Microfluidic system for improving cell recovery

Abstract

This application provides a microfluidic system for improving cell recovery. After droplet generation, a high-precision droplet sorting module separates droplets without DNA tags from those with successfully bound DNA tags. The purpose of sorting is to ensure that unlabeled droplets can be effectively reused in subsequent steps. The sorted droplets without DNA tags are then fused with DNA-tagged droplets by a droplet fusion module, thereby completing the relabeling of cells. This innovative step allows cells that were not effectively labeled during the initial droplet generation process to be reused, significantly improving cell recovery and utilization rates, reducing the initial cell volume required for experiments, avoiding waste of cell samples, especially precious cell samples, and reducing the cost of consumables and repeated experiments.

Description

Technical Field

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

[0002] Currently, single-cell sequencing technologies are mainly divided into two categories: droplet-based single-cell sequencing and plate-based single-cell sequencing. Both technologies are used to analyze gene expression in single cells, but they differ significantly in their operating principles and application scenarios.

[0003] Droplet-based single-cell sequencing, a high-throughput technology, is widely used in the industry. This technique processes single cells and reaction reagents by encapsulating them in tiny, oily droplets. These droplets act as independent reaction chambers, allowing for the parallel processing of thousands to tens of thousands of cells. Each droplet typically contains a unique, barcoded DNA molecule used to identify RNA molecules originating 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 its excellent performance in high-throughput analysis, droplet-based single-cell sequencing technology has significant limitations in practical application, particularly regarding cell recovery rates. For example, droplet encapsulation efficiency is low: a significant limitation of the droplet method lies in the efficiency of droplet encapsulation of DNA-tagged gel beads. Ideally, during droplet generation, each droplet should simultaneously encapsulate a single cell and a DNA-tagged gel bead. However, because current technology cannot guarantee that all droplets successfully encapsulate the DNA-tagged gel bead, some droplets contain only a single cell without the DNA tag. These droplets without the DNA tag cannot be used in subsequent analyses, resulting in a waste of valuable cell samples and further reducing cell recovery rates.

[0005] Currently, even under ideal laboratory conditions, cell recovery rates can only reach around 50%-60%. This means that nearly half of the cell samples are wasted in experiments, especially for precious cell types such as circulating tumor cells (CTCs) or other rare cell types. This waste is particularly severe. This not only limits the application of these technologies in high-value research but also results in a significant waste of resources. Therefore, the market urgently needs a new technology that can effectively improve cell recovery rates, reduce sample waste, and adapt to the complexities of practical operations. Summary of the Invention

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

[0007] This 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 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.

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

[0009] In one embodiment, the portion where the third microchannel connects to the first microchannel forms a diversion port, and the portion where the third microchannel connects to the second microchannel forms a confluence port. The droplet sorting module is disposed at the diversion port, and the droplet fusion module is disposed 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 encapsulated within the cutting oil film; the first intermediate droplet includes a cutting oil film and a single cell and an amplification mixture encapsulated within the cutting oil film; the second intermediate droplet includes a cutting oil film and a DNA tag encapsulated within the cutting oil film; and the second target droplet includes a cutting oil film and a single cell, a DNA tag, and an amplification mixture encapsulated within the cutting oil film.

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

[0012] In one embodiment, the first microchannel 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 through the first droplet recovery channel.

[0013] The second microchannel 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 droplet is generated in the second droplet generation channel, the second intermediate droplet and the first intermediate droplet are mixed and paired in the droplet pairing channel, and the second target droplet is recovered through the second droplet recovery channel.

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

[0015] The first injection channel is connected to the first droplet generation channel and is used to inject single-cell suspension;

[0016] The second injection channel, connected to the first droplet generation channel, is used to inject DNA tags;

[0017] The third injection channel is connected to the first droplet generation channel and is used to inject the amplification mixture;

[0018] The fourth injection channel, connected to the first droplet generation channel, is used to inject cutting oil;

[0019] The fifth injection channel, connected to the second droplet generation channel, is used for injecting the DNA tag; and

[0020] The sixth injection channel, connected to the second droplet generation channel, is used to inject cutting oil.

