Microfluidic chip, micro-particle sorting system, method, device, and storage medium

Abstract

The present application discloses a microfluidic chip and a microparticle sorting system, method, device, and storage medium, relating to the field of biotechnology. In the microfluidic chip, a third liquid pressure control device is provided at the inlet of a sheath liquid injection structure. The third liquid pressure control device is used to generate an injection pressure based on an identification signal obtained when a droplet to be processed passes through the area of a cell transport structure. This injection pressure accelerates the formation and dripping of sheath liquid in the sheath liquid injection structure, thereby selectively injecting the droplets to be processed and obtaining target cell droplets. Simultaneously, a first liquid pressure control device provided at the inlet of the focused flow injection structure simultaneously pressurizes the focused flow injection structure and a second liquid pressure control device provided at the inlet of the sample flow injection structure, respectively. This arrangement accelerates the formation and dripping of sheath liquid in the sheath liquid injection structure, while simultaneously preventing the next droplet to be processed from affecting the sheath liquid injection process of the current droplet to be processed.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a microfluidic chip and a microparticle sorting system, method, device, and storage medium. Background Art

[0002] In related technologies, when sheath fluid is usually dropped onto cell droplets through droplet injection technology, it takes a long time for the gravity of the droplet to be greater than the surface tension and then drip. Therefore, the formation and dripping speed of the sheath fluid in the sheath fluid injection channel is slow. When the inlet pressure of the sheath fluid injection channel is increased alone, a backflow phenomenon will occur, causing the cell droplet to flow back in the direction opposite to the target sorting direction. At the same time, when the flow rate of the next cell droplet is not controlled, the next cell droplet will affect the droplet injection process of the current cell droplet. Summary of the Invention

[0003] The present application aims to address, at least to some extent, one of the technical problems in the related art. To this end, the present application proposes a microfluidic chip, microparticle sorting system, method, apparatus, and storage medium that accelerate the formation and dripping of sheath fluid in a sheath fluid injection structure, while simultaneously ensuring that the next droplet to be processed does not affect the sheath fluid injection process of the current droplet to be processed.

[0004] In a first aspect, an embodiment of the present application provides a microfluidic chip, comprising:

[0005] A focused flow injection structure, wherein a first liquid pressure control device is provided at the inlet of the focused flow injection structure;

[0006] a sample flow injection structure, which is in communication with the focused flow injection structure, and a second liquid pressure control device is provided at the inlet of the sample flow injection structure;

[0007] a cell transport structure, connected to the focused flow injection structure and the sample flow injection structure respectively;

[0008] A sheath liquid injection structure is provided on one side of the cell transport structure and is connected to the cell transport structure. A third liquid pressure control device is provided at the inlet of the sheath liquid injection structure. The third liquid pressure control device is used to generate an injection pressure according to an identification signal obtained when the droplets to be processed pass through the area of the cell transport structure, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure, so as to selectively inject the droplets to be processed in the cell transport structure and obtain target cell droplets. At the same time, the first liquid pressure control device synchronously pressurizes the focused flow injection structure, and the second liquid pressure control device synchronously pressurizes the sample flow injection structure.

[0009] According to some embodiments of the first aspect of the present application, the microfluidic chip also includes a droplet sorting structure, which includes a droplet collection channel and a waste liquid transmission channel. The cell transmission structure is respectively connected to the droplet collection channel and the waste liquid transmission channel. The third liquid pressure control device is also used to generate a sorting pressure according to the identification signal, so that the sorting pressure drains the target cell droplets to the droplet collection channel.

[0010] According to some embodiments of the first aspect of the present application, the microfluidic chip further includes a flow resistance adjustment structure, which is disposed inside the cell transmission structure and close to the sheath fluid injection structure.

[0011] According to some embodiments of the first aspect of the present application, a plurality of distance marks are provided in the cell transport structure, and a flow rate region range is formed between each adjacent two of the plurality of distance marks, and the plurality of flow rate region ranges are used to determine the real-time flow rate of the droplets to be processed.

[0012] In a second aspect, the present invention further provides a microparticle sorting system, comprising:

[0013] a control device, configured to obtain an image to be processed of the droplet to be processed and output an identification signal according to the image to be processed;

[0014] A microfluidic chip includes a focused flow injection structure, a sample flow injection structure, a cell transport structure, and a sheath fluid injection structure. A first liquid pressure control device is provided at the inlet of the focused flow injection structure. The sample flow injection structure is connected to the focused flow injection structure, and a second liquid pressure control device is provided at the inlet of the sample flow injection structure. The cell transport structure is connected to the focused flow injection structure and the sample flow injection structure respectively. The sheath fluid injection structure is provided on one side of the cell transport structure and is connected to the cell transport structure. A third liquid pressure control device is provided at the inlet of the sheath fluid injection structure. The third liquid pressure control device is used to generate an injection pressure based on an identification signal obtained when a droplet to be processed passes through an area of the cell transport structure, so that the injection pressure accelerates the formation and dripping of sheath fluid in the sheath fluid injection structure, thereby selectively injecting the droplets to be processed in the cell transport structure and obtaining target cell droplets. At the same time, the first liquid pressure control device synchronously pressurizes the focused flow injection structure, and the second liquid pressure control device synchronously pressurizes the sample flow injection structure.

[0015] According to some embodiments of the second aspect of the present application, the control device includes:

[0016] a microscopic imaging device, configured to obtain an image of the droplet to be processed and output the image;

[0017] A droplet recognition device is connected to the microscopic imaging device, and is used to receive the image to be processed and obtain detection information based on the image to be processed. The droplet recognition device is also used to generate an identification signal based on a deep learning neural network algorithm and the detection information, and output the identification signal to the first liquid pressure control device, the second liquid pressure control device, and the third liquid pressure control device.

