Multi-path microfluidic cell sorting method and sorting system

Abstract

The invention belongs to the technical field of cell sorting, and particularly relates to a multi-path microfluidic cell sorting method and sorting system. According to the invention, the liquid drops containing single cells are subjected to asymmetric airflow treatment and / or physical field treatment to obtain the multi-path cell liquid drops with different motion trails of any two paths, so that the defect that only single-path cell sorting can be carried out in the prior art is overcome, and the multi-path cell liquid drops with different motion trails can be sorted at the same time; the non-contact physical field is used for treating the multi-path cell liquid drops, the cells in the multi-path cell liquid drops with different movement tracks are guided to move, mechanical damage or chemical pollution possibly caused to the cells in a traditional method is effectively avoided, the adverse effect on cell viability is remarkably reduced, and the cell viability is improved. The single-cell-level high-precision cell screening capacity can be achieved, the cell sorting precision is larger than 99.5%, the highest sorting frequency can reach 1 kHz, and the average survival rate of sorted cells is 94%.

Description

Technical Field

[0001] The invention belongs to the technical field of cell sorting, and in particular relates to a multi-path microfluidic cell sorting method and a sorting system. Background Art

[0002] Cell sorting has important application value in the fields of life sciences, clinical medicine, drug screening and toxicology research. The current mainstream cell sorting method uses flow cytometry based on antibody labeling, which has an extremely fast cell sorting speed, but has defects such as low sorting resolution, low cell activity, bulky device and high reagent consumption. In recent decades, microfluidics has become a mainstream method for precise regulation of single cells at the microscale, and has shown important application potential in the field of cell sorting. Microfluidics systems can achieve efficient, accurate and low-damage processing of cell samples by precisely manipulating microscale continuous fluids or isolated droplets. This unique advantage has greatly promoted the innovation and development of cell sorting technology.

[0003] At present, cell sorting methods based on microfluidic technology include microstructure filtration sorting, acoustic wave sorting, deterministic lateral displacement, etc. Among them, the microstructure filtration sorting method achieves separation based on the three-dimensional size differences of cells by designing a specific microscale filtration structure. However, its versatility in sorting mechanism is limited, and it is difficult to adapt to a variety of cell types and size ranges. At the same time, it faces the challenge of low sorting flux, which affects processing efficiency and wide application.

[0004] Acoustic sorting uses the mechanical effects of sound waves in microfluidic chips to manipulate and sort cells. However, its implementation process is complicated and requires the use of specific piezoelectric substrate materials in the manufacture of microfluidic chips, which not only increases the difficulty of processing, but also leads to a significant increase in manufacturing costs, limiting its feasibility in large-scale industrial applications.

[0005] The deterministic lateral displacement method relies on the difference in cell geometric diameters to achieve lateral movement and separation of cells in a specific array structure within a microfluidic chip. Although its sorting mechanism has a certain degree of accuracy, it also faces the problem of insufficient versatility and is difficult to widely adapt to cell populations with different characteristics. In addition, the complex array structure design can easily lead to blockage of fluid channels, increasing the difficulty of operation and maintenance costs, and affecting the stability and long-term operating efficiency of the sorting system. Summary of the invention

[0006] The purpose of the present invention is to make up for the deficiencies of the prior art and provide a multi-path microfluidic cell sorting method and sorting system, which avoids direct contact with cells in a miniaturized space and achieves high-throughput, high-precision, and low-cell activity-affected cell sorting.

[0007] The present invention provides a multi-path microfluidic cell sorting method, comprising the following steps:

[0008] Asymmetric airflow processing and / or physical field processing are performed on droplets containing single cells to obtain multi-path cell droplets; the motion trajectories of any two paths in the multi-path cell droplets are different;

[0009] Detecting the multi-path cell droplets and collecting detection results;

[0010] According to the detection results, the multi-path cell droplets are processed by using a non-contact physical field to deflect non-target sorted cell droplets in a direction.

[0011] Preferably, the non-contact physical field includes one or more of dielectrophoresis, optical tweezers, magnetic field, electrostatic tweezers and sound waves.

[0012] Preferably, the method for preparing droplets containing single cells comprises: treating a cell suspension using one or more of a gas-liquid copolymerization method, a T-channel method, a flow focusing method, a coaxial flow focusing method and an electrohydrodynamic method to obtain droplets containing single cells.

[0013] Preferably, the detection includes one or more of fluorescence detection, imaging analysis detection and Raman spectroscopy detection.

[0014] Preferably, the method further comprises: recovering the non-target sorted cell droplets.

[0015] Preferably, the sorting method is performed in a microfluidic cell sorting system; the microfluidic cell sorting system comprises a multi-path droplet generation module, a cell detection module, a cell sorting module and a waste liquid recovery module;

[0016] The multi-path droplet generation module comprises a microfluidic chip; the microfluidic chip comprises a substrate and a cell suspension storage module and a single cell droplet generation module located on the substrate; the single cell droplet generation module is an asymmetric airflow processing module and / or a physical field processing module; the outlets of the cell suspension storage module and the single cell droplet generation module meet at a droplet nozzle;

[0017] The cell detection module is connected to the droplet nozzle through the cell inlet; the cell detection module is located below the single cell droplet generation module and is non-contactly connected to the non-contact physical field in the cell sorting module through the cell outlet;

[0018] The waste liquid recovery module runs through the microfluidic chip in parallel and is located on the movement track after the droplet deviates from the running track of the cell sorting module.

