Integrated multifunctional integrated droplet micro-fluidic chip and application thereof

Abstract

The invention relates to an integrated multifunctional integrated liquid drop micro-fluidic chip and application thereof. The integrated multifunctional integrated liquid drop micro-fluidic chip comprises a liquid drop generation area (1), a biochemical reaction incubation area (9) and a liquid drop separation area (10). According to the invention, the integrated function integration of droplet generation, incubation and sorting of single B cells is realized, the high-throughput requirement is met, droplet fusion is effectively reduced, the control is simple and convenient, and the cost is controllable. The invention also has important scientific application value in multiple fields such as stem cell screening, immunotherapy CAR-T cell screening, microbial strain screening, organoid construction and other biosynthesis, disease diagnosis and microbial research.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic technology, and particularly relates to an integrated multifunctional integrated droplet microfluidic chip and applications thereof. Background Art

[0002] Droplet microfluidics is an emerging technology developed in recent years based on microfluidics. Two incompatible liquids are used as dispersed phase and continuous phase respectively. When the two fluid flows meet, the dispersed phase fluid forms tiny volume unit droplets under the shear and extrusion of the continuous phase. Microdroplets have the characteristics of large specific surface area, high throughput, and closed system. The large specific surface area can efficiently transfer substances and energy, greatly reducing the mixing reaction time; each droplet is an independent microreactor, and the sample remains relatively stable in the droplet to avoid cross contamination. These advantages make droplet microfluidics technology have important applications in bioengineering, drug screening, functional material synthesis and other fields.

[0003] Single-cell research and analysis refers to the analysis and measurement of single cells at the molecular or protein level, which can reveal cell heterogeneity and differences between different cells and cell functions. It has great application needs in life science research, drug development, disease occurrence and development, and precision diagnosis and treatment. At present, the main methods for single B cell screening include microwell chip sorting, microfluidic droplet sorting, flow cytometer sorting, and MACS enrichment screening. The basic principle of microwell chip screening is to form microwells or chambers on the chip and introduce individual cells into the microwells for detection reaction and screening. This technology is mainly represented by the Beacon single-cell photoconductive system of Berkeley Lights in the United States, but it has defects such as the screening flux is limited by the number of chip chambers and the high cost of equipment and consumables.

[0004] Droplet microfluidic sorting technology can encapsulate a single B cell and a detection reagent in a droplet of about 100 pl. The antibodies secreted by the B cell react with the detection reagent and then generate a fluorescent signal under laser irradiation. In the droplet sorting chip, the fluorescent signal in the target droplet can trigger a high-voltage output signal, and the droplets are sorted by generating a high-intensity electric field through the electrodes in the chip. The cell sorting flux of droplet microfluidic sorting technology is not limited by the size of the chip, and the cost of equipment and consumables is relatively controllable. The main body of the single-cell droplet sorting microfluidic chip on the market today is divided into three parts: generation, incubation and sorting. It requires a variety of equipment (such as precision pressure control pumps, ovens, lasers, etc.) to be controlled separately to achieve the corresponding functions. The entire implementation process is divided into multiple steps such as droplet generation and transfer into centrifuge tubes for collection, centrifuge tube storage and incubation (oven), and centrifuge tube droplet transfer for sorting. Repeated transfer of droplets is prone to cause droplet fusion, multiple steps, and inconvenient control, which has a certain impact on large-scale market applications. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an integrated multifunctional integrated droplet microfluidic chip and its application, which overcomes the shortcomings of repeated transfer of droplets, easy fusion of droplets, many steps, and inconvenient control, and has the advantages of reducing screening time and meeting high-throughput requirements.

[0006] The present invention provides an integrated multifunctional integrated droplet microfluidic chip, comprising a droplet generation area, a biochemical reaction incubation area and a droplet sorting area; the droplet generation area is provided with an oil phase injection port, a first reagent injection port, a second reagent injection port and a droplet collection port in sequence from one side; a droplet generation area microchannel is provided inside the droplet generation area; the droplet sorting area is provided with an exhaust port, a droplet injection port, a dispersed oil injection port, an invalid droplet collection port and a target droplet collection port in sequence from one side; a droplet sorting area microchannel is provided inside the droplet sorting area; one end of the biochemical reaction incubation area is connected to the droplet collection port, and the other end is connected to the droplet injection port.

[0007] Furthermore, a first cross-shaped intersection and a second cross-shaped intersection are provided between the second reagent injection port and the droplet collection port.

