Microfluidic sorting system and method

Abstract

The invention provides a microfluidic sorting system and method, and relates to the technical field of particle sorting, and the microfluidic sorting system comprises a microfluidic sorting chip, a liquid driving device, an image acquisition device and a controller. According to the microfluidic sorting system and method provided by the invention, the required target particles can be sorted from the to-be-sorted particles on the basis of an image acquisition device and a deep learning technology under the condition that the to-be-sorted particles do not need to be subjected to fluorescent staining, so that the influence and damage to the to-be-sorted particles due to fluorescent staining can be reduced, and the sorting efficiency is improved. According to the method, the accuracy and credibility of subsequent experiments can be improved, the investment cost required by cell sorting can be remarkably reduced, multi-dimensional information such as cell size, roundness, edge characteristics and morphological characteristics can be obtained, the multi-dimensional cell sorting requirements can be better met, target particles can be more accurately and efficiently sorted, and the cell sorting efficiency is improved. The multi-dimensional cell sorting requirement can be better met, and flexible sorting and lossless sorting of cells can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of particle sorting, and in particular to a microfluidic sorting system and method. Background Art

[0002] Microfluidic sorting system is a cell or particle sorting system based on microfluidic technology. Microfluidic sorting system has the advantages of high throughput, automation, integration and low cost, and is widely used in various biomedical research and applications.

[0003] In the related art, the traditional microfluidic sorting system can sort the desired target particles from the particles to be sorted based on the fluorescence-activated cell sorting analysis (Fluorescence-Activated Cell Sorting). However, when using the traditional microfluidic sorting system to sort the desired target particles from the particles to be sorted, it is necessary to first fluorescently stain the particles to be sorted, and then use the optical effect to manipulate and separate the fluorescently stained particles to be sorted, and sort out the desired target particles.

[0004] However, fluorescent staining of cells will change the behavior or activity of the stained cells. Therefore, the target particles sorted by the traditional microfluidic sorting system may have missing or modified features, which will affect the credibility of subsequent experiments. Therefore, how to achieve cell sorting without fluorescent staining of cells is a technical problem that needs to be solved in this field. Summary of the invention

[0005] The present invention provides a microfluidic sorting system and method, which are used to solve the problem that cells need to be fluorescently stained during cell sorting in the prior art, but fluorescent staining of cells will change the behavior or activity of the stained cells, resulting in feature loss or feature modification of the sorted cells, thereby affecting the credibility of subsequent experiments. The system and method can realize cell sorting without fluorescent staining of cells.

[0006] The present invention provides a microfluidic sorting system, comprising: a microfluidic sorting chip, a liquid driving device, an image acquisition device and a controller, wherein the liquid driving device is electrically connected to the image acquisition device; the liquid driving device is used to drive a sample liquid containing particles to be sorted to flow into the microfluidic sorting chip from a second injection port of the microfluidic sorting chip in response to the control of the controller, and to drive a sheath liquid to flow into the microfluidic sorting chip from a first injection port of the microfluidic sorting chip, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip; the liquid driving device is also used to drive a sorting liquid to flow into the microfluidic sorting chip from a third injection port of the microfluidic sorting chip in response to the control of the controller, so that the mixed liquid of the sample liquid and the sheath liquid is impacted by the sorting liquid and flows through the first injection port of the microfluidic sorting chip. A sample outlet flows into a liquid collection bottle, and the particles to be sorted include target particles; the image acquisition device is used to acquire the image of the mixed liquid in the microfluidic sorting chip at a preset frequency, and send the acquired image of the mixed liquid to the controller; the controller is used to control the liquid drive device to drive the sheath liquid and the sample liquid to flow into the microfluidic sorting chip from the first inlet and the second inlet of the microfluidic sorting chip respectively when determining that the cell sorting task is triggered, and the controller is also used to perform image recognition on the image of the mixed liquid when receiving the image of the mixed liquid sent by the image acquisition device, and then determine whether to control the liquid drive device to drive the sorting liquid to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip based on the recognition result of the image of the mixed liquid.

[0007] According to a microfluidic sorting system provided by the present invention, the controller is specifically used to input the image of the mixed liquid into a cell recognition model when receiving the image of the mixed liquid sent by the image acquisition device, obtain the cell recognition result output by the cell recognition model, and then control the liquid driving device to drive the sorting liquid to flow into the microfluidic sorting chip from the third sampling port when it is determined based on the cell recognition result that a predefined sorting condition is met; wherein the cell recognition model is obtained after training based on a sample image and the cell recognition result of the sample image; the cell recognition result of the sample image is the sample image marked with the target particle; and the sorting condition includes that the image of the mixed liquid includes the target particle.

[0008] According to a microfluidic sorting system provided by the present invention, the microfluidic sorting chip comprises: a first flow channel and an annular flow channel; the annular flow channel is provided with two through-holes, namely a first through-hole and a second through-hole; the first through-hole is connected to the first injection port provided outside the annular flow channel, and the second through-hole is connected to the second injection port provided inside the annular flow channel; the first end of the first flow channel is connected to the second through-hole, so that the sheath liquid flowing in from the first injection port covers the sheath liquid flowing in from the second injection port at the second through-hole. The sample liquid flowing into the sample port forms the mixed liquid which flows into the first flow channel; the second end of the first flow channel is respectively connected with the third sample inlet, the first sample outlet and the second sample outlet of the microfluidic sorting chip, so that the mixed liquid at the second end of the first flow channel is not impacted by the sorting liquid flowing in from the third sample inlet, and flows into the waste liquid bottle through the second sample outlet; the mixed liquid is impacted by the sorting liquid flowing in from the third sample inlet at the second end of the first flow channel, and flows into the liquid collection bottle through the first sample outlet.

[0009] According to a microfluidic sorting system provided by the present invention, the liquid driving device includes: a pressurizing device; the pressurizing device is used to pressurize a first closed container containing the sheath liquid and a second closed container containing the sample liquid in response to the control of the controller, so that the sheath liquid and the sample liquid flow into the first injection port and the second injection port respectively through a first pipe and a second pipe under the action of pressure; the pressurizing device is also used to pressurize a third closed container containing the sorting liquid in response to the control of the controller, so that the sorting liquid flows into the third injection port through a third pipe; the first pipe is used to connect the first closed container with the first injection port; the second pipe is used to connect the second closed container with the second injection port; the third pipe is used to connect the third closed container with the third injection port.

[0010] A microfluidic sorting system provided according to the present invention also includes: a second flow channel, a third flow channel, a fourth flow channel, a fifth flow channel and a sixth flow channel; the first flow channel is coaxially arranged with the third flow channel; the second end of the first flow channel is connected to the third sample inlet through the second flow channel, the second end of the first flow channel is connected to the second sample outlet through the third flow channel, and the second end of the first flow channel is connected to the first sample outlet through the fourth flow channel; the first sample inlet is connected to the first flow hole through the fifth flow channel, and the second sample inlet is connected to the second flow hole through the sixth flow channel; the first end of the fourth flow channel is connected to the second end of the first flow channel, and the second end of the fourth flow channel is connected to the first sample outlet, and the diameter of the first end of the fourth flow channel is greater than the diameter of the second end of the fourth flow channel; the diameter of the third flow channel is greater than the diameter of the first end of the fourth flow channel.

[0011] According to a microfluidic sorting system provided by the present invention, the controller is also used to control the first pressure value that the pressurizing device pressurizes into the first closed container based on the radius and specific resistance of the fifth flow channel and the annular flow channel and the density of the sheath fluid. The controller is also used to control the second pressure value that the pressurizing device pressurizes into the second closed container based on the radius and specific resistance of the sixth flow channel and the density of the sample fluid. The controller is also used to control the third pressure value that the pressurizing device pressurizes into the third closed container based on the radius and specific resistance of the second flow channel and the density of the sorting fluid.

[0012] According to a microfluidic sorting system provided by the present invention, the diameter of the flow channel in the microfluidic sorting chip is determined based on the diameter of the microparticles to be sorted.

[0013] According to a microfluidic sorting system provided by the present invention, after controlling the liquid driving device to drive the sorting liquid to flow from the third sampling port into the microfluidic sorting chip, the controller is also used to input the image of the mixed liquid into a cell recognition model when receiving the image of the mixed liquid sent by the image acquisition device, and obtain the cell recognition result output by the cell recognition model, and then control the liquid driving device to stop driving the sorting liquid to flow from the third sampling port into the microfluidic sorting chip when it is determined based on the cell recognition result that the sorting condition is not met.

[0014] According to a microfluidic sorting system provided by the present invention, when the particles to be sorted are cells, the cells to be sorted live in a cell matrix.

