EP-4737900-A2 - MICROFLUIDIC METHOD FOR SINGLE CELL ANALYSIS

EP4737900A2EP 4737900 A2EP4737900 A2EP 4737900A2EP-4737900-A2

Abstract

A first aspect of the present invention is directed to a method for the detection of a compound of interest in a microfluidic system comprising the steps of: a. creating at least one droplet in said microfluidic system, said at least one droplet comprising: i. at least one single cell, ii. one or more first capturing agent, wherein said one or more first capturing agent is capable of binding said single cell as well as said compound of interest, iii. one or more second capturing agent comprising a label, wherein said one or more second capturing agent is capable of binding said compound of interest, b. incubating said at least one droplet capable of generating a detectable event, c. subjecting said at least one droplet to a direct detection, wherein the presence or the relocalization of said detectable event within said at least one droplet determines the presence of said compound of interest. A second aspect of the present invention relates to the use of the method according to the first aspect for monitoring a biological event. A third aspect of the present invention is directed to a method for the detection of a compound of interest in a droplet comprising the steps of: a. providing a microfluidic system comprising: i. at least one inlet, ii. at least one outlet, iii. one or more channels, b. injecting in said microfluidic system a stream of droplets, wherein at least one droplet comprises: i. at least one single cells ii. a plurality of a first capturing agents capable of binding said single cell as well as said compound of interest, and iii. a plurality of second capturing agents, each comprising a label, wherein said plurality of second capturing agents is capable of binding said compound of interest, c. incubating said plurality of droplets under conditions that allow the production of the compound of interest, whereby if the compound of interest is produced by the single cell, it will be captured by said plurality of first and second capturing agents, d. determining the presence of the compound of interest by means of detecting a presence or relocalization of said label. A fourth aspect of the present invention is directed to a microfluidic system comprising: a. at least one inlet, b. at least one outlet, c. one or more channels, d. a module for creating at least one droplet comprising: a. one or more single cell, b.a first capturing agent, c. a second capturing agent. e. a detection module detecting droplet containing cells producing compound of interest, and f. an analysis module configured for the analysis of the signal. A fifth aspect of the present invention relates to the use of a microfluidic system according to the fourth aspect for carrying out the method according to the first or third aspect.

Inventors

Gérard, Annabelle, Patricia, Veronique
Menrath, Vera

Assignees

HiFiBiO SAS

Dates

Publication Date: 20260506
Application Date: 20190418

Claims (15)

A method for the detection of a compound of interest in a microfluidic system, said method comprising: a. creating at least one droplet capable of generating a detectable event, said at least one droplet comprising: i. at least one single cell, ii. one or more first capturing agent, wherein said one or more first capturing agent is capable of binding said at least one single cell as well as said compound of interest, iii. one or more second capturing agent comprising a label, wherein said one or more second capturing agent is capable of binding said compound of interest, b. incubating said at least one droplet capable of generating said detectable event, c. subjecting said at least one droplet to a detection of said detectable event in said microfluidic system, wherein the presence or relocalization of said detectable event within said at least one droplet is indicative of the presence of said compound of interest; and wherein said compound of interest is produced by said at least one single cell.
The method according to claim 1, wherein said one or more first capturing agent binds said at least one single cell before or after creating said at least one droplet.
The method according to any of the claims 1 and 2, wherein said one or more first capturing agent binds said at least one single cell with a density ranging from 10 1 to 10 8 molecules/cell.
The method according to any of the claims 1 to 3, wherein said compound of interest is produced in said at least one droplet with a concentration of 10pM to 100µM.
The method according to any of the claims 1 to 4, wherein said at least one droplet has a volume ranging from 2pL to 10nL.
The method according to any of the claims 1 to 5, wherein said method further comprises measuring cell viability in said at least one droplet after incubation.
The method according to any of the claims 1 to 6, wherein said label is a fluorescent label, an amino-acid based label, a nucleic acid based label, or a barcode label.
The method according to any of the claims 1 to 7, wherein said one or more first capturing agent and said one or more second capturing agent are independently a protein, a peptide, an oligonucleotide, a nucleic acid, a fluorescent conjugate, an enzyme conjugate, a synthetic polymer, or a combination thereof.
The method according to any of the claims 1 to 7, wherein said one or more first capturing agent is an antibody and said one or more second capturing agent is a fluorescent anti-compound of interest antibody.
The method according to claim 9, wherein said one or more first capturing agent is a bifunctional antibody.
The method according to any of the claims 1 to 10, wherein said compound of interest is a cell-secreted compound.
The method according to claim 11, wherein said compound of interest is an antibody, a cytokine, Common b chain (CD131), LIF, OSM, an interferon, TNF, TNF-α, TNF-β, CD153, CD154, LT-β, 4-1BBL, APRIL, CD70, CD132, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRANCE, TGF-β, Tpo, Flt-3L, SCF, M-CSF, MSP, a chemokine, a hormone, or a growth factor.
A method to monitor a biological event, said method comprising: a. creating at least one droplet capable of generating a detectable event, said at least one droplet comprising: i. at least one single cell, ii. one or more first capturing agent, wherein said one or more first capturing agent is capable of binding said at least one single cell as well as a compound of interest, and iii. one or more second capturing agent comprising a label, wherein said one or more second capturing agent is capable of binding said compound of interest, b. incubating said at least one droplet, and c. subjecting said at least one droplet to a detection of said detectable event in a microfluidic system, wherein the presence or relocalization of said detectable event within said at least one droplet is indicative of the presence of said compound of interest; and wherein said compound of interest is produced by said at least one single cell.
A method for the detection of a compound of interest in a droplet, said method comprising: a. providing a microfluidic system comprising: i. at least one inlet, ii. at least one outlet, iii. one or more channels, b. providing a plurality of droplets, wherein at least one droplet comprises: i. at least one single cell, ii. a plurality of first capturing agents capable of binding said at least one single cell as well as a compound of interest, and iii. a plurality of second capturing agents, wherein each second capturing agent comprises a label and is capable of binding said compound of interest, c. incubating said plurality of droplets under conditions that allow the production of the compound of interest, whereby if the compound of interest is produced by said at least one single cell, it is capable of being captured by said plurality of first and second capturing agents, d. injecting the plurality of droplets in said microfluidic system, and e. determining the presence of the compound of interest by detecting, in said microfluidic system, a presence or relocalization of said label.
A microfluidic system comprising: a. at least one inlet, b. at least one outlet, c. one or more channels, d. a module for creating at least one droplet comprising: a.one or more single cell, b.a first capturing agent, c. a second capturing agent, e. a detection module configured to detect said at least one droplet containing said one or more single cells, wherein said one or more single cells produce a compound of interest, and f. an analysis module configured for the analysis of a signal indicative of a presence of the compound of interest in said at least one droplet; wherein said first capturing agent is capable of binding said one or more single cells as well as said compound of interest.