[0021] In one embodiment, the droplet generating device includes multiple injection ports, wherein the first injection channel and the third injection channel are each configured with a separate injection port; the second injection channel and the fifth injection channel share a single injection port, or the second injection channel and the fifth injection channel are each configured with a separate injection port; the fourth injection channel and the sixth injection channel share a single injection port, or the fourth injection channel and the sixth injection channel are each configured with a separate injection port; and / or, the droplet generating device includes a first recovery port connected to the first droplet recovery channel and a second recovery port connected to the second droplet recovery channel, wherein the first recovery port is used to recover the first target droplet, and the second recovery port is used to recover the second target droplet.

[0022] In one embodiment, the inlet of the first injection channel is provided with a filter structure for intercepting tiny particles and slender impurities in the single-cell suspension. The filter structure includes multiple filter units, each filter unit being U-shaped, with the notch of the filter unit facing the injection port of the inlet.

[0023] In one embodiment, the filtration structure employs a filter grid with multiple levels of filter units arranged along the injection direction of the injection port, wherein multiple filter units in each level are spaced apart, and filter units in adjacent levels are staggered; the injection port includes a wide portion and a narrow portion connected sequentially along the injection direction, and the multiple levels of filter units are distributed within the wide portion and the narrow portion.

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

[0025] In summary, this application provides a microfluidic system for improving cell recovery. After droplet generation, a high-precision droplet sorting module separates droplets without DNA tags from those with successfully bound DNA tags. The purpose of sorting is to ensure that unlabeled droplets can be effectively reused in subsequent steps. The sorted droplets without DNA tags are then fused with the DNA-tagged droplets by a droplet fusion module, thereby completing the relabeling of cells. This innovative step allows for the reuse of cells that were not effectively labeled during the initial droplet generation process, significantly improving cell recovery and utilization rates, reducing the initial cell quantity required for experiments, avoiding waste of cell samples, especially precious cell samples, and reducing the cost of consumables and repeated experiments. This advantage is particularly evident when processing rare or precious samples. Furthermore, it significantly reduces data bias caused by cell loss or incomplete labeling, improving the reliability and consistency of experimental data, thereby enhancing the overall success rate and data quality of experiments. Attached Figure Description

[0026] Figure 1 This is a simplified structural diagram of the microfluidic system exemplified in this application.

[0027] Figure 2 This is a detailed structural diagram of the microfluidic system exemplified in this application.

[0028] Figure 3 for Figure 2 An enlarged schematic diagram of part A in the middle.

[0029] Figure 4 for Figure 2 Enlarged schematic diagram of part B.

[0030] Figure 5 for Figure 2 An enlarged schematic diagram of section C.

[0031] Figure 6 for Figure 2 An enlarged schematic diagram of part D in the middle. Detailed Implementation

[0032] Before describing the embodiments in detail, it should be understood that this application is not limited to the detailed structures or element arrangements described below or in the accompanying drawings. This application can be implemented in other ways. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes only and should not be construed as limiting. The terms "comprising," "including," "having," and similar expressions used herein mean to include the items listed thereafter, their equivalents, and other additional items. In particular, when describing "an element," this application does not limit the number of elements to one, but may include multiple elements.

[0033] Please also refer to Figure 1 and Figure 2 As shown, this 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. 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 aforementioned microfluidic system 10, the droplets after the initial fusion are sorted, and the sorted first intermediate droplets without DNA tags are fused a second time. This improves the overall cell recovery rate after the second fusion by utilizing the droplets without DNA tags, building upon the cell recovery rate of the initial fusion. This solves the problem of cell sample waste in existing technologies and is suitable for single-cell sequencing using droplet methods, especially for high-throughput analysis of precious cell samples such as circulating tumor cells and rare immune cells. In the droplet generation process, a microfluidic chip is used as the carrier of the fluid flow and droplets, and equipment such as a droplet generation device, a droplet sorting module, and a droplet fusion module are employed.