[0015] The present invention also provides a microfluidic sorting method based on any of the above-described microfluidic sorting systems, comprising: in the case of determining to trigger a cell sorting task, driving a sample liquid and a sheath liquid containing particles to be sorted to flow into the microfluidic sorting chip from a first injection port and a second injection port of the microfluidic sorting chip, respectively, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip; acquiring an image of a mixed liquid of the sample liquid and the sheath liquid in the microfluidic sorting chip; performing image recognition on the image of the mixed liquid, and then based on the mixed liquid, The recognition result of the image of the mixed liquid is used to determine whether to drive the sorting liquid to flow into the microfluidic sorting chip from the third sample inlet of the microfluidic sorting chip, so that when the sorting liquid is not driven to flow into the microfluidic sorting chip from the third sample inlet of the microfluidic sorting chip, the mixed liquid flows to the waste liquid bottle through the second sample outlet of the microfluidic sorting chip; when the sorting liquid is driven to flow into the microfluidic sorting chip from the third sample inlet of the microfluidic sorting chip, the mixed liquid flows to the liquid collection bottle through the first sample outlet of the microfluidic sorting chip.

[0016] The present invention also provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the computer program, any of the microfluidic sorting methods described above is implemented.

[0017] The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, and when the computer program is executed by a processor, the microfluidic sorting method as described above is implemented.

[0018] The present invention also provides a computer program product, comprising a computer program, wherein when the computer program is executed by a processor, the microfluidic sorting method described above is implemented.

[0019] The present invention provides a microfluidic sorting system and method, wherein the microfluidic sorting system includes a microfluidic sorting chip, a liquid driving device, an image acquisition device, and a controller. Based on the image acquisition device and deep learning technology, the desired target particles can be sorted from the particles to be sorted without fluorescent staining of the particles to be sorted, thereby reducing the influence and damage of the particles to be sorted due to fluorescent staining, improving the accuracy and credibility of subsequent experiments, and significantly reducing the cost of cell sorting. Moreover, compared with the traditional microfluidic sorting system that relies on fluorescent staining, the microfluidic sorting system provided by the present invention is based on the image acquisition device and deep learning technology, and can obtain multi-dimensional information such as cell size, roundness, edge features, and morphological features, which can better meet the multi-dimensional cell sorting requirements, help to more accurately and efficiently sort the target particles, better meet the multi-dimensional cell sorting requirements, and realize flexible and non-destructive sorting of cells. It is particularly suitable for cell recovery and culture for further analysis, and the operation of cell sorting is simple, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a structural schematic diagram of the microfluidic sorting system provided by the present invention.

[0022] Figure 2 It is a schematic diagram of the principle of the microfluidic sorting system provided by the present invention.

[0023] Figure 3 It is a schematic diagram of the structure of the fluid channel in the microfluidic sorting chip in the microfluidic sorting system provided by the present invention.

[0024] Figure 4 This is one of the images of the mixed liquid in the microfluidic sorting system provided by the present invention.

[0025] Figure 5 This is the second image of the mixed liquid in the microfluidic sorting system provided by the present invention.

[0026] Figure 6 It is a working principle diagram of the liquid driving device in the microfluidic sorting system provided by the present invention.

[0027] Figure 7 It is a summary diagram of control parameters in the microfluidic sorting system provided by the present invention.

[0028] Figure 8 This is one of the schematic diagrams of simulation results of the microfluidic sorting chip provided by the present invention.

[0029] Fig. 9 This is the second schematic diagram of the simulation results of the microfluidic sorting chip provided by the present invention.

[0030] Fig.10 It is a schematic diagram of the process of the microfluidic sorting method provided by the present invention.

[0031] Fig.11 It is a structural schematic diagram of the electronic device provided by the present invention.

[0032] Reference numerals: 101: microfluidic sorting system; 102: microfluidic sorting chip; 103: liquid driving device; 104: image acquisition device; 105: controller; 301: first flow channel; 302: annular flow channel; 303: first flow hole; 304: second flow hole; 305: second injection port; 306: first injection port; 307: third injection port; 308: second outlet port; 309: first outlet port; 310: second flow channel; 311: third flow channel; 312: fourth flow channel; 313: fifth flow channel; 314: sixth flow channel. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the description of the invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0035] In the description of the present application, the terms "first", "second", etc. are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those illustrated or described here, and the objects distinguished by "first", "second", etc. are usually a class, and the number of objects is not limited. For example, the first object can be one or more. In addition, in the description of the present application, "and / or" represents at least one of the connected objects, and the character " / " generally represents that the front and back associated objects are in an "or" relationship.

[0036] It should be noted that the conventional microfluidic sorting system in the related art can utilize optical effects to manipulate and separate the fluorescently stained particles to be sorted, thereby sorting out the desired target particles from the particles to be sorted.

[0037] Among them, when the traditional microfluidic sorting system manipulates and separates the fluorescently stained particles to be sorted, the droplets encapsulate different cells or viruses, and through special reactions, the droplets present different fluorescence intensities. The fluorescence signal is collected by the PMT, which converts the optical signal into an electrical signal, which is collected by the FPGA and presented on the software. After the droplets that meet the sorting range are identified, the FPGA will output a TTL level signal, which is amplified by a high-voltage amplifier and output to the microfluidic chip. An uneven electric field is formed on both sides of the chip channel, and the target droplets are polarized under the electric field, thereby moving under the uneven electric field to achieve sorting.

[0038] However, the chemical reagents (such as fixatives, permeabilizers, etc.) used in the process of fluorescent staining of cells may be toxic to cells and affect the normal physiological functions of cells. These chemicals may damage cell membranes, interfere with metabolic processes in cells, or affect cell signal transduction. In addition, the chemical reagents and fixatives used in the fluorescent staining process may change the morphology and size of cells, making the observed cell morphology different from the cell morphology under the actual physiological state. This morphological change may affect the accurate understanding of cell structure and function. During the fluorescent staining process, cells usually need to be fixed and permeabilized. These steps may destroy the activity of cells and cause cells to lose their original functions. In particular, for some cells that need to remain active (such as cells in live cell imaging experiments), fluorescent staining may cause irreversible damage to them.

[0039] Therefore, fluorescent staining of cells will change the behavior or activity of the stained cells, and the target particles sorted by traditional microfluidic sorting systems may have missing or modified features, which will affect the credibility of subsequent experiments. How to achieve cell sorting without fluorescent staining of cells is a technical problem that needs to be solved in this field.

[0040] In addition, traditional microfluidic sorting systems perform cell sorting based on fluorescence-activated cell sorting analysis based entirely on fluorescent biomarkers and / or light scattering intensity without the aid of high-information image information, and lack analysis of the localization and polymorphic characteristics of subcellular organelles, such as cell geometry, nucleus and cytoskeleton shape, etc., which can help improve the accuracy of cell classification and identification, especially for the classification and identification of rare cells and cells without known or unique biomarkers. The cell morphology information and amount of cell information that can be detected based on fluorescence-activated cell sorting analysis are extremely limited.

[0041] In addition, traditional microfluidic sorting systems are usually suitable for oil phase droplets, that is, cells or viruses are encapsulated in droplets, and oil phase is used as the mobile phase. Most cells live in the cell matrix, which is more compatible with the human body environment. Some special cells (such as organoid cells) can only proliferate, differentiate and grow in three dimensions in the cell matrix (such as matrigel and collagen). However, the above special cells cannot be sorted in the oil phase.

[0042] Meanwhile, for large-sized particles such as organoid cells, the diameter of the large-sized particles is about 100-1000 However, traditional microfluidic sorting systems are difficult to sort large-sized particles.

[0043] Combine the following Figure 1-Figure 6 The microfluidic sorting system provided by the present invention is described.

[0044] Figure 1 Schematic diagram of the structure of the microfluidic sorting system provided by the present invention. Figure 1 The microfluidic sorting system provided by the present invention is described. Figure 1 As shown, the microfluidic sorting system 101 includes: a microfluidic sorting chip 102, a liquid driving device 103, an image acquisition device 104 and a controller 105, and the liquid driving device 103 and the image acquisition device 104 are electrically connected; The liquid driving device 103 is used to drive the sample liquid containing the to-be-sorted particles to flow from the second inlet of the microfluidic sorting chip 102 into the microfluidic sorting chip 102 in response to the control of the controller 105, and drive the sheath liquid to flow from the first inlet of the microfluidic sorting chip 102 into the microfluidic sorting chip 102, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip 102. The liquid driving device 103 is also used to drive the sorting liquid to flow from the third inlet of the microfluidic sorting chip 102 into the microfluidic sorting chip 102 in response to the control of the controller 105, so that the mixed liquid of the sample liquid and the sheath liquid flows into the liquid collection bottle through the first outlet 309 of the microfluidic sorting chip 102 under the impact of the sorting liquid, and the to-be-sorted particles include the target particles; The image acquisition device 104 is used to acquire images of the mixed liquid in the microfluidic sorting chip 102 at a preset frequency, and send the acquired images of the mixed liquid to the controller 105; The controller 105 is used to control the liquid driving device 103 to drive the sheath liquid and the sample liquid to flow into the microfluidic sorting chip 102 from the first injection port and the second injection port when determining that the cell sorting task is triggered. The controller 105 is also used to perform image recognition on the image of the mixed liquid when receiving the image of the mixed liquid sent by the image acquisition device 104, and then determine whether to control the liquid driving device 103 to drive the sorting liquid to flow into the microfluidic sorting chip 102 from the third injection port of the microfluidic sorting chip 102 based on the recognition result of the image of the mixed liquid.

