Microfluidic Methods and Systems

Abstract

The present invention provides a microfluidic system, a method for manufacturing the same, and a method for assigning phenotype to genotype using droplets within a microfluidic device. [Solution] The system comprises a) a solid support comprising at least a first group of oligonucleotides, i. each oligonucleotide in the group comprising a first type, a second type and / or further types of nucleic acid sequences, ii. the first type of nucleic acid sequence is a barcode sequence, iii. oligonucleotides comprising the same barcode sequence are grouped into a group of oligonucleotides on the solid support, iv. the first group of oligonucleotides and the further groups of oligonucleotides are spatially separated on the solid support, b) one or more groups of oligonucleotides on the solid support are located in separate reservoirs of the microfluidic system, and c) one or more reservoirs are accessible via channels to fluids, cells, chemicals and / or microdroplets.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and relates to a method for assigning a phenotype to a genotype using droplets in a microfluidic device. The present invention also belongs to the field of microfluidic engineering and encompasses microfluidic devices, methods for manufacturing the same, and their use for performing biological assays.

Background Art

[0002] Recent advances in single-cell analysis methods, such as single-cell RNA-seq methods developed by Klein (Klein et al. 2015, Cell 161(5):1187-1201) and Macosko (Macosko et al. 2015, Cell 161(5):1202-1214), or single-cell epigenetics ChIP-seq methods devised by Rotem (Rotem et al. 2015, Nat. Biotechnol. 33(11):1165-1172), enable dissection of cell populations at a higher throughput than corresponding bulk methods (Jaitin et al. 2014, Sciences 343(6172):776-779). However, sequencing data only allows endpoint measurements of cells or cell lines, and there is an increasing need to include information regarding the dynamic data or phenotype of cells, which is included to complement and enhance the genetic information obtained.

[0003] The underlying methods for functional assays are extensively established and have been adapted by Agresti to single-cell analysis methods (Agresti et al. 2010, PNAS 107(9):4004-4009). Droplet microfluidics offers a range of methods that can address multiple challenges, such as high-throughput screening using elements like single-cell encapsulation, droplet selection, and droplet fusion, for constructing phenotypic assays. For example, Mazutis describes a method for selecting droplets containing B cells that produce antibodies against a target of interest using magnetic beads that capture immunoglobulins (Mazutis et al. 2013, Nat. Prot. 8:870-891). A variation of this method has been published by Eyer, in which a single magnetic bead is replaced with multiple magnetic nanoparticles, ensuring that all cells are analyzable (Eyer et al. 2017, Nat. Biotechnol. 35(10):977-982). These two examples demonstrate antibody binding events within droplets at high throughput.

[0004] In an ideal screening system for drug discovery, the selection of the desired phenotype is not a single-step process, but rather a stepwise selection of phenotypes based on a combination of various phenotypic assays, typically based on endpoint measurements or kinetic binding and / or functional readouts.

[0005] A crucial step in all phenotypic screening is the selection of a reporter system (e.g., an antibody, chemical dye, or genetically encoded fluorescent tag). With fluorescence microscopy, only a relatively small number of reporter systems can be monitored simultaneously in each cell. Multiplexing reporter systems and / or performing additional repeated experiments can increase the number of readouts used to examine cellular responses and provide useful information. However, increasing the number of reporter systems can increase the cost and time required for screening.

[0006] Furthermore, in order to provide high-throughput information in the first step regarding the phenotypic function of intercellular interactions / recognition and / or compound function, and to provide information at the single-cell level in the second step of genotyping analysis, it is necessary to synthesize both phenotype and genotype at the single-cell level based on the information.

[0007] Microfluidics has emerged as a powerful technique for performing a wide range of biological and chemical assays in high throughput. This technique enables high-throughput analysis of complex samples by dividing bulk solutions into numerous independent compartments or microreactors ranging from picoliters to nanoliters in size.

[0008] However, achieving the recovery of individual samples after analysis is difficult using methods known in the art. Furthermore, the mixing of reagents within these devices often requires complex structures or is performed in a single step before compartmentalization, which can prevent the initial reaction product from co-localizing with the starting target.

[0009] In fact, microfluidic methods combining phenotypic screening and single-cell level genotyping analysis lack the precision to identify droplets. In particular, recovering single-cell specific genotypes along with single-cell specific phenotypes is extremely difficult; therefore, a method is highly desirable to screen cells with the desired phenotype and recover specific cell genotype information by combining them with functional readouts at arbitrary selection.

[0010] The methods disclosed herein aim to solve the above-mentioned problems affecting microfluidic methods known in the art.

[0011] The inventors have developed a microfluidic device for carrying out the method disclosed herein, in which single-cell droplets are captured in individual compartments. The single-cell droplets are then selectively fused with other droplets that combine phenotypic information (protein expression levels, cellular pathway activation / activity, ion channel / GPCR activity) and genotype or epigenetic information, thereby enabling the determination of the genotype of a single cell having the desired phenotype. [Overview of the Initiative]

[0012] The present invention relates to a microfluidic system, and the system is a) A solid support comprising at least one group of alkyl groups, i. Each oligonucleotide of the group comprises a first type, a second type and / or further types of nucleic acid sequences, ii. The first type of nucleic acid sequence described above is a barcode sequence, iii. Oligonucleotides containing the same barcode sequence are grouped together in the group of oligonucleotides on the solid support, iv. The first group of oligonucleotides and further groups of oligonucleotides are spatially separated on the solid support, b) One or more of the oligonucleotide groups on the solid support are located in separate reservoirs of the microfluidic system, c) One or more reservoirs are accessible via channels for fluids, cells, chemicals and / or microdroplets. d) Each reservoir containing a group of oligonucleotides on the solid support is also a trap for microfluidic droplets.

[0013] The present invention also relates to a method for attaching oligonucleotides to cells, the method being: a) To provide a microfluidic system according to the present invention, b) Encapsulating the first cell in the first droplet, c) capturing the cells in the reservoir, d) fusing a second droplet containing the dissolving composition with the first droplet, thereby enabling the oligonucleotide of the solid support to adhere to the nucleic acid in the cell.

[0014] The present invention further relates to a method for determining the phenotype and / or genotype of a single cell, wherein the method is: a) To provide a microfluidic device comprising at least one microfluidic channel and at least a collector system including multiple reservoirs, b) Encapsulating at least one cell from a plurality of cells of the first type separately in a droplet of the first type, Optionally, co-encapsulate cells of a second type from multiple second types into each of first type droplets, c) Flowing multiple droplets of a first type through the microfluidic channels of a microfluidic device, capturing the droplets of the first type in each reservoir of the microfluidic device, and optionally analyzing the phenotype of the droplets contained in the reservoir, d) Flowing multiple droplets of a second type through a microfluidic channel and capturing a second droplet of the second type in each reservoir, e) Fusing a first type of droplet with a second type of droplet in the reservoir, f)e) includes carrying out at least one reaction in the fused droplet obtained in f)e) and determining the readout value of the reaction.

