Microfluidic system for improving cell recovery rate

Abstract

According to the microfluidic system capable of improving the cell recovery rate, provided by the invention, after liquid drops are generated, the liquid drops which are not wrapped by DNA tags are separated from the liquid drops which are successfully wrapped by the DNA tags through the high-precision liquid drop sorting module, and the sorting aims at ensuring that the unmarked liquid drops can be effectively reused in subsequent steps. And fusing the sorted liquid drops which are not coated with the DNA labels and the liquid drops coated with the DNA labels again under the action of a liquid drop fusion module, so as to complete the re-labeling of the cells. According to the innovative step, the cells which are not effectively marked in the primary droplet generation process are reused, so that the recovery rate and the utilization rate of the cells are greatly improved, the initial quantity of the cells required by an experiment is reduced, the waste of cell samples, especially precious cell samples, is avoided, and the cost is reduced. The material consumption of samples and the cost of repeated experiments are reduced.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a microfluidic system for improving cell recovery rate. Background Art

[0002] At present, single-cell sequencing technology is mainly divided into two categories: droplet-based single-cell sequencing and plate-based single-cell sequencing. Both technologies are used to analyze the gene expression of single cells, but there are significant differences in operating principles and application scenarios.

[0003] As a high-throughput technology, droplet-based single-cell sequencing is currently widely used in the industry. This technology encapsulates single cells and reaction reagents in tiny oil droplets for processing. These droplets serve as independent reaction chambers and can process thousands to tens of thousands of cells in parallel. Each droplet usually contains a unique barcoded DNA molecule that is used to identify RNA molecules derived from the same cell in subsequent genome sequencing. The main advantages of this technology are its high throughput and relatively low sample input requirements.

[0004] However, despite the excellent performance of droplet-based single-cell sequencing technology in high-throughput analysis, there are significant limitations in actual operation, especially in terms of cell recovery rate. For example, the droplet encapsulation efficiency is low: a significant limitation of the droplet method is the efficiency of droplet encapsulation of DNA-tagged colloids. During the droplet generation process, ideally each droplet should encapsulate a single cell and a DNA-tagged colloid at the same time. However, because the existing technology cannot ensure that all droplets can successfully encapsulate DNA-tagged colloids, some droplets contain only a single cell without a DNA tag. These droplets that are not encapsulated with DNA tags cannot be used in subsequent analysis, resulting in a waste of precious cell samples and further reducing the cell recovery rate.

[0005] At present, under ideal laboratory conditions, the cell recovery rate can only reach about 50%-60%. This means that nearly half of the cell samples are wasted in the experiment, especially for precious cell types such as circulating tumor cells (CTCs) or other rare cell types. This waste is particularly serious. This not only limits the application of these technologies in high-value research, but also causes a lot of waste of resources. Therefore, the market urgently needs a new technology that can effectively improve cell recovery rate, reduce sample waste, and adapt to the complex situations in actual operations. Summary of the invention

[0006] In view of this, the present application provides a microfluidic system for improving cell recovery rate, aiming to effectively solve the problems of low cell recovery rate and waste of cell samples in the existing droplet-based single-cell sequencing technology.

[0007] The present application provides a microfluidic system for improving cell recovery rate, including a microfluidic chip, a droplet sorting module and a droplet fusion module, the microfluidic chip includes a first microfluidic channel and a second microfluidic channel connected to each other, a first target droplet and a first intermediate droplet are generated in the first microfluidic channel, a second intermediate droplet is generated in the second microfluidic channel, the droplet sorting module is used to sort the first intermediate droplet into the second microfluidic channel, and the droplet fusion module is used to fuse the second intermediate droplet with the sorted first intermediate droplet to generate a second target droplet.

[0008] In one embodiment, the microfluidic chip includes a third microfluidic channel, and the third microfluidic channel is connected between the first microfluidic channel and the second microfluidic channel, and is used for allowing the sorted first intermediate droplets to flow into the second microfluidic channel.

[0009] In one embodiment, the portion where the third microfluidic channel is connected to the first microfluidic channel forms a diversion port, the portion where the third microfluidic channel is connected to the second microfluidic channel forms a confluence port, the droplet sorting module is arranged at the diversion port, and the droplet fusion module is arranged downstream of the confluence port.

[0010] In one embodiment, the first target droplet includes a cutting oil film and a single cell, a DNA tag, and an amplification mixture wrapped in the cutting oil film; the first intermediate droplet includes a cutting oil film and a single cell, and an amplification mixture wrapped in the cutting oil film; the second intermediate droplet includes a cutting oil film and a DNA tag wrapped in the cutting oil film; the second target droplet includes a cutting oil film and a single cell, a DNA tag, and an amplification mixture wrapped in the cutting oil film.