[0045] It should be noted that the particles to be sorted are the sorting objects of the microfluidic sorting system 101 provided by the present invention. The particles to be sorted include the required target particles and non-target particles. The microfluidic sorting system 101 provided by the present invention can sort the sample liquid containing the particles to be sorted into a target liquid including the target particles and a waste liquid not including the target particles, thereby realizing the sorting of the particles to be sorted.

[0046] It should be noted that the microparticles in the embodiments of the present invention may be cells, viruses, enzymes, proteins, and other types of microparticles, and the cells may include single cells and organoid cells.

[0047] It is understandable that the particles to be sorted and the target particles in the embodiment of the present invention may be determined based on actual needs.

[0048] For example, the target particles in the embodiment of the present invention are organoid cells of a certain specific morphology, and the particles to be sorted include organoid cells of various morphologies. The microfluidic sorting system 101 provided by the present invention can sort the sample liquid containing organoid cells of various morphologies into a target liquid including the organoid cells of the above-mentioned specific morphology and a waste liquid that does not include the organoid cells of the above-mentioned specific morphology.

[0049] For another example, if the target particles in the embodiment of the present invention are a certain type of organoid cells, and the particles to be sorted include multiple types of organoid cells, the microfluidic sorting system 101 provided by the present invention can sort the sample liquid containing multiple types of organoid cells into a target liquid including the above-mentioned specific type of organoid cells and a waste liquid not including the above-mentioned specific type of organoid cells. In the embodiment of the present invention, the particles to be sorted and the target particles are not specifically limited.

[0050] The microfluidic sorting chip 102 integrates the basic operating units such as sample preparation, reaction, separation, detection, cell culture, sorting, lysis, etc. involved in the fields of chemistry and biology onto a micrometer-scale chip, and utilizes the special properties of the fluid in the microchannel, such as laminar flow effect and diffusion effect, to manipulate and sort cells or particles.

[0051] The microfluidic sorting chip 102 is mainly composed of a chip substrate, a fluid channel, a microstructure, an electrode layer, a dielectric layer, and a hydrophobic layer.

[0052] The chip substrate is the main structure of the microfluidic sorting chip 102, and is usually made of silicon, glass or polymer materials (such as PMMA, PDMS, etc.). These materials have good processing performance and chemical stability, and can withstand the pressure, temperature and chemical environment in microfluidic operations. Tiny fluid channels and reaction chambers are processed on the substrate to control and guide the flow of fluids and to sort cells or particles.

[0053] The fluid channel is the core part of the microfluidic sorting chip 102, which is used to transport and mix fluid samples. The size of these channels is usually in the micron level, which can accurately control the flow direction and speed of the fluid. The design of the channel can be customized according to the specific sorting requirements, such as setting a liquid reservoir, a main channel, a focusing channel, a detection channel, and a collection channel, etc., to achieve accurate sorting and collection of cells.

[0054] Microstructures include micro components such as reaction chambers, valves, pumps, and sensors. In the microfluidic sorting chip 102, these microstructures are used to control the flow and mixing of fluids, and to monitor the physical and chemical changes in the reaction process in real time. For example, valves and pumps can be used to control the flow speed and direction of the fluid to ensure uniform distribution and accurate sorting of cells in the channel. Sensors are used to monitor parameters such as cell concentration and flow rate in the fluid so as to adjust the sorting conditions in a timely manner.

[0055] Optionally, the chip substrate of the microfluidic sorting chip 102 in the embodiment of the present invention is polydimethylsiloxane (PDMS).

[0056] The microfluidic sorting chip 102 in the embodiment of the present invention is configured with three sample inlets and two sample outlets, namely, a first sample inlet, a second sample inlet and a third sample inlet, and a first sample outlet 309 and a second sample outlet.

[0057] The first injection port of the microfluidic sorting chip 102 is used to inject sheath liquid into the microfluidic sorting chip 102. The second injection port of the microfluidic sorting chip 102 is used to inject sample liquid containing particles to be sorted into the microfluidic sorting chip 102. The third injection port of the microfluidic sorting chip 102 is used to inject sorting liquid into the microfluidic sorting chip 102.

[0058] The first sample outlet 309 of the microfluidic sorting chip 102 is used for the outflow of the target liquid including the target particles. The second sample outlet of the microfluidic sorting chip 102 is used for the outflow of the waste liquid not including the target particles.

[0059] The controller 105 in the embodiment of the present invention refers to a device that can control and adjust a circuit or system according to a predetermined program or instruction. The controller 105 is usually composed of a program counter, an instruction register, an instruction decoder, a timing generator, and an operation controller 105. These parts work together to enable the controller 105 to issue commands, coordinate and direct the operation of the entire system.

[0060] The controller 105 in the embodiment of the present invention determines to trigger the cell sorting task when receiving the control instruction for instructing to trigger the cell sorting task. The control instruction for instructing to trigger the cell sorting task may be input by a user.

[0061] Figure 2 Schematic diagram of the principle of the microfluidic sorting system provided by the present invention. Figure 2 As shown, when determining to trigger the cell sorting task, the controller 105 can control the liquid driving device 103 to drive the sample liquid containing the to-be-sorted particles to flow into the microfluidic sorting chip 102 from the second sampling port of the microfluidic sorting chip 102, and control the liquid driving device 103 to drive the sheath liquid to flow into the microfluidic sorting chip 102 from the first sampling port of the microfluidic sorting chip 102.

[0062] The sheath liquid and the sample liquid flowing into the microfluidic sorting chip 102 from the first injection port and the second injection port of the microfluidic sorting chip 102 are mixed into a mixed liquid of the sample liquid and the sheath liquid in the microfluidic sorting chip 102 .

[0063] The image acquisition device 104 can acquire images of the mixed liquid in the microfluidic sorting chip 102 at a preset frequency, and can send the acquired images of the mixed liquid to the controller 105 .

[0064] After receiving the image of the mixed liquid sent by the image acquisition device 104, the controller 105 can perform image recognition on the image of the mixed liquid based on deep learning technology, and when the target particles are recognized in the image of the mixed liquid, determine that the mixed liquid corresponding to the image of the mixed liquid includes the target particles, and then send a sorting signal to the liquid driving device 103, so that the liquid driving device 103 drives the sorting liquid to flow from the third sampling port of the microfluidic sorting chip 102 into the microfluidic sorting chip 102 when receiving the sorting signal, so that the mixed liquid flows to the liquid collecting bottle through the first sampling port 309 of the microfluidic sorting chip 102 under the impact of the sorting liquid, thereby obtaining the target liquid including the target particles in the liquid collecting bottle.

[0065] It can be understood that when the controller 105 does not control the liquid driving device 103 to drive the sorting liquid to flow into the microfluidic sorting chip 102 from the third sampling port of the microfluidic sorting chip 102, the sample liquid is not impacted by the sorting liquid, and the sample liquid will flow directly into the waste liquid bottle through the second sampling port of the microfluidic sorting chip 102.

[0066] It should be noted that the liquid driving device 103 in the embodiment of the present invention can drive the sample liquid, sheath liquid and sorting liquid to flow into the microfluidic sorting chip 102 in a variety of ways. For example, the liquid driving device 103 can drive the sample liquid, sheath liquid and sorting liquid to flow into the microfluidic sorting chip 102 by air pressure driving, gravity driving and mechanical driving.

[0067] It should be noted that the microfluidic sorting chip 102 in the embodiment of the present invention can achieve mixing of the sample liquid and the sheath liquid through the fluid channel, and the mixed liquid of the sample liquid and the sheath liquid flows out through the first sample outlet 309 of the microfluidic sorting chip 102 under the impact of the sorting liquid. The specific structure of the fluid channel in the microfluidic sorting chip 102 is not limited in the embodiment of the present invention.

[0068] Optionally, the image acquisition device 104 in the embodiment of the present invention may include a fluorescence microscope and a high-resolution fast camera (CMOS). After the light enters the detection light path from the objective lens of the fluorescence microscope, it is divided into two parts by the beam splitter on the detection light path. After a part of the light is filtered out by the filter to remove stray light, it is detected by the high-speed camera and converted into an image signal and input into the controller 105.

[0069] It should be noted that the resolution of the high-speed camera and the preset frequency at which the high-speed camera collects images of the mixed liquid in the microfluidic sorting chip 102 in the embodiment of the present invention can be determined based on prior knowledge and / or actual conditions. The above preset frequency and the resolution of the high-speed camera are not specifically limited in the embodiment of the present invention.