[0035] Specifically, the microfluidic chip includes a third microchannel 28, which is connected between the first microchannel 16 and the second microchannel 18, for allowing the sorted first intermediate droplet 22 to flow into the second microchannel 18. The portion where the third microchannel 28 connects to the first microchannel 16 forms a diversion port 30, and the portion where the third microchannel 28 connects to the second microchannel 18 forms a confluence port 32. The droplet sorting module 12 is disposed at the diversion port 30, and the droplet fusion module 14 is disposed 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 encapsulated within 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 encapsulated within the cutting oil film 34; the second intermediate droplet 24 includes a cutting oil film 34 and a DNA tag 38 encapsulated within the cutting oil film 34; and the second target droplet 26 includes a cutting oil film 34 and a single cell 36, a DNA tag 38, and an amplification mixture encapsulated within the cutting oil film 34. Among them, single cell 36 is the cell to be sequenced; the main function of the amplification mixture is to provide the necessary reaction components, promote polymerase chain reaction (DNA amplification), and improve the sensitivity and specificity of the reaction; the amplification mixture usually contains enzymes, primers, and deoxynucleotides. The enzyme, for example, is DNA polymerase, which is responsible for catalyzing DNA synthesis; the primer is a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the onset of nucleotide polymerization and is linked to the reactants by hydrogen bonds; the deoxynucleotides are used to provide the raw materials required for DNA synthesis; DNA tag 38 is used to identify RNA molecules from the same cell in subsequent genome sequencing. DNA tag 38 is, for example, a DNA tag gel ball; the cutting oil is an inert oil with suitable viscosity and surface tension, used to provide a closed oil film layer (cutting oil film 34) in the water-in-oil structure of various droplets. The oil film layer encapsulates the reactant solution, thus serving 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 specific implementation, the DNA tag 38 is made conductive, for example, by carrying a positive or negative charge. The droplet sorting module 12 uses an electric field or magnetic field to separate the first target droplet 20 and the first intermediate droplet 22. Alternatively, a fluorescence detection device can be used to identify and sort droplets that have not successfully encapsulated the DNA tag 38. The droplet sorting module 12 applies an electric field of appropriate strength to both sides of the first microchannel 16, causing the first target droplet 20 containing the DNA tag 38 to separate from the first intermediate droplet 22 without the DNA tag 38 under the influence of the electric field. Due to its conductivity, the DNA tag 38 undergoes electrophoresis under the influence of an electric field, causing the first target droplet 20, which encapsulates the DNA tag 38, to deflect into the first droplet generation channel 40. Meanwhile, the first intermediate droplet 22, which does not contain the DNA tag 38, is not significantly affected by the electric field and is sorted into the third microfluidic channel 28, thus 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, achieving rapid and precise microfluidic separation operations. It has high 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 positioned around the diversion port 30. The module applies an electric field or magnetic field to the first target droplet 20 and the first intermediate droplet 22 in the microfluidic channel for sorting. In the illustrated embodiment, the droplet sorting module 12 employs 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 respectively located on opposite sides of the diversion port 30 near the 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 microchannel 16 includes a first droplet generation channel 40 located upstream of the diversion port 30 and a first droplet recovery channel 42 located downstream of the diversion 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 microchannel 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, and the second intermediate droplet 24 and the first intermediate droplet 22 are mixed and paired in the droplet pairing channel 46. The second target droplet 26 is recovered through the second droplet recovery channel 48.

[0039] In the illustrated embodiment, as Figure 2As shown, the microfluidic system includes a droplet generation device, on which a microfluidic chip is mounted. Ensuring secure connections and no leakage at each interface, the initial pressure of each channel can be calibrated using a pressure monitoring device to ensure stable and uniform pressure distribution. A droplet sorting module 12 and a droplet fusion module 14 can be installed beside the corresponding microchannels of the microfluidic chip. The droplet sorting module 12 is located beside the first microchannel 16, and the droplet fusion module 14 is located beside the second microchannel 18. Specifically, the microfluidic chip includes:

[0040] The 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] The second injection channel 52 is connected to the first droplet generation channel 40 and is used to inject DNA tags.