[0011] In one embodiment, the droplet sorting module includes an electric field module or a magnetic field module, and the electric field module or the magnetic field module is arranged around the diversion port, and the first target droplet and the first intermediate droplet are sorted by applying an electric field or a magnetic field.

[0012] In one embodiment, the first microfluidic channel includes a first droplet generation channel located upstream of the diversion port and a first droplet recovery channel located downstream of the diversion port, the first target droplet and the first intermediate droplet are generated in the first droplet generation channel, and the first target droplet is recovered via the first droplet recovery channel;

[0013] The second microfluidic channel includes a second droplet generation channel located upstream of the confluence, a droplet pairing channel located between the confluence and the droplet fusion module, and a second droplet recovery channel located downstream of the droplet fusion module. The second intermediate droplets are generated in the second droplet generation channel, the second intermediate droplets and the first intermediate droplets are mixed and paired in the droplet pairing channel, and the second target droplets are recovered through the second droplet recovery channel.

[0014] In one embodiment, the microfluidic system includes a droplet generation device, the microfluidic chip is installed on the droplet generation device, the droplet sorting module and the droplet fusion module are installed beside the first microfluidic channel and the second microfluidic channel respectively; the microfluidic chip includes:

[0015] A first injection channel, connected to the first droplet generation channel, for injecting a single cell suspension;

[0016] A second injection channel, connected to the first droplet generation channel, for injecting DNA tags;

[0017] A third injection channel, connected to the first droplet generation channel, for injecting an amplification mixed solution;

[0018] a fourth injection channel connected to the first droplet generation channel and used for injecting cutting oil;

[0019] a fifth injection channel connected to the second droplet generation channel and used for injecting DNA tags; and

[0020] The sixth injection channel is connected to the second droplet generation channel and is used for injecting cutting oil.

[0021] In one embodiment, the droplet generating device includes a plurality of injection holes, the first injection channel and the third injection channel are respectively configured with injection holes; the second injection channel and the fifth injection channel are jointly configured with an injection hole, or the second injection channel and the fifth injection channel are respectively configured with injection holes; the fourth injection channel and the sixth injection channel are jointly configured with an injection hole, or the fourth injection channel and the sixth injection channel are respectively configured with injection holes; and / or, the droplet generating device includes a first recovery hole connected to the first droplet recovery channel and a second recovery hole connected to the second droplet recovery channel, the first recovery hole is used to recover the first target droplet, and the second recovery hole is used to recover the second target droplet.

[0022] In one embodiment, a filtering structure for intercepting tiny particles and slender impurities in a single-cell suspension is disposed in the injection hole of the first injection channel, and the filtering structure includes a plurality of filtering units, the filtering units are concave-shaped, and the notches of the filtering units are disposed toward the injection port of the injection hole.

[0023] In one embodiment, the filtering structure adopts a filtering fence with multiple levels of filtering units arranged along the injection direction of the injection hole, the multiple filtering units of each level are arranged at intervals, and the filtering units of two adjacent levels are arranged in an staggered manner; the injection hole includes a wide part and a narrow part connected in sequence along the injection direction, and the multiple levels of filtering units are distributed in the wide part and the narrow part.

[0024] In one embodiment, a fusion cavity is formed at the connection portion between the droplet pairing channel and the second droplet recovery channel, and the droplet fusion module includes a positive electrode module and a negative electrode module, the positive electrode module includes a positive electrode liquid channel for injecting positive electrode liquid and a positive terminal arranged in the positive electrode liquid channel, the negative electrode module includes a negative electrode liquid channel for injecting negative electrode liquid and a negative terminal arranged in the negative electrode liquid channel, the positive terminal and the negative terminal are respectively arranged on opposite sides of the fusion cavity, and an electric field is formed between the positive terminal and the negative terminal.

[0025] In summary, the present application provides a microfluidic system for improving cell recovery rate. After droplet generation, the droplets that are not wrapped with DNA labels are separated from the droplets that have successfully wrapped with DNA labels by a high-precision droplet sorting module. The purpose of sorting is to ensure that the unlabeled droplets can be effectively reused in subsequent steps. The sorted droplets that are not wrapped with DNA labels and the droplets wrapped with DNA labels are fused again under the action of the droplet fusion module to complete the re-labeling of the cells. This innovative step allows cells that are not effectively labeled during the initial droplet generation process to be reused, greatly improving the cell recovery rate and cell utilization rate, reducing the starting amount of cells required for the experiment, avoiding the waste of cell samples, especially precious cell samples, reducing the consumables of samples and the cost of repeated experiments, especially when dealing with rare or precious samples. This advantage is particularly obvious; and significantly reduces the data deviation caused by cell loss or incomplete labeling, improves the reliability and consistency of experimental data, and thus improves the overall success rate and data quality of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A simplified schematic diagram of the microfluidic system exemplifying the present application.