[0070] It is understandable that using a high-resolution high-speed camera to capture the image of the mixed liquid in the microfluidic sorting chip 102 can ensure that the captured image of the mixed liquid is clear and distortion-free.

[0071] Optionally, in the embodiment of the present invention, the value range of the preset frequency may be 400fps to 600fps, for example, the preset frequency may be 400fps, 500fps or 600fps.

[0072] The microfluidic sorting system in the embodiment of the present invention includes a microfluidic sorting chip, a liquid driving device, an image acquisition device and a controller. Based on the image acquisition device and deep learning technology, the desired target particles can be sorted from the particles to be sorted without fluorescent staining of the particles to be sorted, which can reduce the influence and damage of the particles to be sorted due to fluorescent staining, improve the accuracy and credibility of subsequent experiments, and significantly reduce the cost of cell sorting. Moreover, compared with the traditional microfluidic sorting system that relies on fluorescent staining, the microfluidic sorting system provided by the present invention is based on the image acquisition device and deep learning technology, which can obtain multi-dimensional information such as cell size, roundness, edge features and morphological features, can better meet the multi-dimensional cell sorting needs, help to more accurately and efficiently sort the target particles, can better meet the multi-dimensional cell sorting needs, can realize flexible sorting and non-destructive sorting of cells, is particularly suitable for cell recovery and culture for further analysis, and the operation of cell sorting is simple, with broad application prospects.

[0073] Figure 3 Schematic diagram of the structure of the fluid channel in the microfluidic sorting chip in the microfluidic sorting system provided by the present invention. Figure 3 As shown, as an optional embodiment, the microfluidic sorting chip 102 includes: a first flow channel 301 and an annular flow channel 302; The annular flow channel 302 is provided with two through-holes, namely a first through-hole 303 and a second through-hole 304; The first through hole 303 is connected to a first injection port 306 disposed outside the annular flow channel 302, and the second through hole 304 is connected to a second injection port 305 disposed inside the annular flow channel 302; The first end of the first flow channel 301 is connected to the second flow hole 304, so that the sheath liquid flowing in from the first injection port 306 covers the sample liquid flowing in from the second injection port 305 at the second flow hole 304, forming a mixed liquid flowing into the first flow channel 301; The second end of the first flow channel 301 is respectively connected to the third sample inlet 307, the first sample outlet 309 and the second sample outlet 308 of the microfluidic sorting chip 102, so that the mixed liquid at the second end of the first flow channel 301 is not impacted by the sorting liquid flowing in from the third sample inlet 307, and flows to the waste liquid bottle through the second sample outlet 308, and the mixed liquid is impacted by the sorting liquid flowing in from the third sample inlet 307 at the second end of the first flow channel 301, and flows to the liquid collection bottle through the first sample outlet 309.

[0074] Optionally, the annular flow channel 302 in the embodiment of the present invention may be a circular annular flow channel 302 , an elliptical or rectangular annular flow channel 302 , or a polygonal annular flow channel 302 . Figure 3 The annular flow channel 302 shown is a polygonal annular flow channel 302. The specific shape of the annular flow channel 302 is not limited in the embodiment of the present invention.

[0075] Preferably, the annular flow channel 302 in the embodiment of the present invention is as follows Figure 3 The polygon shown is composed of a rectangle and a triangle.

[0076] As an optional embodiment, the microfluidic sorting chip 102 further includes: a second flow channel 310, a third flow channel 311, a fourth flow channel 312, a fifth flow channel 313 and a sixth flow channel 314; the first flow channel 301 and the third flow channel 311 are coaxially arranged; The second end of the first flow channel 301 is connected to the third sample inlet 307 through the second flow channel 310, the second end of the first flow channel 301 is connected to the second sample outlet 308 through the third flow channel 311, and the second end of the first flow channel 301 is connected to the first sample outlet 309 through the fourth flow channel 312; The first injection port 306 is connected to the first through-hole 303 via the fifth flow channel 313, and the second injection port 305 is connected to the second through-hole 304 via the sixth flow channel 314; The first end of the fourth flow channel 312 is connected to the second end of the first flow channel 301, the second end of the fourth flow channel 312 is connected to the first sample outlet 309, and the diameter of the first end of the fourth flow channel 312 is greater than the diameter of the second end of the fourth flow channel 312; The diameter of the third flow channel 311 is greater than the diameter of the first end of the fourth flow channel 312 .

[0077] Optionally, the cross-sectional shape of the flow channel in the microfluidic sorting chip 102 in the embodiment of the present invention may be circular, rectangular, trapezoidal, semicircular, etc. The cross-sectional shape of the flow channel in the microfluidic sorting chip 102 in the embodiment of the present invention is not limited.

[0078] It can be understood that the flow channels in the microfluidic sorting chip 102 in the embodiment of the present invention include a first flow channel 301 , a second flow channel 310 , a third flow channel 311 , a fourth flow channel 312 , a fifth flow channel 313 and a sixth flow channel 314 .

[0079] As an optional embodiment, the diameter of the flow channel in the microfluidic sorting chip 102 is determined based on the diameter of the microparticles to be sorted.

[0080] It is understandable that the diameter of the flow channel in the microfluidic sorting chip 102 is larger than the diameter of the particle to be sorted. For example, the diameter of the particle to be sorted is 180 In the case of, the diameter of the flow channel in the microfluidic sorting chip 102 can be 300 .

[0081] It should be noted that the flow channel in the microfluidic sorting chip 102 in the embodiment of the present invention can be designed differently to achieve the separation of particles of different sizes (100-1000 μm). For example, when the length of the microfluidic sorting chip 102 and the depth of the flow channel are both 30 In the case of The length of the microfluidic sorting chip 102 and the depth of the flow channel are 300 In the case of That is, by changing the size of the flow channel in the microfluidic sorting chip 102, the sorting of particles of different sizes can be achieved, which can be applicable to various sizes, and is particularly suitable for the sorting of large-sized particles (such as organoid cells).

[0082] Optionally, the diameter of the first end of the fourth flow channel 312 may be determined based on the diameter of the second end of the fourth flow channel 312. For example, the diameter of the first end of the fourth flow channel 312 may be twice the diameter of the second end of the fourth flow channel 312. In the case of .

[0083] Alternatively, if Figure 3 As shown, the first end of the fourth flow channel 312 is funnel-shaped, and the diameter of the first end of the fourth flow channel 312 gradually decreases as it moves away from the second end of the first flow channel 301 to be the same as the diameter of the second end of the fourth flow channel 312. The length of the first end of the fourth flow channel 312 can be determined based on prior knowledge and / or actual conditions, and the length of the first end of the fourth flow channel 312 is not limited in the embodiment of the present invention.

[0084] The design that the diameter of the first end of the fourth flow channel 312 is larger than the diameter of the second end helps to gradually focus and accelerate the fluid during the sorting process, thereby improving the accuracy and efficiency of sorting. At the same time, it can also reduce the diffusion and loss of the fluid in the flow channel, further improving the utilization rate of the reagent.

[0085] Alternatively, if Figure 3 As shown, the first flow channel 301 , the second flow channel 310 , the third flow channel 311 , the fifth flow channel 313 and the sixth flow channel 314 are all straight flow channels, and the fourth flow channel 312 is in a corner shape.

[0086] Alternatively, if Figure 3 As shown, the first flow channel 301 is located on the central axis of the annular flow channel 302 , the fifth flow channel 313 , the sixth flow channel 314 , the first flow channel 301 and the third flow channel 311 are coaxially arranged, and the second flow channel 310 and the first end of the fourth flow channel 312 are coaxially arranged.

[0087] As an optional embodiment, the image acquisition device 104 is used to acquire images of the mixed liquid in the first flow channel 301 at a preset frequency.

[0088] It should be noted that the image acquisition device 104 in the embodiment of the present invention can be arranged near the first flow channel 301, and the image acquisition device 104 can be used to acquire an image of the mixture of the sample liquid and the sheath liquid in the first flow channel 301 at a preset frequency, and send the acquired image of the mixture to the controller 105.

[0089] The microfluidic sorting chip in the embodiment of the present invention realizes the coating of the sample liquid by the sheath liquid through the annular flow channel, and connects the annular flow channel and the third sample inlet, the first sample outlet and the second sample outlet respectively through the two ends of the first flow channel, thereby realizing the diversion of the mixed liquid of the sheath liquid and the sample liquid under the impact of the sorting liquid, and then realizing the sorting of the target particles. The microfluidic sorting chip has a simple structure and a simple manufacturing process, can sort the target particles more accurately and efficiently, can realize flexible and non-destructive sorting of cells, is particularly suitable for cell recovery and culture for further analysis, and has great application prospects.

[0090] As an optional embodiment, the controller 105 is specifically used to input the image of the mixed liquid into the cell recognition model when receiving the image of the mixed liquid sent by the image acquisition device 104, obtain the cell recognition result output by the cell recognition model, and then control the liquid driving device 103 to drive the sorting liquid to flow into the microfluidic sorting chip 102 from the third injection port 307 when it is determined based on the cell recognition result that the predefined sorting condition is met; The cell recognition model is obtained after training based on the sample image and the cell recognition result of the sample image; the cell recognition result of the sample image is the sample image marked with target particles; and the sorting condition includes that the image of the mixed liquid includes the target particles.

