Liquid drop generation method for improving cell recovery rate

Abstract

According to the droplet generation method capable of improving the cell recovery rate, provided by the invention, after the droplets are generated, the droplets which are not wrapped by DNA tags are separated from the successfully wrapped droplets through a high-precision sorting system, and the purpose of sorting is to ensure that the unmarked droplets can be effectively reused in subsequent steps. 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 an electric fusion system, 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 the sample and the cost of repeated experiments are reduced, and the advantage is particularly obvious when rare or precious samples are treated.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a droplet generation method 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 droplet generation method for improving cell recovery rate, aiming to effectively solve the problems of low cell recovery rate and waste of cell samples in existing droplet-based single-cell sequencing technology.

[0007] The present application provides a droplet generation method for improving cell recovery rate, the method comprising: fusing a single cell suspension with a DNA tag to generate a first fused droplet; sorting out a first intermediate droplet that is not wrapped with a DNA tag from the first fused droplet; and performing a second fusion of the first intermediate droplet to generate a second fused droplet.

[0008] In one embodiment, the step of separating the first intermediate droplet without the DNA tag from the first fused droplet further comprises:

[0009] Using a droplet sorting system to sort out a first intermediate droplet from the first fused droplet;

[0010] Recovering and sorting out the first target droplets remaining after the first intermediate droplets are separated;

[0011] The DNA tag is conductive, and the droplet sorting system applies an electric field to the first target droplet and the first intermediate droplet, so that the first target droplet containing the DNA tag is separated from the first intermediate droplet not containing the DNA tag under the action of the electric field.

[0012] In one embodiment, the step of secondary fusion of the first intermediate droplets to generate the second fused droplets further comprises:

[0013] mixing the DNA tag and the cutting oil to produce a second mixed solution;

[0014] Using an electrofusion system to electrofuse the first intermediate droplet with the second mixed liquid to generate a second fused droplet;

[0015] The electric fusion system includes a positive electrode module and a negative electrode module, an electric field is generated between the positive electrode module and the negative electrode module, and the electric field is applied to the first intermediate droplet and the second mixed liquid, so that the first intermediate droplet and the second mixed liquid are fused into the second fused droplet.

[0016] In one embodiment, the positive electrode module includes positive electrode liquid, and the negative electrode module includes negative electrode liquid; the positive electrode liquid and the negative electrode liquid are dispensed into the electrode liquid channel by a syringe; and the electric field is applied with a voltage conversion frequency higher than the droplet passing frequency.

[0017] In one embodiment, the step of fusing the single cell suspension with the DNA tag to generate a first fusion droplet further comprises:

[0018] The single cell suspension, amplification mixture, DNA tag, and cutting oil were respectively injected into different microchannels of the microfluidic chip;

[0019] The single cell suspension, the amplification mixture, and the DNA tag are combined in the microfluidic channel to form a first mixture;

[0020] The first mixed liquid and the cutting oil merge in the microchannel to generate a first fused droplet;

[0021] Wherein, the single cell suspension is filtered before entering the microchannel of the microfluidic chip to intercept tiny particles and elongated impurities in the single cell suspension.

[0022] In one embodiment, the amplification mixture includes enzymes, primers and deoxynucleotides.

[0023] In one embodiment, in the step of fusing the single cell suspension with the DNA tag to generate a first fused droplet, and in the step of secondary fusion of the first intermediate droplet to generate a second fused droplet, cutting oil is injected from the cutting oil inlet on the microfluidic chip and then supplied separately via microchannel diversion.

[0024] In one embodiment, the microfluidic chip is respectively provided with a first recovery droplet channel and a second recovery droplet channel; in the step of selecting the first intermediate droplets not wrapped with DNA tags from the first fused droplets, the first target droplets remaining after selecting the first intermediate droplets enter the first recovery droplet channel and are recovered; in the step of secondary fusion of the first intermediate droplets to generate second fused droplets, the second fused droplets enter the second recovery droplet channel and are recovered.

[0025] In one embodiment, the method further comprises the following steps: selecting a second intermediate droplet that does not contain a DNA tag from the second fused droplet; and fusing the second intermediate droplet again to generate a third fused droplet.

[0026] In one embodiment, the method comprises:

[0027] Observation using a microscope; and / or,

[0028] Use temperature control devices to maintain a stable temperature; and / or,

[0029] Real-time monitoring using high-speed cameras.

