A single-cell droplet generation and dispensing system and method

Abstract

The present application relates to a single cell droplet generation and dispensing system and method, comprising a microdroplet generation device, a detection device and a screening device, and a receiving device; the microdroplet generation device comprises a generation oil droplet main body, a sample channel and an oil phase channel are formed on the generation oil droplet main body, the bottom of the sample channel and the oil phase channel meet to form a detection channel, the bottom of the detection channel is connected with a separation channel and an underside channel; the detection device and the screening device are arranged in the detection channel, the detection device is used for detecting whether the microdroplet is a single cell droplet, if it is a single cell droplet, it is screened into the underside channel through the screening device and enters the rear receiving unit through the underside channel, if it is not a single cell droplet, it enters the separation channel after being screened through the screening device and flows out. The single cell droplet generation and dispensing system solves the technical problem that too many empty microdroplets and multi-cell microdroplets are contained in the existing water-in-oil single cell microdroplets.

Description

Technical Field

[0001] This invention relates to a single-cell droplet generation and dispensing system and method, belonging to the field of microfluidics. Background Technology

[0002] In experiments such as single-cell screening, single-cell culture, and single-cell sequencing, single-cell preparation is a crucial step. Microfluidic technology is widely used in the preparation of single-cell microdroplets.

[0003] Microdroplet technology is a micro / nanotechnology that utilizes the interaction between flow shear force and surface tension within microscale channels to separate continuous fluids into discrete droplets of nanoscale or smaller volume. The principle of water-in-oil microdroplet generation involves introducing two immiscible liquids, such as an oil phase and an aqueous phase, into a microchannel. Under the influence of the microchannel, the aqueous phase distributes as tiny volume modules within the oil phase, forming a series of discrete microdroplets. However, in this technology, the number of cells in each microdroplet follows a Poisson distribution, resulting in a large number of empty droplets (containing no cells) and multicellular droplets (containing two or more cells). These droplets, especially multicellular droplets, can adversely affect subsequent culturing and sequencing processes. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a single-cell droplet generation and dispensing system and method, thereby solving the technical problem that existing water-in-oil single-cell droplets contain too many empty droplets and multi-cell droplets.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] The present invention provides a single-cell droplet generation and dispensing system, including a droplet generation device, a detection device and a screening device, and a receiving device;

[0007] The droplet generating device includes a droplet generating body, on which a sample channel and an oil phase channel are formed. The sample channel is used to pass through a diluted sample solution, and the oil phase channel is used to pass through oil. The bottom of the sample channel and the oil phase channel merge to form a detection channel. The oil phase and the sample solution interact at the merging point to form droplets. The bottom of the detection channel is connected to a separation channel and a lower channel.

[0008] The detection device and the screening device are arranged in the detection channel. The detection device is used to detect whether the droplet is a single-cell droplet. If it is a single-cell droplet, it is screened by the screening device and enters the lower channel and then enters the rear receiving unit through the lower channel. If it is not a single-cell droplet, it is screened by the screening device and then enters the separation channel and flows out.

[0009] Preferably, the sample channel includes a cell solution channel and a diluent channel, which merge at the bottom to form the sample channel. The cell solution channel is used to pass cell solution, and the diluent channel is used to pass diluent. The cell solution is diluted by the diluent in the sample channel to form a sample solution.

[0010] Preferably, the oil phase channel includes two channels, which are symmetrically arranged on both sides of the sample channel. The diluent channel also includes two channels, which are symmetrically arranged on both sides of the cell solution channel.

[0011] Preferably, a droplet storage area is also formed on the detection channel, the droplet storage area is used to store droplets, and the droplet storage area is located upstream of the detection device.

[0012] Preferably, the detection device is a detection imaging device.

[0013] Preferably, the screening device uses dielectrophoresis and pressure adsorption to screen single-cell droplets.

[0014] The preferred pressure adsorption method includes either suction or blowing.