Description

FIELD OF THE INVENTION The present invention is in the field of cellular and molecular biology and is based on methods for detecting a compound of interest produced by a single cell in a droplet. The invention is also related to the field of microfluidics and encompasses microfluidic devices and their use thereof for carrying out biological assays. BACKGROUND During a drug discovery program, one of the step is related to the validation of the drug candidate based on its expected biological effect. On that purpose, either in-vivo or in-vitro models can be used. On one hand, in-vivo experiments have the advantage to address the question on a whole living organism. However, animal models are not necessarily predictive of what would happen in human. Moreover, in-vivo studies are expensive and their use is limited by ethical considerations. On the other hand, in-vitro systems, even though failing to replicate the precise cellular conditions of an organism, can be performed on human cells and are particularly suitable in case of screening process, where a high throughput is needed. These cell-based assays are usually performed in bulk on cells of interest. However, in certain conditions, as it is the case for immune cells, each of them is unique and the need of functional cell-based assays at a single cell level is of great interest. Indeed, measuring immune responses in bulk populations increases the risks to mask the unique behavior or contribution of each single cell, especially when immune response is highly heterogenous, or driven by rare cell populations. Therefore, a single cell-based assay is required to better understand potential variations from cell to cell that would consider individual cell phenotypes. Recent advances in single cell analysis methods have improved biological understandings within single cells by characterizing relationships between cells within a population. Therefore, by determining rare cell events or small changes between individual cells it is possible to address unresolved questions in the field of cancer, immunology, infectious disease, stem cell and developmental biology and neurology. Immune cells protect the host organism against diseases by producing antibodies, chemokines and cytokines. This former class of molecules are group of proteins secreted by innate and adaptive immune cells acting as chemical messengers. Their production by immune cells is due to the body's ability to raise an immune response and therefore has high clinical diagnostic value. Thus, both the study of antibody and cytokine secretion kinetic could give significant information for diagnostics of diseases and personalized therapies. However, the absence of quantitative, single cell, high-throughput systems to analyze individual secreting cells limits investigation on dynamics of the immune response. Recently, droplet based microfluidic systems have attracted significant interest because of their range of applications towards cell biology and based on their ability to control the mechanical, biological and fluidic environment at the single cell level. The technology enables assays to be carried out very rapidly (up to thousands of cells and/or droplet per second). Additionally, the system provides macroscale (pico-or nanoliter volumes of samples and reagents) cell culture experiments where biological samples are confined in droplets, allowing fast detection of high concentration of compound (from pM to µM range). Moreover, the system minimizes sample loss and cross contamination but allows fast mixing, thermal transfer, and chemical reaction. Interestingly, the technology provides the possibility to perform large-scale genotypic and phenotypic screens at the single cell level. In the last few years, different microfluidic devices and systems have been proposed for single-cell analysis (Gross et al. 2015, Int. J. Mol. Sci. 16(8):16897-16919; Reece et al. 2016, Curr. Opin. Biotechnol. 40:90-96). Different methods and techniques have been proposed for cells sorting in microfluidics. Sorting principles are mainly classified in two categories: methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. High purity cell separation and sorting can be achieved using a monoclonal antibody that binds to a cellular component. Widely used antibody-based cell analysis and/or separation techniques include cell panning, magnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS), including fluorescence-activated droplet sorting (FADS). In cell panning technique, cells exhibiting specific antigens can be selectively attached on an antibody-coated surface. Despite this technique can provide high purity, it is affected by some limitations such as high cell loss or impact on cell viability. In other cell panning technique as single cell sorting by flow cytometry, cells secretin