in each micro compartment.

2. The method of claim 1, which is for the quantification of the number of encapsulated target entities of distinct species of target entities out of a plurality of species of target entities, wherein the method comprises the steps of (a) Providing multiple species of detection molecules, each species of detection molecules comprising (i) a target binding site specific for a distinct species of target entity out of a plurality of species of target entities, or at least capable of binding to a target entity our of a plurality of species of target entites and (ii) an enzymatic component that is capable of catalysing a chemical signalling reaction that is different from a chemical signalling reaction catalysed by any other enzymatic component comprised in a detection molecule of another species of detection molecules; (b) Bringing into contact the multiple species of detection molecules and the plurality of species of target entities to allow for a specific binding of the detection molecule to the target entities; (c) Optionally, removing at least any unbound detection molecules; (d) Encapsulating one or more (an unknown number of) target entities of each species of target entities bound with detection molecules into one or more micro-compartments together with multiple species of substrate molecules, wherein each species of substrate molecule is a substrate for the enzymatic component of not more than one species of detection molecule, and which when brought into contact with the corresponding enzymatic component, the enzymatic component catalyses the chemical signalling reaction that generates a distinct detectable signal for each of the chemical signalling reactions; (e) detecting each detectable signal for each micro compartment and thereby quantifying the number of target entities of each species of target entities encapsulated in each micro compartment.

3. A method for the quantification of the number of target entities encapsulated in a micro compartment, the method comprising the steps of: (a) Providing a first detection molecule comprising (i) a target binding site specific for the target entity and (ii) a binding site capable of being bound by a second detection molecule; (b) Providing a second detection molecule comprising (x) a target binding site specific for the first detection molecule, and (y) an enzymatic component capable of catalyzing a chemical signaling reaction; (c) Bringing into contact the first- and the second detection molecule and the target entity to allow for a specific binding of the first detection molecule to the target entity and a specific binding of the second detection molecule to the first detection molecule; (d) Optionally, removing at least any unbound detection molecules; (e) Encapsulating one or more (an unknown number of) target entities bound with first and/or second detection molecules into one or more micro-compartments together with substrate molecules, wherein the substrate molecule is a substrate of the enzymatic component, and which when brought into contact with the enzymatic component renders the enzymatic component to catalyse the chemical signalling reaction; and (f) Quantifying the detectable signal for each micro compartment and thereby quantifying the number of target entities encapsulated in each micro compartment.

4. The method of claim 1, wherein the target entity is a particle, such as a micro or nano particle, a bead, a vesicle, a biological cell, or a cell-accumulation, such as a tissue fragment, spheroid or organism, in particular an embryo or microscopic multicellular organism (worm, plant, fungus, etc.).

5. The method of claim 1, wherein the enzymatic reaction is a colorimetric, chemiluminescent or fluorescent reaction.

6. The method of claim 1, wherein the micro compartment is an aqueous micro compartment such as a plug, a (microfluidic) droplet or a well.

7. The method of claim 1, wherein the substrate molecule is encapsulated within each micro compartment in excess to the concentration of the detection molecule in the same micro compartment.

8. The method of claim 1, wherein each target entity when encapsulated is capable of eliciting one or more detectable assay signals, and wherein the method comprises in step (e) detecting the detectable assay signal, and normalizing the detectable assay signals of two more micro compartments with the detectable signals for each of the two or more micro compartments.

9. A plurality of micro compartments, of which at least 5% of micro compartments have an aqueous phase comprising a target entity bound with detection molecules, wherein the detection molecule comprises (i) a target binding site specific for the target entity and (ii) an enzymatic component that is capable of catalysing a chemical signalling reaction; and an unbound substrate molecule dissolved within the aqueous phase and which is a substrate of the enzymatic component of the detection molecule, and which when brought into contact with the enzymatic component renders the enzymatic component to catalyse the chemical signalling reaction that generates a detectable signal.

10. The plurality of micro compartments of claim 9, wherein the target entity is a particle, such as a micro or nano particle, a bead, a vesicle, a biological cell, or a cell-accumulation, such as a tissue fragment, spheroid or organism, in particular an embryo or microscopic multicellular organism (worm, plant, fungus, etc.).

11. The plurality of micro compartments of claim 9, wherein the enzymatic reaction is a colorimetric, chemiluminescent or fluorescent reaction.