[0030] In summary, the present application provides a method for generating droplets to improve the cell recovery rate. After the droplets are generated, the droplets that are not wrapped with DNA labels are separated from the droplets that have been successfully wrapped by a high-precision sorting system. 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 are fused again with the droplets wrapped with DNA labels under the action of the electric fusion system to complete the re-labeling of the cells. This innovative step allows those 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 sample consumables 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

[0031] Figure 1 This is a schematic diagram of a process for generating droplets to improve cell recovery rate in one embodiment of the present application.

[0032] Figure 2 for Figure 1 Detailed flowchart of step S1 in FIG.

[0033] Figure 3 for Figure 1 Detailed flowchart of step S2 in FIG.

[0034] Figure 4 for Figure 1 Detailed flowchart of step S3 in FIG.

[0035] Figure 5 FIG. 4 is a schematic flow chart of a droplet generation method for improving cell recovery rate in another embodiment of the present application. DETAILED DESCRIPTION

[0036] 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.

[0037] Please refer to Figure 1As shown, the present application provides a droplet generation method for improving cell recovery rate, the method comprising: S1, a single cell suspension is fused with a DNA tag to generate a first fused droplet; S2, a first intermediate droplet that is not wrapped with a DNA tag is sorted out from the first fused droplet; S3, the first intermediate droplet is secondary fused to generate a second fused droplet. In the above droplet generation method, the fused droplets are sorted, and the sorted first intermediate droplets that are not wrapped with a DNA tag are secondary fused, so that on the basis of the cell recovery rate of the initial fusion, the droplets that are not wrapped with a DNA tag after the initial fusion are utilized through secondary fusion, and finally the total cell recovery rate after the secondary fusion is improved, solving the problem of cell sample waste in the prior art. In the process of implementing the above droplet generation method, a microfluidic chip is used as a carrier of liquid flow and droplets, and a droplet generation device, a droplet sorting system, an electrofusion system and other equipment are used. The following will describe the detailed operation process of each step in combination with the above-mentioned carriers and equipment.

[0038] Please refer to Figure 2 As shown, the first fused droplet generation in step S1 further includes the following steps:

[0039] S1.1, inject the single cell suspension, amplification mixture, DNA tag, and cutting oil into different microchannels of the microfluidic chip respectively.

[0040] Among them, the single cell in the single cell suspension is the cell to be sequenced; the main function of the amplification mixture is to provide the necessary reaction components, promote the 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; primers refer 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 tags are used to identify RNA molecules derived from the same cell in subsequent genome sequencing, and DNA tags are, for example, DNA tag colloids; cutting oil is an inert oil with a suitable viscosity and surface tension, which is used to provide a closed oil film layer 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.

[0041] 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.

[0042] S1.2, the single cell suspension, the amplification mixture, and the DNA tag are combined in the microfluidic channel to form a first mixture.

[0043] S1.3, the first mixed liquid and the cutting oil merge in the microchannel to generate a first fused droplet.

[0044] During the fusion process of the first mixed liquid and the cutting oil in the microfluidic channel, droplets encapsulating single cells and DNA tags (first target droplets) are generated, and droplets that fail to encapsulate DNA tags (first intermediate droplets) are also generated. Therefore, the first fused droplets contain both of the above droplets.

[0045] Please refer to Figure 3 As shown, the first intermediate droplet sorting in step S2 further includes the following steps:

[0046] S2.1, using a droplet sorting system to sort out a first intermediate droplet from the first fused droplet.

[0047] In the step of sorting out the first intermediate droplet from the first fused droplet, a droplet sorting system may be used to separate the first target droplet from the first intermediate droplet.

[0048] Specifically, the sorting principle of the droplet sorting system is to use an electric field or a magnetic field to selectively separate different droplets. In the specific implementation process, the DNA tag is designed to be conductive, for example, the DNA tag carries a positive charge or a negative charge. The droplet sorting system is used to apply an electric field of appropriate strength to both sides of the microfluidic channel through which the first fused droplet flows, so that the first target droplet containing the DNA tag is separated from the first intermediate droplet without the DNA tag under the action of the electric field. Due to its conductivity, the DNA tag produces an electrophoretic effect under the action of the electric field, causing the first target droplet wrapped with the DNA tag to deflect into the side branch channel, while the first intermediate droplet without the DNA tag continues to move along the main channel because it is not significantly affected by the electric field, thereby realizing the sorting of the first target droplet and the first intermediate droplet. This electric field-based separation technology can effectively distinguish droplets with different conductivity, realize fast and precise microfluidic separation operations, have a high degree of controllability and flexibility, and is suitable for the sorting and analysis of various complex samples.