[0019] Preferably, the asymmetric airflow processing module includes a first gas phase passage and a second gas phase passage; the first gas phase passage and the second gas phase passage are respectively located on both sides of the cell suspension storage module, and intersect with the outlet of the single cell droplet generation module at a droplet nozzle.

[0020] Preferably, looking down at the plane where the cell suspension storage module is located, with the normal line of the droplet nozzle as the central axis, the angle between the droplet emission direction and the normal line is 32-48°.

[0021] Preferably, the cell detection module comprises a continuous wave laser generator, a first optical fiber, a second optical fiber, a photomultiplier tube and a field programmable gate array which are connected in sequence; the first optical fiber and the second optical fiber are embedded on both sides of the droplet nozzle.

[0022] The present invention also provides a multi-path microfluidic cell sorting system, comprising a multi-path droplet generation module, a cell sorting module, a cell detection module and a waste liquid recovery module;

[0023] The multi-path droplet generation module comprises a microfluidic chip; the microfluidic chip comprises a substrate and a cell suspension storage module and a single cell droplet generation module located on the substrate; the single cell droplet generation module is an asymmetric airflow processing module or a physical field processing module; the outlets of the cell suspension storage module and the single cell droplet generation module meet at a droplet nozzle;

[0024] The cell detection module is connected to the droplet nozzle through the cell inlet; the cell detection module is located below the single cell droplet generation module and is non-contactly connected to the non-contact physical field in the cell sorting module through the cell outlet;

[0025] The waste liquid recovery module runs through the microfluidic chip in parallel and is located on the movement track after the droplet deviates from the running track of the cell sorting module.

[0026] Beneficial effects:

[0027] The present invention provides a multi-path microfluidic cell sorting method, comprising the following steps: asymmetric airflow treatment and / or physical field treatment are performed on droplets containing single cells to obtain multi-path cell droplets; the multi-path cell droplets are treated by a non-contact physical field to deflect non-target sorted cell droplets in a directional manner. The present invention forms multi-path cell droplets through asymmetric airflow treatment and / or physical field treatment, which makes up for the deficiency that the prior art can only carry out single-path cell sorting, and can sort multi-path cell droplets at the same time; and the present invention uses a non-contact physical field to guide the movement of cells in multi-path cell droplets, effectively avoiding mechanical damage or chemical contamination that may be caused to cells in traditional methods, significantly reducing the adverse effects on cell activity, and can achieve high-precision cell screening capabilities at the single-cell level, the cell sorting accuracy is greater than 99.5%, the highest sorting frequency can reach 1kHz, and the average survival rate of cells after sorting is 94%.

[0028] Furthermore, the present invention is based on the above-mentioned multi-path microfluidic cell sorting method, and proposes a multi-path microfluidic cell sorting system by utilizing a path single-cell droplet generation module, a cell sorting module and a waste liquid recovery module. While meeting the miniaturized space requirements, it can avoid direct contact with cells and achieve high-throughput, high-precision, and low-cell activity-affected cell sorting through multi-path microfluidics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0030] Figure 1 This is a schematic diagram of the structure of the multi-path microfluidic cell sorting system of Example 1; wherein, 1-microfluidic chip, 2-cell suspension storage module, 3-gas phase passage, 4-optical fiber, 5-continuous wave laser generator, 6-photomultiplier tube, 7-FPGA, 8-voltage amplifier, 9-high voltage cylindrical electrode, 10-collection tube;

[0031] Figure 2 Schematic diagram of droplet generation based on flow focusing method;

[0032] Figure 3 Schematic diagram of multi-path droplet generation based on asymmetric airflow method;

[0033] Figure 4 This is a schematic diagram of the fluorescent cell detection module detecting cells;

[0034] Figure 5 is a schematic diagram of a high voltage cylindrical electrode having a circular cross section;

[0035] Figure 6 Schematic diagram of cell sorting based on axial dielectrophoresis sorting module;

[0036] Figure 7 Schematic diagram of the structure of recycling microtubules;

[0037] Figure 8 This is a schematic diagram of the structure of a multi-path droplet generation module in a multi-path microfluidic cell sorting system in Example 2; wherein 1-microfluidic chip, 2-cell suspension storage module, 3-gas phase passage, 12-single cell droplet generation channel;

[0038] Fig. 9 Schematic diagram of single-cell droplet generation based on coaxial flow focusing;

[0039] Fig.10 Schematic diagram of single-cell droplet generation based on electrohydrodynamics;

[0040] Fig.11 Schematic diagram of multi-path droplet generation based on electrohydrodynamics;

[0041] Fig.12 is a schematic diagram of a high voltage cylindrical electrode having a square cross section;

[0042] Fig.13 is a schematic diagram of a high voltage cylindrical electrode having a triangular cross section;

[0043] Fig.14 It is a structural schematic diagram of the recovery flow channel;

[0044] Fig.15 It is a schematic diagram of the structure of the recovery baffle. DETAILED DESCRIPTION

[0045] The present invention provides a multi-path microfluidic cell sorting method, comprising the following steps:

[0046] Asymmetric airflow processing and / or physical field processing are performed on droplets containing single cells to obtain multi-path cell droplets; the motion trajectories of any two paths in the multi-path cell droplets are different;

[0047] Detecting the multi-path cell droplets and collecting detection results;

[0048] According to the detection results, the multi-path cell droplets are processed by using a non-contact physical field to deflect non-target sorted cell droplets in a direction.