[0091] Specifically, when the controller 105 receives the image of the mixed liquid sent by the image acquisition device 104, it can first perform image preprocessing on the image of the mixed liquid to obtain the image of the mixed liquid after image preprocessing. The above-mentioned image preprocessing may include image scaling and / or image cropping according to a preset image size, as well as image denoising, image filtering, etc.

[0092] Figure 4 This is one of the images of the mixed liquid in the microfluidic sorting system provided by the present invention. Figure 5 This is the second image of the mixed liquid in the microfluidic sorting system provided by the present invention. Figure 4 These are images of pancreatic ductal adenocarcinoma cells in different gels. Figure 5 Here are images of different cells in collagen.

[0093] After obtaining the image of the mixed liquid after image preprocessing, the image of the mixed liquid after image preprocessing can be input into the cell recognition model, and the cell recognition model can recognize the target particles in the image of the mixed liquid after image preprocessing, and then the cell recognition result output by the cell recognition model can be obtained.

[0094] It should be noted that the cell recognition result output by the cell recognition model in the embodiment of the present invention is an image of the mixed solution labeled with target particles.

[0095] It should be noted that the cell recognition model in the embodiment of the present invention can be constructed based on the YOLO v4 (You Only Look Once, Version 4) network model.

[0096] Among them, the YOLO v4 network model is a deep learning model for target detection. While maintaining a fast detection speed, it greatly improves the detection accuracy and can identify more object categories. The YOLO v4 network model treats the target detection problem as a single regression problem, and implements target detection by dividing the image into grids and predicting the bounding box and category probability on each grid.

[0097] Compared to two-stage deep learning object detectors, the YOLO v4 network model can identify objects in images faster. The YOLO v4 network model uses anchor boxes to detect object categories in images. The YOLO v4 network model predicts the following three properties for each anchor box: Intersection over Union (IoU) - predicts the objectness score of each anchor box; Anchor box offset - optimizes the anchor box position; Class probability - predicts the class label assigned to each anchor box. The YOLO v4 network model runs deep learning on the input image to produce network predictions.

[0098] Optionally, since 32 The convolution kernel of the YOLO v4 network model of 14 is 32, so when the controller 105 receives the image of the mixed liquid sent by the image acquisition device 104, it can Image of a mixture of 730, changes to 448 448, to improve the computational speed of the cell recognition model.

[0099] In the embodiment of the present invention, an image including target particles can be acquired under focusing conditions, and then images with poor image quality, unclear target particles or wrong target particle identification can be deleted, and the retained images can be determined as sample images.

[0100] The target particles are marked in the sample image, and the sample image marked with the target particles can be determined as the cell recognition result of the sample image to obtain a sample set for model training.

[0101] The sample images in the sample set were randomly divided into 70% training subsets, 30% validation subsets and 10% test subsets, and the initial model built based on the YOLO v4 network model was trained, tested and verified to obtain a trained cell recognition model.

[0102] It should be noted that, when the controller 105 receives the image of the mixed liquid sent by the image acquisition device 104, it can store the image data and the image acquisition time in a stack, and can call the image data in the stack according to the timing of image acquisition through the MATLAB image processing program.

[0103] It should be noted that the sorting condition in the embodiment of the present invention may also be that the number of image target particles in the mixed liquid exceeds a preset value.

[0104] The controller 105 can determine whether the predefined sorting conditions are met based on the cell recognition results output by the cell recognition model. If the controller 105 determines that the sorting conditions are met, it can send a sorting signal to the liquid driving device 103, and can also send a sorting signal to the liquid driving device 103 after a delayed sorting delay period.

[0105] It should be noted that since it takes a certain amount of time for the mixed liquid after image acquisition to flow to the second end of the first flow channel 301, and it also takes a certain amount of time for the controller 105 to determine whether to flow to the liquid driving device 103 based on the image of the mixed liquid, if the above two time periods are equal, the controller 105 determines the moment when the sorting condition is met based on the image of the mixed liquid, and the mixed liquid corresponding to the image of the mixed liquid just flows to the second end of the first flow channel 301. The controller 105 can determine the moment when the sorting condition is met based on the image of the mixed liquid as the sorting moment, and then can send a sorting signal to the liquid driving device 103 at the above sorting moment, so that the mixed liquid corresponding to the image of the mixed liquid can flow into the first sample outlet 309 under the impact of the sorting liquid.

[0106] However, when the first flow channel 301 is long or the flow rate of the mixed liquid in the first flow channel 301 is slow, at the moment when the controller 105 determines that the sorting condition is met based on the image of the mixed liquid, the mixed liquid corresponding to the image of the mixed liquid has not flowed to the second end of the first flow channel 301, and it is necessary to delay the sorting delay time before sending the sorting signal to the liquid driving device 103. The sorting delay time in the embodiment of the present invention can be calculated based on the length and diameter of the first flow channel 301, the length and diameter of the second flow channel 310, and the flow rate of the mixed liquid in the first flow channel 301.

[0107] It should be noted that in the embodiment of the present invention, different labels can be added to the target particles in the sample image. After the trained cell recognition model is obtained, the cell recognition model can be instructed to recognize the target particles with the specified labels through control instructions.

[0108] As an optional embodiment, after controlling the liquid driving device 103 to drive the sorting liquid to flow into the microfluidic sorting chip 102 from the third sampling port 307, the controller 105 is also used to input the image of the mixed liquid into the cell recognition model when receiving the image of the mixed liquid sent by the image acquisition device 104, and obtain the cell recognition result output by the cell recognition model, and then control the liquid driving device 103 to stop driving the sorting liquid to flow into the microfluidic sorting chip 102 from the third sampling port 307 when it is determined based on the cell recognition result that the sorting conditions are not met.

[0109] The controller in the embodiment of the present invention can, upon receiving the image of the mixed liquid sent by the image acquisition device 104, input the image of the mixed liquid into the cell recognition model, obtain the cell recognition result output by the cell recognition model, and then, when it is determined based on the cell recognition result that the predefined sorting conditions are met, control the liquid driving device 103 to drive the sorting liquid to flow into the microfluidic sorting chip from the third sampling port. Based on the deep learning technology, it can more accurately and efficiently identify whether the mixed liquid includes target particles, thereby more accurately and efficiently determining the sorting timing for cell sorting, better meeting the multi-dimensional cell sorting requirements, and further improving the accuracy and efficiency of cell sorting.

[0110] As an optional embodiment, the liquid driving device 103 includes: a pressurizing device; The pressurizing device is used to pressurize the first sealed container containing the sheath liquid and the second sealed container containing the sample liquid respectively in response to the control of the controller 105, so that the sheath liquid and the sample liquid flow into the first injection port 306 and the second injection port 305 respectively through the first pipe and the second pipe under the action of pressure. The pressurizing device is also used to pressurize the third sealed container containing the sorting liquid in response to the control of the controller 105, so that the sorting liquid flows into the third injection port 307 through the third pipe; The first pipeline is used to connect the first sealed container with the first injection port 306 ; the second pipeline is used to connect the second sealed container with the second injection port 305 ; the third pipeline is used to connect the third sealed container with the third injection port 307 .

[0111] Figure 6 Schematic diagram of the working principle of the liquid driving device in the microfluidic sorting system provided by the present invention. Figure 6 As shown, the working principle of the liquid driving device 103 in the embodiment of the present invention includes: a certain amount of solution is contained in a closed container, a gas with a certain pressure is introduced into the closed container, and the solution in the closed container will enter the microfluidic sorting chip 102 along the liquid pipeline under the action of pressure.

[0112] Optionally, the pressurizing device in the embodiment of the present invention may include an air source and an air pump, wherein the air source is connected to the air pump, and the air pump increases the pressure in the sealed container by injecting gas from the air source into the sealed container.

[0113] Optionally, the gas source may be nitrogen or an air compressor. The air pump may be a Fluigent air pump.

[0114] The air pump can pressurize the first closed container to a first pressure value, the second closed container to a second pressure value, and the third closed container to a third pressure value by injecting different volumes of gas into the first closed container, the second closed container and the third closed container respectively.

[0115] Optionally, precision pressure sensors are provided at the first injection port 306, the second injection port 305 and the third injection port 307 to monitor the pressure at the first injection port 306, the second injection port 305 and the third injection port 307, so as to adjust the volume of gas injected into the first closed container, the second closed container and the third closed container by the air pump.

[0116] Optionally, a solenoid valve may be provided between the air pump and the sealed container, and the volume of gas input into the sealed container by the air pump may be controlled by the solenoid valve.