[0015] Preferably, the device also includes a microvalve and a pressurized gas forming device. The microvalve is located at the inlet of the detection channel. The microvalve controls whether the droplet storage area is connected to a droplet generating device or a pressure control device. When connected to a gas pressure device, the gas pressure forming device generates gas pressure to force the droplets into the detection channel. When connected to a droplet generating chip, the generated droplets enter the storage area, completing droplet collection. The gas pressure forming device generates gas pressure to force the droplets into the detection channel.

[0016] Another aspect of the present invention provides a method for generating and dispensing single-cell droplets, based on the aforementioned single-cell droplet generation and dispensing system, comprising the following steps:

[0017] The diluent and cell solution are forced into the diluent channel and cell solution channel respectively by a pressure device, and then they are combined in the sample channel to form a diluted sample solution.

[0018] The oil phase is pressed into the oil phase channel, and the oil phase and sample solution interact in the confluence channel to form microdroplets;

[0019] The micro-valve and gas pressure forming device are opened, and the gas pressure formed forces the microdroplets into the microdroplet storage area;

[0020] The detection device uses imaging technology to perform image recognition on the microdroplets, detecting whether they are single-particle microdroplets. If they are single-cell droplets, they are filtered by the screening device and enter the lower channel, then proceed to the receiving unit. If they are not single-cell droplets, they are filtered through the screening channel and flow out through the separation channel. The markers for image recognition can be normal cells, stained cells, or normal components, stained components, fluorescent components, etc., within the cells.

[0021] Preferably, the receiving unit is a container in the form of an orifice plate, but it can also be a container in the form of a test tube.

[0022] The present invention has the following advantages due to the adoption of the above technical solutions:

[0023] This invention uses a pressure device to pressurize diluent and cell solution into the diluent channel and cell solution channel respectively, where they merge in the sample channel to form a diluted sample solution. An oil phase is then pressed into the oil phase channel, where the oil phase and sample solution interact to form microdroplets in the detection channel. A microvalve and a gas pressure generating device are opened, and the generated gas pressure forces the microdroplets into the microdroplet storage area. The detection device uses imaging technology to identify the microdroplets, determining whether they are single-particle microdroplets. If they are single-cell droplets, they are filtered by a screening device and enter the lower channel, then proceed to the receiving unit. If they are not single-cell droplets, they are filtered by the screening device and flow out through the separation channel. The single-cell droplet generation and dispensing device and method provided by this invention can separate empty microdroplets and multi-cell droplets from single-cell droplets, avoiding adverse effects on subsequent culture, sequencing, etc. Attached Figure Description

[0024] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0025] In the attached diagram:

[0026] Figure 1 This is a schematic diagram of an embodiment of a single-cell droplet generation and dispensing system;

[0027] Figure 2 This is a schematic diagram of an embodiment of a droplet generation device employing vacuum screening.

[0028] Figure 3 This is a schematic diagram of another embodiment of the droplet generation device;

[0029] Figure 4 This is a schematic diagram of an embodiment of a droplet generation device using a blow-and-screen method;

[0030] The markings in the attached diagram are as follows:

[0031] 1-Oil droplet generating body, 2-Detection device, 3-Receiving unit, 4-Screening device, 5-Micro valve, 11-Oil phase channel, 12-Sample channel, 13-Detection channel, 121-Cell solution channel, 122-Dilution solution channel, 14-Lower channel, 15-Separation channel. Detailed Implementation

[0032] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0033] This invention provides a single-cell droplet generation and dispensing system, comprising a droplet generation device, a detection device, a screening device, and a receiving device; the droplet generation device, the detection device, the screening device, and the receiving device are connected in series. This single-cell droplet generation and dispensing system solves the technical problem of existing water-in-oil single-cell droplets containing excessive void droplets and multi-cell droplets.

[0034] like Figure 1 As shown, the single-cell droplet generation and dispensing system includes a droplet generation device, a detection device 2, a screening device 4, and a receiving device. The droplet generation device, the detection device 2, the screening device 4, and the receiving device are connected in series.