[0018] In a third aspect, an embodiment of the present application further provides a microparticle sorting method, which is applied to the microparticle sorting system according to any one of the second aspects, characterized in that the method comprises:

[0019] The control device acquires an image of a droplet to be processed and outputs an identification signal according to the image, wherein the droplet to be processed is stored in the cell transport structure;

[0020] The third liquid pressure control device generates an injection pressure according to the identification signal, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure, so as to selectively inject the droplets to be processed in the cell transfer structure and obtain target cell droplets. At the same time, the first liquid pressure control device synchronously pressurizes the focused flow injection structure, and the second liquid pressure control device synchronously pressurizes the sample flow injection structure.

[0021] According to some embodiments of the third aspect of the present application, the microfluidic chip further includes a droplet sorting structure, the droplet sorting structure includes a droplet collection channel and a waste liquid transmission channel, the cell transport structure is respectively connected to the droplet collection channel and the waste liquid transmission channel, and the method further includes:

[0022] The third liquid pressure control device generates a sorting pressure according to the identification signal, so that the sorting pressure guides the target cell droplets to the droplet collection channel.

[0023] In a fourth aspect, an embodiment of the present application further provides another droplet sorting 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, the microparticle sorting method as described in any one of the third aspects above is implemented.

[0024] In a fifth aspect, an embodiment of the present application further provides a computer-readable storage medium, wherein the computer-readable storage medium stores computer-executable instructions, and the computer-executable instructions are used to enable a computer to execute the microparticle sorting method as described in any one of the third aspects above.

[0025] According to one or more technical solutions provided in the embodiments of this application, at least the following beneficial effects are achieved:

[0026] A first liquid pressure control device is provided at the inlet of the focused flow injection structure, a second liquid pressure control device is provided at the inlet of the sample flow injection structure, and a third liquid pressure control device is provided at the inlet of the sheath liquid injection structure. The third liquid pressure control device is configured to generate an injection pressure based on an identification signal, so that the injection pressure accelerates the formation and dripping of sheath liquid in the sheath liquid injection structure, thereby selectively injecting the droplets to be processed and obtaining target cell droplets. Simultaneously, the first liquid pressure control device and the second liquid pressure control device simultaneously pressurize the focused flow injection structure and the sample flow injection structure based on the identification signal. The focused flow injection structure, the sample flow injection structure, and the sheath liquid injection structure are synchronously pressurized, so that the flow rate in the cell transport structure approaches zero. At this time, the next droplet to be processed will briefly stop, without affecting the sheath liquid injection process of the current droplet to be processed and causing no droplet loss. The synchronous pressurization of the focused flow injection structure, the sample flow injection structure, and the sheath liquid injection structure does not cause backflow.

[0027] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0029] Figure 1 This is a schematic diagram of the structure of a microfluidic chip provided in one embodiment of the present application;

[0030] Figure 2 This is a schematic diagram of a microfluidic chip with multiple distance markers provided in one embodiment of the present application;

[0031] Figure 3 is a schematic diagram of a droplet sorting process provided by another embodiment of the present application;

[0032] Figure 4 This is a schematic diagram of a droplet sorting process simulation provided by another embodiment of the present application;

[0033] Figure 5 This is a structural schematic diagram of the droplet sorting direction provided by another embodiment of the present application;

[0034] Figure 6 is a schematic structural diagram of a microfluidic chip provided in another embodiment of the present application;

[0035] Figure 7 It is a flow chart of a microparticle separation method provided in another embodiment of the present application.

[0036] Reference numerals:

[0037] Microfluidic chip 100;

[0038] Focused flow injection structure 110, first liquid pressure control device 111;

[0039] Sample flow injection structure 120, second liquid pressure control device 121;

[0040] Sheath liquid injection structure 130, third liquid pressure control device 131, sheath liquid injection channel 132;

[0041] Droplet collection channel 140, droplet collection pool 141, waste liquid transmission channel 142, waste liquid pool 143;

[0042] Cell transport structure 150 and flow resistance adjustment structure 151. DETAILED DESCRIPTION

[0043] The following describes in detail embodiments of the present application. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and are not to be construed as limiting the present invention.

[0044] In the description of this application, it should be understood that descriptions involving orientations, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they cannot be understood as limitations on this application.

[0045] In the description of this application, terms such as "greater than," "less than," and "exceed" are understood to exclude the number indicated, while terms such as "above," "below," and "within" are understood to include the number indicated. The terms "first" and "second" are used solely to distinguish technical features and are not to be construed as indicating or implying relative importance, or as implicitly specifying the number or order of the technical features indicated.

[0046] In the description of this application, unless otherwise explicitly defined, terms such as "dispose," "install," and "connect" should be interpreted broadly. For example, they can refer to fixed connection, detachable connection, or integral connection; they can refer to mechanical connection or electrical connection; they can refer to direct connection or indirect connection through an intermediate medium; and they can refer to internal communication between two components. Those skilled in the art can reasonably determine the specific meanings of the above terms in this application based on the specific content of the technical solution.

[0047] In fields such as single-cell sequencing, cell cloning, drug screening, cancer screening, and stem cell transplantation, it is often necessary to isolate individual cells from a sample. Due to the heterogeneity between cells, analyzing only the cell population often loses a large amount of individual information, and the group signal can mask the signal released by a single cell. Single-cell analysis provides a method to intuitively understand the connection between gene expression and cell state, so the research on devices for obtaining single cells is of great significance.

