Integrated multifunctional integrated droplet microfluidic chip and application thereof

Abstract

The application relates to an integrated multifunctional integrated droplet microfluidic chip and application thereof, the integrated multifunctional integrated droplet microfluidic chip comprising a droplet generation area (1), a biochemical reaction incubation area (9) and a droplet sorting area (10). The application realizes the integrated function of single B cell droplet generation, incubation and sorting, meets the high-throughput demand, effectively reduces droplet fusion, is simple to operate and controllable in cost. The application also has important scientific application value in the fields of stem cell screening, immunotherapy CAR-T cell screening, microbial strain screening, organoid construction and other biological synthesis, disease diagnosis and microbial research.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics technology, and specifically relates to an integrated multifunctional droplet microfluidic chip and its applications. Background Technology

[0002] Droplet microfluidics is an emerging technology developed in recent years within the field of microfluidics. Two immiscible liquids serve as the dispersed and continuous phases, respectively. When these two fluids meet, the dispersed phase forms tiny droplets under the shearing and compression of the continuous phase. Microdroplets possess characteristics such as large specific surface area, high throughput, and closed system design. The large specific surface area enables efficient mass transfer and energy transfer, significantly reducing mixing reaction time. Each droplet acts as an independent microreactor, maintaining relative sample stability within the droplet and avoiding cross-contamination. These advantages make droplet microfluidics an important technology for applications in bioengineering, drug screening, and the synthesis of functional materials.

[0003] Single-cell research analysis refers to the analysis and measurement of individual cells at the molecular or protein level. It can reveal cellular heterogeneity and differences in cell types and functions, and has significant application needs in life science research, drug development, disease progression, and precision medicine. Currently, single-cell B-cell screening methods mainly include microarray sorting, microfluidic droplet sorting, flow cytometry sorting, and MACS enrichment screening. The basic principle of microarray sorting is to form microwells or chambers on a chip and introduce individual cells into the microwells for detection and screening. This technology is mainly represented by the Beacon single-cell photoconductive system from Berkeley Lights in the United States. However, it has drawbacks such as limited screening throughput due to the number of chip chambers and high equipment and consumable costs.

[0004] Droplet microfluidic sorting technology can encapsulate individual B cells and detection reagents within droplets of approximately 100 μL. Antibodies secreted by the B cells bind to the detection reagents, and fluorescence signals are generated under laser irradiation. In the droplet sorting chip, the fluorescence signal within the target droplet triggers a high-voltage output signal, generating a high-intensity electric field through electrodes within the chip to sort the droplets. The cell sorting throughput of droplet microfluidic sorting technology is not limited by chip size, and the cost of equipment and consumables is relatively controllable. Currently, single-cell droplet sorting microfluidic chips on the market mainly consist of three parts: generation, incubation, and sorting. Multiple devices (such as precision pressure control pumps, ovens, and lasers) are required to individually control the corresponding functions. The entire process involves multiple steps, including droplet generation and transfer into centrifuge tubes for collection, centrifuge tube preservation and incubation (in an oven), and transfer of droplets from the centrifuge tubes for sorting. Repeated droplet transfer can easily lead to droplet fusion, and the numerous steps and inconvenient operation significantly impact 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 droplet microfluidic chip and its application, which overcomes the disadvantages of repeated droplet transfer causing droplet fusion, multiple steps, and inconvenient operation, and has the advantages of reducing screening time and meeting high throughput requirements.

[0006] This invention provides an integrated multifunctional droplet microfluidic chip, comprising a droplet generation region, a biochemical reaction incubation region, and a droplet sorting region. The droplet generation region is provided with an oil phase inlet, a first reagent inlet, a second reagent inlet, and a droplet collection port sequentially from one side. A droplet generation region microchannel is provided inside the droplet generation region. The droplet sorting region is provided with an exhaust port, a droplet inlet, a dispersed oil inlet, an invalid droplet collection port, and a target droplet collection port sequentially from one side. A droplet sorting region microchannel is provided inside the droplet sorting region. One end of the biochemical reaction incubation region is connected to the droplet collection port, and the other end is connected to the droplet inlet.

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

[0008] Furthermore, one side of the microchannel in the droplet sorting area is provided with a negative electrode metal electrode liquid injection port and a positive electrode metal electrode liquid injection port.

[0009] Furthermore, the microchannels in the droplet sorting area are Y-shaped, causing the target droplets to flow towards the target droplet collection port and other droplets to flow towards the invalid droplet collection port in the electric field.