[0049] As an embodiment, the present invention utilizes one or more of gas-liquid copolymerization, T-channel method, flow focusing method, coaxial flow focusing method and electrohydrodynamic method to process the cell suspension to obtain droplets containing single cells. As another embodiment, the present invention utilizes gas-liquid copolymerization to process the cell suspension to obtain droplets containing single cells. The cell suspension of the present invention contains cells to be sorted. The present invention has no strict requirements on the gas-liquid copolymerization, T-channel method, flow focusing method, coaxial flow focusing method and electrohydrodynamic method, all of which belong to conventional operating methods in the art.

[0050] After obtaining the droplets containing single cells, the present invention performs asymmetric airflow processing and / or physical field processing on the droplets containing single cells to obtain multi-path cell droplets; the motion trajectories of any two paths in the multi-path cell droplets are different.

[0051] As an embodiment, the present invention performs asymmetric airflow treatment on the droplets containing single cells. As an embodiment, the present invention uses asymmetric airflow and / or physical field to treat droplets containing single cells, which can regulate the movement direction and speed of droplets containing single cells, forming multi-path cell droplets, making up for the deficiency of the prior art that can only carry out single-path cell sorting, and performing subsequent sorting on multi-path cell droplets. The present invention has no strict requirements on the specific steps of the asymmetric airflow treatment and / or physical field treatment, which are all routine operations in the field.

[0052] As an embodiment, after obtaining the multi-path cell droplets, the present invention detects the multi-path cell droplets to obtain detection results. The present invention has no strict requirements on the detection method, and conventional methods in the field can be used. Fluorescence-activated cell method can be used, but it is also applicable to any technical means that can detect cells in real time or perform imaging analysis to determine cell characteristics, specifically including but not limited to imaging analysis method and Raman spectroscopy detection method. As an embodiment, the detection described in the present invention is fluorescence detection. The present invention uses fluorescence detection as an example for explanation in the embodiments, but it cannot be understood as the entire protection scope of the present invention. The present invention has no strict requirements on the specific methods of the fluorescence-activated cell method, imaging analysis method and Raman spectroscopy detection method, and conventional methods in the field can be used.

[0053] After obtaining the detection results, the present invention processes the multi-path cell droplets using a non-contact physical field based on the detection results to deflect the non-target sorted cell droplets in a direction.

[0054] As an embodiment, the non-contact physical field of the present invention includes one or more of dielectrophoresis, optical tweezers, magnetic field, electrostatic tweezers and sound waves; as another embodiment, the non-contact physical field of the present invention is dielectrophoresis. The present invention utilizes a non-contact physical field, and non-target sorted cell droplets will be deflected under the action of the non-contact physical field, while the sorted single cell droplets are not affected. The present invention has no strict requirements on the parameters of the non-contact physical field. According to the test results, it is sufficient to determine the required sorted cells and make routine adjustments.

[0055] As an embodiment, the dielectrophoresis of the present invention utilizes a columnar electrode. The columnar electrode can realize the application of a non-contact physical field to a multi-path droplet group flowing through within the entire axial length, so as to directional deflect the droplets of non-target sorted cells, and its deflection efficiency is not limited to the specific cross-sectional shape and type of the columnar electrode, the cross-section is specific but not limited to circular, square, triangular, hexagonal, etc., the electrode type is specific but not limited to copper, chromium, aluminum alloy, etc., and the deflection implementation method is not limited to the specific non-uniform electric field layout generated in the actual operation of the system.

[0056] The present invention utilizes a non-contact physical field to guide the movement of cells in multi-path cell droplets, effectively avoiding mechanical damage or chemical contamination that may be caused to cells in traditional methods, significantly reducing the adverse effects on cell activity, and achieving high-precision cell screening capabilities at the single-cell level. The cell sorting accuracy is >99.5%, the highest sorting frequency can reach 1kHz, and the average survival rate of cells after sorting is 94%.

[0057] As an embodiment, the present invention collects deflected cell droplets of non-target sorting. As an embodiment, the present invention can set microtubes, flow channels, baffles or other microstructure elements on the movement path of non-target sorting cell droplets after deflection, and combine physical means such as vacuum negative pressure to achieve effective capture of deflected waste droplets and discharge the deflected cell droplets of non-target sorting. The present invention has no strict requirements on the collection method of the cell droplets that have not been deflected, and can be collected by conventional methods in the field.