[0035] like Figure 2 and Figure 3 As shown, the droplet generating device includes a droplet generating body 1, on which a sample channel 12 and an oil phase channel 11 are formed. The sample channel 12 is used for passing cell solution, and the oil phase channel 11 is used for passing oil. The bottom of the sample channel 12 and the oil phase channel 11 converge to form a detection channel 13. The oil phase and the sample solution interact at the convergence point to form droplets. The bottom of the detection channel 13 is connected to a separation channel and a lower channel. The detection device 13 and a screening device 4 are disposed within the detection device 2. The detection device 2 is used to detect whether the droplets are single-cell droplets. If they are single-cell droplets, they are screened by the screening device 4 and enter the lower channel 14, and then enter the receiving unit 3 through the lower channel 14. If they are not single-cell droplets, they are screened by the screening device 4 and then enter the separation channel 15 to flow out.

[0036] As one implementation method, such as Figure 2 As shown, in order to improve the efficiency of droplet generation, the sample channel 12 may include a cell solution channel 121 and a diluent channel 122. The bottom of the cell solution channel 121 and the diluent channel 122 merge to form the sample channel 12. The cell solution channel 121 is used for passing cell solution, and the diluent channel 122 is used for passing diluent. The cell solution is diluted by the diluent in the sample channel 12 to form a sample solution.

[0037] As another implementation method, the sample solution can be prepared first with diluent and cell solution, and then injected into the sample channel. Both of these methods are within the protection scope of this invention.

[0038] The cell solution channel 121 and diluent channel 122 may be provided in one or more or in multiple groups, rather than being limited to one or two.

[0039] Preferably, in order to improve the efficiency of droplet generation, the oil phase channel 122 includes two channels, which are symmetrically arranged on both sides of the sample channel 121. The diluent channel 122 also includes two channels, which are symmetrically arranged on both sides of the cell solution channel 121.

[0040] The oil phase channel 122 and sample channel 121 can be set as one or two, or as multiple or multiple groups.

[0041] Preferably, a droplet storage area 4 is also formed on the detection channel 12. The droplet storage area 4 is used to store droplets and is located upstream of the detection device 2. Since the droplet generation rate is very fast while the detection rate is relatively slow, a cavity for storing droplets, i.e., the droplet storage area 4, can be added. After the droplets have been generated, the system switches to detection mode (controlled by a micro-valve). Under gas pressure, the droplets are forced into the detection channel 13.

[0042] The detection device 13 is a detection imaging device. In the detection area, each microdroplet is photographed using microscopic imaging technology. Then, the image processing method is used to identify whether the microdroplet is a single-cell microdroplet. If it is a single-cell microdroplet, the screening device 4 is activated to draw the single-cell droplet into the lower channel 14 and into the receiving unit 3; otherwise, it flows directly into the separation channel 15.

[0043] The screening device 4 uses dielectrophoresis and pressure adsorption to screen single-cell droplets.

[0044] like Figure 2 and Figure 4As shown, the pressure adsorption method includes either suction or blowing.

[0045] The single-cell droplet generation and dispensing system further includes a microvalve 5 and a pressurized gas forming device. The microvalve 5 is located at the inlet of the detection channel 13, and the gas pressure forming device generates gas pressure to force the droplets into the detection channel. The microvalve can control whether the droplet storage area is connected to the droplet generation chip or the pressure control device. When connected to the gas pressure device, the gas pressure forming device generates gas pressure to force the droplets into the detection channel; when connected to the droplet generation chip, the generated droplets enter the storage area, completing the droplet collection.

[0046] The receiving unit 3 can be a container in the form of a perforated plate or a container in the form of a test tube.