[0117] The liquid driving device in the embodiment of the present invention can accurately pressurize the closed container containing sheath liquid, sample liquid and sorting liquid by responding to the control of the controller, ensuring that the liquid flows stably into the corresponding sampling port under the action of pressure, thereby realizing high-precision control of the liquid flow rate in the microfluidic sorting chip. By accurately controlling the flow rate of the sheath liquid, sample liquid and sorting liquid in the microfluidic sorting chip, the analysis throughput and efficiency of the microfluidic sorting chip can be improved. The use of a closed container and a piping system can effectively prevent the contamination and volatilization of the sheath liquid, sample liquid and sorting liquid during the flow process, thereby ensuring the purity and stability of the sheath liquid, sample liquid and sorting liquid. The liquid driving device has a relatively simple structure and is relatively easy to maintain, thereby reducing the operating cost of the microfluidic sorting system and improving the reliability and service life of the microfluidic sorting system.

[0118] As an optional embodiment, the controller 105 is also used to control the first pressure value that the pressurizing device applies to the first closed container based on the radius and specific resistance of the fifth flow channel 313 and the annular flow channel 302 and the density of the sheath fluid. The controller 105 is also used to control the second pressure value that the pressurizing device applies to the second closed container based on the radius and specific resistance of the sixth flow channel 314 and the density of the sample fluid. The controller 105 is also used to control the third pressure value that the pressurizing device applies to the third closed container based on the radius and specific resistance of the second flow channel 310 and the density of the sorting fluid.

[0119] It should be noted that the pressurizing device in the embodiment of the present invention can generate different air pressures in response to the control of the controller 105 .

[0120] Based on the radius and specific resistance of the fifth flow channel 313 and the annular flow channel 302, the density of the sheath fluid and the expected flow rate of the sheath fluid in the fifth flow channel 313 and the annular flow channel 302, the controller 105 can calculate the first pressure value for pressurizing the first closed container, and then control the pressurizing device to apply the first pressure value of air pressure to the first closed container, so that the sheath fluid flows from the first closed container into the first injection port 306.

[0121] The first pressure value is calculated by the following formula:

[0122] in, Indicates the first pressure value; represents the radius of the annular flow channel 302; represents the radius of the fifth flow channel 313; represents the specific resistance of the annular flow channel 302; represents the specific resistance of the fifth flow channel 313; represents the density of the sheath fluid; 313 and 302. The expected flow rate of the sheath fluid in the fifth flow channel 313 and the annular flow channel 302 is shown.

[0123] Based on the radius and specific resistance of the sixth flow channel 314, the density of the sample liquid and the expected flow rate of the sample liquid in the sixth flow channel 314, the controller 105 can calculate the second pressure value for pressurizing the second closed container, and then control the pressurizing device to apply air pressure of the second pressure value to the second closed container, so that the sample liquid flows from the second closed container into the second sampling port 305.

[0124] The second pressure value is calculated by the following formula:

[0125] in, Indicates the second pressure value; represents the radius of the sixth flow channel 314; represents the specific resistance of the sixth flow channel 314; Indicates the density of the sample liquid; represents the expected flow rate of the sample liquid in the sixth flow channel 314 .

[0126] Based on the radius and specific resistance of the second flow channel 310, the density of the sorting liquid and the expected flow rate of the sorting liquid in the second flow channel 310, the controller 105 can calculate the third pressure value for pressurizing the third closed container, and then, when it is determined that the sorting conditions are met, control the pressurizing device to apply air pressure of the third pressure value to the third closed container, so that the sorting liquid flows from the third closed container into the third sampling port 307.

[0127] The third pressure value is calculated by the following formula:

[0128] in, Indicates the third pressure value; represents the radius of the second flow channel 310; represents the specific resistance of the second flow channel 310; Indicates the density of the sorting liquid; represents the expected flow rate of the separation liquid in the second flow channel 310 .

[0129] It should be noted that the expected flow rates of the sheath fluid in the fifth flow channel 313 and the annular flow channel 302 , the expected flow rate of the sample fluid in the sixth flow channel 314 , and the expected flow rate of the sorting fluid in the second flow channel 310 may be determined based on prior knowledge and / or actual conditions.

[0130] The microfluidic sorting chip in the embodiment of the present invention highly integrates multiple components such as sample inlets, sample outlets, flow channels and flow holes on a tiny chip, realizing highly integrated sample processing. This design not only reduces the size of the chip, but also improves the portability and operability of the chip. The microfluidic sorting chip modularizes the functions of coating, mixing, sorting and collecting sample liquids through different flow channels and connection structures, so that each functional unit can work independently without interfering with each other. This design improves the flexibility and versatility of the chip, making it convenient for users to select and adjust according to experimental needs.

[0131] The sheath liquid flowing into the microfluidic sorting chip in the embodiment of the present invention coats the sample liquid flowing in from the second inlet at the second flow hole to form a mixed liquid, which helps to reduce the diffusion and loss of the sample liquid in the flow channel and improve the accuracy of sorting. At the same time, the sheath liquid can also protect and stabilize the sample liquid to prevent it from being damaged during the sorting process. The mixed liquid is impacted by the sorting liquid flowing in from the third inlet at the second end of the first flow channel, thereby realizing the sorting of the particles to be sorted. This impact method is efficient and fast, and can significantly improve the sorting efficiency. At the same time, by adjusting the flow rate and impact strength of the sorting liquid, it is possible to achieve accurate sorting of particles of different sizes and properties.

[0132] The flow channel design in the microfluidic sorting chip in the embodiment of the present invention has been carefully optimized, including the size, curvature and connection structure of the flow channel, to ensure smooth flow and uniform distribution of the fluid in the flow channel, which helps to reduce the resistance and loss of the fluid in the flow channel and improve the utilization and sorting efficiency of the fluid.

[0133] Due to the integrated and miniaturized design of the microfluidic sorting chip in the embodiment of the present invention, the required amount of reagents is greatly reduced, which helps to reduce the experimental cost and reduce the pollution to the environment. In addition, by accurately controlling the amount and flow rate of the reagents, the accuracy and reliability of the experiment can be further improved.

[0134] The microfluidic sorting chip in the embodiment of the present invention is small in size, easy to carry and store, and can be used in different experimental environments, which improves the flexibility and convenience of the experiment. Due to the highly integrated and automated design of the microfluidic sorting chip, the experimental operation becomes simple and easy to understand. The user only needs to add samples and reagents according to the specified steps to automatically complete the sorting operation. This design reduces the requirements for the professionalism and experience accumulation of the operator, allowing more people to use this chip for experiments and research.

[0135] Figure 7 is a summary diagram of the control parameters in the microfluidic sorting system provided by the present invention. Figure 7 As shown, the control parameters in the microfluidic sorting system 101 include sample loading parameters, image parameters and sorting parameters. Sample loading parameters include a first pressure value of the pressurizing device pressurizing the first closed container, a second pressure value of the pressurizing device pressurizing the second closed container and a third pressure value of the pressurizing device pressurizing the third closed container. Image parameters may include a preset frequency, image size and labels of target particles. Sorting parameters may include sorting delay time and sorting time.

[0136] In the microfluidic sorting system 101 provided by the present invention, below the bright field light source is the microfluidic sorting chip 102, and the flow of the sample liquid, sheath liquid and sorting liquid in the microfluidic sorting chip 102 is controlled by the liquid driving device 103. Below the microfluidic sorting chip 102 is an optical module, which is composed of an objective lens and a filter. The fluorescent field light source is located in the middle of the image acquisition device 104, and the controller 105 is on the right. The controller 105 can be divided into two parts: an upper computer and a lower computer. The upper computer is mainly used to control the image acquisition device 104 to acquire images, store and process the image of the mixed liquid. The lower computer is implemented by a printed circuit board (PCB), and mainly drives the liquid driving device 103 and the optical path switching device to work by receiving control instructions issued by the upper computer. The microfluidic sorting system 101 has the advantages of simple operation, fast detection and high precision.

[0137] As an optional embodiment, when the particles to be sorted are cells, the cells to be sorted live in a cell matrix.

[0138] The particles to be sorted in the embodiment of the present invention exist in the cell matrix, and can achieve water phase sorting of the particles to be sorted. It is better suitable for the forces that cannot be sorted in the oil phase, and can be suitable for the sorting of various biological components (such as cells, viruses, enzymes, proteins, etc.) in the water phase at the same time.

[0139] As an optional embodiment, the microfluidic sorting system 101 further includes: a display device; the display device is used to display the image of the mixed liquid and other contents in response to the control of the controller 105.

[0140] By performing physical simulation on the structure of the microfluidic sorting chip 102 in the microfluidic sorting system 101, it can be verified that the microfluidic sorting chip 102 has no structural defects and other potential design defects in fluid mechanics, and the design of the microfluidic sorting chip 102 can be optimized to ensure the stability and reliability of the microfluidic sorting system 101 in practical applications. Figure 8 This is one of the schematic diagrams of simulation results of the microfluidic sorting chip provided by the present invention. Fig. 9 FIG2 is a second schematic diagram of the simulation results of the microfluidic sorting chip provided by the present invention. Figure 8 and Fig. 9 shown.