[0058] As an embodiment, the sorting method of the present invention is carried out in a microfluidic cell sorting system; the microfluidic cell sorting system includes a multi-path droplet generating module, a cell detection module, a cell sorting module and a waste liquid recovery module; the multi-path droplet generating module includes a microfluidic chip; the microfluidic chip includes a substrate and a cell suspension storage module and a single cell droplet generating module located on the substrate; the single cell droplet generating module is an asymmetric airflow processing module or a physical field processing module; the outlets of the cell suspension storage module and the single cell droplet generating module meet at a droplet nozzle; the cell detection module is connected to the droplet nozzle through a cell inlet; the cell detection module is located below the single cell droplet generating module, and is contactlessly connected to the non-contact physical field in the cell sorting module through a cell outlet; the waste liquid recovery module runs through the microfluidic chip in parallel, and is located on the motion trajectory after the droplet deviates from the running trajectory of the cell sorting module.

[0059] As an embodiment, the microfluidic cell sorting system of the present invention includes a multi-path droplet generation module; the multi-path droplet generation module includes a microfluidic chip; the microfluidic chip includes a substrate and a single cell droplet generation module located on the substrate. As an embodiment, the material on the substrate is polydimethylsiloxane (PDMS); as another embodiment, the substrate is formed by bonding two layers of polydimethylsiloxane with a thickness of 5 mm.

[0060] As an embodiment, the single-cell droplet generation module of the present invention is an asymmetric airflow processing module or a physical field processing module; the outlets of the cell suspension storage module and the single-cell droplet generation module meet at a droplet nozzle. As an embodiment, the asymmetric airflow processing module of the present invention includes a first gas phase passage and a second gas phase passage; the first gas phase passage and the second gas phase passage are respectively located on both sides of the cell suspension storage module, and meet at the droplet nozzle with the outlet of the single-cell droplet generation module. As an embodiment, the first gas phase passage and the second gas phase passage of the present invention are respectively connected to an external fluid source. As another embodiment, the thickness of the first gas phase passage is 150 μm. As an embodiment, looking down at the plane where the cell suspension storage module of the present invention is located, with the normal of the droplet nozzle as the central axis, the angle between the droplet emission direction and the normal is 32 to 48°; as another embodiment, looking down at the plane where the cell suspension storage module is located, with the normal of the droplet nozzle as the central axis, the angle between the droplet emission direction and the normal is 32°. The present invention utilizes the gas phases of the first gas phase passage and the second gas phase passage on both sides of the cell suspension storage module to shear the cell mixture to produce single cell droplets; adopts an asymmetric airflow method to adjust the speed of the airflow input of the first gas phase passage and the second gas phase passage respectively, so as to change the emission direction and emission speed of the droplets, thereby realizing the generation of multi-path droplets. This method can control the direction and speed of the droplets while generating single cell droplets, and the maximum emission speed can reach 5m / s.

[0061] As an embodiment, the physical field processing module includes a non-contact physical field and other modules. As an embodiment, the non-contact physical field is located at the droplet nozzle. As an embodiment, the non-contact physical field includes one or more of electricity, magnetism and light. As an embodiment, the thickness of the third gas phase passage is 150 μm. As an embodiment, the intersection includes a vertical intersection or a coaxial intersection.

[0062] As an embodiment, the other modules are a third gas phase passage or an electric field. As an embodiment, the outlets of the third gas phase passage and the cell suspension storage module of the present invention meet at a droplet nozzle. As an embodiment, the electric field of the present invention is connected to the cell suspension storage module through an electrode. The present invention shears the cell mixture of the cell suspension storage module through the gas phase of the third gas phase passage to produce single-cell droplets. After the single-cell droplets are produced, a non-contact physical field is used to apply an external force to the single-cell droplets with an initial velocity to change the velocity direction of the single-cell droplets, thereby achieving multi-path droplet generation. The present invention is based on electrohydrodynamics, and an external electric field is used to push the cell mixture in the cell suspension storage module out of the droplet nozzle to produce single-cell droplets. After the single-cell droplets are produced, a non-contact physical field is used to apply an external force to the single-cell droplets with an initial velocity to change the velocity direction of the single-cell droplets, thereby achieving multi-path droplet generation.

[0063] As an embodiment, the cross-sectional shape of the droplet nozzle is a rectangle. As an embodiment, the length×width of the rectangle is 30-50 μm×30-50 μm; as another embodiment, the length×width of the rectangle is 30 μm×30 μm.

[0064] As an embodiment, the microfluidic cell sorting system of the present invention includes a cell sorting module; the cell sorting module is non-contactly connected to the droplet nozzle of the multi-path droplet generation module. As an embodiment, the cell sorting module is an axial dielectrophoresis sorting module or a non-contact physical field sorting module; as another embodiment, the cell sorting module is an axial dielectrophoresis sorting module. The cell sorting module of the present invention can directional deflect droplets.

[0065] As an embodiment, the axial dielectrophoresis sorting module includes a voltage amplifier and a high-voltage columnar electrode connected in sequence; the high-voltage columnar electrode runs through the microfluidic chip in parallel, and is located below the single-cell droplet generation module, and is non-contactly connected to the cell outlet of the cell detection module; the voltage amplifier is connected to the cell detection module electrode. The present invention utilizes a high-voltage columnar electrode to achieve the application of a non-contact physical field for a multi-path droplet group flowing through the entire axial length, so as to directional deflect the droplets of non-target sorted cells, and its deflection efficiency is not limited to the specific cross-sectional shape and type of the columnar electrode, and the cross-section includes but is not limited to circular, square, triangular, hexagonal, etc., and the electrode type is specific but not limited to copper, chromium, aluminum alloy, etc. As an embodiment, the cross-section of the high-voltage columnar electrode is circular, the surface is smooth, and the geometric symmetry is good. As an embodiment, the diameter of the circle is 600μm.