[0015] The present invention further relates to a method for manufacturing the system according to the present invention.

[0016] The present invention also relates to a kit comprising the microfluidic system of the present invention and, optionally, instructions for carrying out the method of the present invention. [Brief explanation of the drawing]

[0017] [Figure 1]A 3D diagram of the device of the present invention is shown, including a first cell droplet captured in a reservoir, with a second reagent droplet in contact with the first cell droplet and localized beneath an array of barcoded oligos. The reservoir is configured such that the first droplets do not come into contact with each other, and the second and further droplets do not come into contact with each other, so that fusion can occur only between the two first and second droplets that are captured in the reservoir and humidify locally spatially arranged oligos. The barcodes are configured to come into contact with a single second droplet. [Figure 2] This is a 2D diagram of two devices exhibiting two different functions. The array section is designed to regularly spot oligos (2) onto a slide surface (1). The second device, referred to as the fluid engineering device (3), is designed to organize droplets introduced into a fluid system. This device is also used to manipulate various types of droplets. [Figure 3] Figure 2 shows 2D diagrams of both assembled devices as described. Next, both are combined to organize droplets according to oligos spotted on the slide surface. Oligos are used to react specifically with any type of substance introduced into the droplet, typically cells or cell lysates. [Figure 4]This is the manufacturing process of a fully assembled device. Both devices described in Figure 2 are manufactured individually. (1) The array slide is ordered from a subcontractor that prepares different oligo spots on the slice surface. The oligo composition can be adapted to any type of reaction performed in droplets. For the microfluidic part, the manufacturing starts with the production of the (19) SU8 mold. The initial device is drawn using any type of 3D software, usually AUTOCAD. Next, a mask is printed, followed by the photoactivation of SU8 (resin) in the recessed part of the printed mask. Next, an organic solvent is used to remove the excess resin. The 3D SU8 mold (19) containing the same design represents a convex bottom area. This process is performed multiple times to create multiple layers of SU8 resin with different designs. This is used to generate various features on the microfluidic device and create the organization or manipulation of different types of droplets. a) Non-polymerized PDMS is molded on the SU8 mold (19), wrapping the shape of the SU8 mold. After baking, the PDMS becomes firm, and the shape of the SU8 is replicated as a recess in the PDMS piece. b) The PDMS is removed from the SU8 mold, constituting the PDMS mold (20). c) Onto the PDMS mold, the COC polymer is hot embossed onto the PDMS surface. The plastic wraps the PDMS surface and replicates the recessed design on the COC surface. d) After removing the COC piece from the PDMS mold, the COC piece becomes a microfluidic device with known fluid properties. e) Subsequently, both the array part and the COC fluid part are assembled using any type of sealing (thermal sealing, double tape, adhesive, resin, etc.). [Figure 5] The array slide (1) spotted with oligos is composed of three types of arrays in this example. (8) corresponds to the first type of array. (9) corresponds to the second type of array. (10) corresponds to the third type of array. The three different arrays are used for different functions. In this example, (8) is used as a specific sequence for capturing mRNA by reverse transcription. (9) is different for each spot and is used as a known identifier. (10) is used for further molecular biology reactions. [Figure 6]2D diagram of an assembled array and a microfluidic device. A flow of droplets (25, 26, 27) of a first type containing at least one or more cells is introduced into the fluid chamber. The droplets of the first type contain any type of reagent suitable for phenotypic analysis. The droplets of the first type are individualized within a single compartment by buoyancy. Next, a flow of droplets of a second type is introduced into the fluid chamber. Droplets of the second type containing reagents for molecular biology reactions are configured to contact the droplets of the first type. The droplets of the first type and the droplets of the second type are fused (29) by applying any suitable technique. The fused droplets containing cells, lysis agents, and molecular biology reagents are brought into contact with the oligos spotted on the slide surface. Subsequently, the oligos are released using any type of oligo cleavage. In such an example, the molecular biology reaction begins with the lysis of cells in the presence of molecular biology reagents and the release of the spotted oligos. [Figure 7] Microfluidic workflow according to one aspect of the present invention. [Figure 8] Example of a microfluidic device and droplets captured in a reservoir. Cell droplets (small droplets) are captured in the first reservoir. Reagent droplets (larger droplets) are captured by two pillars and the two droplets are physically and temporarily placed at the location where the oligonucleotides are spotted. The fusion of the two droplets and the wetting of the droplets on the oligonucleotide surface mix the three reservoirs of cell droplets, reagent droplets, and oligonucleotides. [Figure 9]A fully assembled prototype of the chip. The complete array consists of six different fluid chambers (5) including spots and cavities for capturing droplets. Droplets are injected through the chip using a first inlet (connector) channel (3). Excess oil or droplets are discharged from the chambers (5) using an outlet channel (4). Carrier oil is injected through a second inlet channel (1). For droplet fusion, 10% PFO must be injected into the chambers (5) using a third inlet channel (2). Droplets are captured in cavities organized within the fluid chambers (5). Other fluid chambers are also present in the entire chip and can be used independently (6, 7, 8, 9, 10). [Modes for carrying out the invention]

[0018] The present invention relates to a microfluidic system, and the system is a) A solid support comprising at least one group of alkyl groups, i. Each oligonucleotide of the group comprises a first type, a second type and / or further types of nucleic acid sequences, ii. The first type of nucleic acid sequence described above is a barcode sequence, iii. Oligonucleotides containing the same barcode sequence are grouped together in the group of oligonucleotides on the solid support, iv. The first group of oligonucleotides and further groups of oligonucleotides are spatially separated on the solid support, b) One or more of the oligonucleotide groups on the solid support are located in separate reservoirs of the microfluidic system, c) One or more reservoirs are accessible via channels for fluids, cells, chemicals and / or microdroplets. d) Each reservoir containing a group of oligonucleotides on the solid support is also a trap for microfluidic droplets.

[0019] In the context of the present invention, the term “microfluidic system” means a device comprising at least one microfluidic channel. The channel can be fabricated by any method known in the art, including milling, etching, ablation, embossing, or molding of a material (such as glass, silicon, ceramic paper, hydrogel, or polymers such as PDMS, TPE, PS, PEGDA, PFEP / PFA / PFPE, PU, ​​PMMA, PC, COP, or COC and composites thereof).

[0020] Microfluidic systems may also include sorting systems. Microfluidic cell sorting systems are known to those skilled in the art and have been described, for example, by Wyatt Schields (Wyatt Schields et al. 2015, Lab Chip 15(5):1230-1249).

[0021] In the context of the present invention, the term "oligonucleotide" refers to oligomers or polymers of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), as well as non-natural oligonucleotides. Non-natural oligonucleotides are species that contain oligomers or polymers containing nucleic acid base sequences not found in nature, or functional equivalents of naturally occurring nucleic acid bases, sugars, or sugar bonds.