[0049] S2.2, recovering and sorting out the first target droplets remaining after the first intermediate droplets are separated.

[0050] After the first intermediate droplets are separated from the first fused droplets, the remaining first target droplets can enter the first recovery droplet channel and the first recovery hole of the microfluidic chip along the side branch channel and be recovered.

[0051] Please refer to Figure 4 As shown, the second fused droplet generation in step S3 further includes the following steps:

[0052] S3.1, mix the DNA tag and the cutting oil to produce a second mixed solution.

[0053] The DNA tag and the cutting oil merge in the microfluidic channel, and the cutting oil wraps the DNA tag to form a second mixed solution.

[0054] S3.2, using an electrofusion system to electrofuse the first intermediate droplet with the second mixed liquid to generate a second fused droplet.

[0055] The sorted first intermediate droplet without DNA tags is electrofused with the second mixed liquid with DNA tags through an electrofusion system.

[0056] The first intermediate droplet is fused with the second mixed liquid using an electric fusion system. Under the action of an electric field, the first intermediate droplet and the second mixed liquid are fused by utilizing the different conductivity and permeability between them. This is because polarized charges are continuously accumulated at the droplet interface. When the polarized charges accumulate to a certain extent, they will induce interaction between the droplets, causing the droplets to deform and fuse. Specifically, the electric fusion system includes a positive electrode module and a negative electrode module. An electric field is generated between the positive electrode module and the negative electrode module, wherein the positive electrode module is, for example, a positive electrode liquid, and the negative electrode module is, for example, a negative electrode liquid. The electric field is applied to the first intermediate droplet and the second mixed liquid, causing the first intermediate droplet and the second mixed liquid to fuse into a second fused droplet.

[0057] During the secondary electrofusion process, the electrode liquid (positive electrode liquid and negative electrode liquid) in the electrofusion system can be slowly dispensed and filled into the electrode liquid channel through a high-precision syringe so that no bubbles are generated; the application of the electric field converts the voltage at a frequency higher than the frequency of the droplet passing through, so that the secondary electrofusion step successfully fuses the sorted unlabeled first intermediate droplet with the second mixed liquid through 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 efficiency of the experiment and significantly improved the data quality. By ensuring that each droplet contains the required labeling through secondary electrofusion, this solution significantly reduces data deviations caused by cell loss or incomplete labeling, greatly improves the success rate of the experiment, and ensures the reliability and consistency of the data.

[0058] The second target droplet can enter the second recovery droplet channel and the second recovery hole of the microfluidic chip and be recovered.

[0059] It should be noted that when the cell sample is extremely precious and there is an extremely high requirement for cell recovery rate, by analogy, sorting and re-fusion can be continued. For details, please refer to Figure 5 As shown, the droplet generation method for improving cell recovery rate of the present application may also include: S4, sorting out a second intermediate droplet that does not contain a DNA tag from the second fused droplet; S5, the second intermediate droplet is fused again to generate a third fused droplet. The specific details of step S4 are similar to those of step S2, and the specific details of step S5 are similar to those of step S3, which will not be repeated here.

[0060] In addition, during the above method, a microscope can be used to observe the process of droplet generation, sorting and fusion; a 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; a 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.

[0061] Preferably, cutting oil is required in both the generation of the first fused droplet in step S1 and the generation of the second fused droplet in step S3. The cutting oil injected from the same cutting oil inlet on the microfluidic chip can be supplied separately via microchannel diversion, thereby ensuring the consistency of the outer oil film of various droplets and improving the reliability of the data.

[0062] Since the method of this 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 in clinical research and precision medicine that requires 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 and improves the applicability and research value of the technology.