[0066] As an implementation mode, the voltage amplifier provides a voltage of 0 to 4 kV. The present invention generates a non-uniform electric field in three-dimensional space through a voltage amplifier. Due to the different relative dielectric constants between droplets and air, single-cell droplets are subjected to dielectrophoretic forces near the electrodes of the axial dielectrophoretic sorting module, and non-sorted cells are deflected toward the electrodes. Non-sorted cells enter the waste liquid recovery module and leave the system. Since a columnar electrode is used, each section of the columnar electrode can generate a similar non-uniform electric field, and the trajectory offset of multi-path droplets fluctuates by less than 10 μm, so that the collection tube can fully collect multi-path non-sorted cells.

[0067] As an embodiment, the microfluidic cell sorting system of the present invention includes a cell detection module. As an embodiment, the cell detection module is connected to the droplet nozzle through a cell inlet, and is located between the multipath droplet generation module and the cell sorting module, and is connected to the cell sorting module electrode. As an embodiment, the cell detection module of the present invention includes a continuous wave laser generator, a first optical fiber, a second optical fiber, a photomultiplier tube and a field programmable gate array connected in sequence; the first optical fiber and the second optical fiber are embedded on both sides of the droplet nozzle. As an embodiment, the outer diameters of the first optical fiber and the second optical fiber are 225μm, respectively, and the inner diameters of the first optical fiber and the second optical fiber are 200μm, respectively. As an embodiment, the power of the continuous wave laser generator is 100mW, and the wavelengths are 405nm, 473nm, 532nm, and 640nm, respectively. The outer sides of the first optical fiber and the second optical fiber of the present invention are respectively provided with a grounded metal layer to ensure the subsequent normal generation of a non-uniform electric field. The present invention utilizes a continuous wave laser generator to generate extremely low power consumption and cell-mild laser, irradiates single cell droplets through optical fibers and outputs the fluorescence signal of the cell. The generated fluorescence signal is transmitted to a photomultiplier tube through the optical fiber on the other side of the droplet nozzle, and the optical signal is converted into an electrical signal for detection and judgment by a field programmable gate array (FPGA).

[0068] The microfluidic cell sorting system of the present invention includes a waste liquid recovery module; the waste liquid recovery module runs through the microfluidic chip in parallel, and is located on the motion trajectory of the single cell droplet after it deviates from the running trajectory of the cell sorting module. As an embodiment, the waste liquid recovery module includes a collecting tube and a vacuum pump connected in sequence; the collecting tube runs through the microfluidic chip in parallel, and is located on the motion trajectory of the single cell droplet after it deviates from the running trajectory of the cell sorting module. As an embodiment, the collecting tube includes any one or more of a recovery microtube, a recovery flow channel, and a recovery flow channel. As an embodiment, the recovery microtube can be the remaining part after cutting off one-third of the microcircular tube, and the incision is facing the deviated droplet to fully recover the waste droplet. The vacuum pump of the present invention provides a continuous vacuum negative pressure for the collecting tube to ensure the collection and emptying of the deviated droplets in the collecting tube, and will not affect the motion trajectory of the sorted single cell droplets.

[0069] The present invention provides a multi-path microfluidic cell sorting system, including a multi-path droplet generating module, a cell sorting module, a cell detection module and a waste liquid recovery module; the multi-path droplet generating module includes a microfluidic chip; the microfluidic chip includes a substrate and a cell suspension storage module and a single cell droplet generating module located on the substrate; the single cell droplet generating module is an asymmetric airflow processing module or a physical field processing module; the outlets of the cell suspension storage module and the single cell droplet generating module meet at a droplet nozzle; the cell detection module is connected to the droplet nozzle through a cell inlet, and is non-contactly connected to the cell sorting module through a cell outlet; the waste liquid recovery module runs through the microfluidic chip in parallel, and is located on the motion trajectory after the droplet deviates from the running trajectory of the cell sorting module. The specific structure of the multi-path microfluidic cell sorting system has been recorded above and will not be repeated here. The system provided by the present invention can avoid direct contact with cells while meeting the requirements of miniaturization space, and realize high-throughput, high-precision, and low-cell activity-affected cell sorting through multi-path microfluidics. The cell sorting accuracy is >99.5%, and the highest sorting frequency can reach 1kHz. The average survival rate of cells after sorting is 94%. Further, the present invention uses an axial dielectrophoresis sorting module as a cell sorting module; the axial dielectrophoresis sorting module includes a voltage amplifier and a high-voltage columnar electrode connected in sequence; the high-voltage columnar electrode runs through the microfluidic chip in parallel and is located below the single-cell droplet generation module, constructing a non-uniform electric field environment in the axial dimension. This unique design not only optimizes the electric field distribution, but also realizes efficient sorting of multi-path cell droplets, and improves the sorting processing flux; the overall structure of the system is compact, occupies very little space, has a wide range of cell compatibility, strong adaptability and versatility, and is suitable for sorting various types of cells.