[0048] Microfluidics can reduce the size of particles to a few microns to tens of microns, and achieve the separation and printing of single cells by precisely controlling the movement of fluids. Compared with traditional printing, microfluidics requires less sample volume, has higher sensitivity, and has a high throughput, which makes it more suitable for processing certain more precious samples. In addition, microfluidics methods usually do not damage the biological activity of particles in the sample, so microfluidic sorting and printing technology is increasingly favored by researchers.

[0049] In related technologies, when sheath fluid is dropped onto cell droplets through droplet injection technology, it takes a long time for the gravity of the droplet to be greater than the surface tension and then drip. Therefore, the formation and dripping speed of the sheath fluid in the sheath fluid injection channel is slow. When the inlet pressure of the sheath fluid injection channel is increased alone, a backflow phenomenon will occur, causing the cell droplet to flow back in the direction opposite to the target sorting direction. At the same time, if the flow rate of the next cell droplet is not controlled, the next cell droplet will sometimes affect the droplet injection process of the current cell droplet.

[0050] Based on the above situation, the embodiment of the present application provides a microfluidic chip 100 and a microparticle sorting system, method, and storage medium, which can accelerate the formation and dripping of sheath liquid in the sheath liquid injection structure 130, and at the same time, the next droplet to be processed will not affect the sheath liquid injection process of the current droplet to be processed.

[0051] The embodiments of the present application are further described below with reference to the accompanying drawings.

[0052] The first embodiment of the present application specifically provides a microfluidic chip 100, referring to Figure 1The microfluidic chip 100 includes a focused flow injection structure 110, a sample flow injection structure 120, a cell transport structure 150, and a sheath liquid injection structure 130. A first liquid pressure control device 111 is provided at the inlet of the focused flow injection structure 110; the sample flow injection structure 120 is connected to the focused flow injection structure 110, and a second liquid pressure control device 121 is provided at the inlet of the sample flow injection structure 120; the cell transport structure 150 is respectively connected to the focused flow injection structure 110 and the sample flow injection structure 120; the sheath liquid injection structure 130 is provided at the cell transport structure 150. One side is connected to the cell transmission structure 150, and a third liquid pressure control device 131 is provided at the inlet of the sheath liquid injection structure 130. The third liquid pressure control device 131 is used to generate an injection pressure according to the identification signal, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure 130, so as to selectively inject the droplets to be processed in the cell transmission structure 150 and obtain target cell droplets. At the same time, the first liquid pressure control device 111 and the second liquid pressure control device 121 synchronously pressurize the focused flow injection structure 110 and the sample flow injection structure 120 respectively.

[0053] In this embodiment, a first liquid pressure control device 111 is provided at the inlet of the focused flow injection structure 110, a second liquid pressure control device 121 is provided at the inlet of the sample flow injection structure 120, and a third liquid pressure control device 131 is provided at the inlet of the sheath liquid injection structure 130. The third liquid pressure control device 131 is used to generate an injection pressure according to an identification signal, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure 130, so as to selectively inject the droplets to be treated and obtain target cell droplets; at the same time, the first liquid pressure control device 111 synchronously pressurizes the focused flow injection structure 110 according to the identification signal, and the second liquid pressure control device 121 synchronously pressurizes the sample flow injection structure 120 according to the identification signal. The focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 are pressurized synchronously, so that the flow rate in the cell transmission structure 150 is close to 0. At this time, the next droplet to be processed will stay for a short time, which will not affect the sheath liquid injection process of the current droplet to be processed and will not cause the loss of droplets. The focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 are pressurized synchronously, and no backflow phenomenon will occur.

[0054] Specifically, the focused flow injection structure 110 includes an inlet of the focused flow injection structure 110 and a sheath liquid injection channel 132. The first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 receive identification signals obtained based on the droplets to be processed. Accordingly, the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 respectively synchronously pressurize the focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 to generate injection pressure and increase the flow rate of the sheath liquid injection channel 132, thereby generating a large flow rate to quickly form sheath liquid and drip it onto the droplets to be processed to obtain target cell droplets, and also prevent the lateral structure in the cell transmission structure 150 from generating backflow phenomenon, wherein the target cell droplets are droplets containing microparticles.

[0055] It should be noted that if the droplets to be processed are obtained as encapsulated cell droplets according to the identification signal, the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 respectively synchronously pressurize the focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 to generate injection pressure, increase the flow rate of the sheath liquid injection channel 132, thereby generating a large flow rate to quickly form sheath liquid and drip it onto the droplets to be processed to obtain target cell droplets, wherein the target cell droplets are droplets of encapsulated sheath liquid; if the droplets to be processed are obtained as cell-free or target-free encapsulated cell droplets according to the identification signal, the pressure of the sheath liquid injection channel 132 remains unchanged, the third liquid pressure control device 131 does not generate injection pressure, and the sheath liquid will not be injected into the cell-free or target-free cell droplets.

[0056] It should be noted that the sheath fluid can be a buffer or culture medium, the liquid flowing into the inlet of the sample flow injection structure 120 can be a suspension of cells or particles, and the liquid flowing into the inlet of the focused flow injection structure 110 can be a cell culture medium. This embodiment of the present application does not limit this. For example, in experiments corresponding to particles, the cell culture medium is water.