[0022] In one embodiment, the oligonucleotide may comprise one or more nucleic acid sequences selected from the group consisting of a first type, a second type, and / or a third type. In one embodiment, the nucleic acid sequence of the first type may be a barcode sequence. As used herein, the barcode sequence is used to identify a nucleic acid molecule, and sequencing may reveal a specific barcode bound to the target nucleic acid molecule. In the context of the present invention, it is sufficient for at least a portion of the barcode sequence to be recognized in a sequence-specific event in order to identify the target oligonucleotide.

[0023] In the system according to the present invention, the barcode sequence of each group is known, and the position on the solid support is known.

[0024] In the system according to the present invention, at least a portion of the system is optically transparent, enabling optical analysis of the cell(s) captured in the reservoir. Ideally, the transparent portion is adjacent to the oligonucleotide group.

[0025] In the system according to the present invention, each group of oligonucleotides is 10 4 ~10 11 Contains oligonucleotides between the individual cells. The group is approximately 10 9 It is preferable to have (+ / -25%) units.

[0026] In the system according to the present invention, the cell trap is a cavity with dimensions of approximately 10 μm to 200 μm (+ / - 25%). This dimension is set to accommodate droplets containing one or two cells, or in some embodiments, three or more cells, preferably small cells such as bacteria and large cells such as nerve cells.

[0027] In the context of this invention, the term “cell” refers to any eukaryotic cell. Eukaryotic cells include, but are not limited to, epithelial cells, immune cells (such as lymphocytes, neutrophils, and monocytes / macrophages), hematopoietic cells, bone marrow cells, osteoblasts, cardiomyocytes, hepatocytes, and nerve cells. Furthermore, as used herein, unless otherwise specified, the term “cell” refers to a “single cell.”

[0028] In the context of this invention, the term “reservoir” refers to any physical location of a material (e.g., fluid, cell, particle, droplet) such that the material is temporarily or permanently stored / placed in a particular location within the device. Reservoirs may or may not hinder the flow, connection, interaction, contact, and communication of the material with one another.

[0029] In one embodiment of the present invention, it is understood that the group of oligonucleotides on the solid support is not physically located within a reservoir, but rather should be interpreted as being located on the solid support in correspondence with the reservoir. Therefore, there is no reservoir on the solid support containing the group of oligonucleotides. This is also evident from the figures provided herein.

[0030] In another embodiment of the present invention, the oligonucleotide group may be physically conceived within a reservoir.

[0031] In the system according to the present invention, the spatial separation of the oligonucleotide group is at least 100 nm and less than or equal to 1,000 μm (+ / - 25%).

[0032] The inventors have found that this spatial separation is essential to avoid contamination between different spotted DNA or different reservoirs (droplets). Such contamination can lead to misassignment of phenotypic / genotype linkages, or assignment to multiple droplets, resulting in an inability to accurately identify phenotypic / genotype linkages. Another parameter to consider is droplet size. In this regard, reducing spatial separation beyond the scope of the claims would impair chemical, mechanical, and physical events or reactions occurring within the droplets, such as the efficiency of the reverse transcription (RT) reaction.

[0033] In the system according to the present invention, oligonucleotides in a group include nucleic acid sequences of a second type of nucleic acid sequence which may be a universal sequence, a further type of sequence which may be a hybridizing sequence or a primer sequence, and a further type of sequence which may be a hybridizing sequence. The oligonucleotides in each group are identical. See Figure 5. They are typically attached to the 5' prime end. Ideally, the oligonucleotides have different sequence portions that serve different purposes, such as i) barcoding, ii) priming, or iii) hybridizing.

[0034] The present invention also relates to a method for attaching oligonucleotides to cellular biomolecules, the method being: a) To provide a microfluidic system according to the present invention, b) Encapsulating the first cell in the first droplet, c) capturing the cell droplets in the reservoir, d) fusing a second droplet containing the dissolving composition with the first droplet, thereby enabling the oligonucleotide of the solid support to adhere to the nucleic acid in the cell.

[0035] Strictly speaking, oligonucleotides do not adhere to the cell surface. They adhere to nucleic acids and / or biomolecules within the cell. This guides the cell to the oligonucleotide. As used herein, the expression “attaching oligonucleotides to biomolecules within the cell” refers to the process of “binding” or “hybridizing” the oligonucleotide to a selected target biomolecule within the cell. As used herein, the term “biomolecule” refers to any oligonucleotide, single-stranded or double-stranded DNA or RNA. These oligonucleotides then bind to the biomolecule within the cell, preferably nucleic acids. Nucleic acids may be selected from DNA, RNA, tRNA, mRNA, genomic DNA, ribosomal RNA, chromatin, etc. The cell may or may not be dissolved / lysed during the binding process. In a preferred embodiment in which the oligonucleotide binds to a nucleic acid derived from the cell, the cell is lysed, and the bound nucleic acid is then further analyzed.

[0036] In this specification, “droplet” generally refers to a measure of volume. In the context of the present invention, “droplet” refers to an isolated portion of a first fluid surrounded by a second fluid. As used in connection with the processes of the present invention, the term “droplet” includes first, second, third, and fourth types of droplets, such as a single cell, a reagent or fused droplet, or a droplet containing multiple such droplets.

[0037] The "droplets" may have an average volume of less than 5 nL, for example, less than 4 nL, less than 3 nL, preferably less than 3 nL. In some embodiments, the average volume is less than 3 nL, less than 2.5 nL, less than 2 nL, less than 1.5 nL, less than 1 nL, less than 0.5 nL (e.g., 0.1 nL to 3 nL, 0.5 nL to 3 nL, 1 nL to 3 nL), typically 1 pL, 10 pL, 20 pL, 30 pL, 50 pL, 0.1 nL, 0.5 nL, 1 nL, 1.2 nL, 1.4 nL, 1.6 nL, 1.8 nL, 2.0 nL, 2.2 nL, 2.4 nL, 2.6 nL, 2.8 nL, 3 nL.

[0038] Therefore, the "fused droplet" may have an average volume of less than 10 nL. In some embodiments, the average volume is less than 9nL, less than 8nL, less than 7nL, less than 6nL, less than 5nL, less than 4nL, less than 3nL, less than 2nL, less than 1nL, less than 0.5nL, for example, 0.1nL to 10nL, 0.1nL to 8nL, 0.1nL to 6nL, 0.1nL to 5nL (for example, 0.1nL to 3nL, 0.5nL to 5nL, 0.5nL to 3nL, 1nL to 3nL), typically 0.1nL, 0.5nL, 1nL, 1.2nL, 1.4nL, 1.6nL, 1.8nL, 2.0nL, 2.2nL, 2.4nL, 2.6nL, 2.8nL, 3nL, 4nL, or 5nL (for example, 11pL to 8000pL).

[0039] After the fusion of the first droplet and the second droplet occurs, it is preferable to perform a reaction step selected from the group including cell-cell interaction, exposure to one or more substances, exposure to one or more dyes or one or more antibodies, cell lysis, nucleic acid ligation, nucleic acid amplification, nucleic acid hybridization, nucleic acid sequencing, and / or reporter or viability assay.