[0027] Figure 2 Schematic diagram of the detailed structure of the illustrative microfluidic system of this application.

[0028] Figure 3 for Figure 2 Enlarged schematic diagram of part A.

[0029] Figure 4 for Figure 2 Schematic diagram of the enlarged portion B.

[0030] Figure 5 for Figure 2 Enlarged schematic diagram of part C.

[0031] Figure 6 for Figure 2 Enlarged schematic diagram of part D in the figure. DETAILED DESCRIPTION

[0032] Before describing the embodiments in detail, it should be understood that the present application is not limited to the detailed structure or component arrangement described below or in the drawings in the present application. The present application may be an embodiment implemented in other ways. Moreover, it should be understood that the words and terms used herein are only used for descriptive purposes and should not be interpreted restrictively. The words "including", "comprising", "having" and the like used herein are meant to include the items listed thereafter, their equivalents and other additional items. In particular, when describing "a certain element", the present application does not limit the number of the element to one, but may also include multiple elements.

[0033] Please also refer to Figure 1 and Figure 2 As shown, the present application provides a microfluidic system 10 for improving cell recovery rate, including a microfluidic chip, a droplet sorting module 12 and a droplet fusion module 14, wherein the microfluidic chip includes a first microchannel 16 and a second microchannel 18 connected to each other, a first target droplet 20 and a first intermediate droplet 22 are generated in the first microchannel 16, and a second intermediate droplet 24 is generated in the second microchannel 18. The droplet sorting module 12 is used to sort the first intermediate droplet 22 into the second microchannel 18, and the droplet fusion module 14 is used to fuse the second intermediate droplet 24 with the sorted first intermediate droplet 22 to generate a second target droplet 26.

[0034] In the above-mentioned microfluidic system 10, the droplets after the initial fusion are sorted, and the sorted first intermediate droplets without DNA tags are subjected to secondary fusion, so that on the basis of the cell recovery rate of the initial fusion, the droplets without DNA tags after the initial fusion are utilized through secondary fusion, and finally the total cell recovery rate after the secondary fusion is improved, which solves the problem of cell sample waste in the prior art, is suitable for single-cell sequencing using the droplet method, and is particularly suitable for processing precious cell samples, such as high-throughput analysis of circulating tumor cells, rare immune cells, etc. In the process of implementing the above-mentioned droplet generation, the microfluidic chip is used as a carrier of liquid flow and droplets, and equipment such as a droplet generation device, a droplet sorting module, and a droplet fusion module are used.

[0035] Specifically, the microfluidic chip includes a third microfluidic channel 28, which is connected between the first microfluidic channel 16 and the second microfluidic channel 18, and is used for the sorted first intermediate droplet 22 to flow into the second microfluidic channel 18. The portion where the third microfluidic channel 28 is connected to the first microfluidic channel 16 forms a diversion port 30, and the portion where the third microfluidic channel 28 is connected to the second microfluidic channel 18 forms a confluence port 32. The droplet sorting module 12 is arranged at the diversion port 30, and the droplet fusion module 14 is arranged downstream of the confluence port 32.

[0036] Please also refer to Figures 3 to 5 As shown, the first target droplet 20 includes a cutting oil film 34 and a single cell 36, a DNA tag 38, and an amplification mixture wrapped in the cutting oil film 34; the first intermediate droplet 22 includes a cutting oil film 34 and a single cell 36, and an amplification mixture wrapped in the cutting oil film 34; the second intermediate droplet 24 includes a cutting oil film 34 and a DNA tag 38 wrapped in the cutting oil film 34; the second target droplet 26 includes a cutting oil film 34 and a single cell 36, a DNA tag 38, and an amplification mixture wrapped in the cutting oil film 34. Among them, the single cell 36 is the cell to be sequenced; the main function of the amplification mixture is to provide necessary reaction components, promote polymerase chain reaction (amplify DNA), and improve the sensitivity and specificity of the reaction; the amplification mixture usually contains enzymes, primers and deoxynucleotides, the enzyme is, for example, DNA polymerase, which is responsible for catalyzing the synthesis of DNA; primer refers to a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the beginning of nucleotide polymerization and is connected to the reactant in the form of hydrogen bonds; deoxynucleotides are used to provide the raw materials required for synthesizing DNA; DNA label 38 is used to identify RNA molecules derived from the same cell in subsequent genome sequencing, and the DNA label 38 is, for example, a DNA label gel ball; cutting oil is an inert oil with suitable viscosity and surface tension, which is used to provide a closed oil film layer (cutting oil film 34) in the oil-in-water structure of various droplets, and the reactant solution is wrapped in the oil film layer, so that it can serve as an independent reaction chamber.