[0070] In order to further illustrate the present invention, a multi-path microfluidic cell sorting method and sorting system provided by the present invention are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0071] Example 1

[0072] A kind of Figure 1 The multi-path microfluidic cell sorting system shown in the invention comprises a multi-path droplet generating module, a fluorescent cell detection module, an axial dielectrophoresis sorting module and a waste liquid recovery module; the fluorescent cell detection module, the axial dielectrophoresis sorting module and the waste liquid recovery module are respectively connected to the multi-path droplet generating module, the fluorescent cell detection module is connected to the droplet nozzle of the multi-path droplet generating module through a cell inlet, the outlet (droplet nozzle) of the multi-path droplet generating module is the fluorescent cell detection module, the axial dielectrophoresis sorting module is connected to the fluorescent cell detection module, specifically, it is non-contactly connected to the axial dielectrophoresis sorting module through the cell outlet of the fluorescent cell detection module.

[0073] The multi-path droplet generation module includes a microfluidic chip 1. The microfluidic chip 1 includes a substrate, a cell suspension storage module 2 and a single cell droplet generation module, the cell suspension storage module 2 and the single cell droplet generation module are respectively located on the substrate; the substrate is formed by bonding two layers of polydimethylsiloxane (PDMS) with a thickness of 5 mm; the single cell droplet generation module is composed of two gas phase passages 3, the cell suspension storage module 2 is a cell suspension storage chamber, and the gas phase passage 3 is used for focusing and generating single cell droplets, and the thickness is 150 μm; the cell suspension storage module 2 and the gas phase passage 3 are respectively connected to an external fluid source, and the two gas phase passages 3 are divided into two sides located at the cell suspension storage module 2, and the outlets of the cell suspension storage module 2 and the gas phase passage 3 meet at the droplet nozzle, and the cross-sectional shape of the droplet nozzle is rectangular, and the length × width is 30 μm × 30 μm. The multi-path droplet generation module of this embodiment uses a flow focusing structure to generate single cell droplets by shearing the cell mixture through the gas phase of the gas phase passages 3 on both sides of the cell suspension storage module 2. The schematic diagram of single cell droplet generation is shown in FIG. Figure 2 As shown; by adopting the asymmetric airflow method, the speed of the airflow input on both sides of the gas phase passage 3 is adjusted respectively, so that the emission direction and emission speed of the droplets can be changed, thereby realizing the generation of multi-path droplets. This method can control the direction and speed of the droplets while generating single-cell droplets. In this example, the angle of the droplet emission direction is 32° (looking down at the plane where the cell suspension storage module 2 is located, with the normal of the droplet nozzle as the central axis, the angle between the droplet emission direction and the normal can reach ±16°), and the maximum emission speed can reach 5m / s. The schematic diagram of multi-path droplet generation is shown in Figure 3 shown.

[0074] The fluorescent cell detection module includes an optical fiber 4, a continuous wave laser generator 5, a photomultiplier tube 6 and a field programmable gate array (FPGA) 7 connected in sequence. The optical fiber 4 is embedded on both sides of the droplet nozzle, with an outer diameter of 225μm and an inner diameter of 200μm. There is a grounded metal layer on its outer side to ensure the subsequent normal generation of a non-uniform electric field. The left side of the optical fiber 4 embedded on one side of the droplet nozzle is connected to the continuous wave laser generator 5, and the right side of the optical fiber 4 embedded on the other side of the droplet nozzle is connected to the field programmable gate array (FPGA) 7 through the photomultiplier tube 6. The continuous wave laser generator 5 generates extremely low power consumption and cell-mild laser, irradiates single-cell droplets through the optical fiber 4 and outputs the fluorescence signal of the cell. The generated fluorescence signal is transmitted to the photomultiplier tube 6 through the optical fiber 4 on the other side of the droplet nozzle, and the optical signal is converted into an electrical signal and detected and judged by the field programmable gate array (FPGA) 7, as shown in the schematic diagram. Figure 4 As shown; the power of the continuous wave laser generator 5 is 100mW, and the wavelengths are 405nm, 473nm, 532nm, and 640nm respectively. The power of the continuous wave laser generator 5 is adjusted until the laser power at the detection droplet is 1mW to avoid affecting the cell activity.

[0075] The axial dielectrophoresis sorting module includes a voltage amplifier 8 and a high-voltage columnar electrode 9 connected in sequence. The high-voltage columnar electrode 9 runs through the microfluidic chip 1 and is located obliquely below the single-cell droplet generation module. The center distance (referring to the distance from the center of the high-voltage cylindrical electrode to the multi-path droplet emission plane) is 415 μm. The cross-section of the high-voltage columnar electrode 9 is circular with a diameter of 600 μm, as shown in the schematic diagram. Figure 5 As shown; one side of the voltage amplifier 8 is connected to the high-voltage cylindrical electrode 9, providing a voltage of 0 to 4 kV to generate a non-uniform electric field in three-dimensional space, and the other side is connected to the field programmable gate array (FPGA) 7. Due to the difference in relative dielectric constants between droplets and air, single-cell droplets are subjected to dielectrophoretic force near the electrode of the axial dielectrophoresis sorting module, and non-sorted cells will be deflected toward the electrode, as shown in the schematic diagram Figure 6 As shown, the non-sorted cells enter the collection tube 10 and leave the system. Since a columnar electrode is used, each section of the columnar electrode can generate a similar non-uniform electric field, and the trajectory deviation of the multi-path droplets is less than 10 μm, so that the collection tube can fully collect the multi-path non-sorted cells.