[0141] As for the software design of the controller 105, a driver is written in the software part to realize the seamless connection between the controller 105 and the liquid driving device 103, ensuring that the sample liquid, sheath liquid and sorting liquid flow into the microfluidic sorting chip 102 accurately, quickly and safely. A user-friendly visual interface is developed to enable operators to intuitively control and monitor the entire sorting process.

[0142] The performance of the microfluidic sorting system 101 provided by the present invention is verified through sorting experiments, including sorting efficiency, accuracy, repeatability, etc. According to the experimental feedback, the design, hardware configuration and software algorithm of the microfluidic sorting system 101 are continuously adjusted and optimized until the microfluidic sorting system 101 reaches the expected performance indicators.

[0143] The microfluidic sorting system 101 provided by the present invention can determine the diameter of the flow channel in the microfluidic sorting chip 102 based on the diameter of the particles to be sorted, and is suitable for sorting particles of different sizes to be sorted, for example, it can realize the sorting of single cells and organoid cells respectively.

[0144] The microfluidic sorting system 101 provided by the present invention uses an air pump pressurization method to control the flow of sheath liquid, sample liquid and sorting liquid in the microfluidic sorting chip 102, which can realize water phase sorting and is suitable for sorting various biological components (such as cells, viruses, enzymes, proteins, etc.) in the water phase at the same time.

[0145] The microfluidic sorting system 101 provided by the present invention has the characteristics of multiple parameters, can provide high-content images of cell morphology and cell-to-cell interactions, and can quickly analyze and sort cell populations in combination with image features.

[0146] The microfluidic sorting system 101 provided by the present invention develops real-time imaging technology to achieve high-speed, real-time imaging, and flexible sorting of the microfluidic sorting system 101 at the cell level.

[0147] The microfluidic sorting system 101 provided by the present invention is based on deep collection to assist in processing high-throughput data, and is particularly suitable for various omics analyses and massive database research.

[0148] The microfluidic sorting system 101 provided by the present invention can achieve flexible sorting without loss, and is particularly suitable for cell recovery and culture for further analysis.

[0149] In the microfluidic sorting system provided by the present invention, the microfluidic sorting chip and the liquid driving device are closely combined to form a complete sorting system. This highly integrated design makes the system more compact, lightweight, easy to carry and operate. The microfluidic sorting system realizes the automatic inflow and mixing of sample liquid, sheath liquid and sorting liquid through the pressurization of the liquid driving device. The user only needs to set the relevant parameters, and the microfluidic sorting system can automatically complete the sorting process, greatly reducing the difficulty of operation and labor intensity. The microfluidic sorting system can adjust the flow rate, pressure and other parameters of the sample liquid, sheath liquid and sorting liquid according to different experimental requirements to adapt to different sorting conditions and particle characteristics. The design of the microfluidic sorting chip is easy to expand and modify, and more sample inlets, sample outlets and flow channels can be added to meet more complex sorting requirements. At the same time, the liquid driving device can also be upgraded and expanded as needed to improve the performance and function of the system. The structural design of the microfluidic sorting system is reasonable and easy to disassemble and clean. This helps to extend the service life of the system and keep it in good working condition. The liquid drive device adopts mature technical means such as pressurizing device and air pump, which has stable and reliable performance. The microfluidic sorting system can also be equipped with corresponding safety protection measures, such as pressure sensor, overcurrent protection, etc., to ensure the safe operation of the system.

[0150] Fig.10 1 is a schematic diagram of the flow chart of the microfluidic sorting method provided by the present invention. The microfluidic sorting method provided by the present invention is implemented based on the microfluidic sorting system 101 as described above. Fig.10 As shown, the method includes the following steps: Step 1001, when determining to trigger a cell sorting task, driving a sheath liquid and a sample liquid containing particles to be sorted to flow into a microfluidic sorting chip from a first injection port and a second injection port of a microfluidic sorting chip, respectively, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip; Step 1002, acquiring an image of a mixture of sample liquid and sheath liquid in a microfluidic sorting chip; Step 1003, performing image recognition on the image of the mixed liquid, and then based on the recognition result of the image of the mixed liquid, determining whether to drive the sorting liquid to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, so that when the sorting liquid is not driven to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, the mixed liquid flows to the waste liquid bottle through the second sampling port of the microfluidic sorting chip, and when the sorting liquid is driven to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, the mixed liquid flows to the liquid collection bottle through the first sampling port of the microfluidic sorting chip.

[0151] It should be noted that the execution subject of the microfluidic sorting method in the embodiment of the present invention may be the controller 105 in the microfluidic sorting system 101. The microfluidic sorting method provided by the present invention is implemented based on the microfluidic sorting system 101 described above. The specific execution steps of the microfluidic sorting method can refer to the contents of the above embodiments, which will not be repeated in the embodiments of the present invention.

[0152] In the embodiment of the present invention, when determining that a cell sorting task is triggered, sheath fluid and sample fluid containing to-be-sorted particles are driven to flow into the microfluidic sorting chip from the first and second inlets of the microfluidic sorting chip respectively, so that the sample fluid and the sheath fluid are mixed in the microfluidic sorting chip, an image of the mixed fluid of the sample fluid and the sheath fluid in the microfluidic sorting chip is obtained, image recognition is performed on the image of the mixed fluid, and then based on the recognition result of the image of the mixed fluid, it is determined whether to drive the sorting fluid to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, so that when the sorting fluid is not driven to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, the mixed fluid flows to the waste liquid bottle through the second outlet of the microfluidic sorting chip, and when the sorting fluid is driven to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, the mixed fluid flows to the liquid collection bottle through the first outlet of the microfluidic sorting chip, so that the cell sorting task is triggered. Image acquisition and deep learning technology can achieve the sorting of required target particles from the particles to be sorted without fluorescent staining of the particles to be sorted, which can reduce the impact and damage of fluorescent staining on the particles to be sorted, improve the accuracy and credibility of subsequent experiments, and significantly reduce the cost of cell sorting. Moreover, compared with the traditional microfluidic sorting system that relies on fluorescent staining, the microfluidic sorting system provided by the present invention is based on image acquisition device and deep learning technology, can obtain multi-dimensional information such as cell size, roundness, edge characteristics and morphological characteristics, can better meet the multi-dimensional cell sorting needs, help to more accurately and efficiently sort the target particles, can better meet the multi-dimensional cell sorting needs, can realize flexible and non-destructive sorting of cells, is particularly suitable for cell recovery and culture for further analysis, and the operation of cell sorting is simple, with broad application prospects.

[0153] Fig.11An example of a physical structure diagram of an electronic device is shown in FIG. Fig.11 As shown, the electronic device may include: a processor (processor) 1110 , a communication interface (Communications Interface) 1120 , a memory (memory) 1130 and a communication bus 1140 , wherein the processor 1110 , the communication interface 1120 , and the memory 1130 communicate with each other via the communication bus 1140 . The processor 1110 can call the logic instructions in the memory 1130 to execute the microfluidic sorting method, which includes: in the case of determining that the cell sorting task is triggered, driving the sheath liquid and the sample liquid containing the to-be-sorted particles to flow into the microfluidic sorting chip from the first inlet and the second inlet of the microfluidic sorting chip respectively, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip; acquiring an image of the mixed liquid of the sample liquid and the sheath liquid in the microfluidic sorting chip; performing image recognition on the image of the mixed liquid, and then determining whether to drive the sorting liquid to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip based on the recognition result of the image of the mixed liquid, so that when the sorting liquid is not driven to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, the mixed liquid flows to the waste liquid bottle through the second outlet of the microfluidic sorting chip, and when the sorting liquid is driven to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, the mixed liquid flows to the liquid collection bottle through the first outlet of the microfluidic sorting chip.

[0154] In addition, the logic instructions in the above-mentioned memory 1130 can be implemented in the form of a software functional unit and can be stored in a computer-readable storage medium when it is sold or used as an independent product. Based on such an understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art or the part of the technical solution, can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including a number of instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk, etc. Various media that can store program codes.

[0155] On the other hand, the present invention also provides a computer program product, which includes a computer program. The computer program can be stored on a non-transitory computer-readable storage medium. When the computer program is executed by a processor, the computer can execute the microfluidic sorting method provided by the above methods, the method comprising: in the case of determining to trigger the cell sorting task, driving the sheath fluid and the sample fluid containing the particles to be sorted to flow into the microfluidic sorting chip from the first injection port and the second injection port of the microfluidic sorting chip respectively, so that the sample fluid and the sheath fluid are mixed in the microfluidic sorting chip; obtaining the microfluidic sorting An image of a mixture of sample liquid and sheath liquid in a chip; performing image recognition on the image of the mixture, and then determining whether to drive the sorting liquid to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip based on the recognition result of the image of the mixture, so that when the sorting liquid is not driven to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, the mixed liquid flows to the waste liquid bottle through the second outlet of the microfluidic sorting chip, and when the sorting liquid is driven to flow into the microfluidic sorting chip from the third inlet of the microfluidic sorting chip, the mixed liquid flows to the liquid collection bottle through the first outlet of the microfluidic sorting chip.