[0040] This is the essence of the present invention. Once a single cell is placed in the trap, it can be analyzed. The analysis is supported by (1) phenotypic analysis of the cell(s) using microscopic readouts, and (2) spatial barcoding of oligonucleotides that can be bound to a solid support and adhere to the nucleic acids of the single cell. In the context of the present invention, the term “spatial barcode” refers to a specific location of a barcode on the surface of a microfluidic chip or slide.

[0041] Preferably, and more preferably, the phenotype of one or more cells in one or more reservoirs is analyzed. a. Before the droplets fuse, b. After the droplets have fused, c. Before the reaction according to claim 4, or d. The phenotypic analysis is performed after the reaction according to claim 4.

[0042] Preferably, the barcode of the oligonucleotide attached to the solid support is used to identify specific cells in a particular reservoir. Oligonucleotides can also be used in reactions such as PCR. In this case, the amplification product includes barcodes and sequences from a single cell. The phenotype of the cell can then be bound to the barcode, thereby binding its position on the solid support.

[0043] Ideally, phenotypic analysis would include at least one method selected from the group consisting of fluorescence imaging, bright-field microscopy, fluorescence microscopy, confocal microscopy, time-lapse analysis, sequencing, qPCR, isothermal amplification, and, for example, RTqPCR.

[0044] The present invention further relates to a method for determining the phenotype and / or genotype of a single cell, wherein the method is: a) To provide a microfluidic system comprising at least one microfluidic channel and at least a collector system including multiple reservoirs, b) Encapsulating at least one cell from a plurality of cells of the first type separately in a droplet of the first type, Optionally, co-encapsulate cells of a second type from multiple second types into each of first type droplets, c) Flowing multiple droplets of a first type through the microfluidic channels of a microfluidic device, capturing the droplets of the first type in each reservoir of the microfluidic device, and optionally analyzing the phenotype of the droplets contained in the reservoir, d) Flowing multiple droplets of a second type through a microfluidic channel and capturing a second droplet of the second type in each reservoir, e) Fusing a first type of droplet with a second type of droplet in the reservoir, f)e) includes carrying out at least one reaction in the fused droplet obtained in f)e) and determining the readout value of the reaction.

[0045] The microfluidic method for assigning a genotype to a given phenotype of interest, disclosed herein, offers several advantages over methods known in the art. One advantage of the method according to the present invention is that it allows for phenotypic evaluation (including, but not limited to, functional readouts for agonist and / or antagonist assays) based on interaction, recognition, labeling, staining, imaging and / or microscopy, while preserving the precise phenotype / genotype relationship of each individual cell, followed by genotyping assays (including measurement of internal messenger molecules). A further advantage of the method is that it provides improved reliability by using a two-step phenotypic measurement. Finally, the method is characterized by its excellent versatility, as it can be adapted to perform various functional assays by adding a second phenotypic droplet to a first phenotypic droplet.

[0046] The aforementioned advantages are disclosed below in the embodiments and features that characterize the present invention. Embodiments of the present invention are provided in the Examples and Figures section.

[0047] According to one aspect of the present invention, a microfluidic method is provided for assigning a genotype to a desired phenotype in at least one droplet, the method comprising the step of encapsulating at least one cell from a plurality of cells of a first type within a plurality of droplets of the first type, each droplet of the first type containing a single cell or not containing a cell. Optionally, cells of a second type may be co-encapsulated together with cells of the first type within droplets of the first type. The method according to the present invention further comprises injecting and / or flowing such droplets of the first type, containing a single cell of the first type and, optionally, an additional single cell of the second type, into a channel of a microfluidic device. The microfluidic device further comprises at least one collector system including a plurality of reservoirs. Thus, droplets of the first type may be captured separately within such reservoirs. Optionally, imaging or microscopy, not limited to, can be used to analyze the droplets of the first type within the reservoirs to determine the phenotype of the first type of cell or of the first and second types of cells. Further methods for determining phenotypes by the method of the present invention are described herein.

[0048] Subsequently, a second type of droplet containing reagents for performing one or more reactions is injected and / or flowed into a channel of a microfluidic device, and as a result, the second type of droplet may be separately captured inside each of the reservoirs of the microfluidic device. Thus, each reservoir of the microfluidic device contains one droplet of the first type and one droplet of the second type. The first type of droplet may be fused or merged with the second type of droplet according to methods known in the art. After the fusion of the droplets, one or more reactions may be initiated or occur, resulting in one or more detectable readouts or signals. Such readouts may be genotyping reactions, phenotyping reactions, or a combination of both. Thus, in one embodiment of the present invention, the second droplet contains reagents necessary for genotyping and / or phenotyping reactions.

[0049] A reservoir in a microfluidic device may contain multiple oligonucleotides attached to the bottom and a solid support. Such oligonucleotides can be classified into at least a first group, each group being spatially separated from other groups contained in other reservoirs of the device. Groups of oligonucleotides contained within the same reservoir may contain the same nucleic acid sequence of the first type, which may be a barcode sequence. Different reservoirs in a microfluidic device may contain the same or different barcode sequences. In one embodiment, each reservoir contains oligonucleotides having a barcode specific to the reservoir, enabling the identification of oligonucleotides and / or nucleic acids attached to the oligonucleotides contained within or located within the same particular reservoir. Thus, the method according to the present invention facilitates the association of a particular reservoir with a particular barcode, and therefore with a particular phenotype of cells detected within the reservoir. Thus, once the genotype of cells trapped within a particular reservoir is determined, the detected barcode sequence can be associated with the phenotype detected in that particular reservoir.

[0050] Those skilled in the art will be familiar with techniques for preparing microfluidic droplets. Techniques for encapsulating cells within microfluidic droplets are described, for example, in Mazutis, et al. 2013, Nat. Protocol 8:870-891. In one example, the droplets are prepared in a separate microfluidic device before injection.

[0051] To carry out a method according to one aspect of the present invention, the microfluidic chip further comprises at least one collector system including a plurality of reservoirs, traps, or cavities. In the context of the present invention, the terms “reservoir,” “trap,” and “cavity” may be used interchangeably herein. In the context of the present invention, at least one droplet moves into one of the plurality of reservoirs by buoyancy, hydrodynamic or physical force. Preferably, the droplet collection step is carried out by buoyancy.

[0052] Further features of a microfluidic chip for carrying out a method according to one aspect of the present invention are provided later in this section.

[0053] The methods disclosed herein encompass the fluidization of droplets containing a first type of single cell, and optionally, a second and / or third type of single cell. Cell type is a classification used to identify cells based on morphological or phenotypic characteristics. As used herein, the term “fluidization” refers to multiple droplets flowing within a microfluidic chip containing single cells. The cells may be of the first, second, or third type, depending on their cell type, specific genetic or gene expression differences, their origin, or specific cellular function.