[0042] The third injection channel 54 is connected to the first droplet generation channel 40 and is used to inject the amplification mixture.

[0043] The fourth injection channel 56 is connected to the first droplet generation channel 40 and is used to inject cutting oil;

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

[0045] The sixth injection channel 60 is connected to the second droplet generation channel 44 and is used to inject 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, thereby improving the success rate of the water-in-oil structure (droplet).

[0047] Furthermore, the droplet generation device includes multiple injection ports for injecting raw materials required for cell sequencing. The first injection channel 50 and the third injection channel 54 are each configured with a separate injection port. The second injection channel 52 and the fifth injection channel 58 share a single injection port, which ensures the consistency of DNA tags in various droplets and improves data reliability; alternatively, the second injection channel 52 and the fifth injection channel 58 can each be configured with a separate injection port. The fourth injection channel 56 and the sixth injection channel 60 share a single injection port, which ensures the consistency of oil film cutting by various droplets and improves data reliability; alternatively, the fourth injection channel 56 and the sixth injection channel 60 can each be configured with a separate injection port. 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 each configured with a separate injection port, while the fourth injection channel 56 and the sixth injection channel 60 share a single injection port.

[0048] More specifically, the droplet generation device includes:

[0049] The first injection port 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 port 62. The single cell suspension first enters the first injection channel 50 through the first injection port 62 and then enters the first droplet generation channel 40 through the first injection channel 50.

[0050] The second injection port 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 port 64. The DNA tag first enters the second injection channel 52 through the second injection port 64 and then enters the first droplet generation channel 40 through the second injection channel 52.

[0051] The third injection port 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 port 66. The amplification mixture first enters the third injection channel 54 through the third injection port 66 and then enters the first droplet generation channel 40 through the third injection channel 54.

[0052] The fourth injection port 68, the fourth injection channel 56 and the sixth injection channel 60 are respectively connected to the fourth injection port 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 port 68. 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 port 68. The cutting oil first enters the fourth injection channel 56 and the sixth injection channel 60 through the fourth injection port 68. 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 port 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 port 70. The DNA tag first enters the fifth injection channel 58 through the fifth injection port 70 and then enters the second droplet generation channel 44 through the fifth injection channel 58.

[0054] like Figures 3 to 5 As shown, the single-cell suspension, amplification mixture, and DNA tag 38 converge in the first droplet generation channel 40 to form a mixture, ensuring an appropriate ratio of amplification mixture to single-cell suspension to guarantee the effectiveness of the reaction and to ensure that DNA tag 38 is fully mixed with the single-cell suspension and amplification mixture. The mixture and cutting oil fuse into droplets in the first droplet generation channel 40, ensuring that the cutting oil and mixture meet in the first droplet generation channel 40 and generate stable water-in-oil 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, which encapsulates the single cell 36 and DNA tag 38, is generated by a cutting oil film 34, while a first intermediate droplet 22 that fails to encapsulate the DNA tag 38 is also generated. At the diversion port 30, the droplet sorting module 12 uses an electric or magnetic field 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, while 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 in the second droplet generation channel 44 to form the second intermediate droplet 24. The second intermediate droplet 24 continues to flow forward and mixes and pairs with the first intermediate droplet 22 sorted from the third microchannel 28. The electric field applied by the droplet fusion module 14 causes the first intermediate droplet 22 and the second intermediate droplet 24 to electro-fuse and 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 a first droplet recovery channel 42 and a second recovery hole 74 connected to a second droplet recovery channel 48. The first recovery hole 72 is used to recover a first target droplet 20 and is connected, for example, to the end of the first droplet recovery channel 42. The second recovery hole 74 is used to recover a second target droplet 26 and is connected, for example, to the end of the second droplet recovery channel 48.