[0156] On the other hand, the present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, which is implemented when the computer program is executed by a processor to execute the microfluidic sorting method provided by the above methods, the method comprising: in the case of determining to trigger the cell sorting task, driving the sheath liquid and the sample liquid containing the particles to be sorted to flow into the microfluidic sorting chip from the first injection port and the second injection port of the microfluidic sorting chip respectively, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip; obtaining an image of the mixed liquid of the sample liquid and the sheath liquid in the microfluidic sorting chip Image; perform image recognition on the image of the mixed liquid, and then determine whether to drive the sorting liquid to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip based on the recognition result of the image of the mixed liquid, so that when the sorting liquid is not driven to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, the mixed liquid flows to the waste liquid bottle through the second sampling port of the microfluidic sorting chip, and when the sorting liquid is driven to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, the mixed liquid flows to the liquid collection bottle through the first sampling port of the microfluidic sorting chip.

[0157] The device embodiments described above are merely illustrative, wherein the units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. Ordinary technicians in this field can understand and implement it without paying creative labor.

[0158] Through the description of the above implementation methods, those skilled in the art can clearly understand that each implementation method can be implemented by means of software plus a necessary general hardware platform, and of course, can also be implemented by hardware. Based on this understanding, the above technical solution is essentially or the part that contributes to the prior art can be embodied in the form of a software product, and the computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, a disk, an optical disk, etc., including a number of instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in each embodiment or some parts of the embodiments.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic sorting system, characterized in that: include: A microfluidic sorting chip, a liquid driving device, an image acquisition device and a controller, wherein the liquid driving device and the image acquisition device are electrically connected; The liquid driving device is used to drive the sample liquid containing the to-be-sorted particles to flow from the second inlet of the microfluidic sorting chip into the microfluidic sorting chip in response to the control of the controller, and drive the sheath liquid to flow from the first inlet of the microfluidic sorting chip into the microfluidic sorting chip, so that the sample liquid and the sheath liquid are mixed in the microfluidic sorting chip. The liquid driving device is also used to drive the sorting liquid to flow from the third inlet of the microfluidic sorting chip into the microfluidic sorting chip in response to the control of the controller, so that the mixed liquid of the sample liquid and the sheath liquid flows into the liquid collection bottle through the first outlet of the microfluidic sorting chip under the impact of the sorting liquid, and the to-be-sorted particles include target particles. The image acquisition device is used to acquire images of the mixed liquid in the microfluidic sorting chip at a preset frequency, and send the acquired images of the mixed liquid to the controller; The controller is used to control the liquid driving device to drive the sheath liquid and the sample liquid to flow into the microfluidic sorting chip from the first injection port and the second injection port respectively when determining to trigger the cell sorting task. The controller is also used to perform image recognition on the image of the mixed liquid when receiving the image of the mixed liquid sent by the image acquisition device, and then determine whether to control the liquid driving device to drive the sorting liquid to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip based on the recognition result of the image of the mixed liquid.

2. The microfluidic sorting system according to claim 1, characterized in that: The controller is specifically used to input the image of the mixed liquid into the cell recognition model when receiving the image of the mixed liquid sent by the image acquisition device, obtain the cell recognition result output by the cell recognition model, and then control the liquid driving device to drive the sorting liquid to flow into the microfluidic sorting chip from the third injection port when it is determined that the predefined sorting condition is met based on the cell recognition result; Wherein, the cell recognition model is obtained after training based on a sample image and a cell recognition result of the sample image; the cell recognition result of the sample image is the sample image marked with the target particles; and the sorting condition includes that the image of the mixed liquid includes the target particles.

3. The microfluidic sorting system according to claim 1, characterized in that: The microfluidic sorting chip comprises: a first flow channel and an annular flow channel; The annular flow channel is provided with two through-flow holes, namely a first through-flow hole and a second through-flow hole; The first through-hole is communicated with the first injection port disposed outside the annular flow channel, and the second through-hole is communicated with the second injection port disposed inside the annular flow channel; The first end of the first flow channel is connected to the second flow hole, so that the sheath liquid flowing in from the first injection port covers the sample liquid flowing in from the second injection port at the second flow hole, forming the mixed liquid flowing into the first flow channel; The second end of the first flow channel is respectively connected to the third sample inlet, the first sample outlet and the second sample outlet of the microfluidic sorting chip, so that the mixed liquid is not impacted by the sorting liquid flowing in from the third sample inlet at the second end of the first flow channel, and flows into the waste liquid bottle through the second sample outlet. The mixed liquid is impacted by the sorting liquid flowing in from the third sample inlet at the second end of the first flow channel, and flows into the liquid collection bottle through the first sample outlet.

4. The microfluidic sorting system according to claim 3, characterized in that: The liquid driving device comprises: a pressurizing device; The pressurizing device is used to pressurize the first sealed container containing the sheath liquid and the second sealed container containing the sample liquid in response to the control of the controller, so that the sheath liquid and the sample liquid flow into the first injection port and the second injection port respectively through the first pipe and the second pipe under the action of pressure. The pressurizing device is also used to pressurize the third closed container containing the separation liquid in response to the control of the controller, so that the separation liquid flows into the third injection port through the third pipeline; The first pipeline is used to connect the first sealed container with the first injection port; the second pipeline is used to connect the second sealed container with the second injection port; the third pipeline is used to connect the third sealed container with the third injection port.

5. The microfluidic sorting system according to claim 4, characterized in that: Also includes: a second flow channel, a third flow channel, a fourth flow channel, a fifth flow channel and a sixth flow channel; the first flow channel and the third flow channel are coaxially arranged; The second end of the first flow channel is connected to the third sample inlet through the second flow channel, the second end of the first flow channel is connected to the second sample outlet through the third flow channel, and the second end of the first flow channel is connected to the first sample outlet through the fourth flow channel; The first injection port is connected to the first flow hole through the fifth flow channel, and the second injection port is connected to the second flow hole through the sixth flow channel; The first end of the fourth flow channel is connected to the second end of the first flow channel, the second end of the fourth flow channel is connected to the first sample outlet, and the diameter of the first end of the fourth flow channel is greater than the diameter of the second end of the fourth flow channel; A diameter of the third flow channel is greater than a diameter of the first end of the fourth flow channel.

6. The microfluidic sorting system according to claim 5, characterized in that: The controller is also used to control the first pressure value that the pressurizing device applies to the first closed container based on the radius and specific resistance of the fifth flow channel and the annular flow channel and the density of the sheath fluid. The controller is also used to control the second pressure value that the pressurizing device applies to the second closed container based on the radius and specific resistance of the sixth flow channel and the density of the sample fluid. The controller is also used to control the third pressure value that the pressurizing device applies to the third closed container based on the radius and specific resistance of the second flow channel and the density of the sorting fluid.

7. The microfluidic sorting system according to claim 6, characterized in that: The diameter of the flow channel in the microfluidic sorting chip is determined based on the diameter of the microparticles to be sorted.

8. The microfluidic sorting system according to claim 2, characterized in that: After controlling the liquid driving device to drive the sorting liquid to flow from the third sampling port into the microfluidic sorting chip, the controller is also used to input the image of the mixed liquid into a cell recognition model when receiving the image of the mixed liquid sent by the image acquisition device, and obtain the cell recognition result output by the cell recognition model, and then control the liquid driving device to stop driving the sorting liquid to flow from the third sampling port into the microfluidic sorting chip when it is determined based on the cell recognition result that the sorting condition is not met.

9. The microfluidic sorting system according to any one of claims 1 to 8, characterized in that: When the particles to be sorted are cells, the cells to be sorted live in a cell matrix.

10. A microfluidic sorting method implemented based on the microfluidic sorting system according to any one of claims 1 to 9, characterized in that: include: When the cell sorting task is determined to be triggered, the sheath fluid and the sample fluid containing the to-be-sorted particles are driven to flow into the microfluidic sorting chip from the first injection port and the second injection port of the microfluidic sorting chip, respectively, so that the sample fluid and the sheath fluid are mixed in the microfluidic sorting chip; Acquiring an image of a mixture of the sample liquid and the sheath liquid in the microfluidic sorting chip; Image recognition is performed on the image of the mixed liquid, and then based on the recognition result of the image of the mixed liquid, it is determined whether to drive the sorting liquid to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, so that when the sorting liquid is not driven to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, the mixed liquid flows to the waste liquid bottle through the second sampling port of the microfluidic sorting chip, and when the sorting liquid is driven to flow into the microfluidic sorting chip from the third sampling port of the microfluidic sorting chip, the mixed liquid flows to the liquid collection bottle through the first sampling port of the microfluidic sorting chip.

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