[0063] In summary, the present application provides a method for generating droplets to improve the cell recovery rate. After the droplets are generated, the droplets that are not wrapped with DNA labels are separated from the droplets that have been successfully wrapped by a high-precision sorting system. 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 are fused again with the droplets wrapped with DNA labels under the action of the electric fusion system to complete the re-labeling of the cells. This innovative step allows those 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 sample consumables 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.

[0064] 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 method for generating droplets for improving cell recovery rate, characterized in that: The method comprises: fusing a single cell suspension with a DNA tag to generate a first fused droplet; sorting out a first intermediate droplet not encapsulating the DNA tag from the first fused droplet; and fusing the first intermediate droplet for a second time to generate a second fused droplet.

2. The method for generating droplets for improving cell recovery rate according to claim 1, characterized in that: The step of selecting the first intermediate droplet without the DNA tag from the first fused droplet further comprises: Using a droplet sorting system to sort out a first intermediate droplet from the first fused droplet; Recovering and sorting out the first target droplets remaining after the first intermediate droplets are separated; The DNA tag is conductive, and the droplet sorting system applies an electric field to the first target droplet and the first intermediate droplet, so that the first target droplet containing the DNA tag is separated from the first intermediate droplet not containing the DNA tag under the action of the electric field.

3. The method for generating droplets for improving cell recovery rate according to claim 1, characterized in that: The step of secondary fusion of the first intermediate droplets to generate second fused droplets further comprises: mixing the DNA tag and the cutting oil to produce a second mixed solution; Using an electrofusion system to electrofuse the first intermediate droplet with the second mixed liquid to generate a second fused droplet; The electric fusion system includes a positive electrode module and a negative electrode module, an electric field is generated between the positive electrode module and the negative electrode module, and the electric field is applied to the first intermediate droplet and the second mixed liquid, so that the first intermediate droplet and the second mixed liquid are fused into the second fused droplet.

4. The method for generating droplets for improving cell recovery rate according to claim 3, characterized in that: The positive electrode module includes positive electrode liquid, and the negative electrode module includes negative electrode liquid; the positive electrode liquid and the negative electrode liquid are dispensed into the electrode liquid channel through a syringe; and the electric field is applied with a voltage conversion frequency higher than the droplet passing frequency.

5. The method for generating droplets for improving cell recovery rate according to any one of claims 1 to 4, characterized in that: The step of fusing the single cell suspension with the DNA tag to generate a first fusion droplet further includes: The single cell suspension, amplification mixture, DNA tag, and cutting oil were respectively injected into different microchannels of the microfluidic chip; The single cell suspension, the amplification mixture, and the DNA tag are combined in the microfluidic channel to form a first mixture; The first mixed liquid and the cutting oil merge in the microchannel to generate a first fused droplet; Wherein, the single cell suspension is filtered before entering the microchannel of the microfluidic chip to intercept tiny particles and elongated impurities in the single cell suspension.

6. The method for generating droplets for improving cell recovery rate according to claim 5, characterized in that: The amplification mixture comprises enzyme, primer and deoxynucleotide.

7. The method for generating droplets for improving cell recovery rate according to claim 5, characterized in that: In the step of fusing the single cell suspension with the DNA tag to generate a first fused droplet, and in the step of secondary fusion of the first intermediate droplet to generate a second fused droplet, cutting oil is injected from the cutting oil inlet on the microfluidic chip and then supplied separately via microchannel diversion.

8. The method for generating droplets for improving cell recovery rate according to claim 5, characterized in that: The microfluidic chip is respectively provided with a first recovery droplet channel and a second recovery droplet channel; in the step of selecting the first intermediate droplet not wrapped with the DNA tag from the first fused droplet, the first target droplet remaining after selecting the first intermediate droplet enters the first recovery droplet channel to be recovered; in the step of secondary fusion of the first intermediate droplet to generate the second fused droplet, the second fused droplet enters the second recovery droplet channel to be recovered.

9. The method for generating droplets for improving cell recovery rate according to claim 1, characterized in that: The method further comprises the following steps: selecting a second intermediate droplet without a DNA tag from the second fused droplet; and fusing the second intermediate droplet again to generate a third fused droplet.

10. The method for generating droplets for improving cell recovery rate according to any one of claims 1 to 4, characterized in that: The method comprises: Observation using a microscope; and / or, Use temperature control devices to maintain a stable temperature; and / or, Real-time monitoring using high-speed cameras.

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