[0056] The droplet fusion module 14 utilizes the principle of electrofusion to fuse the first intermediate droplet 22 and the second intermediate droplet 24. Under the influence of an electric field, the difference in conductivity and permeability between the first intermediate droplet 22 and the second intermediate droplet 24 causes them to fuse. This is because polarization charges continuously accumulate at the droplet interface. When the polarization charges accumulate to a certain level, they induce interactions between the droplets, thereby causing the droplets to deform and fuse. For details, please refer to [further details]. Figure 2 and Figure 5 As shown, a fusion cavity 76 is formed at the connection between the droplet pairing channel 46 and the second droplet recovery channel 48. The droplet fusion module 14 is disposed adjacent to the fusion cavity 76. The droplet fusion module 14 can generate an electric field around the fusion cavity 76 and use the electric field to make the first intermediate droplet 22 and the second intermediate droplet 24 electrically fuse within the fusion cavity 76. Specifically, the droplet fusion module 14 includes a positive electrode module 78 and a negative electrode module 80. 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. 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. The positive terminal 84 and the negative terminal 88 are respectively disposed on opposite sides of the fusion cavity 76, and an electric field is formed between the positive terminal 84 and the negative terminal 88. The positive electrode 84 is formed, for example, at one end of the positive electrode liquid channel 82, and the negative electrode 88 is formed, for example, on the side wall of the negative electrode liquid channel 86.

[0057] Preferably, the fusion cavity 76 includes multiple interconnected sub-cavities. In the flow direction, each sub-cavity includes an enlarging end with a gradually increasing cross-section, a straight section with a constant cross-section, and a shrinking section with a gradually decreasing cross-section. The positive end 84 and the negative end 88 have protrusions corresponding to the shrinking section and the two ends of the fusion cavity 76, respectively. The droplet generating device includes a positive electrode liquid inlet 90 and two negative electrode liquid inlets 92. The positive electrode liquid channel 82 is connected to the positive electrode liquid inlet 90 at the opposite end to the positive end 84. The positive electrode liquid inlet 90 and one of the negative electrode liquid inlets 92 are located on one side of the fusion cavity 76, and the other negative electrode liquid inlet 92 is located on the other side of the fusion cavity 76. One end of the negative electrode liquid channel 86 is connected to one of the negative electrode liquid inlets 92, and the other end is connected to the other negative electrode liquid inlet 92.

[0058] During the secondary electrofusion process, the electrode solutions (positive and negative electrode solutions) in the droplet fusion module 14 are slowly dispensed and filled into the positive electrode solution channel 82 and negative electrode solution channel 86 through the positive electrode solution inlet 90 and negative electrode solution inlet 92 respectively using high-precision syringes, preventing the formation of air bubbles. The electric field strength and duration can be adjusted to ensure successful fusion of the first intermediate droplet 22 and the second intermediate droplet 24. The electric field is applied at a frequency higher than the droplet passage frequency for voltage conversion. Thus, the secondary electrofusion, through the action of the electric field, enables the sorted unlabeled first intermediate droplet 22 and the second intermediate droplet 24 to fuse successfully. This not only improves the labeling success rate but also ensures that each cell is utilized to the maximum extent. The application of this technology greatly improves experimental efficiency and significantly enhances data quality. By ensuring that each droplet contains the required label through secondary electrofusion, this scheme significantly reduces data bias caused by cell loss or incomplete labeling, greatly improves the experimental success rate, and guarantees the reliability and consistency of the data.

[0059] During the preparation of single-cell suspensions, although most impurities are removed through pretreatment filtration, some microscopic impurities (such as irregular particles and filaments) may still enter the microchannels. These impurities can obstruct flow within the microchannels, thereby reducing the cell encapsulation rate in the droplets. This not only increases the risk of experimental failure but also prevents the effective recovery of cells, directly affecting the final data quality. Therefore, the single-cell suspension can be filtered before entering the microchannels of the microfluidic chip to intercept tiny particles and elongated impurities. Filtration of the single-cell suspension can be achieved through a filter structure placed before the single-cell suspension inlet on the microfluidic chip.