[0037] In this embodiment, the sorting principle of the droplet sorting module 12 is to selectively separate different droplets using an electric field or a magnetic field. In the specific implementation process, the DNA tag 38 is set to be conductive, for example, the DNA tag 38 is allowed to carry a positive charge or a negative charge, and the droplet sorting module 12 uses an electric field or a magnetic field to sort the first target droplet 20 and the first intermediate droplet 22. In addition, the droplets that have not successfully wrapped the DNA tag 38 can also be identified and sorted out by a fluorescence detection device. The droplet sorting module 12 applies an electric field of appropriate strength to both sides of the first microfluidic channel 16, so that the first target droplet 20 containing the DNA tag 38 is separated from the first intermediate droplet 22 without the DNA tag 38 under the action of the electric field. The DNA tag 38 produces an electrophoretic effect under the action of an electric field due to its conductivity, causing the first target droplet 20 wrapped with the DNA tag 38 to be deflected and enter the first droplet generation channel 40, while the first intermediate droplet 22 without the DNA tag 38 is not significantly affected by the electric field and is sorted into the third microfluidic channel 28, thereby achieving the sorting of the first target droplet 20 and the first intermediate droplet 22. This electric field-based separation technology can effectively distinguish droplets with different conductivity, achieve fast and accurate microfluidic separation operations, have a high degree of controllability and flexibility, and is suitable for the sorting and analysis of various complex samples. Specifically, the droplet sorting module 12 includes an electric field module or a magnetic field module, which is arranged around the diversion port 30, and is sorted by applying an electric field or a magnetic field to the first target droplet 20 and the first intermediate droplet 22 in the microfluidic channel. In the illustrated embodiment, the droplet sorting module 12 adopts an electric field module, which includes a positive electrode portion 12a and a negative electrode portion 12b. The positive electrode portion 12a and the negative electrode portion 12b are arranged on opposite sides of the diversion port 30 near one end of the first droplet generation channel 40, and an electromagnetic field is formed between the positive electrode portion 12a and the negative electrode portion 12b.

[0038] Further, the first microfluidic channel 16 includes a first droplet generation channel 40 located upstream of the branch port 30 and a first droplet recovery channel 42 located downstream of the branch port 30. The first target droplet 20 and the first intermediate droplet 22 are generated in the first droplet generation channel 40, and the first target droplet 20 is recovered through the first droplet recovery channel 42. The second microfluidic channel 18 includes a second droplet generation channel 44 located upstream of the confluence port 32, a droplet pairing channel 46 located between the confluence port 32 and the droplet fusion module 14, and a second droplet recovery channel 48 located downstream of the droplet fusion module 14. The second intermediate droplet 24 is generated in the second droplet generation channel 44, the second intermediate droplet 24 and the first intermediate droplet 22 are mixed and paired in the droplet pairing channel 46, and the second target droplet 26 is recovered through the second droplet recovery channel 48.

[0039] In the embodiment shown, Figure 2As shown, the microfluidic system includes a droplet generation device, and the microfluidic chip is installed on the droplet generation device to ensure that each interface is firmly connected and there is no leakage. The initial pressure of each flow channel can be calibrated by a pressure monitoring device to ensure that the pressure is stable and evenly distributed. The droplet sorting module 12 and the droplet fusion module 14 can be installed next to the corresponding microchannel of the microfluidic chip. The droplet sorting module 12 is arranged next to the first microchannel 16, and the droplet fusion module 14 is arranged next to the second microchannel 18. Specifically, the microfluidic chip includes:

[0040] A first injection channel 50 is connected to the first droplet generation channel 40 and is used to inject a single cell suspension, wherein the single cell 36 in the single cell suspension is a cell to be sequenced;

[0041] A second injection channel 52, connected to the first droplet generation channel 40, for injecting DNA tags;

[0042] A third injection channel 54, connected to the first droplet generation channel 40, for injecting the amplification mixture;

[0043] a fourth injection channel 56 connected to the first droplet generation channel 40 and used for injecting cutting oil;

[0044] a fifth injection channel 58 connected to the second droplet generation channel 44 and used for injecting DNA tags; and

[0045] The sixth injection channel 60 is connected to the second droplet generation channel 44 and is used for injecting cutting oil.

[0046] Preferably, the connection of the fourth injection channel 56 is located downstream of the connection of the first injection channel 50, the second injection channel 52, and the third injection channel 54, and the connection of the sixth injection channel 60 is located downstream of the fifth injection channel 58, so that the cutting oil finally enters the microchannel to improve the success rate of the oil-in-water structure (droplet).