[0047] Another embodiment of the present invention provides a method for generating and dispensing single-cell droplets, based on the single-cell droplet generation and dispensing system, comprising the following steps:

[0048] The diluent and cell solution are respectively forced into the diluent channel 123 and the cell solution channel 121 by a pressure device, and then merged in the sample channel 12 to form a diluted sample solution;

[0049] The oil phase is pressed into the oil phase channel 13, and the oil phase and sample solution react within the detection channel 13 to form microdroplets;

[0050] Open the micro-valve 5 and the gas pressure forming device, and the formed gas pressure will force the microdroplets into the microdroplet storage area 4;

[0051] The detection device 2 uses imaging technology to identify the microdroplets and detect whether the microdroplets are single-particle microdroplets. If they are single-cell droplets, they are filtered by the screening device and enter the lower channel and then enter the rear receiving unit 3 through the lower channel. If they are not single-cell droplets, they are filtered by the screening device 4 and then enter the separation channel 15 to flow out.

[0052] The markers for image recognition can be normal cells, stained cells, or intracellular staining components, fluorescent components, etc.

[0053] The single-cell droplet generation and dispensing device and method provided by this invention can separate empty droplets and multi-cell droplets from single-cell droplets, avoiding adverse effects on subsequent culture, sequencing, etc.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A single-cell droplet generation and dispensing system, characterized in that, It includes a droplet generation device, a detection device and a screening device, as well as a receiving device; The droplet generating device includes a droplet generating body, on which a sample channel and an oil phase channel are formed. The sample channel is used to pass through a diluted sample solution, and the oil phase channel is used to pass through oil. The bottom of the sample channel and the oil phase channel merge to form a detection channel. The oil phase and the sample solution interact at the merging point to form droplets. The bottom of the detection channel is connected to a separation channel and a lower channel. The detection device and the screening device are arranged in the detection channel. The detection device is used to detect whether the droplet is a single-cell droplet. If it is a single-cell droplet, it is screened by the screening device and enters the lower channel and then enters the receiving unit through the lower channel. If it is not a single-cell droplet, it is screened by the screening device and then enters the separation channel and flows out. It also includes a microvalve and a pressurized gas forming device. The microvalve is located at the inlet of the detection channel. The first end of the microvalve is connected to the gas pressure forming device, the second end is connected to the droplet generating device, and the third end is connected to the droplet storage area. The microvalve controls whether the droplet storage area is connected to the droplet generating chip or to a pressure control device. When connected to the gas pressure device, when the droplet storage area is connected to the gas pressure forming device, the gas pressure generated by the gas pressure forming device forces the droplets into the detection channel. When the droplet storage area is connected to the droplet generating device, the generated droplets enter the droplet storage area, completing the droplet collection. The diluent and cell solution are forced into the diluent channel and cell solution channel respectively by a pressure device, and then they are combined in the sample channel to form a diluted sample solution. The oil phase is pressed into the oil phase channel, and the oil phase and sample solution interact in the confluence channel to form microdroplets; The micro-valve and gas pressure forming device are opened, and the gas pressure formed forces the microdroplets into the microdroplet storage area. The detection device performs image recognition on the microdroplets using imaging technology.

2. The single-cell droplet generation and dispensing system according to claim 1, characterized in that, The sample channel includes a cell solution channel and a diluent channel, which merge at the bottom to form the sample channel. The cell solution channel is used to pass cell solution, and the diluent channel is used to pass diluent. The cell solution is diluted by the diluent in the sample channel to form a sample solution.

3. The single-cell droplet generation and dispensing system according to claim 1, characterized in that, The oil phase channel includes two channels, which are symmetrically arranged on both sides of the sample channel. The diluent channel also includes two channels, which are symmetrically arranged on both sides of the cell solution channel.

4. The single-cell droplet generation and dispensing system according to claim 1, characterized in that, A droplet storage area is also formed on the detection channel, which is used to store droplets and is located upstream of the detection device.

5. The single-cell droplet generation and dispensing system according to claim 1, characterized in that, The detection device is a detection imaging device.

6. The single-cell droplet generation and dispensing system according to claim 1, characterized in that, The screening device uses dielectrophoresis and pressure adsorption to screen single-cell droplets.

7. The single-cell droplet generation and dispensing system according to claim 6, characterized in that, The pressure adsorption method includes either suction or blowing.

8. The single-cell droplet generation and dispensing system according to claim 1, wherein the receiving unit is a test tube-shaped container in the form of a well plate.

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