[0054] In the methods disclosed herein, the first type of droplet may comprise the first type of encapsulated cell, or the first and second types of co-encapsulated cell.

[0055] In further embodiments, the droplets do not contain cells but contain biomolecules derived from cells or fractions thereof.

[0056] The second type of droplet may contain reagents for performing, inducing, enabling, or supporting a reaction or detectable event within the fused droplet, which may be obtained by fusing the first type of droplet with the second type of droplet.

[0057] In the context of the present invention, the first type of cell may be a bacterial cell (e.g., E. coli and B. subtilis). It may also be, but is not limited to, an epithelial cell, an immune cell (e.g., lymphocytes, neutrophils and monocytes / macrophages), a hematopoietic cell, a myeloid cell, an osteoblast, a cardiomyocyte, a hepatocyte and a nerve cell, or a eukaryote such as a yeast (e.g., Saccharomyces and Pichia). It may also be an insect cell. It may also be a eukaryotic or prokaryotic cell, or a virus, or a pseudoparticle (e.g., a small molecule aggregate as a DNA-forming particle, DNA complex, or DNA aggregate). There are no limitations. Preferred cells include immune cells such as B cells, T cells, NK cells, NKT cells, macrophages, or dendritic cells.

[0058] In the context of the present invention, the desired phenotype may be the presence of a surface marker, a change in the composition of the surface marker, activation or blocking activity, intracellular modification, production of molecules such as metabolites, peptides, and proteins, cell viability, cell interactions, cell replacement, and other cellular behaviors.

[0059] In the context of the present invention, the target genotype may be DNA such as transcript mRNA, tRNA, siRNA, miRNA, piRNA, genomic DNA, or mitochondrial DNA; epigenomics such as modified DNA; chromatin structure; modified RNA; or the structural organization of these molecules.

[0060] In another embodiment, the first type of cell may be a reporter cell. In contrast, the second type of cell may be a secretory cell, preferably an antibody-secreting cell, wherein the antibody is against a membrane target presented by the reporter cell. Thus, in the context of the present invention, the first or second type of cell may have the first phenotype. Similarly, the third type of cell may have the second phenotype.

[0061] According to another embodiment of one aspect of the present invention, the first type of cell may be an antibody-secreting cell, and the second type of cell may be a reporter cell. As used herein, the term “reporter cell” refers to a cell containing a reporter gene, protein or lipid, or chemical compound that ultimately acts on the reporter system to produce the functional effect of the drug.

[0062] According to another embodiment of one aspect of the present invention, the first type of cell may be a T cell, and the second type of cell may be an antibody-presenting cell.

[0063] As used herein, the term “reporter cell” refers to a cell containing a reporter gene, protein, lipid, or chemical compound that, when expressed, produces a reporter signal that is readily measurable, for example, by biological assays, immunoassays, radioimmunoassays, or by colorimetric, fluorescence, or chemiluminescence methods.

[0064] According to one embodiment of one aspect of the present invention, a single cell containing a cell droplet or a co-encapsulated cell droplet has a volume in the range of 10 pL to 10 nL.

[0065] In one embodiment of the method according to the present invention, each of a first type of cell contained in a droplet can be distinguished from another cell of a second type contained in the droplet by using a labeling system such as calcein AM for secretory cells and CellTracker Red for reporter cells. Further selection means may be expressed by using secondary fluorescently labeled detection reagents, such as AlexaFluor647 labeling, Fc-specific anti-IgG F(ab')2 (red fluorescence), or indirect detection (e.g., by streptavidin, or a reagent conjugated with biotin) to visualize the binding of immunoglobulins to targets on reporter cells.

[0066] One embodiment of the present invention enables high-throughput analysis of complex intercellular interactions, such as T cells or plasmablasts that secrete antibodies against a membrane-presented target, and antigen-presenting cells co-encapsulated with them.

[0067] The key is to perform cell assays in droplets and measure compound-induced functional responses, including, but not limited to, calcium flow, cyclic AMP, beta-arrestin recruitment, internalization, cytokine secretion, chemokine secretion, receptor dimerization, actin polymerization, cell division, cell cycle blockade or phosphorylation, MAP kinase activation, apoptosis, necrosis, granulation, dimerization assays, and overexpression and presentation of specific molecules on and / or within the cell surface.

[0068] Secretory cells and reporter cells can be co-encapsulated, and the number of such co-encapsulated cells can be estimated using a Poisson distribution. In the context of the present invention, the co-encapsulation process is performed by making the lambda value of the Poisson distribution of the reporter cell greater than 0.5 to achieve a co-encapsulation rate of more than 50% of secretory cells and reporter cells in the droplet. Alternatively, the same result or higher performance can be achieved using a specially designed device.

[0069] In the context of the present invention, the encapsulation or co-encapsulation of a first type of cell, or first and second types of cells, in a first type of droplet may be performed within the same chip where the analysis is performed, off-chip, in a different chip, or within a microfluidic device. Off-chip may refer to a separate area outside the microfluidic chip. As a result, in one embodiment, multiple droplets can be manipulated or analyzed by storing them off-chip, for example, in a test tube, and then reinjecting the multiple droplets into the microfluidic chip.

[0070] The method according to the present invention may include at least one incubation step, which can be timed to allow the occurrence of a first or second detectable event or reaction.

[0071] As used herein, the terms “detectable event,” “detectable reaction,” or “reaction” refer to any chemical-mechanical-physical event or reaction that can be observed and / or detected. Depending on the phenotypic assay, at least one single cell can be assayed for selected parameters using any suitable assay method that may be qualitative and / or quantitative. Suitable detection methods may include spectroscopic, electrical, hydrodynamic, imaging, microscopic, reporter assays, methods for detecting luminescence or fluorescence, and / or biological methods. The terms “detectable event,” “detectable reaction,” “reaction,” or “assay” may be used interchangeably herein.

[0072] Reactions or chemical-mechanical-physical events may include staining or absence of staining of cells with dyes or any other reagent known to those skilled in the art, amplification reactions, real-time or qPCR reactions, reverse transcription reactions, ligation, viability assays or toxicity assays, sequencing reactions, antibody detection and / or binding to antigens, fluorescence reactions or reporter assays, cell death assays, molecular secretion, cell-cell interactions, cell-to-cell exchange of substances, changes in morphological reactions, viscosity and / or aggregation measurements, synthesis of molecular products, fluorescence emission, and the like.

[0073] As reported above, one of the advantages of the method disclosed herein is its versatility. Thus, a further flow of a third type of droplet containing reagents used in a second reaction or reaction step can also be injected into a microfluidic chip and come into contact with at least one droplet containing at least one cell, which is collected in a cavity or reservoir of a collector system, to generate at least one fused droplet containing a first phenotype and a second phenotype.

[0074] According to another embodiment of one aspect of the present invention, the third type of single-cell droplet has a volume in the range of 10 pL to 10 nL, preferably 50 pL to 1 nL.