[0057] It should be noted that the first liquid pressure control device 111, the second liquid pressure control device 121, and the third liquid pressure control device 131 are syringe pumps or pressure pumps, respectively, which increase the flow rate of the liquid in the channel. The first liquid pressure control device 111 is connected to the inlet of the focused flow inlet mechanism 110, the second liquid pressure control device 121 is connected to the inlet of the sample flow inlet mechanism 120, and the third liquid pressure control device 131 is connected to the inlet of the sheath fluid inlet mechanism 130.

[0058] It should be noted that Figure 4The first of the three figures from top to bottom shows the normal single cell detection state. If the detected particle is a non-target particle, the program will make a decision to discard the example and the sheath flow channel pressure will not be changed. The second figure shows that when the particle is a target particle, the program increases the sheath flow channel pressure to allow the particle to enter the print port. The third figure shows that after further increasing the sheath flow, the droplet collection channel and waste liquid transmission channel are completely closed. The next detected particle enters the detection area and the flow is stagnant until the particle prints low, at which time the next detection cycle begins. Figure 4 The waste liquid port in the figure represents the waste liquid pool, and the print port represents the droplet collection pool 141. When the sheath liquid injection process is not synchronously pressurized in the focused flow injection structure 110, the sample injection structure, and the sheath liquid injection structure 130, it takes a long time for the gravity of the droplets in the sheath liquid injection structure 130 to be greater than the surface tension and then drip; when the inlet pressure of the sheath liquid injection structure 130 is increased alone, a backflow phenomenon will occur; when the focused flow injection structure 110, the sample flow injection structure 120, and the sheath liquid injection structure 130 are synchronously pressurized, and during the synchronous pressurization process, the flow field distribution is the same as the injection state without synchronous pressurization, but the flow rate of the core channel is close to 0, which means that the next droplet to be processed will stay for a short time, which will not affect the sheath liquid injection process of the current droplet to be processed, and will not cause the loss of the droplet to be processed. After the sheath liquid injection of the current droplet to be processed is completed, the pressure of the sheath liquid injection channel 132 and the core channel pressure are restored to normal for the next sheath liquid selective injection. Among them, Figure 4 The gray liquid in the figure represents the core flow, i.e., the combination of the cell solution and the focused sheath fluid. The black liquid represents the sheath fluid. Both the focused flow injection structure and the sample flow injection structure consist of an inlet and a channel. The core channel is the channel in the focused flow injection structure 110 and the sample flow injection structure 120.

[0059] In the related technologies, microfluidic sorting methods include active sorting methods, methods using fluorescent signals and printing methods based on inkjet printing. Traditional active sorting methods require the introduction of external physical fields such as acousto-optical, electromagnetic and other external physical fields. The cell characteristics that can be expressed by fluorescent signals are limited, and fluorescent staining will cause damage to cells. The sorted cells cannot be used for subsequent clinical applications. External field equipment is usually required, and the external physical field is likely to affect the biological activity of rare cells and increase production and operating costs. Specific flow parameters need to be adjusted, otherwise the systems cannot complete the correct connection work. The inkjet printing-based method is uncontrollable. The continuous inflow of cells during the printing process will affect the printing results. Secondly, continuous extrusion will cause fatigue damage to the device. Finally, the external piezoelectric drive system will increase the sensitivity requirements of the entire system, and will affect integration while increasing manufacturing costs. Therefore, we propose a low-cost, non-invasive, fluid dynamics-based single-cell online sorting and printing microfluidic chip 100, which can realize multi-index sorting and real-time printing of target cell droplets in the flowing sample stream, i.e., the droplets to be processed. The microfluidic chip 100 has the characteristics of high precision, low cost, no pollution, and great application prospects.

[0060] It is understandable that, referring to Figure 1 The microfluidic chip 100 also includes a droplet sorting structure, which includes a droplet collection channel 140 and a waste liquid transmission channel 142. The cell transmission structure 150 is connected to the droplet collection channel 140 and the waste liquid transmission channel 142 respectively. The third liquid pressure control device 131 is also used to generate a sorting pressure according to the identification signal, so that the sorting pressure can drain the target cell droplets to the droplet collection channel 140.

[0061] It should be noted that the microfluidic chip 100 further includes a waste liquid pool 143 and a droplet collection pool 141 . The droplet collection channel 140 is connected to the droplet collection pool 141 , and the waste liquid transmission channel 142 is connected to the waste liquid pool 143 .

[0062] It should be noted that, referring to Figure 3The cell culture medium in the focused flow injection structure 110 encapsulates the cell suspension in the sample flow injection structure 120 at the droplet encapsulation structure to obtain encapsulated cell droplets. The encapsulated cell droplets enter the inlet of the cell transport structure 150 and flow to the single cell recognition zone, where they are identified and an identification signal is generated. The third liquid pressure control device 131 generates an injection pressure based on the identification signal. The injection pressure accelerates the formation of sheath fluid in the sheath fluid injection structure 130 and causes it to drip into the cell transport structure 150, thereby selectively injecting the encapsulated cell droplets and obtaining target cell droplets. The single cell recognition zone is the area of the cell transport structure 150 near the sheath fluid injection structure 130, and the droplet encapsulation structure is the connection point with the inlet of the cell transport structure 150, the channel of the focused flow injection structure 110, and the outlet of the sample flow injection structure 120.