[0060] In the illustrated embodiment, as Figure 6 As shown, the first sample inlet 62 is equipped with a filter structure 94 for intercepting tiny particles and slender impurities in the single-cell suspension, ensuring 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 using a microscope to ensure that there are no obvious particulate impurities after filtration. The filter structure 94 includes multiple filter units 96, which are designed, for example, in a U-shape, with the notch 98 of the filter unit 96 facing the injection port of the first sample inlet 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 and the opening portion 102 are connected through a connecting port 104. The opening portion 102 gradually increases in size from the end near 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 V-shaped.

[0061] Preferably, the filter structure 94 employs a multi-stage filter grid with multiple filter layers arranged along the injection direction of the first injection port 62. The multiple filter layers are spaced apart, and each filter layer includes multiple filter units 96, which are spaced apart and staggered between adjacent filter layers. Further, the first injection port 62 includes a wide portion 106 and a narrow portion 108 connected sequentially along the injection direction. The multi-stage filter layers are distributed within the wide portion 106 and the narrow portion 108. The filter structure at the wide portion 106 removes larger particles, ensuring smooth liquid flow. The filter structure at the narrow portion 108 further filters fine impurities, ensuring the single-cell suspension flowing into the microchannel is pure and free of impurities, thereby improving cell encapsulation efficiency.

[0062] In some embodiments, when cell samples are extremely precious and there is a very high requirement for cell recovery rate, 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 can be used as the first droplet generation channel of the next microfluidic system to further sort the first target droplet 20 or the second target droplet 26 and subsequently re-fuse them. The specific details and structure are similar to the microfluidic system described above, and will not be repeated here.

[0063] Furthermore, the microfluidic system 10 of this application may also include a microscope, a temperature control device, and a high-speed camera. The microscope can be used to observe the processes of droplet generation, sorting, and fusion; the temperature control device can be used to maintain temperature stability during droplet generation, sorting, and fusion, promoting stable experimental progress; and the high-speed camera can be used to monitor the processes of droplet generation, sorting, and fusion in real time, enabling researchers to observe and record the experimental process in real time, and to adjust instrument parameters promptly when problems are detected, ensuring the smooth progress of the experiment.

[0064] Because the microfluidic system 10 of this application provides higher cell recovery rates and data quality, it is particularly suitable for complex applications of single-cell sequencing technology, such as clinical research and precision medicine analyses requiring extremely high data accuracy. 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, improving the applicability and research value of the technology.

[0065] In summary, this application provides a microfluidic system for improving cell recovery. After droplet generation, a high-precision droplet sorting module separates droplets without DNA tags from those with successfully bound DNA tags. The purpose of sorting is to ensure that unlabeled droplets can be effectively reused in subsequent steps. The sorted droplets without DNA tags are then fused with the DNA-tagged droplets by a droplet fusion module, thereby completing the relabeling of cells. This innovative step allows for the reuse of cells that were not effectively labeled during the initial droplet generation process, significantly improving cell recovery and utilization rates, reducing the initial cell quantity required for experiments, avoiding waste of cell samples, especially precious cell samples, and reducing the cost of consumables and repeated experiments. This advantage is particularly evident when processing rare or precious samples. Furthermore, it significantly reduces data bias caused by cell loss or incomplete labeling, improving the reliability and consistency of experimental data, thereby enhancing the overall success rate and data quality of experiments.

[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, and not by the preceding description. Any changes within the literal meaning and equivalent scope of the claims should fall within the scope of those claims.

Claims

1. A microfluidic system for improving cell recovery rate, characterized in that, The system includes a microfluidic chip, a droplet sorting module, and a droplet fusion module. The microfluidic chip includes 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. The droplet fusion module is used to fuse the second intermediate droplet with the sorted first intermediate droplet to generate a second target droplet. The first target droplet includes a cutting oil film and a single cell, a DNA tag, and an amplification mixture encapsulated within the cutting oil film. The first intermediate droplet includes a cutting oil film and a single cell, a DNA tag, and an amplification mixture encapsulated within the cutting oil film. The second intermediate droplet includes a cutting oil film and a DNA tag encapsulated within the cutting oil film. The second target droplet includes a cutting oil film and a single cell, a DNA tag, and an amplification mixture encapsulated within the cutting oil film.