[0008] Furthermore, a negative metal electrode liquid infusion port and a positive metal electrode liquid infusion port are provided on one side of the microchannel of the droplet sorting area.

[0009] Furthermore, the microchannel in the droplet sorting area is bifurcated in a Y shape, so that the target droplets flow to the target droplet collection port in the electric field, and the other droplets flow to the invalid droplet collection port.

[0010] Furthermore, a laser positioning point is provided on one side of the invalid droplet collection port.

[0011] Furthermore, the exhaust port is connected with a pinch valve.

[0012] Furthermore, the oil phase used in the oil phase inlet is fluorinated oil.

[0013] Furthermore, the reagents used in the first reagent injection port and the second reagent injection port include one or more of cells, microorganisms, fluorescent microspheres, and magnetic beads.

[0014] Furthermore, the droplet generation area, the biochemical reaction incubation area and the droplet sorting area are connected in a zigzag shape.

[0015] The present invention also provides an integrated multifunctional integrated droplet microfluidic chip for use in single B cell screening, stem cell screening, immunotherapy CAR-T cell screening, microbial strain screening or organoid construction.

[0016] Beneficial Effects

[0017] The present invention realizes the integrated function integration of droplet generation, incubation and sorting of single B cells, meets the high-throughput requirements, effectively reduces droplet fusion, is easy to operate and has controllable costs. In the screening of monoclonal antibodies, single B cell screening technology can greatly improve the efficiency of antibody preparation. Compared with traditional hybridoma fusion technology, it can greatly reduce the screening time and improve the screening success rate, becoming the development direction of monoclonal antibody preparation technology in the future. In addition, the analysis system based on the integrated multifunctional integrated droplet microfluidic chip also has important scientific application value in multiple fields such as stem cell screening, immunotherapy CAR-T cell screening, microbial strain screening, organoid construction, biosynthesis, disease diagnosis and microbial research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic structural diagram of the integrated multifunctional integrated droplet microfluidic chip of the present invention.

[0019] Figure 2 This is a structural diagram of the microchannels in the droplet generation area of ​​the integrated multifunctional integrated droplet microfluidic chip of the present invention.

[0020] Figure 3 This is a structural diagram of the microchannel in the droplet sorting area of ​​the integrated multifunctional integrated droplet microfluidic chip of the present invention.

[0021] Figure 4 This is a diagram showing an application example of the integrated multifunctional droplet microfluidic chip of the present invention.

[0022] Figure 1-Figure 4 The accompanying drawings are denoted as follows:

[0023] 1. Droplet generation area;

[0024] 2. Oil phase inlet;

[0025] 3. First reagent injection port;

[0026] 4. Second reagent injection port;

[0027] 5. Droplet collection port;

[0028] 6. Microchannel in droplet generation area;

[0029] 7. The first “X” shaped intersection;

[0030] 8. The second "cross" intersection;

[0031] 9. Biochemical reaction incubation area;

[0032] 10. Droplet sorting area;

[0033] 11. Exhaust port;

[0034] 12. Droplet injection port;

[0035] 13. Dispersed oil inlet;

[0036] 14. Invalid droplet collection port;

[0037] 15. Target droplet collection port;

[0038] 16. Pinch valve;

[0039] 17. Droplet sorting area microchannel;

[0040] 18. Negative metal electrode liquid filling port;

[0041] 19. Positive metal electrode liquid filling port;

[0042] 20. Laser positioning point. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0044] Example 1

[0045] Depend on Figure 1 As shown, this embodiment provides an integrated multifunctional integrated droplet microfluidic chip, including a droplet generation area 1, a biochemical reaction incubation area 9 and a droplet sorting area 10; the droplet generation area 1 is provided with an oil phase injection port 2, a first reagent injection port 3, a second reagent injection port 4 and a droplet collection port 5 in sequence from one side; a droplet generation area microchannel 6 is provided inside the droplet generation area 1; the droplet sorting area 10 is provided with an exhaust port 11, a droplet injection port 12, a dispersed oil injection port 13, an invalid droplet collection port 14 and a target droplet collection port 15 in sequence from one side; a droplet sorting area microchannel 17 is provided inside the droplet sorting area 10; one end of the biochemical reaction incubation area 9 is connected to the droplet collection port 5, and the other end is connected to the droplet injection port 12. The droplet generation area 1, the biochemical reaction incubation area 9 and the droplet sorting area 10 are connected in a zigzag shape.