[0063] It should be noted that, referring to Figure 3 and Figure 5 After obtaining the target cell droplet, the target cell droplet is located in the cell transport structure 150 and flows toward the droplet sorting structure. The third liquid pressure control device 131 regenerates the sorting electric field according to the recognition signal. The sorting electric field guides the target cell droplet to the droplet collection channel 140 to complete the sorting of single cell droplets. For example, refer to Figure 1 The droplet collection channel 140 is provided below the waste liquid transmission channel 142. If the droplets to be processed are encapsulated cell droplets according to the identification signal, the third liquid pressure control device 131 reduces the pressure of the sheath liquid injection channel 132, so that the target cell droplets flow into the droplet collection channel 140. After the target cell droplets flow into the droplet collection channel 140, the pressures of the core channel and the sheath liquid injection channel 132 are rapidly increased simultaneously, so that the target cell droplets in the droplet collection channel 140 can flow into the droplet collection pool 141 more quickly. Figure 5 The waste liquid port in the figure represents the waste liquid pool, and the printing port represents the droplet collection pool 141.

[0064] It should be noted that the flow rate in the droplet collection channel 140 is n times the flow rate of the droplet collection channel 140 when not pressurized synchronously (here n corresponds to the case of synchronous pressurization of n times the original pressure).

[0065] It is understandable that the microfluidic chip 100 further includes a flow resistance adjustment structure 151 . The flow resistance adjustment structure 151 is disposed inside the cell transport structure 150 and close to the sheath fluid injection structure 130 .

[0066] It should be noted that by using the flow resistance adjustment structure 151 to increase the flow resistance of the cell transport structure 150, the impact of changes in the flow field on the core flow during the sheath fluid injection process will be reduced, thereby improving the stability of the entire process. Moreover, the addition of this structure will not have a negative impact on the focusing effect and the sorting process. Adding the flow resistance adjustment structure 151 in front of the sheath fluid injection channel 132 to realize the function of a one-way valve can increase the pressure and flow rate of the sheath fluid injection channel 132, further accelerating the formation and dripping of sheath fluid in the sheath fluid injection structure 130. In this embodiment, a multi-stage flow resistance adjustment structure 151 similar to a baffle is embedded in the cell transport structure 150.

[0067] Reference Figure 2 It can be understood that a plurality of distance marks are provided in the cell transport structure 150, and a flow rate region range is formed between two adjacent distance marks in the plurality of distance marks, and the plurality of flow rate region ranges are used to determine the real-time flow rate of the droplets to be processed.

[0068] It should be noted that the multiple flow rate regions are used to obtain the real-time flow rate of the droplets to be processed according to the position information of the droplets to be processed in the cell transport structure 150, such as Figure 2 The figure shows 11 distance markers, forming 10 ROIs (Regions of Interest), which can detect droplets in 10 flow rate ranges. For example, when the ROI area is between 0 and 1, the corresponding flow rate is 3 mm / s, and when the ROI area is between 9 and 10, the corresponding flow rate is 30 mm / s. By adding distance markers, it can be applied to sorting processes with different flow rates (3 mm / s-30 mm / s) by simply adjusting the driving pressure and the position of the detection area. This improves the universality of the entire system, reduces equipment and operating costs, and improves the detection and sorting accuracy of droplets to be processed.

[0069] It should be noted that Figure 2 The arrows in the cell transport structure 150 represent the flow direction of the droplets to be processed, the black area in the middle represents the various flow channels in the microfluidic chip 100, and the conical marks represent distance marks, and there are eleven distance marks in total.

[0070] In this embodiment, real-time image capture of the droplet to be processed is performed in the single-cell identification zone to generate an image to be processed. Based on the image, the cell outline, timestamp, and current location of the droplet to be processed are obtained. The image is then transmitted to an inference engine equipped with a deep learning classifier for target classification, generating an identification signal. This engine performs inference in just 3 milliseconds and simultaneously records the current timestamp and current location of the droplet to be processed. This information is then used to calculate the real-time flow rate of the droplet to be processed. Based on the identification signal and flow rate of the droplet to be processed, instructions are then issued to the focused flow injection mechanism 110, the sample flow injection mechanism 120, and the sheath fluid injection mechanism 130. Based on the received identification signal, if the droplet to be processed is an encapsulated cell droplet, a decision execution time is calculated based on the distance between the sheath fluid transmission channel outlet and the single-cell identification zone, and a sorting decision is made. This causes the third liquid pressure control device 131 to generate an injection pressure, thereby generating a high flow rate to rapidly form sheath fluid, which then drips onto the droplet to be processed, resulting in a target cell droplet. The flow rate refers to the flow velocity of the droplet to be processed. Through this setting, the microfluidic chip 100 can adapt to changes in any internal and external parameters (driving flow rate, channel size, solution viscosity, etc.), and realize the detection and sorting of single cells through automatic calculation. The microfluidic chip 100 uses an active sorting method, but does not introduce an external physical field. It only relies on the principles of fluid mechanics to achieve selective injection and sorting of the droplets to be processed. It has the advantages of low processing difficulty, low cost, low operability, and can be well connected with external equipment, with high integration.

[0071] It should be noted that Figure 6 The first figure in FIG corresponds to a plurality of distance marker figures of the flow velocity measurement area.

[0072] It should be noted that Figure 6 The upper and lower figures in FIG respectively represent a top view and a side view of the microfluidic chip 100. Figure 6 、 Figure 3 The cone marks in the middle indicate distance markers. Figure 6 The inverted cone-shaped structure located at the lower right of the side view represents the waste liquid pool 143 or the droplet collection pool 141.