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

3. The microfluidic system for improving cell recovery rate as described in claim 2, characterized in that, The portion where the third microchannel connects to the first microchannel forms a diversion port, and the portion where the third microchannel connects to the second microchannel forms a confluence port. The droplet sorting module is located at the diversion port, and the droplet fusion module is located downstream of the confluence port.

4. The microfluidic system for improving cell recovery rate as described in claim 3, characterized in that, The droplet sorting module includes an electric field module or a magnetic field module, which is disposed around the diversion port to sort the first target droplet and the first intermediate droplet by applying an electric field or magnetic field.

5. The microfluidic system for improving cell recovery rate as described in claim 3, characterized in that, The first microchannel 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 through the first droplet recovery channel. The second microchannel 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 droplet is generated in the second droplet generation channel, the second intermediate droplet and the first intermediate droplet are mixed and paired in the droplet pairing channel, and the second target droplet is recovered through the second droplet recovery channel.

6. The microfluidic system for improving cell recovery rate as described in claim 5, characterized in that, The microfluidic system includes a droplet generation device, a microfluidic chip mounted on the droplet generation device, and a droplet sorting module and a droplet fusion module respectively mounted beside the first microchannel and the second microchannel; the microfluidic chip includes: The first injection channel is connected to the first droplet generation channel and is used to inject single-cell suspension; The second injection channel, connected to the first droplet generation channel, is used to inject DNA tags; The third injection channel is connected to the first droplet generation channel and is used to inject the amplification mixture; The fourth injection channel, connected to the first droplet generation channel, is used to inject cutting oil; The fifth injection channel, connected to the second droplet generation channel, is used for injecting the DNA tag; and The sixth injection channel, connected to the second droplet generation channel, is used to inject cutting oil.

7. The microfluidic system for improving cell recovery rate as described in claim 6, characterized in that, The droplet generating device includes multiple injection ports, wherein the first injection channel and the third injection channel are each configured with a separate injection port; the second injection channel and the fifth injection channel share a single injection port, or the second injection channel and the fifth injection channel are each configured with a separate injection port; the fourth injection channel and the sixth injection channel share a single injection port, or the fourth injection channel and the sixth injection channel are each configured with a separate injection port; and / or, the droplet generating device includes a first recovery port connected to the first droplet recovery channel and a second recovery port connected to the second droplet recovery channel, wherein the first recovery port is used to recover the first target droplet, and the second recovery port is used to recover the second target droplet.

8. The microfluidic system for improving cell recovery rate as described in claim 7, characterized in that, The first injection channel has a filter structure in the inlet for intercepting tiny particles and slender impurities in the single-cell suspension. The filter structure includes multiple filter units, each filter unit is U-shaped, and the notch of the filter unit faces the injection port of the inlet.

9. The microfluidic system for improving cell recovery rate as described in claim 8, characterized in that, The filtration structure employs a filter grid with multiple levels of filter units arranged along the injection direction of the injection port. Multiple filter units in each level are spaced apart, and filter units in adjacent levels are staggered. The injection port includes a wide portion and a narrow portion connected sequentially along the injection direction, and the multiple levels of filter units are distributed within the wide portion and the narrow portion.

10. The microfluidic system for improving cell recovery rate as described in claim 5, characterized in that, A fusion cavity is formed at the connection between the droplet pairing channel and the second droplet recovery channel. 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 disposed on the positive electrode liquid channel. The negative electrode module includes a negative electrode liquid channel for injecting negative electrode liquid and a negative terminal disposed on the negative electrode liquid channel. The positive terminal and the negative terminal are respectively disposed 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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