[0047] Further, the droplet generating device includes a plurality of injection holes, and the injection holes are used to inject raw materials required for cell sequencing. The first injection channel 50 and the third injection channel 54 are respectively configured with injection holes separately. The second injection channel 52 and the fifth injection channel 58 are jointly configured with an injection hole, which can ensure the consistency of DNA labels in various droplets and improve the reliability of data, or the second injection channel 52 and the fifth injection channel 58 are respectively configured with injection holes separately. The fourth injection channel 56 and the sixth injection channel 60 are jointly configured with an injection hole, which can ensure the consistency of the cut oil film of various droplets and improve the reliability of data, or the fourth injection channel 56 and the sixth injection channel 60 are respectively configured with injection holes separately. In this embodiment, the first injection channel 50, the second injection channel 52, the third injection channel 54 and the fifth injection channel 58 are respectively configured with injection holes separately, and the fourth injection channel 56 and the sixth injection channel 60 are jointly configured with an injection hole.

[0048] More specifically, the droplet generating device comprises:

[0049] The first injection hole 62 is connected to the first injection channel 50. For example, one end of the first injection channel 50 is connected to the first droplet generation channel 40, and the other end is connected to the first injection hole 62. The single cell suspension first enters the first injection channel 50 through the first injection hole 62, and then enters the first droplet generation channel 40 through the first injection channel 50;

[0050] The second injection hole 64 is connected to the second injection channel 52. For example, one end of the second injection channel 52 is connected to the first droplet generation channel 40, and the other end is connected to the second injection hole 64. The DNA tag first enters the second injection channel 52 through the second injection hole 64, and then enters the first droplet generation channel 40 through the second injection channel 52;

[0051] The third injection hole 66 is connected to the third injection channel 54. For example, one end of the third injection channel 54 is connected to the first droplet generation channel 40, and the other end is connected to the third injection hole 66. The amplification mixed solution first enters the third injection channel 54 from the third injection hole 66, and then enters the first droplet generation channel 40 from the third injection channel 54;

[0052] The fourth injection hole 68, the fourth injection channel 56 and the sixth injection channel 60 are respectively connected to the fourth injection hole 68, for example, one end of the fourth injection channel 56 is connected to the first droplet generation channel 40, and the other end is connected to the fourth injection hole 68, and one end of the sixth injection channel 60 is connected to the second droplet generation channel 44, and the other end is connected to the fourth injection hole 68, and the cutting oil first enters the fourth injection channel 56 and the sixth injection channel 60 respectively from the fourth injection hole 68, and the cutting oil in the fourth injection channel 56 enters the first droplet generation channel 40, and the cutting oil in the sixth injection channel 60 enters the second droplet generation channel 44;

[0053] The fifth injection hole 70 is connected to the fifth injection channel 58. For example, one end of the fifth injection channel 58 is connected to the second droplet generation channel 44, and the other end is connected to the fifth injection hole 70. The DNA tag first enters the fifth injection channel 58 from the fifth injection hole 70, and then enters the second droplet generation channel 44 from the fifth injection channel 58.

[0054] like Figures 3 to 5 As shown, the single cell suspension, the amplification mixture, and the DNA tag 38 converge in the first droplet generation channel 40 to form a mixture, ensuring that the ratio of the amplification mixture to the single cell suspension is appropriate to ensure the effectiveness of the reaction, and ensuring that the DNA tag 38 is fully mixed with the single cell suspension and the amplification mixture. The mixture and the cutting oil merge into droplets in the first droplet generation channel 40, ensuring that the cutting oil and the mixture meet in the first droplet generation channel 40 and generate stable oil-in-water droplets. The flow rate and pressure of the cutting oil can be adjusted to ensure the uniformity and stability of droplet generation. During the fusion process, a first target droplet 20 in which the cutting oil film 34 encapsulates the single cell 36 and the DNA tag 38 will be generated, and a first intermediate droplet 22 that fails to encapsulate the DNA tag 38 will also be generated. The droplet sorting module 12 uses an electric field or a magnetic field at the diversion port 30 to separate the first intermediate droplet 22 from the first target droplet 20. The first target droplet 20 is diverted to the first droplet recovery channel 42 for recovery, and the first intermediate droplet 22 is diverted to the third microchannel 28 and flows into the second microchannel 18. The DNA tag 38 and the cutting oil merge and fuse into the second intermediate droplet 24 in the second droplet generation channel 44. The second intermediate droplet 24 continues to flow forward and mixes with the first intermediate droplet 22 sorted in the third microchannel 28. The electric field applied by the droplet fusion module 14 causes the first intermediate droplet 22 to be electrically fused with the second intermediate droplet 24 to generate the second target droplet 26. Finally, the second target droplet 26 flows into the second droplet recovery channel 48 for recovery.

[0055] In the illustrated embodiment, the droplet generating device includes a first recovery hole 72 connected to the first droplet recovery channel 42 and a second recovery hole 74 connected to the second droplet recovery channel 48, the first recovery hole 72 is used to recover the first target droplet 20, and the first recovery hole 72 is, for example, connected to the end of the first droplet recovery channel 42, and the second recovery hole 74 is used to recover the second target droplet 26, and the second recovery hole 74 is, for example, connected to the end of the second droplet recovery channel 48.