[0046] The materials of the droplet generation area 1 and the droplet sorting area 10 can be glass-like inorganic materials, or organic materials such as PDMS and plastics, which have good biocompatibility and are processed and formed under certain process conditions. Chambers and microchannels are distributed in the generation area and the sorting area, and the height of the microchannel is maintained at 40-100 microns. The oil phase used in the oil phase injection port 2 is fluorinated oil, and the reagents used in the first reagent injection port 3 and the second reagent injection port 4 can be various reagents such as cells, microorganisms, fluorescent microspheres, and magnetic beads.

[0047] The biochemical reaction incubation area 9 is where the droplets stay for biochemical reactions. The droplets are stacked and accumulated in the incubation area to maintain a stable state. The biochemical reaction incubation area 9 can be made of glass, PDMS or plastic, or a silica gel composite, with a certain volume to store droplets and oil phase, and the incubation area outlet is connected with the droplet inlet 12 of the droplet sorting area 10.

[0048] The droplet generation area 1 and the biochemical reaction incubation area 9 are connected by sealing and fixing at the droplet collection port 5, so that the generated droplets quickly enter the biochemical reaction incubation area 9 from the connection site. The sealing and fixing method can be packaged according to the selected material properties. The biochemical reaction incubation area 9 is connected to the droplet injection port 12 of the droplet sorting area 10 by sealing and fixing. After the biochemical reaction is completed, the droplets enter the droplet sorting area 10 from the droplet injection port 12. The sealing and fixing method can be packaged according to the selected material properties.

[0049] Depend on Figure 2 As shown, a first "cross" intersection 7 and a second "cross" intersection 8 are also provided between the second reagent injection port 4 and the droplet collection port 5. The first reagent and the second reagent are respectively pushed into the first "cross" intersection 7 through a syringe from the first reagent injection port 3 and the second reagent injection port 4, and the two reagents meet to form a laminar flow. The oil phase is pushed into the second "cross" intersection 8 from the oil phase injection port 2 through a syringe to form droplets with the mixed liquid phase. The droplet size can be changed by adjusting the flow rate of the reagent and the oil phase.

[0050] Depend on Figure 3 As shown, a negative metal electrode liquid infusion port 18 and a positive metal electrode liquid infusion port 19 are provided on one side of the droplet sorting zone microchannel 17. The droplet inlet 12 and the dispersed oil inlet 13 of the droplet sorting zone intersect in a "Y" shape, and the dispersed oil flow rate is adjusted to keep the droplets at a stable interval and flow rate.

[0051] The droplet sorting area microchannel 17 is Y-shaped and bifurcated. The electrode is used to generate an electric field. The target droplet is deflected by the dielectrophoretic force in the electric field and enters the target droplet collection port 15, while the remaining droplets not affected by the dielectrophoretic force enter the invalid droplet collection port 14. A laser positioning point 20 is provided on one side of the invalid droplet collection port 14.

[0052] The exhaust port 11 of the droplet sorting area 10 is connected to the pinch valve 16, which can effectively reduce the flow resistance in the microchannel cavity of the droplet sorting area, which is conducive to the droplets entering the sorting area. When the droplets are generated and incubated, the pinch valve 16 is kept open, and when the droplets move to the junction of the incubation area and the sorting area, the pinch valve 16 is closed, and the droplets are slowly injected into the sorting area.

[0053] Depend on Figure 4As shown, this embodiment also provides a method for using an integrated multifunctional integrated droplet microfluidic chip, using cells and fluorescent microspheres as the first reagent and the second reagent, and fluorinated oil as the oil phase, forming a mixed laminar flow of cells and fluorescent microspheres in a microchannel, and further forming droplets under the wrapping of fluorinated oil. The target droplets contain both cells and fluorescent microspheres, the microsphere droplets only contain fluorescent microspheres, and the blank droplets contain neither cells nor fluorescent microspheres. After the biochemical reaction of cells and fluorescent microspheres in the droplets of the biochemical reaction incubation area is completed, the droplets enter the sorting area, and the target droplets enter the target droplet collection channel under the action of the dielectrophoretic force, and the microsphere droplets and blank droplets enter the invalid droplet channel, and finally the sorting of single cells is achieved.

[0054] The specific steps include:

[0055] (1) The pinch valve 16 is opened, and the fluorescent microsphere solution and the cell fluid are pushed into the first cross-shaped intersection 7 from the first reagent injection port 3 and the second reagent injection port 4 through a syringe to form a laminar flow. The fluorinated oil is pushed into the second cross-shaped intersection 8 from the oil phase injection port 2 through a syringe to form droplets with the liquid phase. The droplet size is 45-50 μm.