[0073] In a second aspect, the embodiment of the present application further provides a microparticle sorting system, which includes a control device and a microfluidic chip 100, wherein the control device is used to obtain an image of a droplet to be processed and output an identification signal according to the image to be processed; Figure 2The microfluidic chip 100 includes a focused flow injection structure 110, a sample flow injection structure 120, a cell transport structure 150 and a sheath liquid injection structure 130. A first liquid pressure control device 111 is provided at the inlet of the focused flow injection structure 110. The sample flow injection structure 120 is connected to the focused flow injection structure 110, and a second liquid pressure control device 121 is provided at the inlet of the sample flow injection structure 120. The cell transport structure 150 is respectively connected to the focused flow injection structure 110 and the sample flow injection structure 120. The sheath liquid injection structure 130 is provided on one side of the cell transport structure 150 and is connected to the cell transport structure 150 is connected, and a third liquid pressure control device 131 is provided at the inlet of the sheath liquid injection structure 130. The third liquid pressure control device 131 is used to generate an injection pressure according to the identification signal, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure 130, so as to selectively inject the droplets to be processed in the cell transmission structure 150 and obtain target cell droplets. At the same time, the first liquid pressure control device 111 and the second liquid pressure control device 121 synchronously pressurize the focused flow injection structure 110 and the sample flow injection structure 120, respectively, wherein the droplets to be processed are stored in the cell transmission structure 150.

[0074] It should be noted that if the droplets to be processed are obtained as encapsulated cell droplets according to the identification signal, the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 respectively synchronously pressurize the focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 to generate injection pressure, increase the flow rate of the sheath liquid injection channel 132, thereby generating a large flow rate to quickly form sheath liquid and drip it onto the droplets to be processed to obtain target cell droplets, wherein the target cell droplets are droplets of encapsulated sheath liquid; if the droplets to be processed are obtained as cell-free or target-free encapsulated cell droplets according to the identification signal, the pressure of the sheath liquid injection channel 132 remains unchanged, the third liquid pressure control device 131 does not generate injection pressure, and the sheath liquid will not be injected into the cell-free or target-free cell droplets.

[0075] In the present application, the focused flow injection structure 110 includes an inlet of the focused flow injection structure 110 and a sheath liquid injection channel 132. The first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 receive identification signals obtained based on the droplets to be processed. Accordingly, the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 respectively synchronously pressurize the focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 to generate injection pressure and increase the flow rate of the sheath liquid injection channel 132, thereby generating a large flow rate to quickly form sheath liquid and drip it onto the droplets to be processed to obtain target cell droplets, and also prevent the lateral structure in the cell transmission structure 150 from generating backflow.

[0076] It is understandable that, referring to Figure 2 , the control device includes:

[0077] A microscopic imaging device, the microscopic imaging device is used to obtain an image of the droplet to be processed and output the image to be processed;

[0078] The droplet recognition device is connected to the microscopic imaging device. The droplet recognition device is used to receive the image to be processed and obtain detection information based on the image to be processed. The droplet recognition device is also used to generate an identification signal based on the deep learning neural network algorithm and the detection information, and output the identification signal to the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131.

[0079] In some embodiments, the microscopic imaging device includes a microscope, a camera, and optical components, wherein the optical components include a mercury lamp, a filter, and other optical components for providing fluorescence excitation signals to identify the droplets to be processed; the control device also includes hardware control software, and the computer is used to run the control software and complete related calculations, and coordinate and control the work of various related hardware.

[0080] It should be noted that the detection information includes the cell outline of the droplet to be processed, the timestamp and the current position information of the droplet to be processed.

[0081] Specifically, the droplet recognition device includes a deep learning classifier. Real-time image capture of the droplets to be processed is performed in the single-cell recognition zone to obtain a processed image. Based on the processed image, the cell outline, timestamp, and current location of the droplet to be processed are obtained. The processed image is then transmitted to an inference engine deployed with the deep learning classifier for target classification, generating an identification signal. The engine's inference time is only 3ms. The current timestamp and current location of the droplet to be processed are simultaneously recorded to calculate the real-time flow rate of the droplet to be processed. Based on the identification signal and flow rate of the droplet to be processed, instructions are then issued to the focused flow injection mechanism 110, the sample flow injection mechanism 120, and the sheath fluid injection mechanism 130. Based on the received identification signal, if the droplet to be processed is an encapsulated cell droplet, the decision execution time is calculated based on the distance between the sheath fluid transmission channel outlet and the single-cell recognition zone, and a sorting decision is made, causing the third liquid pressure control mechanism 131 to generate an injection pressure, thereby generating a high flow rate to quickly form sheath fluid, which drips onto the droplet to be processed to obtain the target cell droplet.

[0082] It should be noted that the droplet recognition device of the present application combines a deep learning neural network algorithm to perform multi-indicator (size, shape, color, type, internal structure distribution, etc.) classification detection of droplets to be processed, and is deployed on an inference framework to improve real-time inference efficiency. It is a non-invasive, label-free, and efficient detection and classification method with more promising clinical applications.

[0083] In a third aspect, the present application also provides a method for sorting microparticles, which is applied to the microparticle sorting system as described in any one of the second aspects, referring to Figure 7 , the method includes but is not limited to the following steps:

[0084] Step S100 , the control device acquires an image of the droplet to be processed and outputs a recognition signal according to the image, wherein the droplet to be processed is stored in the cell transmission structure 150 ;

[0085] In step S200, the third liquid pressure control device 131 generates an injection pressure according to the identification signal, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure 130, so as to selectively inject the droplets to be processed in the cell transfer structure 150 and obtain the target cell droplets. At the same time, the first liquid pressure control device 111 and the second liquid pressure control device 121 synchronously pressurize the focused flow injection structure 110 and the sample flow injection structure 120 respectively.