[0056] The droplet fusion module 14 uses the principle of electric fusion to fuse the first intermediate droplet 22 and the second intermediate droplet 24. Under the action of the electric field, the first intermediate droplet 22 and the second intermediate droplet 24 are fused by taking advantage of the different conductivity and permeability between them. This is because polarized charges accumulate continuously at the droplet interface. When the polarized charges accumulate to a certain extent, they will induce interaction between the droplets, thereby causing the droplets to deform and fuse. For details, please also refer to Figure 2 and Figure 5 As shown, a fusion chamber 76 is formed at the connection portion between the droplet pairing channel 46 and the second droplet recovery channel 48, and the droplet fusion module 14 is disposed adjacent to the fusion chamber 76. The droplet fusion module 14 can form an electric field around the fusion chamber 76, and utilize the electric field to cause the first intermediate droplet 22 and the second intermediate droplet 24 to be electrically fused in the fusion chamber 76. Specifically, the droplet fusion module 14 includes a positive electrode module 78 and a negative electrode module 80, wherein the positive electrode module 78 includes a positive electrode liquid channel 82 for injecting positive electrode liquid and a positive terminal 84 disposed in the positive electrode liquid channel 82, and the negative electrode module 80 includes a negative electrode liquid channel 86 for injecting negative electrode liquid and a negative terminal 88 disposed in the negative electrode liquid channel 86, and the positive terminal 84 and the negative terminal 88 are disposed on opposite sides of the fusion chamber 76, respectively, and an electric field is formed between the positive terminal 84 and the negative terminal 88. The positive terminal 84 is, for example, formed at one end of the positive electrode liquid channel 82 , and the negative terminal 88 is, for example, formed on a side wall of the negative electrode liquid channel 86 .

[0057] Preferably, the fusion chamber 76 includes a plurality of interconnected sub-cavities. In the flow direction, the sub-cavity includes an enlarged end with a gradually increasing cross section, a straight section with a constant cross section, and a reduced section with a gradually decreasing cross section. The positive terminal 84 and the negative terminal 88 are respectively formed with protrusions corresponding to the reduced sections and the two ends of the fusion chamber 76. The droplet generation device includes a positive electrode liquid injection hole 90 and two negative electrode liquid injection holes 92. The other end of the positive electrode liquid channel 82 opposite to the positive terminal 84 is connected to the positive electrode liquid injection hole 90. The positive electrode liquid injection hole 90 and one of the negative electrode liquid injection holes 92 are located on one side of the fusion chamber 76, and the other negative electrode liquid injection hole 92 is located on the other side of the fusion chamber 76. One end of the negative electrode liquid channel 86 is connected to one of the negative electrode liquid injection holes 92, and the other end is connected to the other negative electrode liquid injection hole 92.

[0058] During the secondary electrofusion process, the electrode liquid (positive electrode liquid and negative electrode liquid) in the droplet fusion module 14 can be slowly dispensed from the positive liquid injection hole 90 and the negative liquid injection hole 92 through a high-precision syringe to fill the positive liquid channel 82 and the negative liquid channel 86, respectively, so that no bubbles are generated, and the electric field strength and duration can be adjusted to ensure that the first intermediate droplet 22 and the second intermediate droplet 24 are successfully fused; the application of the electric field is converted to a voltage at a frequency higher than the droplet passing frequency, so that the secondary electrofusion successfully fuses the sorted unlabeled first intermediate droplet 22 and the second intermediate droplet 24 through the action of the electric field, which not only improves the success rate of labeling, but also ensures that each cell is maximized. The application of this technology has greatly improved the experimental efficiency and significantly improved the data quality. By ensuring that each droplet contains the required label through secondary electrofusion, this solution significantly reduces the data deviation caused by cell loss or incomplete labeling, the success rate of the experiment has been greatly improved, and the reliability and consistency of the data have also been guaranteed.

[0059] When preparing a single-cell suspension, although most impurities have been removed by pre-treatment filtration, some micro impurities (such as irregular particles and fiber filaments) may still enter the microchannel. These impurities will cause poor flow in the microchannel, thereby reducing the encapsulation rate of cells in the droplets. This not only increases the risk of experimental failure, but also causes the cells to be unable to be effectively recovered, directly affecting the final data quality. Therefore, the single-cell suspension can be filtered before entering the microchannel of the microfluidic chip to intercept tiny particles and slender impurities in the single-cell suspension. Filtration of the single-cell suspension can be achieved by setting a filtration structure in front of the single-cell suspension inlet on the microfluidic chip.