[0075] According to one embodiment of one aspect of the present invention, the fusion droplet has a volume in the range of 20 pL to 10 nL, preferably 50 pL to 1 nL.

[0076] According to another embodiment of one aspect of the present invention, a second or third type of single-cell droplet may include one or more dyes for staining cells, a reagent for a sequencing reaction including a fluorescent substrate, a reverse transcription reagent, a lysis buffer, a PCR or qPCR reagent, a reporter and / or a reagent for a viability assay, and / or a reagent for detecting antibody binding.

[0077] Sequencing and / or reverse transcription reactions can analyze genes representing the entire genome or transcriptome of lysed cells, or panels of RNA or DNA used as indicators of effector function, or random sets of RNA or DNA, or epigenetic information (proteins, DNA, RNA and structural arrangements), RNA and DNA combinations, or proteins from the cells or compartments.

[0078] A first type of droplet, which includes cells having a first phenotype and optionally co-encapsulated cells having a second phenotype collected or captured in a reservoir, may optionally be imaged and then brought into contact with a stream of a second type of droplet containing reagents for performing a genotyping reaction, thereby promoting the capture of the second type of droplet in the reservoir and subsequent fusion with the first type of droplet. After the fusion of the first and second type of droplets, the genotyping reaction may occur.

[0079] According to one embodiment of the present invention, a second type of droplet may contain reagents for at least a first reaction. In another embodiment, a second type of droplet may contain reagents for the first and second reactions. In yet another embodiment, a second type of droplet may contain reagents for the first, second, and at least a third reaction. The first, second, and any further reactions may be carried out in a sequential or parallel manner within the fused droplet.

[0080] According to one embodiment of the present invention, a third type of droplet may contain at least a reagent for a second reaction. The third type of droplet can be flowed through a microfluidic channel into a reservoir containing a fused droplet obtained by fusing a first type of droplet and a second type of droplet, both of which are trapped in the same reservoir. The third type of droplet may then be trapped in the reservoir containing the fused droplet after the first and / or second reactions have occurred within the fused droplet. The third type of droplet may be fused with the “fused droplet” in the reservoir, and the second and / or third reactions may occur.

[0081] According to another embodiment of the present invention, a second type of droplet may contain reagents for chromatin digestion (including, but not limited to, MNAse, DNAse, and tagmantase), and a third type of droplet may contain reagents for a sequencing reaction including a ligase (or transposase) and buffer reagents, thereby enabling the capture of desired chromatin fragments when the droplets come into contact with a surface or solid support on which barcoded DNA has been spotted. These chromatin fragments may represent mono, di, tri, or arrays of nucleosomes. They may represent digested DNA ranging in length from 10 bp to several Mb.

[0082] In another embodiment of the present invention, the desired phenotype may include the production of antibodies having effector functions (binding, cross-reactivity, specificity, agonist, antagonist, allosteric regulator), including activation / inhibition of downstream signaling cascades from reporter cells; the production of cytokines and / or granules (e.g., perforin, granzymes) and / or the expression of cell surface markers (e.g., CD69, CD137, CD40L, OX40, PD1), respectively, induced by TCR-MHC peptide complexes from T cells and APCs, which may include activation / inhibition of cellular metabolism (e.g., production of interleukins, cytokines, chemokines, apoptosis, or necrosis).

[0083] Reagents for performing genotyping reactions are known to those skilled in the art. Generally, such reagents may, but are not limited to, a fluorescent substrate, a reverse transcription reagent and a lysis buffer, as well as any source of a barcoded library, oligonucleotides, primers, barcodes, polymerases, ligases, transposases and amplification reagents. As used herein, the term “genotyping” refers to the process of determining the nucleic acid sequence of a single cell using biochemical methods, and / or the structural characteristics of the cell genome / transcriptome.

[0084] Methods for fusing droplets are also known in the art, as described, for example, by Mazutis (Mazutis et al. 2012, Lab Chip 12, 1800-1806). These methods may include the addition of a surfactant such as perfluorooctanol, the provision of a specific microfluidic channel shape, and / or the application of an electric field or acoustic waves. In the context of the present invention, the fusing step is preferably carried out by applying an electric field. The fusing step is carried out in a predetermined area of ​​the chip, and droplets in contact with the predetermined area can be selectively fusing. The terms “fuse” and “fuse” may be used interchangeably herein.

[0085] In the context of the present invention, droplet fusion is achieved by applying an electric field having a frequency in the range of 2 kHz to 40 kHz and a voltage in the range of 500 V to 20000 V for the time required to achieve a fusion efficiency of 80% to 100% between the two droplets involved in the event. Higher or lower frequencies and voltages can also be applied depending on, for example, the surface tension between the droplets, the concentration of the surfactant, the volume of the droplets to be fused, etc.

[0086] According to other embodiments, fusion is performed by laser / photo-induced, chemical, and acoustic fusion, but is not limited to these. According to one embodiment of one aspect of the present invention, fusion step (i) is performed by an electrical configuration comprising a plurality of electrodes. In the context of the present invention, the plurality of electrodes are preferably fabricated on a glass array chip in row and column form from indium tin oxide with a thickness of 300 to 600 angstroms. The electrodes can be structured by photolithography and by sputtering indium tin oxide onto the glass chip. According to a further embodiment of one aspect of the present invention, fusion step (i) is performed by an electrical configuration comprising a plurality of electrodes arranged in row form above and column form below a microfluidic system, or vice versa. An exemplary device for generating a concentrated electric field may be an antistatic gun.

[0087] The inventors have found that droplets can be selectively fused by activating a defined combination of row and column indices. This procedure is particularly advantageous because it provides an additional selection step to the screening process.

[0088] Furthermore, selective droplet fusion is used to release and / or make accessible to the first droplet a second or third droplet contents that potentially possess the desired phenotype and genotypic information. Additionally, the selection of functional antibodies for further processing, such as subsequent sequencing and cloning, expression, and validation, increases the probability of obtaining a true hit with desirable characteristics for secondary screening.

[0089] Following droplet fusion, one or more of the following reactions may be initiated and / or performed within the droplet: fluorescent staining of cells or components of cells, sequencing or sequence capture reactions, amplification or ligation reactions, reporter assays, etc.

[0090] The detection of the first and / or second detectable events according to the present invention may include the use of staining, dyes, labels, enzymes, substrates, cofactors and / or specific binding partners (SBPs). Those skilled in the art will know which method is suitable depending on the phenotype to be detected. In the context of the present invention, the detection of the second detectable event is preferably performed by using a spectroscopic method that yields a mapping of the desired phenotype contained in at least one fusion droplet located in at least one reservoir for each reservoir.

[0091] According to another embodiment of one aspect of the present invention, the fusion step (i) is controlled by electrowetting.