12. The plurality of micro compartments of claim 9, wherein the enzymatic component or enzyme is a peroxidase, or a fragment thereof, or a luciferase or a fragment.

13. The plurality of micro compartments of claim 9, wherein the substrate molecule is encapsulated within each micro compartment in excess to the concentration of the detection molecule in the same micro compartment.

14. The plurality of micro compartments of claim 14, wherein the micro compartment is an aqueous micro compartment such as a plug, a (microfluidic) droplet or a well

15. The plurality of micro compartments of claim 14, wherein the micro compartment is a plug formed in a three-phase system preferably composed of an aqueous phase, a fluorinated oil phase as a carrier phase, and a mineral oil phase as a spacer between individual plugs of the plurality of micro compartment.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0079] The figures show:

[0080] FIG. 1: shows the working principle of the procedure of the invention. a) embodiment of the invention with a single type of encapsulated entity of cell: cells to be encapsulated are stained with a binder directed to an expressed surface antigen (top schematic). The bottom schematic shows exemplary three separate possible compartment states after encapsulation. Version A shows a situation where no cell is encapsulated, version B shows a situation where only one cell is encapsulated, and version C shows the situation where two cells are encapsulated. Below the schematic compartments, the expected fluorescent signal is shown in the below prophetic graphs. b) embodiment of the invention similar to a), but with two different types of encapsulated entities (cells).

[0081] FIG. 2: shows the experimental testing of the method of the invention using an existing microfluidic personalized medicine platform described by previously (Eduati F, et al. (2018) Nature Communications 9(1):1-13.). a) the fluorescence signal in the microcompartment increases linear with the number of encapsulated cells. b) shows an example for a readout of the assay. Each compartment consists of cells which are co-encapsulated with drugs and two substrates one for caspase 3 indicating apoptosis (wavelength I) and substrate 2 (emission at wavelength II not equal to wavelength I), which reacts with the antibody-coupled enzymes on the cell membrane enabling to quantify the cell number in each compartment. c) The encapsulated drug kills the cells and the fluorescence signal of the cells undergoing apoptosis increases linear with the fluorescence intensity of substrate II for cell counting. Thus, apoptosis signal is linear with the number of cells. d) The apoptosis signal can be normalized to the number of cells in the microcompartments (=peaks in b)).

EXAMPLES

[0082] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0083] The examples show:

Example 1: Normalization of Encapsulated Cell Numbers in Microfluidic Screens

[0084] The invention enables the normalization to the cell number for cell assays, e.g. caspase readout in plates and high-throughput droplet microfluidic experiments. Furthermore, it is describing the established procedure to dissect primary colorectal cancer biopsies (from colon, rectum and liver metastasis) using human tumor dissection kit (miltenyi) and the approach to combine caspase 3 with caspase 9 substrate for obtaining a Caspase readout indicating which of the tested drugs and combinations thereof show the strongest effect of killing the screened cancer cells. The general principle of the method of the invention is depicted in FIG. 1.

[0085] In brief, cells are stained with a binder directed to an expressed surface antigen. The binder can be an antibody or antibody combination, which is functionalized with an enzyme. Unbound binder is removed by washing. In the next step, stained cells and the reporter substrate are co-encapsulated into aqueous microcompartments and incubated. Upon reaction of the substrate and enzyme the fluorescence intensity changes. For non-endpoint measurements, this change in fluorescence intensity is linear with the number of cells due to linearity between enzyme activity and species number (FIG. 1A).

[0086] In addition, the cell quantification assay can also be applied to quantify the number of a specific cell type I within a mixture of different types of cells, when one subpopulation does not bind the enzyme-linked binder due to the fact, that the target-antigen is not expressed or due to the specificity of the binder to a certain species. Cells out of a mixture with a cell type II from another species will not contribute to the fluorescence signal unless the cells of cell type II are labelled with the same enzyme or another enzyme converting another substrate, allowing to count both cell types in a multiplexed fashion (FIG. 1B).