[0060] In the embodiment shown, Figure 6 As shown, a filtering structure 94 for intercepting tiny particles and elongated impurities in the single-cell suspension is provided in the first injection hole 62 to ensure uniform distribution of cells in the suspension. After preparing the required single-cell suspension and filtering, the quality of the single-cell suspension can be checked with a microscope to ensure that there are no obvious particulate impurities after filtering. The filtering structure 94 includes a plurality of filtering units 96, and the filtering unit 96 is designed to be, for example, in a concave shape, and the notch 98 of the filtering unit 96 is arranged toward the injection port of the first injection hole 62. Optionally, the inner wall of the notch 98 is arc-shaped, for example, the notch 98 includes an arc portion 100 and an opening portion 102 connected to one side of the arc portion 100, the arc portion 100 is connected to the opening portion 102 through a connecting port 104, and the size of the opening portion 102 gradually increases from one end close to the arc portion 100 to the end away from the arc portion 100, for example, the opposite side walls of the opening portion 102 are in an eight-shaped shape.

[0061] Preferably, the filter structure 94 adopts a filter fence with multiple filter layers arranged along the injection direction of the first injection hole 62, the multiple filter layers are arranged at intervals, each filter layer includes a plurality of filter units 96, the multiple filter units 96 are arranged at intervals, and the filter units 96 of two adjacent filter layers are arranged alternately. Further, the first injection hole 62 includes a wide portion 106 and a narrow portion 108 connected in sequence along the injection direction, and the multiple filter layers are distributed in the wide portion 106 and the narrow portion 108. The filter structure at the wide portion 106 can remove larger particulate matter to ensure the smoothness of liquid flow, and the filter structure at the narrow portion 108 further filters fine impurities to ensure that the single cell suspension flowing into the microchannel is pure and free of impurities, thereby improving the cell encapsulation rate.

[0062] In some embodiments, when the cell sample is extremely precious and the cell recovery rate is extremely high, the first target droplet 20 or the second target droplet 26 can also be sorted and subsequently re-fused. For example, the first droplet recovery channel or the second droplet recovery channel is used as the first droplet generation channel of the next microfluidic system to re-sort the first target droplet 20 or the second target droplet 26 and perform subsequent re-fusion operations. The specific details and structure are similar to those of the above-mentioned microfluidic system and will not be repeated here.

[0063] In addition, the microfluidic system 10 of the present application may also include a microscope, a temperature control device, and a high-speed camera. The microscope can be used to observe the process of droplet generation, sorting, and fusion; the temperature control device can be used to keep the temperature stable during the process of droplet generation, sorting, and fusion to promote the stable progress of the experimental process; the high-speed camera can be used to monitor the process of droplet generation, sorting, and fusion in real time, so that researchers can observe and record the experimental process in real time, and adjust the instrument parameters in time when problems are found to ensure the smooth progress of the experimental process.

[0064] Since the microfluidic system 10 of the present application provides higher cell recovery rate and data quality, it is particularly suitable for complex application scenarios of single-cell sequencing technology, such as analysis with extremely high data accuracy requirements in clinical research and precision medicine. Through more efficient sample utilization and reliable data acquisition, this technical solution expands the application of single-cell sequencing in a wider range of fields and improves the applicability and research value of the technology.

[0065] In summary, the present application provides a microfluidic system for improving cell recovery rate. After droplet generation, the droplets that are not wrapped with DNA labels are separated from the droplets that have successfully wrapped with DNA labels by a high-precision droplet sorting module. The purpose of sorting is to ensure that the unlabeled droplets can be effectively reused in subsequent steps. The sorted droplets that are not wrapped with DNA labels and the droplets wrapped with DNA labels are fused again under the action of the droplet fusion module to complete the re-labeling of the cells. This innovative step allows cells that are not effectively labeled during the initial droplet generation process to be reused, greatly improving the cell recovery rate and cell utilization rate, reducing the starting amount of cells required for the experiment, avoiding the waste of cell samples, especially precious cell samples, reducing the consumables of samples and the cost of repeated experiments, especially when dealing with rare or precious samples. This advantage is particularly obvious; and significantly reduces the data deviation caused by cell loss or incomplete labeling, improves the reliability and consistency of experimental data, and thus improves the overall success rate and data quality of the experiment.

[0066] The concepts described herein may be implemented in other forms without departing from their spirit and characteristics. The specific embodiments disclosed should be considered illustrative rather than restrictive. Therefore, the scope of this application is determined by the appended claims rather than by the preceding description. Any changes within the literal meaning and equivalent scope of the claims should fall within the scope of these claims.

Claims

1. A microfluidic system for improving cell recovery rate, characterized in that: The invention comprises a microfluidic chip, a droplet sorting module and a droplet fusion module. The microfluidic chip comprises a first microchannel and a second microchannel connected to each other. A first target droplet and a first intermediate droplet are generated in the first microchannel, and a second intermediate droplet is generated in the second microchannel. The droplet sorting module is used to sort the first intermediate droplet into the second microchannel, and the droplet fusion module is used to fuse the second intermediate droplet with the sorted first intermediate droplet to generate a second target droplet.