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

[0011] Furthermore, the exhaust port is connected to a clamp valve.

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

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

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

[0015] This invention also provides an integrated multifunctional 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] This invention integrates the functions of single-B cell droplet generation, incubation, and sorting, meeting high-throughput requirements, effectively reducing droplet fusion, and offering simple operation and controllable costs. In monoclonal antibody screening, single-B cell screening technology can greatly improve antibody preparation efficiency, significantly reducing screening time and increasing the success rate compared to traditional hybridoma fusion technology, becoming the future development direction of monoclonal antibody preparation technology. Furthermore, the analysis system based on the integrated multifunctional droplet microfluidic chip also has significant scientific application value in multiple fields such as stem cell screening, CAR-T cell screening for immunotherapy, microbial strain screening, organoid construction, disease diagnosis, and microbial research. Attached Figure Description

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

[0019] Figure 2 This is a diagram of the microchannel structure of the droplet generation region of the integrated multifunctional droplet microfluidic chip of the present invention.

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

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

[0022] Figures 1-4 The labels in the attached figures are as follows:

[0023] 1. Droplet generation region;

[0024] 2. Oil phase inlet;

[0025] 3. First reagent inlet;

[0026] 4. Second reagent inlet;

[0027] 5. Droplet collection port;

[0028] 6. Microchannels in the droplet generation region;

[0029] 7. The first crossroads;

[0030] 8. The second "+" shaped intersection;

[0031] 9. Biochemical reaction incubation area;

[0032] 10. Droplet sorting area;

[0033] 11. Exhaust port;

[0034] 12. Droplet inlet;

[0035] 13. Dispersion oil inlet;

[0036] 14. Ineffective droplet collection port;

[0037] 15. Target droplet collection port;

[0038] 16. Pinch valve;

[0039] 17. Droplet sorting area microchannel;

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

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

[0042] 20. Laser positioning point. Detailed Implementation

[0043] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0044] Example 1

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

[0046] The droplet generation zone 1 and droplet sorting zone 10 can be made of glass-like inorganic materials or organic materials such as PDMS and plastics, possessing good biocompatibility, and are manufactured through specific process conditions. Both the generation and sorting zones contain chambers and microchannels, with the microchannel height maintained at 40-100 micrometers. The oil phase inlet 2 uses fluorinated oil, while the first reagent inlet 3 and the second reagent inlet 4 can use various reagents such as cells, microorganisms, fluorescent microspheres, and magnetic beads.

[0047] The biochemical reaction incubation zone 9 is a place where droplets reside and undergo biochemical reactions. The droplets accumulate in layers within the incubation zone, maintaining a stable state. The biochemical reaction incubation zone 9 can be made of materials such as glass, PDMS, or plastic, or it can be a silicone-based composite material. It has a certain volume to store droplets and the oil phase. The outlet of the incubation zone is connected to the droplet inlet 12 of the droplet sorting zone 10.

[0048] The droplet generation zone 1 and the biochemical reaction incubation zone 9 are connected by a sealed connection at the droplet collection port 5, allowing the generated droplets to quickly enter the biochemical reaction incubation zone 9 from the connection point. The sealing method can be customized based on the properties of the selected material. The biochemical reaction incubation zone 9 is also connected to the droplet inlet 12 of the droplet sorting zone 10 by a sealed connection. After the biochemical reaction is complete, the droplets enter the droplet sorting zone 10 through the droplet inlet 12. The sealing method can be customized based on the properties of the selected material.

[0049] Depend on Figure 2 As shown, a first cross-shaped junction 7 and a second cross-shaped junction 8 are also provided between the second reagent inlet 4 and the droplet collection port 5. The first reagent and the second reagent are respectively injected into the first cross-shaped junction 7 through the first reagent inlet 3 and the second reagent inlet 4 via syringe. The two reagents meet to form a laminar flow. The oil phase is injected into the second cross-shaped junction 8 through the oil phase inlet 2 via 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, the microchannel 17 of the droplet sorting zone is provided with a negative electrode metal electrode liquid filling port 18 and a positive electrode metal electrode liquid filling port 19 on one side. The flow channels of the droplet inlet 12 and the dispersion oil inlet 13 of the droplet sorting zone intersect in a "Y" shape, and the flow rate of the dispersion oil is adjusted to keep the droplets at a stable interval and flow rate.