[0076] The waste liquid recovery module includes a collection tube 10 and a vacuum pump 11 connected in sequence. The collection tube 10 runs through the microfluidic chip 1, is located on the movement trajectory of the waste droplets after they are offset, and is connected to the vacuum pump 11. When the waste liquid recovery module is working, the vacuum pump 11 is always on to provide a continuous vacuum negative pressure for the collection tube 10, ensuring the collection and emptying of the offset droplets in the collection tube 10 without affecting the movement trajectory of the sorted single cell droplets. In this embodiment, one third of the micro-circular tube is cut off to obtain a recovery micro-tube, which is the collection tube 10, as shown in the schematic diagram Figure 7 As shown, the collection tube 10 is cut toward the offset droplets to fully recover the waste droplets, as shown in the schematic diagram Figure 6 shown.

[0077] The cell sorting efficiency of the multi-path microfluidic cell sorting system of this embodiment is high. The microfluidic system is designed based on the principle of fluorescence-activated cell sorting, and FPGA is used to improve the detection speed of cell optical signals to achieve automated high-speed sorting; it has little effect on cell activity, and the dielectrophoresis technology is used to sort cells to avoid direct contact with cells and gently manipulate cells; multi-path cell sorting is achieved, and multi-path droplets are generated by gas-liquid flow focusing, combined with the spatially inhomogeneous electric field generated by the columnar electrodes, and multi-path single-cell droplets are detected and sorted, which can achieve high-throughput simultaneous sorting of multiple cells; the product has high compatibility, the entire system occupies little space, can be directly integrated with other microfluidic systems, and is suitable for biological in situ printing. The cell sorting accuracy is greater than 99.5%, and the maximum sorting frequency can reach 1kHz. The average survival rate of cells after sorting is 94%, which is basically the same as the survival rate of the control group without electric field sorting.

[0078] Example 2

[0079] A multi-path microfluidic cell sorting system similar to Example 1, the only difference being that the multi-path droplet generation module comprises a microfluidic chip 1. The microfluidic chip 1 comprises a substrate, a cell suspension storage module 2 and a single cell droplet generation module; the single cell droplet generation module is composed of two gas phase passages 3 and a single cell droplet generation channel 12; the single cell droplet generation channel 12 and the cell suspension storage module 2 intersect vertically; the outlets of the two gas phase passages 3 and the single cell droplet generation channel 12 intersect at a liquid nozzle, and the schematic diagram of the path single cell droplet generation module is as shown in FIG. Figure 8 As shown; after the single-cell droplet is generated, a non-contact physical field is used to apply an external force to the single-cell droplet with an initial velocity to change the velocity direction of the single-cell droplet, thereby realizing multi-path droplet generation. The schematic diagram of multi-path droplet generation is shown in Fig.11 shown.

[0080] Example 3

[0081] A multi-path microfluidic cell sorting system similar to Example 1, the only difference being that the single-cell droplet generation module is composed of a gas phase passage 3, a cell suspension storage module 2 and a non-contact physical field, wherein the non-contact physical field includes but is not limited to a magnetic field, an electric field, and light; the cell suspension storage module 2 and the gas phase passage 3 converge at the droplet nozzle through coaxial flow focusing, and the gas phase of the gas phase passage 3 shears the cell mixture to generate a single-cell droplet. The schematic diagram of the single-cell droplet generation is shown in FIG. Fig. 9 As shown; after the single-cell droplet is generated, a non-contact physical field is used to apply an external force to the single-cell droplet with an initial velocity to change the velocity direction of the single-cell droplet, thereby realizing multi-path droplet generation. The schematic diagram of multi-path droplet generation is shown in Fig.11 shown.

[0082] Example 4

[0083] A multi-path microfluidic cell sorting system similar to Example 1, the only difference being that the single-cell droplet generation module does not contain a gas phase passage 3, and is composed of a cell suspension storage module 2, a first external electric field and a non-contact physical field, wherein the non-contact physical field includes but is not limited to a magnetic field, an electric field, and light; based on electrohydrodynamics, a cell mixture is pushed out of a droplet nozzle through an external strong electric field to generate a single-cell droplet, and a schematic diagram of single-cell droplet generation is shown in FIG. Fig.10 As shown; after the single-cell droplet is generated, a non-contact physical field is used to apply an external force to the single-cell droplet with an initial velocity to change the velocity direction of the single-cell droplet, thereby realizing multi-path droplet generation. The schematic diagram of multi-path droplet generation is shown in Fig.11 shown.