2. The microfluidic system for improving cell recovery rate according to claim 1, characterized in that: The microfluidic chip includes a third microfluidic channel, which is connected between the first microfluidic channel and the second microfluidic channel and is used for allowing the sorted first intermediate droplets to flow into the second microfluidic channel.

3. The microfluidic system for improving cell recovery rate according to claim 2, characterized in that: The portion where the third microfluidic channel is connected to the first microfluidic channel forms a branch port, the portion where the third microfluidic channel is connected to the second microfluidic channel forms a confluence port, the droplet sorting module is arranged at the branch port, and the droplet fusion module is arranged downstream of the confluence port.

4. The microfluidic system for improving cell recovery rate according to claim 3, characterized in that: The droplet sorting module includes an electric field module or a magnetic field module, and the electric field module or the magnetic field module is arranged around the diversion port, and is used to apply an electric field or a magnetic field to the first target droplet and the first intermediate droplet for sorting.

5. The microfluidic system for improving cell recovery rate according to claim 3, characterized in that: The first microfluidic channel comprises a first droplet generation channel located upstream of the diversion port and a first droplet recovery channel located downstream of the diversion port, the first target droplet and the first intermediate droplet are generated in the first droplet generation channel, and the first target droplet is recovered through the first droplet recovery channel; The second microfluidic channel includes a second droplet generation channel located upstream of the confluence, a droplet pairing channel located between the confluence and the droplet fusion module, and a second droplet recovery channel located downstream of the droplet fusion module. The second intermediate droplets are generated in the second droplet generation channel, the second intermediate droplets and the first intermediate droplets are mixed and paired in the droplet pairing channel, and the second target droplets are recovered through the second droplet recovery channel.

6. The microfluidic system for improving cell recovery rate according to claim 5, characterized in that: The microfluidic system comprises a droplet generating device, the microfluidic chip is mounted on the droplet generating device, the droplet sorting module and the droplet fusion module are respectively mounted beside the first microfluidic channel and the second microfluidic channel; the microfluidic chip comprises: a first injection channel, connected to the first droplet generation channel, for injecting a single cell suspension; A second injection channel, connected to the first droplet generation channel, for injecting DNA tags; A third injection channel, connected to the first droplet generation channel, for injecting an amplification mixed solution; a fourth injection channel connected to the first droplet generation channel and used for injecting cutting oil; a fifth injection channel connected to the second droplet generation channel and used for injecting DNA tags; and The sixth injection channel is connected to the second droplet generation channel and is used for injecting cutting oil.

7. The microfluidic system for improving cell recovery rate according to claim 6, characterized in that: The droplet generating device includes a plurality of injection holes, the first injection channel and the third injection channel are respectively configured with injection holes; the second injection channel and the fifth injection channel are jointly configured with an injection hole, or the second injection channel and the fifth injection channel are respectively configured with injection holes; the fourth injection channel and the sixth injection channel are jointly configured with an injection hole, or the fourth injection channel and the sixth injection channel are respectively configured with injection holes; and / or the droplet generating device includes a first recovery hole connected to the first droplet recovery channel and a second recovery hole connected to the second droplet recovery channel, the first recovery hole is used to recover the first target droplet, and the second recovery hole is used to recover the second target droplet.

8. The microfluidic system for improving cell recovery rate according to claim 7, characterized in that: A filtering structure for intercepting tiny particles and elongated impurities in a single-cell suspension is arranged in the injection hole of the first injection channel. The filtering structure includes a plurality of filtering units, each of which is concave in shape, and the concave opening of the filtering unit is arranged toward the injection port of the injection hole.

9. The microfluidic system for improving cell recovery rate according to claim 8, characterized in that: The filtering structure adopts a filtering fence with multiple levels of filtering units arranged along the injection direction of the injection hole, the multiple filtering units of each level are arranged at intervals, and the filtering units of two adjacent levels are arranged in an alternating manner; the injection hole includes a wide part and a narrow part connected in sequence along the injection direction, and the multiple levels of filtering units are distributed in the wide part and the narrow part.

10. The microfluidic system for improving cell recovery rate according to claim 5, characterized in that: A fusion cavity is formed at the connection portion between the droplet pairing channel and the second droplet recovery channel, and the droplet fusion module includes a positive electrode module and a negative electrode module, the positive electrode module includes a positive electrode liquid channel for injecting positive electrode liquid and a positive terminal arranged in the positive electrode liquid channel, the negative electrode module includes a negative electrode liquid channel for injecting negative electrode liquid and a negative terminal arranged in the negative electrode liquid channel, the positive terminal and the negative terminal are respectively arranged on opposite sides of the fusion cavity, and an electric field is formed between the positive terminal and the negative terminal.

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