[0092] As used herein, the term “electrowetting” refers to the use of an electric field to alter the wettability of a droplet to a chip surface in order to control the movement and / or shape of the droplet. In the context of the present invention, electrowetting can be used to control the spreading of fused droplets on a chip surface without the need to utilize pumps, valves, channels, and / or other similar fluid handling mechanisms. Examples of electrowetting can be found, for instance, in Pollack et al. 2000, Applied Physics Letters, 77, 1725 (describes a microactuator for rapid manipulation of individual microdroplets, which transfers droplets (0.7-1.0 μl) of 100 mM KCl solution between adjacent electrodes at a voltage of 40-80 V, achieving a maximum electrode switching speed of 20 Hz and an average speed of 30 mm / sec for repeated droplet transfer); and Fouillet et al., Proceedings of ASME ICNMM2006 4 International Conference on Nanochannels, Microchannels and Minichannels June 19-21, 2006, Limerick, Ireland; Paper No. ICNMM2006-96020 (describes the use of dielectric electrowetting (EWOD) in real-time PCR (polymerase chain reaction) in a 64 nl microfluidic droplet).

[0093] Controlling the behavior of fused droplets by electrowetting is important because it can enable the incorporation of barcode nucleotide sequences spotted on the surface of a microfluidic chip into the droplets. The droplets enter through hydrophilic contact with the slide containing the spotted DNA. The contents of the droplets then come into contact with the spotted DNA, triggering a reaction.

[0094] In some embodiments, the fusion droplet contains a specific enzyme capable of cleaving a particular DNA site within the spotted DNA. This reaction is used to release the barcoded DNA within the fusion droplet.

[0095] In one embodiment, the present invention provides a microfluidic chip or device comprising two inlets and one outlet, 2,000 spatial barcodes (up to 200k), and a corresponding reservoir (potentially including a droplet maker design and nozzle integrated into the device).

[0096] In the context of the present invention, a microfluidic chip or device may include different inlets and outlets, as well as different combinations of inlets and outlets. Therefore, a microfluidic chip or device may have at least one inlet and one outlet.

[0097] As used herein, the term “corresponding” refers to a determined location on the chip surface of a spot containing a barcode. In the context of the present invention, the location is preferably defined on the area of ​​the chip surface opposite the reservoir.

[0098] According to another embodiment, each spot is 10 5 Contains an oligonucleotide density exceeding [a certain value].

[0099] According to another embodiment, each spot has a diameter in the range of 10 to 200 μm, preferably in the range of 50 to 150 μm, and more preferably in the range of 60 to 80 μm.

[0100] As used herein, the term “spot” refers to a defined area on the first and / or second surface of a microfluidic chip, where the second droplet comes into contact with the first droplet, and a coalescence / fusion event is triggered by activating a plurality of electrodes positioned on the first and / or second surface of the microfluidic chip by controlling the physical or chemical parameters of the fluid, such as temperature or ionic forces.

[0101] In one embodiment, an electric field is used to fuse at least one droplet of a first type with at least one droplet of a second type. In another embodiment, the fusion step results in a fusion efficiency of 80% to 100%, preferably 90% to 100%, between the first and second type droplets.

[0102] The microfluidic chip disclosed herein offers the advantage of compartmentalizing reactions in separate and distinct regions of the microfluidic chip by the coalescence of selected microfluidic droplets. Thus, the microfluidic chip according to the present invention enables improved control of biological assays that may occur simultaneously in different regions of the chip.

[0103] Polydimethylsiloxane (PDMS) is a two-component polymer comprising a base elastomer and a curing agent. The standard mixing ratio of PDMS is 10 parts base elastomer to 1 part curing agent. In one embodiment, the first polymer solution contains the elastomer and curing agent in a 5:1 ratio. The inventors have found that this ratio provides desirable mechanical properties to the mold.

[0104] When droplets fuse using the electrical configuration according to the present invention, oligonucleotides can be cleaved from the chip surface by any suitable method. Preferably, the oligonucleotides are cleaved by photocutting.

[0105] In one embodiment, the barcode sequence may be specific to one or more reservoirs of the microfluidic device, thus facilitating the identification of single cells captured and analyzed within each reservoir. By customizing and specifically selecting barcodes spotted at specific locations on the solid support of the microfluidic device, the method facilitates the identification of specific phenotypes detected at those specific locations and their association with genetic information obtained by the analytical methods described herein. Thus, the phenotype of a single cell captured within a specific reservoir of the microfluidic device can be associated with the genotype of that single cell.

[0106] As used herein, the term “nucleic acid” generally refers to at least one molecule or chain of DNA, RNA, miRNA, or a derivative or mimic thereof, comprising at least one nucleic acid base, such as a naturally occurring purine or a pyrimidine base found in DNA or RNA. The term “nucleic acid” encompasses the term “oligonucleotide.” The nucleic acids herein may also be attached to one or more proteins.

[0107] In this specification, "RNA" refers to, but is not limited to, functional RNA such as mRNA, tRNA, rRNA, catalytic RNA, siRNA, miRNA, piRNA, ncRNA, lncRNA, and antisense RNA. In one preferred embodiment, RNA refers to mRNA.

[0108] The term "oligonucleotide" refers to at least one molecule with a length of approximately 3 to approximately 500 nucleic acid bases. For example, an oligonucleotide may have a length of at least 3 nucleic acid bases, at least 10 nucleic acid bases, at least 30 nucleic acid bases, at least 50 nucleic acid bases, or at least 100 nucleic acid bases. In some cases, an oligonucleotide may have a length of 100 nucleic acid bases or less, 50 nucleic acid bases or less, etc. Any combination of these is also possible, and for example, the length of an oligonucleotide may be between 3 and 300 nucleic acid bases, preferably between 3 and 200 nucleic acid bases, and more preferably between 3 and 100 nucleic acid bases.

[0109] When carried out in droplets, the method according to the present invention further includes a step of recovering or collecting the fused droplets at the outlet of the channel after the reaction step carried out in the reservoir.

[0110] In another aspect, a method for manufacturing a microfluidic system according to the present invention is disclosed herein, the method being a. Mask generation including fluid device design, b. A resin, preferably photoactivated SU8, for reproducing the recessed design printed on the mask into a raised design. c. Remove excess resin using a suitable solvent for non-photoactivatable resins. d. Polymer molding of microfluidic systems on resin, preferably on SU8 type (PDMS), e. Polymer reaction for solidification, usually PDMS polymerization. f. Removing the molded and solidified polymer from the mold. g. COC hot embossed on solidified polymer (PDMS), h. Remove the COC from the mold. i. Preferably, the process includes assembling the array containing the oligos and the COC fluid portion using thermal sealing, double-sided tape, or any other sealing technique. [Examples]

[0111] Cell sequences are captured from two model cell lines: Jurkat (T cell type) and Ramos (B cell type).

[0112] To encapsulate and sort Jurkat and Ramos cells, reverse transcription (RT) was performed in a microfluidic chamber and pre-spotted slide assembly provided by Arbor Bioscience, as described in patent application WO2018167218A1.