[0051] The droplet sorting microchannel 17 is Y-shaped and bifurcated. The electrodes generate an electric field, in which the target droplet is deflected by the dielectric force and enters the target droplet collection port 15, while the remaining droplets, which are not affected by the dielectric 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 vent 11 of the droplet sorting zone 10 is connected to the pinch valve 16. The vent 11 can effectively reduce the flow resistance in the microchannel cavity of the droplet sorting zone, which is beneficial for droplets to enter the sorting zone. During droplet generation and incubation, the pinch valve 16 is kept open. When the droplet moves to the junction of the incubation zone and the sorting zone, the pinch valve 16 is closed, and the droplet is slowly injected into the sorting zone.

[0053] Depend on Figure 4As shown, this embodiment also provides a method for using an integrated multifunctional droplet microfluidic chip. Cells and fluorescent microspheres are used as the first and second reagents, respectively, with fluorinated oil as the oil phase. Cells and fluorescent microspheres are mixed in a laminar flow within the microchannel, further forming droplets under the coating of fluorinated oil. Target droplets contain both cells and fluorescent microspheres, microsphere droplets contain only fluorescent microspheres, and droplets containing neither cells nor fluorescent microspheres are blank droplets. After the biochemical reaction of cells and fluorescent microspheres in the droplets in the biochemical reaction incubation zone is completed, the droplets enter the sorting zone. Target droplets enter the target droplet collection channel under the action of dielectrophoresis, while microsphere droplets and blank droplets enter the ineffective droplet channel, ultimately achieving single-cell sorting.

[0054] Specifically, the steps include the following:

[0055] (1) Open the clamp valve 16. The fluorescent microsphere solution and cell fluid are pushed into the first "+" shaped junction 7 through the syringe from the first reagent inlet 3 and the second reagent inlet 4, respectively, to form a laminar flow. The fluorinated oil is pushed into the second "+" shaped junction 8 through the syringe from the oil phase inlet 2 to form droplets with the liquid phase. The droplet size is 45-50 micrometers.

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

[0057] (3) After the droplet generation is completed, close the liquid and oil phase syringes in the generation zone. At this time, the droplets in the incubation zone remain due to the biochemical reaction, and the droplets and oil separate into layers, with the droplets on the upper layer and the oil on the lower layer. The upper layer of droplets stably forms a stacked structure.

[0058] (4) After the biochemical reaction is completed, open the oil phase injector in the generation zone and inject oil 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 clamp valve 16. When the droplets enter the droplet sorting zone 10, the syringe pushes the dispersing oil into the dispersing oil inlet 13 of the droplet sorting zone 10. The dispersing oil and the droplets meet at the “Y”-shaped junction. By controlling the flow rate of the dispersing oil, the dispersing oil keeps the droplets at a certain interval, which facilitates droplet sorting.

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

[0061] (7) When the high voltage amplifier is turned on, the droplet reaches the sorting site and is subjected to the dielectric force. The target droplet is deflected and enters the target droplet collection port 15, while the microsphere droplet and blank droplet do not deflect and continue to flow into the invalid droplet collection port 14.

[0062] (8) After the target droplet is collected, turn off the dispersing oil injector and leave the target droplet for later use.

Claims

1. An integrated multifunctional droplet microfluidic chip, characterized in that: It includes a droplet generation zone (1), a biochemical reaction incubation zone (9), and a droplet sorting zone (10); the droplet generation zone (1) is provided with an oil phase inlet (2), a first reagent inlet (3), a second reagent inlet (4), and a droplet collection port (5) from one side; the droplet generation zone (1) is provided with a droplet generation zone microchannel (6) inside; the droplet sorting zone (10) is provided with an exhaust port (11), a droplet inlet (12), a dispersed oil inlet (13), an ineffective droplet collection port (14), and a target liquid from one side. Drop collection port (15); the droplet sorting area (10) is provided with a droplet sorting area microchannel (17); 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); a first cross-shaped junction (7) and a second cross-shaped junction (8) are also provided between the second reagent injection port (4) and the droplet collection port (5); the droplet generation area (1), the biochemical reaction incubation area (9) and the droplet sorting area (10) are connected in a zigzag pattern.

2. The integrated multifunctional droplet microfluidic chip according to claim 1, characterized in that: The droplet sorting microchannel (17) is provided with a negative electrode metal electrode liquid inlet (18) and a positive electrode metal electrode liquid inlet (19) on one side.

3. The integrated multifunctional droplet microfluidic chip according to claim 2, characterized in that: The droplet sorting microchannel (17) is Y-shaped and bifurcates, causing the target droplet to flow to the target droplet collection port (15) in the electric field, while other droplets flow to the invalid droplet collection port (14).

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

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

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

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

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