[0084] Embodiments 5 to 8

[0085] The multi-path microfluidic cell sorting systems of Examples 5 to 8 are similar to the multi-path microfluidic cell sorting systems of Examples 1 to 4, with the only difference being that the cross section of the high-voltage columnar electrode 9 is square, as shown in the schematic diagram. Fig.12 shown.

[0086] Examples 9 to 12

[0087] The multi-path microfluidic cell sorting systems of Examples 9 to 12 are similar to the multi-path microfluidic cell sorting systems of Examples 1 to 4, with the only difference being that the cross section of the high-voltage columnar electrode 9 is a triangle, as shown in the schematic diagram. Fig.13 shown.

[0088] Examples 13 to 24

[0089] The multi-path microfluidic cell sorting systems of Examples 13 to 24 are similar to the multi-path microfluidic cell sorting systems of Examples 1 to 12, with the only difference being that the structure of the collecting tube 10 is different, specifically the recycling channel, as shown in the schematic diagram. Fig.14 shown.

[0090] Embodiments 25 to 36

[0091] The multi-path microfluidic cell sorting systems of Examples 25 to 36 are similar to the multi-path microfluidic cell sorting systems of Examples 1 to 12, with the only difference being that the structure of the collection tube 10 is different, specifically the recovery baffle, as shown in the schematic diagram. Fig.15 shown.

[0092] It can be seen from the above content that the present invention can avoid direct contact with cells while meeting the miniaturized space requirements, thereby achieving high-throughput, high-precision, and low-cell activity-affected cell sorting.

[0093] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A multi-path microfluidic cell sorting method, characterized in that: The steps include: Asymmetric airflow processing and / or physical field processing are performed on droplets containing single cells to obtain multi-path cell droplets; the motion trajectories of any two paths in the multi-path cell droplets are different; Detecting the multi-path cell droplets and collecting detection results; According to the detection results, the multi-path cell droplets are processed by using a non-contact physical field to deflect non-target sorted cell droplets in a direction.

2. The sorting method according to claim 1, characterized in that: The non-contact physical field includes one or more of dielectrophoresis, optical tweezers, magnetic fields, electrostatic tweezers and sound waves.

3. The sorting method according to claim 1, characterized in that: The method for preparing droplets containing single cells comprises: treating a cell suspension using one or more of a gas-liquid copolymerization method, a T-channel method, a flow focusing method, a coaxial flow focusing method and an electrohydrodynamic method to obtain droplets containing single cells.

4. The sorting method according to claim 1, characterized in that: The detection includes one or more of fluorescence detection, imaging analysis detection and Raman spectroscopy detection.

5. The sorting method according to claim 1, further comprising: The non-target sorted cell droplets are recovered.

6. The separation method according to any one of claims 1 to 5, characterized in that: The sorting method is carried out in a microfluidic cell sorting system; the microfluidic cell sorting system comprises a multi-path droplet generation module, a cell detection module, a cell sorting module and a waste liquid recovery module; The multi-path droplet generation module comprises a microfluidic chip; the microfluidic chip comprises a substrate and a cell suspension storage module and a single cell droplet generation module located on the substrate; the single cell droplet generation module is an asymmetric airflow processing module and / or a physical field processing module; The outlets of the cell suspension storage module and the single cell droplet generation module meet at a droplet nozzle; The cell detection module is connected to the droplet nozzle through the cell inlet; the cell detection module is located below the single cell droplet generation module and is non-contactly connected to the non-contact physical field in the cell sorting module through the cell outlet; The waste liquid recovery module runs through the microfluidic chip in parallel and is located on the movement track after the droplet deviates from the running track of the cell sorting module.

7. The sorting method according to claim 6, characterized in that: The asymmetric airflow processing module includes a first gas phase passage and a second gas phase passage; the first gas phase passage and the second gas phase passage are respectively located on both sides of the cell suspension storage module, and intersect with the outlet of the single cell droplet generation module at a droplet nozzle.

8. The sorting method according to claim 6, characterized in that: Looking down at the plane where the cell suspension storage module is located, with the normal line of the droplet nozzle as the central axis, the angle between the droplet emission direction and the normal line is 32-48°.

9. The sorting method according to claim 6, characterized in that: The cell detection module comprises a continuous wave laser generator, a first optical fiber, a second optical fiber, a photomultiplier tube and a field programmable gate array which are connected in sequence; the first optical fiber and the second optical fiber are embedded on both sides of the droplet nozzle.

10. A multi-path microfluidic cell sorting system, characterized in that: It includes a multi-path droplet generation module, a cell sorting module, a cell detection module and a waste liquid recovery module; The multi-path droplet generation module comprises a microfluidic chip; the microfluidic chip comprises a substrate and a cell suspension storage module and a single cell droplet generation module located on the substrate; the single cell droplet generation module is an asymmetric airflow processing module or a physical field processing module; the outlets of the cell suspension storage module and the single cell droplet generation module meet at a droplet nozzle; The cell detection module is connected to the droplet nozzle through the cell inlet; the cell detection module is located below the single cell droplet generation module and is non-contactly connected to the non-contact physical field in the cell sorting module through the cell outlet; The waste liquid recovery module runs through the microfluidic chip in parallel and is located on the movement track after the droplet deviates from the running track of the cell sorting module.

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