[0113] protocol 1. Cell preparation - Collect Jurkat and Ramos cells, spin wash twice with 1 mL of 1×PBS at 300 g for 6 minutes, and then resuspend the cells in 500 μL of 1×PBS. - Label Jurkat cells with CellTrace FarRed (0.5 μL). - Label Ramos cells with CellTrace Far Red+Yellow (0.25 μL + 0.25 μL). - Incubate in a dark place at room temperature for 30 minutes. - Add RPMI medium containing 10% HI-FBS, and rotate and wash twice with 1×PBS. - Resuspend Jurkat cells in 30 μL of PBS, and resuspend Ramos cells in 200 μL of PBS. - Counting cells: Jurkat: 4 mLn / mL Ramos: 70 mLn / mL - Preparation of cell mixture (λ=1): [Table 1] - An integrated droplet generator and sorter are used to encapsulate and sort cells according to parameters. Aqueous phase: 50 μL / hour, oil 1: 500 μL / hour, oil 2 (spacer): 600 μL / hour. Selection parameters: Red channel, 6000Hz amplitude, 300V, 200μs delay, selection based on 2ms selection time. - Once approximately 30,000 droplets have been sorted, connect the collection outlet to the chamber and seal the waste channel with an Eppendorf tube to stop the aqueous phase. Invert the chamber and collect the droplets rising in the tube until they reach the inside of the chamber, then place the chamber on a microscope stand to observe the filling. - Reduce the oil flow rate to approximately 300 μL / hour. - Once most droplet traps are occupied, flush out any remaining droplets and the clamp outlet, move the chamber to the imaging station, and image in brightfield, TRITC, and Cy5 channels: Jurkat cells - red only, Ramos - yellow (low in red, but still detectable in red).

[0114] 2 RT mix: [Table 2]

[0115] - The droplets are encapsulated with a flow rate of 200 μL / hour + 600 μL / hour of water and oil until they reach the end of the outlet. - Connect the tip outlet to the fluid chamber (the tip assembled according to the present invention) and increase the oil flow rate to 1500 μL / hour until the droplet reaches the center of the tip (stop the water flow). - Reduce the oil flow rate to 200 μL / hour to wash away unwanted droplets. Once there are no excess droplets in the chamber, fuse the droplets with an anti-static gun, trigger for 1 minute, and then fuse the droplets to the surface with 10% PFO at a rate of 200 μL / hour. - Before fusion: - Clamp the tube with a 1.5 mL Eppendorf tube and transfer it to a thermal incubator (with plate adapter). - Incubation program execution: 10 minutes at 37°C, 1.5 hours at 52°C, 1 hour at 4°C. - Elute cDNA from the chamber by injecting AMPure 1.0× into 100 μL of TE buffer, 100 μL of 10% PFO, and 100 μL of TE buffer, followed by 20 μL of water.

[0116] The above process is also shown in Figure 7.

Claims

1. A microfluidic system, wherein the system is v. A solid support comprising at least one group of oligonucleotides, i. Each oligonucleotide in the group comprises a first type, a second type, and / or further types of nucleic acid sequences, ii. The first type of nucleic acid sequence is a barcode sequence, iii. Oligonucleotides containing the same barcode sequence are grouped together in the group of oligonucleotides on the solid support. iv. The first group of oligonucleotides and the further group of oligonucleotides are spatially separated on the solid support, vi. One or more of the oligonucleotide groups on the solid support are located in separate reservoirs of the microfluidic system. vii. The one or more reservoirs are accessible via channels to fluids, cells, chemicals and / or microdroplets, viiii. Each reservoir containing a group of oligonucleotides on the solid support is also a trap for microfluidic droplets in the system.

2. The system according to claim 1, wherein the barcode sequence of each group is known and the position on the solid support is known.

3. The system according to claim 1 or 2, wherein at least a portion of the system is optically transparent, enabling optical analysis of cells captured in the reservoir.

4. Each of the aforementioned groups of oligonucleotides is 10 ４ ~10 １１ The system according to claims 1 to 3, comprising an oligonucleotide molecule between the units.

5. The system according to claims 1 to 4, wherein the cell trap is a cavity with dimensions of 10 and 100 μm.

6. The system according to claims 1 to 5, wherein the spatial separation of each oligonucleotide group is at least 100 nm and 1,000 μm or less.

7. The system according to claims 1 to 6, wherein the oligonucleotides in the group include nucleic acid sequences of a second type of nucleic acid sequence which may be a universal sequence and a further type of sequence which is a hybridized sequence.

8. A method for attaching oligonucleotides to cells, biomolecules of said cells, or preferably nucleic acids contained in said cells, wherein the method is: a) To provide a microfluidic system according to any one of claims 1 to 7, b) Encapsulating the first cell in the first droplet, c) capturing the cells in the reservoir, d) The method comprising fusing a second droplet containing the dissolving composition with the first droplet, thereby enabling the oligonucleotide of the solid support to adhere to the nucleic acid in the cell.

9. The method according to claim 8, wherein, after the fusion of the first droplet and the second droplet occurs, a reaction step is performed, selected from the group consisting of intercellular interaction, exposure to one or more substances, exposure to one or more dyes or one or more antibodies, cell lysis, nucleic acid ligation, nucleic acid amplification, nucleic acid hybridization, nucleic acid sequencing and / or reporter or viability assay.

10. Furthermore, the phenotype of one or more cells in the one or more reservoirs is analyzed. a. Before fusing the droplets, b. After fusing the droplets, c. Before the reaction according to claim 9, d. The method according to claim 8, wherein the phenotypic analysis is performed after the reaction according to claim 9.

11. The method according to claims 8 to 10, wherein the barcode of the oligonucleotide attached to the solid support is used to identify a specific cell in a specific reservoir.

12. The method according to claims 8 to 11, wherein the oligonucleotide attached to the solid support is used in the reaction step described in claim 9.

13. The method according to claim 10, wherein the analysis of the phenotype includes at least one method selected from the group consisting of fluorescence imaging, bright-field microscopy, fluorescence microscopy, confocal microscopy, sequencing, and qPCR.

14. A kit comprising a microfluidic system according to claims 1 to 7, and optionally, instructions for carrying out the method according to claims 8 to 13.

15. A method for manufacturing a microfluidic system according to claims 1 to 7, wherein the method is: a. Generation of a mask including the design of the fluid device, b. A resin, preferably a photoactivated SU8, for reproducing the recessed design printed on the mask in a raised manner. c. Remove excess resin using a suitable solvent for non-photoactivatable resins. d. Polymer molding (PDMS) of the microfluidic system on the resin, preferably on the SU8 type. e. Polymer reaction for solidification, usually PDMS polymerization. f. Removing the molded and solidified polymer from the mold. g. COC hot embossed on solidified polymer (PDMS), h. Removing the COC from the mold. i. The method comprising the step of assembling the array containing the oligo and the COC fluid portion, preferably using thermal sealing, double-sided tape or any other sealing technique.

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