[0087] In order to test the invention, the following assay-specific reagents were used: [0088] Reagents amplex red assay [0089] Amplex red (Thermofisher, Art.no. A12222_5 mg) [0090] DMSO [0091] ?2-microglobulin antibody (Biolegend, clone 2M2, Art.no. 316305) [0092] anti-C298 antibody (Biolegend, clone LNH-94, Art.no. 341704) [0093] m-IgG BP-HRP (Santa Cruz Biotechnology, Art.no. se-516102) [0094] anti-mouse H-2 antibody (Biolegend, clone M1/42, Art.no. 125502) [0095] anti-mouse CD45 antibody (Biolegend, clone 30-F11, Art.no. 103101) [0096] HRP Goat anti-rat IgG antibody (Biolegend, clone Poly4054, Art.no. 103101)

Example 1

[0097] Reagents Caspase Assay [0098] caspase-3 substrate (Z-Asp-Glu-Val-Asp)z-Rhodaminelio (BACCHEM, Art.no. 4048381.0025) [0099] caspase-9 substrate (AC-Leu-Glu-His-Asp-AFC trifluoroacetate salt, BACCHEM, Art.no. 4028699.0005) [0100] DMSO (Sigma Aldrich) [0101] 5? Reaction buffer (50 mM PIPES, pH 7.4, 10 mM EDTA, 0.5% CHAPS all from Sigma) [0102] DTT (1 M, Cleland's reagents, Roche, Art no. 10197777001) [0103] Distillated water (dH2O) [0104] Cascade Blue? hydrazide tritium salt (10 mM in DMSO) (Art.no. C3239, Invitrogen) [0105] Mineral oil (Sigma, Art.no. M8410-1L) [0106] 3M? Fluorinated FC-40 (IOLITEC Ionic liquids technology GmbH) [0107] 1H,1H12H,2H-Perfluorooetanol (PFO, Abcr GmbH, Art.no. AB125017)

[0108] After preparation of the microfluidic setup, the cells for encapsulation the first detection antibody is added to the cells to allow binding to the cell surface (see Table i) and incubate for 15 min at 4? C. to allow for a sufficient surface staining. Then the stained cells are washed to remove unbound antibody. As a second step a HRP conjugated secondary antibody specific for the first antibody is added to the stained cells and incubated

TABLE-US-00001 TABLE 1 Successful staining protocol of the cell membrane in example 1 Add 1 ?l Species Add ? 2 ?l first secondary of cell antibody to 1 ml washing antibody to 1 ml washing origin staining buffer step staining buffer step Mouse anti-mouse H-2 14 ml Anti-rat-HRP 14 ml antibody staining staining anti-mouse CD45 buffer buffer antibody Human anti-human C298 14 ml Anti-mouse-HRP 14 ml antibody staining staining anti-human ?2- buffer buffer microglobulin Time 15 5 15 5 [min]

[0109] The stained cells are then encapsulated and signal detection is performed as described before (Eduati et al. 2018 Nature Communications volume 9, Article number: 2434, of which the Methods section is incorporated herein by reference in its entirety). Results of normalized signals are shown in FIG. 2.

Example 2: [Prophetic] Functional Antibody Screens

[0110] In general, all assays carried out in either large aqueous compartments or compartments with multiple cells require a normalization for the number of cells. As another example the same kind of assay could be used for functional antibody screens involving more than one target cell: Single antibody expressing cells (e.g. plasma cells) can be encapsulated together with multiple target cells (e.g. cancer cells) into aqueous compartments for identifying antibodies that can trigger apoptosis in the target cells. In this type of assay one could stain the target cells with an enzyme-linked antibody to enable normalization of the functional antibody assay (e.g. induction of intracellular Ca-flux upon agonistic action of antibodies on GPCRs) to the number of cells per compartment. Only this way, the most potent therapeutic antibodies can be isolated (note that antibody discovery is a multi-billion dollar business in which even slight technical improvements have significant commercial impact).

Example 3: [Prophetic] Multiplex Screens

[0111] Assays involving different cell types (multiplex screens) are even more prone to biases based on cell number variation (and variation of the relative cell numbers). For example, assays in cancer immunology often include T-cells, target cells and antibody presenting cells. For being able to carry out such types of assays at high throughput in aqueous compartments, one has to be able to measure the relative cell numbers accurately, which can be achieved using the described assay. A different enzyme labelled antibody can be used for each cell type (particularly with a different enzyme for each cell type) so that the relative cell numbers can be easily detected by determining the substrate turnover for the different enzymes (e.g. using substrate-1 for enzyme-1, conjugated to antibodies used for staining cell type 1, substrate-2 for enzyme-2, conjugated to antibodies used for staining cell type 2, etc.)