[0086] Specifically, the focused flow injection structure 110 includes an inlet of the focused flow injection structure 110 and a sheath liquid injection channel 132. The first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 receive identification signals obtained based on the droplets to be processed. Accordingly, the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 synchronously pressurize the focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 respectively to generate injection pressure and increase the flow rate of the sheath liquid injection channel 132, thereby generating a very high flow rate to quickly form sheath liquid and drip it onto the droplets to be processed to obtain target cell droplets, and also prevent the lateral structure in the cell transmission structure 150 from generating backflow.

[0087] It should be noted that if the droplets to be processed are obtained as encapsulated cell droplets according to the identification signal, the first liquid pressure control device 111, the second liquid pressure control device 121 and the third liquid pressure control device 131 respectively synchronously pressurize the focused flow injection structure 110, the sample flow injection structure 120 and the sheath liquid injection structure 130 to generate injection pressure, increase the flow rate of the sheath liquid injection channel 132, thereby generating a large flow rate to quickly form sheath liquid and drip it onto the droplets to be processed to obtain target cell droplets, wherein the target cell droplets are droplets of encapsulated sheath liquid; if the droplets to be processed are obtained as cell-free or target-free encapsulated cell droplets according to the identification signal, the pressure of the sheath liquid injection channel 132 remains unchanged, the third liquid pressure control device 131 does not generate injection pressure, and the sheath liquid will not be injected into the cell-free or target-free cell droplets.

[0088] It is understandable that, referring to Figure 1 and Figure 7 The microfluidic chip 100 further includes a droplet sorting structure, the droplet sorting structure includes a droplet collection channel 140 and a waste liquid transmission channel 142, the cell transmission structure 150 is respectively connected to the droplet collection channel 140 and the waste liquid transmission channel 142, and the method further includes:

[0089] In step S300 , the third liquid pressure control device 131 generates a sorting pressure according to the identification signal, so that the sorting pressure guides the target cell droplets to the droplet collection channel 140 .

[0090] It should be noted that the microfluidic chip 100 further includes a waste liquid pool 143 and a droplet collection pool 141 . The droplet collection channel 140 is connected to the droplet collection pool 141 , and the waste liquid transmission channel 142 is connected to the waste liquid pool 143 .

[0091] It should be noted that, referring to Figure 3The cell culture medium in the focused flow injection structure 110 encapsulates the cell suspension in the sample flow injection structure 120 at the droplet encapsulation structure to obtain encapsulated cell droplets. The encapsulated cell droplets enter the inlet of the cell transport structure 150 and flow to the single cell recognition zone, where they are identified and an identification signal is generated. The third liquid pressure control device 131 generates an injection pressure based on the identification signal. The injection pressure accelerates the formation of sheath fluid in the sheath fluid injection structure 130 and causes it to drip into the cell transport structure 150, thereby selectively injecting the encapsulated cell droplets and obtaining target cell droplets. The single cell recognition zone is the area of the cell transport structure 150 near the sheath fluid injection structure 130, and the droplet encapsulation structure is the connection point with the inlet of the cell transport structure 150, the channel of the focused flow injection structure 110, and the outlet of the sample flow injection structure 120.

[0092] It should be noted that, referring to Figure 3 After obtaining the target cell droplets, the target cell droplets are located in the cell transmission structure 150 and flow toward the droplet sorting structure. The third liquid pressure control device 131 regenerates the sorting electric field according to the identification signal. The sorting electric field guides the target cell droplets to the droplet collection channel 140, completing the sorting of single cell droplets.

[0093] In addition, the fourth embodiment of the present application further provides a droplet sorting device, which includes: a memory, a processor, and a computer program stored in the memory and executable on the processor.

[0094] The processor and the memory may be connected via a bus or other means.

[0095] The memory, as a non-transient computer-readable storage medium, can be used to store non-transient software programs and non-transient computer executable programs. In addition, the memory may include a high-speed random access memory and may also include a non-transient memory, such as at least one disk storage device, a flash memory device, or other non-transient solid-state storage device. In some embodiments, the memory may optionally include a memory remotely arranged relative to the processor, and these remote memories may be connected to the processor via a network. Examples of the above-mentioned network include, but are not limited to, the Internet, an intranet, a local area network, a mobile communication network, and combinations thereof.

[0096] The non-transient software program and instructions required to implement the particle sorting method of the third embodiment of the present invention are stored in the memory. When executed by the processor, the particle sorting method of the present invention is executed, for example, the method described above is executed. Figure 7 Method steps S100 to S300 in .

[0097] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate, i.e., they may be located in one location or distributed across multiple network units. Some or all of the modules may be selected based on actual needs to achieve the objectives of this embodiment.

[0098] In addition, an embodiment of the present application further provides a computer-readable storage medium, which stores computer-executable instructions. The computer-executable instructions are executed by a processor or controller, for example, by a processor in the above-mentioned device embodiment, so that the above-mentioned processor can execute the particle sorting method in the above-mentioned embodiment, for example, execute the above-mentioned Figure 7 Method steps S100 to S300 in .

[0099] Those skilled in the art will appreciate that all or some of the steps and systems in the method disclosed above can be implemented as software, firmware, hardware, and appropriate combinations thereof. Some physical components or all physical components can be implemented as software executed by a processor, such as a central processing unit, a digital signal processor, or a microprocessor, or implemented as hardware, or implemented as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, and the computer-readable medium can include computer storage media (or non-transitory media) and communication media (or temporary media). As known to those skilled in the art, the term computer storage media is included in any method or technology for storing information (such as computer-readable instructions, data structures, program modules, or other data) and is volatile and non-volatile, removable, and non-removable. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVD), or other optical disk storage, magnetic cassettes, magnetic tapes, disk storage, or other magnetic storage devices, or any other medium that can be used to store desired information and can be accessed by a computer. Furthermore, as is well known to those skilled in the art, communication media typically embodies computer-readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave or other transport mechanism, and may include any information delivery media.