[0056] (2) The droplets flow through the droplet generation zone microchannel 6 and enter the biochemical reaction incubation zone 9. The droplets accumulate in the incubation zone and move upward to the 1 / 2 position of the incubation zone, maintaining a stable state.

[0057] (3) After the droplet generation is completed, the liquid phase and oil phase syringes in the generation area are closed. At this time, the droplets in the incubation area stay due to the biochemical reaction, and the droplets and oil are separated into layers, with the droplets in the upper layer and the oil in the lower layer. The droplets in the upper layer are stable and form a stacked shape.

[0058] (4) After the biochemical reaction is completed, the oil phase syringe in the generation zone is opened, and the oil product is injected into the incubation zone again. As the oil in the incubation zone increases, the droplets gradually move upward and slowly move to the binding site with the sorting zone.

[0059] (5) Close the pinch valve 16. When the droplets enter the droplet separation zone 10, the dispersion oil is pushed into the dispersion oil inlet 13 of the droplet separation zone 10 by a syringe. The dispersion oil and the droplets meet at the "Y"-shaped intersection. By controlling the dispersion oil flow rate, the dispersion oil keeps the droplets at a certain interval, which is convenient for droplet separation.

[0060] (6) Turn on the laser, and the laser positioning point 20 is located at the first positioning point near the bifurcation in the flow channel.

[0061] (7) Turn on the high-voltage amplifier, and the droplets reach the sorting site. Under the action of the dielectrophoretic force, the target droplets are deflected and enter the target droplet collection port 15, while the microsphere droplets and blank droplets are not deflected and continue to flow into the invalid droplet collection port 14.

[0062] (8) After the target droplets are collected, close the dispersed oil syringe and keep the target droplets for later use.

Claims

1. An integrated multifunctional droplet microfluidic chip, characterized in that: The invention comprises a droplet generation area (1), a biochemical reaction incubation area (9) and a droplet sorting area (10); the droplet generation area (1) is provided with an oil phase injection port (2), a first reagent injection port (3), a second reagent injection port (4) and a droplet collection port (5) in sequence from one side; a droplet generation area microchannel (6) is provided inside the droplet generation area (1); the droplet sorting area (10) is provided with an exhaust port (11), a droplet injection port (12), a dispersed oil injection port (13), an invalid droplet collection port (14) and a target droplet collection port (15) in sequence from one side; a droplet sorting area microchannel (17) is provided inside the droplet sorting area (10); one end of the biochemical reaction incubation area (9) is connected to the droplet collection port (5), and the other end is connected to the droplet injection port (12).

2. The integrated multifunctional integrated droplet microfluidic chip according to claim 1, characterized in that: A first cross-shaped intersection (7) and a second cross-shaped intersection (8) are also provided between the second reagent injection port (4) and the liquid droplet collection port (5).

3. The integrated multifunctional integrated droplet microfluidic chip according to claim 1, characterized in that: A negative metal electrode liquid infusion port (18) and a positive metal electrode liquid infusion port (19) are provided on one side of the droplet sorting area microchannel (17).

4. The integrated multifunctional integrated droplet microfluidic chip according to claim 3, characterized in that: The droplet sorting zone microchannel (17) is bifurcated in a Y shape, so that the target droplets flow toward the target droplet collection port (15) in the electric field, and the other droplets flow toward the invalid droplet collection port (14).

5. The integrated multifunctional integrated droplet microfluidic chip according to claim 1, characterized in that: A laser positioning point (20) is provided on one side of the ineffective liquid droplet collection port (14).

6. The integrated multifunctional integrated droplet microfluidic chip according to claim 1, characterized in that: The exhaust port (11) is connected to a pinch valve (16).

7. The integrated multifunctional integrated droplet microfluidic chip according to claim 1, characterized in that: The oil phase used in the oil phase injection port (2) is fluorinated oil; the reagents used in the first reagent injection port (3) and the second reagent injection port (4) include one or more of cells, microorganisms, fluorescent microspheres, and magnetic beads.

8. The integrated multifunctional integrated droplet microfluidic chip according to claim 1, characterized in that: The droplet generation area (1), the biochemical reaction incubation area (9) and the droplet sorting area (10) are connected in a zigzag shape.

9. An application of the integrated multifunctional integrated droplet microfluidic chip as claimed in claim 1 in single B cell screening, stem cell screening, immunotherapy CAR-T cell screening, microbial strain screening or organoid construction.

Images