[0100] It should be noted that the embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the embodiments of the present application are not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the technical field without departing from the purpose of the embodiments of the present application.

[0101] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "particularly," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, illustrative uses of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0102] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and intent of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A microfluidic chip, characterized in that: include: A focused flow injection structure, wherein a first liquid pressure control device is provided at the inlet of the focused flow injection structure; a sample flow injection structure, which is in communication with the focused flow injection structure, and a second liquid pressure control device is provided at the inlet of the sample flow injection structure; a cell transport structure, connected to the focused flow injection structure and the sample flow injection structure respectively; A sheath liquid injection structure is provided on one side of the cell transport structure and is connected to the cell transport structure. A third liquid pressure control device is provided at the inlet of the sheath liquid injection structure. The third liquid pressure control device is used to generate an injection pressure according to an identification signal obtained when the droplet to be processed passes through the area of the cell transport structure, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure, so as to selectively inject the droplets to be processed in the cell transport structure and obtain target cell droplets. At the same time, the first liquid pressure control device synchronously pressurizes the focused flow injection structure and the second liquid pressure control device synchronously pressurizes the sample flow injection structure, so that the next droplet to be processed in the cell transport structure stays briefly.

2. The microfluidic chip according to claim 1, characterized in that The microfluidic chip also includes a droplet sorting structure, which includes a droplet collection channel and a waste liquid transmission channel. The cell transmission structure is connected to the droplet collection channel and the waste liquid transmission channel respectively. The third liquid pressure control device is also used to generate a sorting pressure according to the identification signal, so that the sorting pressure drains the target cell droplets to the droplet collection channel.

3. The microfluidic chip according to claim 1, characterized in that The microfluidic chip further comprises a flow resistance regulating structure, which is arranged inside the cell transmission structure and close to the sheath fluid injection structure.

4. The microfluidic chip according to claim 1, characterized in that A plurality of distance marks are provided in the cell transport structure, and a flow rate region range is formed between two adjacent distance marks in the plurality of distance marks. The plurality of flow rate region ranges are used to determine the real-time flow rate of the droplets to be processed.

5. A microparticle sorting system, characterized in that: include: a control device, configured to obtain an image of the droplet to be processed and output an identification signal according to the image; The microfluidic chip includes a focused flow injection structure, a sample flow injection structure, a cell transport structure and a sheath liquid injection structure. The inlet of the focused flow injection structure is provided with a first liquid pressure control device. The sample flow injection structure is connected to the focused flow injection structure, and the inlet of the sample flow injection structure is provided with a second liquid pressure control device. The cell transport structure is respectively connected to the focused flow injection structure and the sample flow injection structure. The sheath liquid injection structure is provided on one side of the cell transport structure and is connected to the cell transport structure. The inlet of the sheath liquid injection structure is provided with a third liquid. The pressure control device, the third liquid pressure control device is used to generate an injection pressure according to the identification signal obtained when the droplet to be processed passes through the area of the cell transport structure, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure, so as to selectively inject the droplets to be processed in the cell transport structure and obtain target cell droplets. At the same time, the first liquid pressure control device synchronously pressurizes the focused flow injection structure and the second liquid pressure control device synchronously pressurizes the sample flow injection structure, so that the next droplet to be processed in the cell transport structure stays briefly.

6. The microparticle sorting system according to claim 5, characterized in that: The control device comprises: a microscopic imaging device, configured to obtain an image of the droplet to be processed and output the image; A droplet recognition device is connected to the microscopic imaging device, and is used to receive the image to be processed and obtain detection information based on the image to be processed. The droplet recognition device is also used to generate an identification signal based on a deep learning neural network algorithm and the detection information, and output the identification signal to the first liquid pressure control device, the second liquid pressure control device, and the third liquid pressure control device.

7. A microparticle sorting method, applied to the microparticle sorting system according to any one of claims 5 to 6, characterized in that: The method comprises: The control device acquires an image of a droplet to be processed and outputs an identification signal according to the image, wherein the droplet to be processed is stored in the cell transport structure; The third liquid pressure control device generates an injection pressure according to the identification signal, so that the injection pressure accelerates the formation and dripping of the sheath liquid in the sheath liquid injection structure, so as to selectively inject the droplets to be processed in the cell transfer structure and obtain target cell droplets. At the same time, the first liquid pressure control device synchronously pressurizes the focused flow injection structure, and the second liquid pressure control device synchronously pressurizes the sample flow injection structure.

8. The microparticle separation method according to claim 7, characterized in that: The microfluidic chip further includes a droplet sorting structure, the droplet sorting structure including a droplet collection channel and a waste liquid transmission channel, the cell transmission structure being in communication with the droplet collection channel and the waste liquid transmission channel, respectively, and the method further including: The third liquid pressure control device generates a sorting pressure according to the identification signal, so that the sorting pressure guides the target cell droplets to the droplet collection channel.

9. A droplet sorting device, characterized in that: include: A memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the microparticle sorting method according to any one of claims 7 to 8 when executing the computer program.

10. A computer-readable storage medium, characterized in that The computer-readable storage medium stores computer-executable instructions, and the computer-executable instructions are used to enable a computer to execute the microparticle sorting method according to any one of claims 7 to 8.

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